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nyse:bmy
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Juno Therapeutics
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Jun 1st, 2021 12:00AM
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Nov 4th, 2016 12:00AM
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https://www.uspto.gov?id=US11020429-20210601
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Vectors and genetically engineered immune cells expressing metabolic pathway modulators and uses in adoptive cell therapy
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Provided are cells, e.g., engineered immune cells, expressing recombinant or engineered molecules involved in metabolic pathways, such as those that promote or inhibit one or more metabolic steps, reactions, or pathways, for example, in T cells. Such molecules include those that induce or repress a particular functional outcome or metabolic event, for example, one that promotes differentiation or reprogramming into a particular phenotypic state, such as memory, long-lived, activated or activatable, non-exhausted, phenotype or stem-like phenotype. The cells generally further express an immune receptor, such as an antigen receptor, which may be an engineered receptor, such as a CAR or recombinant TCR, or may be a natural immune receptor. Also provided are cells, such as T cells, expressing such molecules and combinations thereof, compositions comprising such cells, nucleic acids such as vectors encoding the same, and methods of administration to subjects in adoptive cell therapy.
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11020429
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1. An engineered immune cell, comprising:
(a) a genetically engineered receptor that specifically binds to a ligand; and
(b) a recombinant molecule or a functional and/or catalytically-active portion or variant thereof, wherein the recombinant molecule is involved in or capable of modulating a metabolic pathway and is under the control of a constitutive promoter, enhancer, or transactivator.
2. The engineered immune cell of claim 1, wherein the metabolic pathway is or comprises triacylglyceride (TAG) synthesis, TAG storage, glycerol phosphate pathway, glycerophospholipid synthesis and/or glycerol uptake.
3. The engineered immune cell of claim 1, wherein the recombinant molecule is or comprises a Glycerol kinase (GYK), a Glycerol-3-phosphate acetyltransferase (GPAT), a Monoacylglycerol O-acetyltransferase (MOGAT), a DAG O-acetyltransferase (DGAT), an acylglycerolphosphate acyltransferase (AGPAT), or a Lipin.
4. The engineered immune cell of claim 3, wherein the molecule is or comprises a GYK, a GPAT1, a MOGAT1, or a DGAT1.
5. The engineered immune cell of claim 1, wherein the recombinant molecule is or comprises an aquaporin-9 (AQP9).
6. The engineered immune cell of claim 1, wherein the recombinant molecule is or comprises a palmitoyltransferase.
7. The engineered immune cell of claim 1, wherein the metabolic pathway comprises oxidative phosphorylation (OXPHOS), generation or accumulation of reactive oxygen species (ROS), cellular respiration, spare respiratory capacity (SPC) and/or mitochondrial respiratory capacity.
8. The engineered immune cell of claim 1,
wherein the recombinant molecule does not promote or enhance glycolysis.
9. The engineered immune cell of claim 1, wherein the recombinant molecule is capable of interacting with or associating with, directly or indirectly, a CR6 interacting factor (CRIF), a lymphocyte expansion molecule (LEM) or a 39S subunit.
10. The engineered immune cell of claim 1, wherein the recombinant molecule is or comprises a lymphocyte expansion molecule (LEM).
11. The engineered immune cell of claim 1, wherein the recombinant molecule is a molecule that is upregulated or activated in response to antigen-receptor signaling, IL-17-mediated signaling, IL-15-mediated signaling, TRAF-mediated signaling, TRAF6-mediated signaling, IL-7-mediated signaling, IL-21-mediated signaling, low-oxygen conditions, succinate, release of reactive oxygen species (ROS), mTOR-induced signaling, or a functional variant thereof.
12. The engineered immune cell of claim 1, wherein the recombinant molecule comprises a hypoxia-induced factor (HIF).
13. The engineered immune cell of claim 12, wherein the recombinant molecule comprises a HIF1-alpha.
14. The engineered immune cell of claim 1, wherein
the recombinant molecule is capable of promoting generation of a glycolysis metabolite.
15. The engineered immune cell of claim 1, wherein the recombinant molecule comprises a phosphoenolpyruvate carboxykinase 1 (PCK1).
16. The engineered immune cell of claim 1, wherein the recombinant molecule is or interacts with GLUT4 or SGK-1.
17. The engineered immune cell of claim 1,
wherein the recombinant molecule is capable of promoting said metabolic pathway; and/or
wherein an outcome of the metabolic pathway is enhanced in the engineered immune cell compared to a reference cell substantially identical to the engineered immune cell, but not expressing the recombinant molecule.
18. The engineered immune cell of claim 1, wherein the recombinant molecule is a nucleic acid or a protein capable of interfering with expression, activity, or stability of a negative regulator of the metabolic pathway, or a molecule that stabilizes the expression or longevity of a molecule that promotes said pathway.
19. The engineered immune cell of claim 17, wherein the recombinant molecule is a nucleic acid and the nucleic acid is an RNAi, siRNA, or shRNA molecule.
20. The engineered immune cell of claim 1, further comprising a disruption in expression or function of an immune checkpoint molecule, wherein the disruption in expression thereby promotes activation, proliferation, expansion, or reduced exhaustion, of the engineered immune cell and/or is capable of reducing generation of or longevity of memory T cells or central memory T cells (TCM).
21. The engineered immune cell of claim 20, wherein the immune checkpoint molecule comprises a PD-1, PD-L1, TIM3, CTLA4 or an adenosine receptor.
22. The engineered immune cell of claim 1, wherein persistence of the engineered immune cell and/or of reprograming in favor of memory T cell, central memory T cell (TCM), T memory stem cells (TSCM), and/or undifferentiated phenotype cells, and/or reduction in exhaustion phenotype, and/or reduction in regulatory T cells, is enhanced or increased in the engineered immune cell as compared to a cell substantially the same as the engineered immune cell not comprising the recombinant molecule, under the same conditions.
23. The engineered immune cell of claim 22, wherein the conditions comprise activation via an antigen receptor, a TCR, an ITAM-containing signaling molecule, cytokine signaling, TNFR signaling, and/or adoptive transfer to a subject containing cells expressing the ligand.
24. The engineered immune cell of claim 1, wherein the cell is a T cell, a natural killer (NK) cell or an iPS-derived cell.
25. The engineered immune cell of claim 24, wherein the cell is a T cell and the T cell is a CD8+ T cell or a CD4+ T cell.
26. The engineered immune cell of claim 1, wherein the genetically engineered receptor that specifically binds to a ligand is a chimeric antigen receptor (CAR).
27. The engineered immune cell of claim 1, wherein the genetically engineered receptor that specifically binds to a ligand is a transgenic T cell receptor (TCR).
28. The engineered immune cell of claim 1, wherein the recombinant molecule is ectopically expressed in the cell.
29. A nucleic acid molecule(s), comprising:
a nucleotide sequence encoding a genetically engineered receptor that specifically binds to a ligand; and
a nucleotide sequence encoding a recombinant molecule that is involved in or capable of modulating a metabolic pathway, or a functional and/or catalytically-active portion or variant thereof.
30. The nucleic acid molecule(s) of claim 29, wherein the genetically engineered receptor that specifically binds to a ligand is a chimeric antigen receptor (CAR).
31. The nucleic acid molecule(s) of claim 29, wherein the genetically engineered receptor that specifically binds to a ligand is a transgenic T cell receptor (TCR).
32. A vector, comprising the nucleic acid molecule(s) of claim 29.
33. An engineered immune cell, comprising the nucleic acid molecules(s) of claim 29.
34. A composition comprising the engineered immune cell of claim 1.
35. A composition comprising the engineered immune cell of claim 33.
36. A method of treatment, comprising administering the engineered immune cell of claim 1 to a subject having a disease or condition.
37. The method of claim 36, wherein the disease or condition is a cancer, a tumor, an autoimmune disease or disorder, or an infectious disease.
38. A method of treatment, comprising administering the engineered immune cell of claim 33 to a subject having a disease or condition.
39. The method of claim 38, wherein the disease or condition is a cancer, a tumor, an autoimmune disease or disorder, or an infectious disease.
40. The engineered immune cell of claim 1, wherein the recombinant molecule does not promote or enhance glycolysis in T cells.
41. The engineered immune cell of claim 1, wherein the recombinant molecule does not promote or enhance glycolysis under conditions under which the recombinant molecule promotes or enhances FAS, FAO, OXPHOS, ROS accumulation or generation, cellular respiration, or respiratory capacity.
42. The engineered immune cell of claim 1, wherein the recombinant molecule is a molecule that is differentially expressed or activated under nutrient-rich versus nutrient-poor conditions, or under hypoxic vs. normoxic conditions, or in effector or vs naive or central memory T cells and/or in exhausted vs non-exhausted T cells, and/or in terminally differentiated T cells vs. non-terminally differentiated T cells.
43. The engineered immune cell of claim 14, wherein the engineered immune cells exhibit increased generation of said glycolysis metabolite compared to reference cells substantially identical to the engineered immune cells but not expressing the recombinant molecule, under the same conditions.
44. The engineered immune cell of claim 1,
wherein the recombinant molecule is capable of inhibiting said metabolic pathway; and/or
wherein an outcome of the metabolic pathway is inhibited or reduced in the engineered immune cell compared to a reference cell substantially identical to the engineered immune cell, but not expressing the recombinant molecule.
45. The engineered immune cell of claim 10, wherein the cell is a T cell.
46. The engineered immune cell of claim 10, wherein the genetically engineered receptor that specifically binds to a ligand is a CAR.
47. The engineered immune cell of claim 46, wherein the cell is a T cell.
48. The engineered immune cell of claim 10, wherein the genetically engineered receptor that specifically binds to a ligand is a TCR.
49. The engineered immune cell of claim 48, wherein the cell is a T cell.
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49
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Stage of International Application No. PCT/US2016/060734 filed Nov. 4, 2016, which claims priority from U.S. provisional application No. 62/251,615 filed Nov. 5, 2015, entitled “Vectors and Genetically Engineered Immune Cells Expressing Metabolic Pathway Modulators and Uses in Adoptive Cell Therapy,” the contents of which are incorporated by reference in its their entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042003700SeqList.txt, created on Apr. 27, 2018, which is 248,039 bytes in size. The information in electronic format of the Sequence Listing is incorporated by reference in its entirety.
FIELD
Provided are cells, e.g., engineered immune cells, expressing recombinant or engineered molecules that promote desirable effects and outcomes upon administration of such cells in adoptive cell therapy, for example, in treatment of tumors. Such molecules generally are molecules involved in metabolic pathways, such as those that promote or inhibit one or more metabolic steps, reactions, or pathways, for example, in T cells. Such molecules include those that induce or repress a particular functional outcome or metabolic event, for example, one that promotes differentiation or reprogramming into a particular phenotypic state, such as memory, long-lived, activated or activatable, non-exhausted, phenotype or stem-like phenotype. The cells generally further express an immune receptor, such as an antigen receptor, which may be an engineered receptor, such as a CAR or recombinant TCR, or may be a natural immune receptor. Also provided are cells, such as T cells, expressing such molecules and combinations thereof, compositions comprising such cells, nucleic acids such as vectors encoding the same, and methods of administration to subjects in adoptive cell therapy.
BACKGROUND
Various strategies are available for producing and administering engineered cells for adoptive therapy. For example, strategies are available for engineering immune cells expressing genetically engineered antigen receptors, such as CARs, and administering compositions containing such cells to subjects. Improved strategies are needed to improve efficacy of the cells, for example, improving the persistence and/or survival of the cells upon administration to subjects. Provided are methods, cells, compositions, kits, and systems that meet such needs.
SUMMARY
Provided are cells, e.g., engineered cells, such as engineered immune cells, generally T cells or NK cells or cells engineered to contain signaling components involved in antigen or target recognition, and/or the delivery of signals thereby, by T cells or NK cells. The cells may be derived from an immune cell compartment such as blood or peripheral blood mononuclear cell (PBMC) sample and optionally selection or enrichment, and/or may be generated via differentiation from less differentiated cells such as HSC or iPSC populations. The cells are generally primary cells from a subject to be treated or a subject of the same species.
The cells generally are engineered to express a recombinant, non-natural, engineered or ectopically expressed molecule, or a functional or catalytically active portion and/or variant of such molecule. The molecule (and/or portion or variant thereof) may be a protein, polypeptide or nucleic acid molecule. In some embodiments, the protein is an enzyme, such as an enzyme that catalyzes one or more metabolic reactions and/or an adapter or other molecule that interacts with or recruits or directs localization of such a molecule. The molecule which plays a role in or interacts with component(s) of one or more metabolic pathways or steps or reactions thereof or events.
The cells generally further include a receptor, such as an antigen receptor or other ligand-binding receptor, such as a natural or endogenously expressed receptor and/or a recombinant or engineered receptor. The receptor may be a TCR or chimeric receptor, such as a chimeric antigen receptor (CAR). The cells may be tumor infiltrating lymphocytes or contain TCRs derived therefrom and optionally modified, e.g., to enhance their recognition or function. In some embodiments, the cells are useful in adoptive cell therapy or other treatment, such as for tumors, cancers, or other proliferative or dysplastic disorders or diseases, and/or infectious disease or autoimmunity.
The molecule (e.g., recombinant, engineered, exogenous or ectopically expressed molecule or variant or portion thereof) in some embodiments is involved with, e.g., promotes or inhibits or otherwise influences, a metabolic event, pathway, reaction, or step, and/or interacts with a molecule involved in such pathway or a metabolite thereof. In some embodiments, the molecule is capable of promoting said metabolic pathway or step or reaction thereof or a metabolic event, directly or indirectly. In some embodiments, the molecule is capable of inhibiting said metabolic pathway. In some embodiments, the molecule is or comprises an enzyme; and/or wherein the recombinant molecule is or comprises an adapter or other molecule that is capable of interacting with a component of the pathway or reaction. The molecule may comprise more than one such molecule, e.g., two or more such molecules, which may impact or be involved with one or more such pathway.
In some embodiments, the metabolic pathway or event comprises lipid metabolism. The molecule is involved in fatty acid synthesis, fatty acid storage and/or fatty acid uptake; and/or the metabolic pathway or event or reaction or step comprises fatty acid uptake, fatty acid synthesis (FAS), and/or fatty acid oxidation (FAO).
In some embodiments, the metabolic pathway or event comprises oxidative phosphorylation (OXPHOS). In some embodiments, it comprises a reactive oxygen species (ROS)-induced signal.
In some embodiments, the pathway or event or step comprises or involves glycolysis or a component or metabolite thereof.
In some embodiments, the pathway or event or step comprises or involves mitochondrial biogenesis.
In some embodiments, the pathway or event or step comprises or involves generation of energy or ATP in a process that occurs within the mitochondria or via mitochondrial proteins, or occurs independently of glucose, or occurs via a pathway other than glycolysis.
In some embodiments, the pathway or event or step comprises or involves the generation of ATP in a glucose-low environment, in a nutrient-poor environment, in a hypoxic environment.
In some embodiments, the pathway or event or step comprises or involves e.g., the molecule influences, e.g., promotes, a catabolic molecular profile, optionally in the engineered cell, as compared to a reference cell substantially similar to the engineered cell but not comprising the recombinant molecule.
In some embodiments, the pathway or event or step comprises or involves, e.g., the molecule influences, e.g., promotes an anabolic molecular profile, optionally in the engineered cell, as compared to a reference cell substantially similar to the engineered cell but not comprising the recombinant molecule.
In some embodiments, the pathway or event or step comprises or involves, e.g., the molecule influences wherein the metabolic pathway or event comprises glutaminolysis, TCA cycle, glucose metabolism, amino acid or nucleotide metabolism, or beta-oxidation.
In some embodiments, the molecule promotes or enhances FAS or FAO or fatty acid storage, and/or TAG synthesis or storage, or glycerol import.
In some embodiments, the pay, event or step or reaction is or comprises triacylglyceride (TAG) synthesis, TAG storage, glycerol phosphate pathway, glycerophospholipid synthesis and/or glycerol uptake.
In some embodiments, the molecule is involved in and/or is capable of promoting glycerol transport, is or comprises a glycerol transporter, is a TAG synthase molecule, is or comprises a glycerol kinase, or is or comprises an acyltransferase.
In some embodiment, the molecule is or comprises a Glycerol kinase (GYK).
In some embodiment, the molecule is or comprises a Glycerol-3-phosphate acetyltransferase mitochondrial (GPAT). In some embodiments, the molecule is or comprises a GPAT1.
In some aspects, the molecule is or comprises a GYK.
In some embodiment, the molecule is or comprises a Monoacylglycerol O-acetyltransferase (MOGAT). In some aspects, it comprises a MOGAT1.
In some embodiment, the molecule is or comprises a DAG O-acetyltransferase (DGAT). In some aspects, the molecule is a DGAT1.
In some embodiment, the molecule is or comprises an acylglycerolphosphate acyltransferase (AGPAT).
In some embodiment, the molecule is or comprises a Lipin. In some aspects, it includes a Lipin 1.
In some embodiments, the molecule is or comprises or interacts with a GPAT2, an AGPAT1, an AGPAT2, an AGPAT3, an AGPAT6, a MOGAT2, a Lipin1, and/or a DGAT2.
In some embodiments, the molecule comprises an amino acid sequence selected from among any of SEQ ID NO: 4, 7, 8 and 9; and a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 4, 7, 8 and 9. In some embodiments, the molecule comprises an amino acid sequence encoded by a nucleotide sequence selected from among any of SEQ ID NO: 76, 77, 78 and 80; and a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 76, 77, 78 and 80 that encodes a functional protein, variant, or fragment thereof.
In some embodiments, the molecule is or comprises a glycerol transporter or functional portion thereof. In some aspects the molecule is or comprises an AQP, such as an APQ9
In some embodiments, the molecule is a molecule that encodes or inhibits or promotes or stabilizes or destabilizes expression or activity any of the foregoing molecules. In some embodiments, it is a molecule that interacts with directly or indirectly, acts downstream of or compensates for, any of the aforementioned molecules.
In some embodiments, the molecule is or comprises a palmitoyltransferase. In some aspects, such a molecule is or comprises a carnitine palmitoyltransferase (CPT), such as CPTI.
In some embodiments, the metabolic pathway or event comprises oxidative phosphorylation (OXPHOS), generation or accumulation of reactive oxygen species (ROS), cellular respiration, spare respiratory capacity (SPC) and/or mitochondrial respiratory capacity.
In some of any of the foregoing embodiments, the molecule does not promote or enhance glycolysis, for example, does not promote or enhance glycolysis in T cells or a particular subtype thereof. In some aspects, the recombinant molecule does not promote or enhance glycolysis under conditions under which the molecule promotes or enhances FAS, FAO, OXPHOS, ROS accumulation or generation, cellular respiration, or respiratory capacity.
In some embodiments, the recombinant molecule is capable of binding to or interacting with an OXPHOS complex protein, is capable of binding to or interacting with a mitochondrial membrane protein, promotes, acts upstream of, or is required for, optionally in lymphocytes, optionally in a population of T cells, the translation and/or insertion of one or more OXPHOS proteins into a mitochondrial membrane.
In some embodiments, the molecule comprises a mitochondrial protein.
The engineered immune cell of any of claims 1-19, wherein the recombinant molecule is capable of interacting with or associating with, directly or indirectly, a CR6 interacting factor (CRIF), optionally CRIF1, a LEM or a 39S subunit, optionally MRPL23.
In some embodiments, the molecule is or comprises a lymphocyte enhancer molecule (LEM).
In some embodiments, the molecule comprises an amino acid sequence set forth in SEQ ID NO:69 or a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:69. In some embodiments, the molecule comprises an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NO:70 or a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:70 that encodes a functional protein, variant, or fragment thereof.
In some embodiments, the molecule is or comprises CRIF, e.g., CRIF1 optionally, CRIF1.
In some embodiments, the molecule is capable of promoting or enhancing the metabolic pathway, reaction or step under conditions of hypoxia, low glucose, poor vascularization, and/or ROS;
In some embodiments, the recombinant molecule is capable of promoting or enhancing the metabolic pathway, reaction or step in the engineered cell under conditions of hypoxia, low glucose, poor vascularization, and/or ROS, at a level increased as compared to a reference cell, substantially identical to the engineered cell but not comprising the molecule.
In some embodiments, the molecule is or comprises NADH ubiquinone oxidoreductase chain (ND), such as ND chain 1 (ND1), a ubiquinol cytochrome c oxidoreductase chain (UQRC), e.g., chain 2 (UQRC2), and/or a cytochrome c oxidoreductase (COX), such as COX1.
In some embodiments, the molecule is involved in or promotes a pathway resulting in the generation of energy or ATP, which optionally is independent of the presence of glucose or independent of glycolysis.
In some embodiments, the metabolic pathway, step, reaction, or event is or comprises a glycolysis pathway, a reaction thereof, and/or a reaction or pathway that metabolizes a metabolite of the glycolysis pathway, optionally under glucose-low, hypoxic, or nutrient-deprived conditions.
In some embodiments, the recombinant molecule is a molecule that is upregulated or activated in response to a particular signal, such as for example, antigen-receptor signaling, IL-17-mediated signaling, IL-15-mediated signaling, TRAF-mediated signaling, TRAF6-mediated signaling, IL-7-mediated signaling, IL-21-mediated signaling, low-oxygen conditions, succinate, release of reactive oxygen species (ROS), mTOR-induced signaling, or a functional variant thereof, and/or is differentially expressed or activated under nutrient-rich versus nutrient-poor conditions, or under hypoxic vs. normoxic conditions, or in effector or vs naïve or central memory T cells and/or in exhausted vs non-exhausted T cells, and/or in terminally differentiated T cells vs. non-terminally differentiated T cells.
In some embodiments, the recombinant molecule comprises a hypoxia-induced factor (HIF), for example, a HIF1-alpha.
In some embodiments, the molecule comprises an amino acid sequence set forth in SEQ ID NO:10 or a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:10. In some embodiments, the molecule comprises an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NO:79 or a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:79 that encodes a functional protein, variant, or fragment thereof.
In some embodiments, the molecule is capable of promoting generation of a glycolysis metabolite, such as PEP.
In some embodiments, the recombinant molecule comprises a phosphoenolpyruvate carboxykinase 1 (PCK1).
In some embodiments, the molecule comprises an amino acid sequence set forth in SEQ ID NO:12 or a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:12. In some embodiments, the molecule comprises an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NO:81 or a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:81 that encodes a functional protein, variant, or fragment thereof.
In some embodiments, the recombinant molecule is or interacts with GLUT4. In some embodiments, the recombinant molecule is or interacts with SGK1.
Also provided are vectors for producing the cells, such as vectors encoding the recombinant receptor and/or the molecule, and combinations of such vectors and compositions containing the same.
In some embodiments, the engineered cells exhibit increased generation of said glycolysis metabolite compared to reference cells substantially identical to the engineered cells but not expressing the recombinant molecule, under the same conditions.
In some embodiments, he molecule is capable of promoting said metabolic pathway, event, or step or reaction; the molecule is capable of inhibiting said metabolic pathway, event, or step or reaction; in some embodiments, the pathway, event or step or reaction is enhanced in the engineered cell compared to a reference cell substantially identical to the engineered cell, but not expressing the recombinant molecule; in some embodiments, the molecule is capable of inhibiting said metabolic pathway, event, or step or reaction.
In some embodiments, an outcome of the metabolic pathway, event, or step is inhibited or reduced in the engineered cell compared to a reference cell substantially identical to the engineered cell, but not expressing the molecule.
In some embodiments, the molecule, e.g. recombinant molecule, is a nucleic acid or protein capable of interfering with expression, activity, or stability of a negative regulator of the metabolic pathway or event or step or reaction, or a molecule that stabilizes the expression or longevity of a molecule that promotes said pathway, event, step or reaction. In some aspects, it is or comprises an RNAi, siRNA, or shRNA molecule.
In some embodiments, the cell further contains a disruption in expression and/or function of an immune checkpoint molecule, wherein the disruption or expression thereby promotes activation, proliferation, expansion, or reduced exhaustion, of the immune cell and/or is capable of reducing generation of or longevity of memory T cells or central memory T cells. The engineered immune cell of claim 31, wherein the checkpoint molecule comprises a PD-1, PD-L1, TIM3, CTLA4 or an adenosine receptor.
In some embodiments, persistence of the engineered cell and/or of reprogramming in favor of memory T cell, central memory T cell, Tscm, and/or undifferentiated phenotype cells, and/or reduction in exhaustion phenotype, and/or reduction in regulatory T cells, is enhanced or increased in the cell as compared to a cell substantially the same as the engineered cell without the recombinant molecule, under the same conditions.
In some embodiments, such conditions comprise one or more of activation via an antigen receptor, such as an artificial or natural receptor, a TCR, an ITAM-containing signaling molecule, cytokine signaling, TNFR signaling, and/or adoptive transfer to a subject containing cells expressing the ligand.
The cells in some embodiments are T cells, such as CD8+ and/or CD4+ T cells. In some embodiments, the molecule is different depending on whether the cell is CD4+ or CD8+. In some embodiments, the composition comprising the cells contains the molecule only in a certain subset of cells, such as in CD8+ T cells and not in CD4+ T cells, for example, for a molecule involved in a pathway known to be important for metabolic signature in CD8+ cells.
The cells generally contain a ligand-binding receptor such as an antigen-receptor, which may be natural or recombinant or engineered or artificially or ectopically expressed. In some embodiments, the ligand-binding receptor is an antigen receptor such as a non-TCR such as a chimeric antigen receptor the ligand-binding receptor and/or is a TCR, such as a functional portion thereof.
In some embodiments, the cells also include or express a recombinant truncated cell surface receptor. In some embodiments, the cell surface receptor is selected from among a modified and/or truncated form of EGFR (tEGFR), ErbB2/HER2/neu (Her2t), CD34 and/or NGFR. In some embodiments, the cell surface receptor comprises an amino acid sequence selected from among: any of SEQ ID NO: 55, 74 and 83; and a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 55, 74 and 83. In some embodiments, the cell surface marker comprises an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NO:74 or a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:74 that encodes a functional protein, variant, or fragment thereof.
Also provided are nucleic acid molecules that encode the recombinant receptor and/or the molecule, and vectors containing said nucleic acid molecules. For example, in some embodiment, the nucleic acid molecule includes the nucleotide sequence set forth in any of SEQ ID NOS: 85, 87, 89, 91, 93, 95 and 97 or a sequence of nucleotides that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to 85, 87, 89, 91, 93, 95 and 97 that encodes a functional protein, variant, or fragment thereof.
In some embodiments, provided herein are a nucleic acid molecule(s), that include a nucleotide sequence encoding a genetically engineered receptor that specifically binds to a ligand; and a nucleotide sequence encoding a molecule that is involved in or capable of modulating a metabolic pathway or a functional and/or catalytically-active portion or variant thereof. In some embodiments, the encoded genetically engineered receptor and encoded molecule involved in or capable of modulating a metabolic pathway are any as present in any of the cells provided herein. In some embodiments, the nucleic acid molecule is a single polynucleotide.
In some embodiments, the nucleic acid molecule further includes at least one promoter operatively linked to control expression of the genetically engineered receptor and the molecule involved in or capable of modulating a metabolic pathway. For example, in some embodiments, the nucleotide sequence encoding the genetically engineered receptor is operatively linked to a first promoter and the nucleotide sequence encoding the molecule involved in or capable of modulating a metabolic pathway is operatively linked to a second promoter, which first and second promoter can be the same or different.
In some embodiments, the nucleotide sequence encoding the genetically engineered receptor and the nucleotide sequence encoding the molecule involved in or capable of modulating a metabolic pathway are separated by an internal ribosome entry site (IRES), a self-cleaving peptide or a peptide that causes ribosome skipping, optionally a T2A, P2A, E2A and/or F2A peptide, and the first and second chimeric receptor are expressed under the control of the same promoter. In some embodiments, the peptide that causes ribosome skipping comprises an amino acid sequence selected from among: any of SEQ ID NO: 54 and 64-68; and a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 54 and 64-68. In some embodiments, nucleotide sequence encoding the peptide that causes ribosome skipping is selected from among a nucleotide sequence set forth in SEQ ID NO:71 and a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:71 that encodes a functional protein, variant, or fragment thereof.
In some embodiments, exemplary encoded molecule that is involved in or capable of modulating a metabolic pathway include an amino acid sequence selected from among: any of SEQ ID NO: 4, 7-10, 12 and 69; and a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 4, 7-10, 12 and 69. In some embodiments, the nucleotide sequence encoding the molecule that is involved in or capable of modulating a metabolic pathway is selected from among: any of SEQ ID NO: 70 and 76-81; and a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 70 and 76-81 that encodes a functional protein, variant, or fragment thereof.
In some embodiments, the nucleic acid molecule can further include a nucleotide sequence encoding a recombinant truncated cell surface receptor. In some embodiments, the cell surface receptor is selected from among a modified and/or truncated form of EGFR (tEGFR), ErbB2/HER2/neu (Her2t), CD34 and/or NGFR, such as those having an amino acid sequence selected from among: any of SEQ ID NO: 55, 74 and 83; and a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 55, 74 and 83. In some embodiments, the nucleotide sequence encoding the cell surface receptor is set forth in SEQ ID NO:74 or is a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:74 that encodes a functional protein, variant, or fragment thereof.
In some embodiments, the nucleotide sequence provided herein encodes an amino acid sequence selected from among: any of SEQ ID NO: 85, 87, 89, 91, 93, 95 and 97; and a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 85, 87, 89, 91, 93, 95 and 97. In some embodiments, the nucleotide sequence is selected from among: any of SEQ ID NO: 84, 86, 88, 90, 92, 94 and 96; and a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 84, 86, 88, 90, 92, 94 and 96 that encodes a functional protein, variant, or fragment thereof.
Also provided herein are vectors that include any of the nucleic acid molecules provided herein. In some embodiments, the vector is a viral vector, such as a retroviral vector, a lentiviral vector or a gammaretroviral vector. Also provided herein are engineered cells, e.g., engineered immune cells, that contain any of the nucleic acid molecules described herein or any of the vectors described herein.
Also provided are compositions containing any of the engineered cells provided herein. In some embodiments, the cells are CD4+ or CD8+ cells. In some embodiments, the cells are CD4+ cells and the composition further comprises CD8+ cells that are genetically engineered with the ligand-binding receptor but do not express the recombinant molecule. In some embodiments, the cells are CD4+ cells and the composition further comprises CD8+ cells that are genetically engineered with the ligand-binding receptor and express the recombinant molecule. In some embodiments, the cells are CD8+ cells and the composition further comprises CD4+ cells that are genetically engineered with the ligand-binding receptor but do not express the recombinant molecule. In some embodiments, the cells are CD8+ cells and the composition further comprises CD4+ cells that are genetically engineered with the ligand-binding receptor and express the recombinant molecule. In some embodiments, the CD4+ cells express a different ligand-binding receptor than the CD8+ cells. In some embodiments, the only difference in the ligand-binding receptor expressed in the CD4+ cell compared to the CD8+ cell is the different costimulatory signaling domain. In some embodiments, the different costimulatory signaling domain is or comprises a cytoplasmic signaling domain of a CD28, a 4-1BB, or an ICOS molecule, or is a functional variant of a signaling portion thereof.
Also provided are methods of treatment that includes administering any of the engineered cells provided or any of the compositions provided herein, to a subject having a disease or condition. In some embodiments, the engineered cells employed in the methods contain ligand-binding receptor that specifically binds to a ligand or antigen associated with the disease or condition. In some embodiments, the disease or condition is a cancer, a tumor, an autoimmune disease or disorder, or an infectious disease.
In some embodiments, the engineered cells exhibit increased or longer expansion and/or persistence in the subject than in a subject administered the same or about the same dosage amount of a reference cell composition. In some embodiments, there is an increase or greater number of memory T cells or a memory T cell subset and/or an increased or longer persistence of memory T cells or a memory T cell subset in the subject derived from the administered the engineered cells compared to the number or persistence of the memory T cells or memory T cell subset in a subject derived from a reference cell composition administered at the same or about the same dosage.
In some embodiments, the memory T cells or memory T cell subset are CD62L+. In some embodiments, the memory T cells or memory T cell subset are central memory T cells (TCM), long-lived memory T cells or T memory stem cells (Tscm). In some embodiments, the memory T cells or memory T cell subset also exhibit a phenotyp that includes CD127+; and/or any one or more of CD45RA+, CD45RO−, CCR7+ and CD27+ and any one or more of t-betlow, IL-7Ra+, CD95+, IL-2Rβ+, CXCR3+ and LFA-1+.
In some embodiments, the memory T cells or memory T cell subset are CD8+.
In some embodiments, the number of memory T cells or a memory T cell subset derived from the administered genetically engineered cells comprises an increase or greater percentage of central memory T cells (Tcm), long-lived memory T cells or T memory stem cells (Tscm) compared to the number of such cells derived from a reference cell composition administered at the same or about the same dosage.
In some embodiments, there is an increase or greater number of non-terminally differentiated T cells in the subject derived from the administered genetically engineered T cells compared to the number of the non-terminally differentiated cells in a subject derived from a reference cell composition administered at the same or about the same dosage amount.
In some embodiments, the genetically engineered cells in the subject derived from the administered genetically engineered cells exhibit an increase in activation or proliferation upon restimulation ex vivo in the presence of a stimulatory agent or agent compared to the activation or proliferation of genetically engineered cells in a subject derived from a reference cell composition administered at the same or about the same dosage when restimulated ex vivo in the presence of the same stimulatory agent or agents. In some embodiments, the stimulatory agent or agents comprise an antigen, an anti-CD3/anti-CD28 antibody or comprises an IL-2, IL-15 and/or IL-7 cytokine. In some embodiments, the increase is at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, or 5-fold.
In some embodiments, there is a decreased or reduced expression of an exhaustion marker genetically engineered cells in the subject derived from the administered genetically engineered T cells compared to the expression of the exhaustion marker in genetically engineered cells in a subject administered the same or about the same dosage amount of a reference cell composition. In some embodiments, the exhaustion marker is selected from among CD244, CD160 and PD-1. In some embodiments, the expression is decreased or reduced 1.2-fold, 1.5-fold, 2.0-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more.
In some embodiments, the increase or decrease is observed or is present within a month, within two months, within six months or within one year of administering the cells.
Also provided are methods for making and using the cells and/or compositions, such as uses in adoptive cell therapy, in a subject in need thereof, such as in a subject having a tumor. The cell therapy may be autologous or allogeneic. In some embodiments, the compositions and/or engineered cells are used in treating a disease or a condition is a subject. In some embodiments, the engineered cells or the engineered cells in the composition contain a ligand-binding receptor that specifically binds to a ligand or antigen associated with the disease or condition. In some embodiments, the disease or condition is a cancer, a tumor, an autoimmune disease or disorder, or an infectious disease. In some embodiments, the cancer or tumor may be a solid tumor and/or a blood tumor such as a leukemia or lymphoma.
Expression of the molecule may be constitutive and/or conditional, e.g., inducible and/or repressible expression, such as inducible upon activation, stress, hypoxia, decreased vasculature, TME, checkpoint molecule upregulation, proliferation, expression of activation marker, low glucose, or other conditions.
DETAILED DESCRIPTION
Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
I. Engineered Cells Expressing Metabolic Pathway-Modulating Molecules
Provided are cells, including immune cells, which cells are engineered to express or contain molecules, such as recombinant, engineered and/or ectopically expressed molecules or a functional and/or catalytically-active portion or variant thereof, which are involved in or capable of modulating, e.g., promoting, inducing, enhancing, inhibiting, preventing, or carrying out or facilitating, a metabolic pathway or event, or a step or reaction thereof, or capable of associating with a metabolite or component of such pathway.
Adoptive cell therapies (including those involving the administration of cells expressing chimeric receptors specific for a disease or disorder of interest, such as chimeric antigen receptors (CARs) and/or other recombinant antigen receptors, as well as other adoptive immune cell and adoptive T cell therapies) can be effective in the treatment of cancer and other diseases and disorders. In certain contexts, available approaches to adoptive cell therapy may not always be entirely satisfactory.
In some contexts, optimal efficacy can depend on the ability of the administered cells to recognize and bind to a target, e.g., target antigen, to traffic, localize to and successfully enter appropriate sites within the subject, tumors, and environments thereof, to become activated, expand, to exert various effector functions, including cytotoxic killing and secretion of various factors such as cytokines, to persist, including long-term, to differentiate, transition or engage in reprogramming into certain phenotypic states (such as effector, long-lived memory, less-differentiated, and effector states), to provide effective, robust, and rapid recall responses, e.g., following clearance, rest, and subsequent re-exposure to target ligand or antigen, and avoid or reduce exhaustion, anergy, terminal differentiation, and/or differentiation into a suppressive state.
Maximizing exposure (including by improving expansion and persistence) of the cell product in the subject, disease, or condition can improve efficacy and therapeutic outcomes. For example, greater and/or longer degree of exposure to the CAR-expressing cells can improve treatment outcomes, including patient survival and remission, even in individuals with severe or significant tumor burden. Nonetheless, exposure can be limited by host immune responses against the recombinant receptors expressed by the administered cells, which may prematurely eliminate the cells. Once such a host immune response develops, either acquired or innate, it may not be feasible or effective to attempt to increase exposure or provide retreatment of subjects by administering a subsequent dose of cells expressing the same recombinant receptor. Once such an immune response has developed against the receptor, administration of such a second or subsequent dose of cells expressing the same receptor or one with similar immunogenic epitopes may result in rapid elimination of the cells before they have had a chance to expand and/or persist to an effective or substantial degree. Provided are embodiments that address these challenges.
In the context of certain diseases such as tumors, particularly solid tumors, optimal efficacy can further depend on the cells' ability to achieve such outcomes even in the context of various environmental factors in locations such as in locations other than immune organs and/or within environments or microenvironments of certain tissues, such as within a tumor microenvironment (TME), which are often unfavorable.
Such environmental factors and conditions can include various metabolic and immunosuppressive signatures, biosensors, and conditions, including nutrient-poor, immunosuppressive, and hypoxic conditions, increased expression of immune checkpoint molecules, increased numbers of regulatory cells, altered availability of certain various metabolites, growth factors, and nutrients (such as lactate, which may be increased in the context of hypoxic conditions, glucose, amino acids (e.g., decreased in the context of amino acid starvation), nucleic acids, acetyl-CoA and NAD+, citrate, post-translational modification, reactive oxygen species (ROS), ATP, lipids, glycogen), increased expression of amino acid-catabolic enzymes such as IDO, TDO, ARG-1, depletion of certain amino acids, poor vascularization, increased or decreased levels of inflammatory cells, cytokines and other factors.
Alterations in metabolism and metabolic pathways and signatures can impact the ability of T cells to properly engage in activation, proliferation, differentiation, homeostasis, persistence, dedifferentiation, and transition or reprogramming into various desirable states such as long-lived memory and undifferentiated populations, such as central memory T (TCM) cells and T memory stem cells (TSCM) cells, and the ability to avoid terminal differentiation, anergy, and exhaustion. In addition to adopting different cell surface expression and other phenotypic profiles (e.g., different expression levels of various activation, differentiation and checkpoint markers and differing levels of secretion of various factors), as T cells at different phenotypic states exhibit distinct metabolic profiles. See, e.g., Wang & Green, 2012 Nature Immunology 13(10) 907-15; Mockler et al., 2012, Fronteirs in Oncology, 4(107), Buck et al., 2015 J. Exp. Med., 212(9) 1345-60, O'Sullivan & Pearce, 2014 Trends in Immunology, 36(2) 71-80.
For example, in some contexts, memory, e.g., CD8+, long-lived, and/or central memory T cells, as compared to other subtypes, memory T cells, exhibit distinct metabolic signatures and/or engage in different responses to various metabolism-related signals and conditions. Such memory cells generally differ in the pathways preferentially involved in the generation of ATP and/or energy. The same may be true in comparing other subtypes. For example, whereas naïve T cells generally do not exhibit considerable net growth and thus do not engage in substantial nutrient uptake or glycolysis, upon activation, activated and effector T cells generally grow considerably and exhibit increased nutrient uptake and glycolysis. Exhaustion or anergy, on the other hand, may be linked to an inability to take up certain nutrients and/or to enter glycolysis, steps thereof, or other energy-generating metabolic events or pathways.
Memory cells, and particularly long-lived and less differentiated memory phenotype cells, generally do not engage in aerobic glycolysis as highly as other subtypes, and instead may preferentially rely on other pathways as primary sources of energy, such as those carried out within mitochondria, e.g., OXPHOS.
They may also drive such pathways in part via lipids produced by their increased catabolism of intracellular fatty acids, via fatty acid oxidation (FAO), in the mitochondria. Such cells in some contexts engage in a comparatively high level of biosynthesis of lipids for use in such reactions, e.g., fatty acid synthesis (FAS), such as events generally observed in adipose cells, such as triacylglyceride (TAG) synthesis. This process may be driven in some contexts via signals that promote importation of glycerol into the mitochondria, mitochondrial biosynthesis, or other components of such pathways. In some contexts, such events are induced by signals known to promote memory- or long-lived phenotypes, such as certain cytokines and other stimuli. Memory T cells may also maintain substantial spare respiratory capacity (SRC) and have increased mitochondrial mass, both of which confer a metabolic advantage for survival and recall following antigenic challenge. The molecule in some embodiments is involved with or interacts with one or more components or metabolites of a lipid metabolism pathway or step or reaction thereof, for example, lipid synthesis, lipid oxidation, and/or lipid storage. For example, it may include fatty acid uptake, fatty acid synthesis (FAS), and/or fatty acid oxidation (FAO). In some embodiments, the metabolic pathway, event, step or reaction is or comprises triacylglyceride (TAG) synthesis, TAG storage, glycerol phosphate pathway, glycerophospholipid synthesis and/or glycerol uptake.
A mutation stabilizing a molecule, lymphocyte activating molecule (LEM) that associated and/or promoted OXPHOS outcomes in T cells was shown to enhance both expansion without terminal differentiation/exhaustion and the emergence and/or persistence of functional CD8+ memory cells, which exhibited functional recall responses, e.g., following clearance and rechallenge with antigen. Okoye et al. Science. 2015 May 29; 348(6238):995-1001. In some embodiments, the provided molecule is a molecule involved in OXPHOS or related pathways or molecules. T cell subsets and different T cell activation/differentiation states also differ in their ability to respond or the types of responses exhibited in environments of particular metabolite levels, such as those observed in a tumor microenvironment, e.g., hypoxia, nutrient deprivation, glucose-poor condition, or poor vasculature. The ability to reprogram or differentiate into one or more T cell phenotypic states may also depend upon the ability to react or react in a desirable way to such conditions. For example, the inability to induce glycolysis or generation of glycolysis metabolites can result in exhaustion or anergy in response to stimulation through the TCR. For example, deficiencies in pathways involving hypoxia-induced factors, such as HIF1-alpha-related pathways and components thereof, have been linked to preference towards an exhausted phenotype in nutrient-poor or hypoxic conditions, whereas expression of certain molecules involved in such pathways can inhibit exhaustion and promote persistent expansion and survival of effector cells, rather than terminal differentiation. Doedens et al. Nat Immunol. 2013 November; 14(11):1173-82. In some embodiments, the molecule is involved with or interacts with a molecule involved with a pathway or metabolic signature induced by or that compensates for hypoxic environment.
Glucose-depravation, such as in the TME, can dampen glycolysis and thereby limit the ability of T cells to effectively respond, proliferate, and adopt effector phenotypes in response to antigen. Insufficient levels of the glycolysis metabolite phosphoenolpyruvate (PEP) can lead to insufficient NFAT and calcium signaling. Expression of molecules involved in promoting the generation of such metabolites such as phosphoenolpyruvate carboxykinase 1 (PCK1) can rescue such events and avoid exhaustion. See Ho et al. Cell. 2015 Sep. 10; 162(6):1217-28. Signals received via the antigen receptor and other signaling molecules such as cytokines and costimulatory molecules can promote pathways that allow these events even in such poor conditions. Such signals can be dampened or prevented by signaling through certain immune checkpoint molecules such as PD-1 (e.g., amino acid sequence set forth in SEQ ID NO:26, nucleic acid sequence set forth in SEQ ID NO:47). Yet the deletion or genetic absence of PD-1 or other such molecules in T cells in vivo may in some contexts—despite preventing an exhausted phenotype and permitting primary expansion and activation and other effector functions—may also negatively impact the long-lived memory compartment, either by preventing reprogramming or persistence. In some embodiments, provided are cells expressing molecules that inhibit exhaustion and promote expansion, while also enhancing or promoting persistence and the generation of functional long-lived memory cells and survival thereof. In some embodiments, the molecule is involved with or interacts with component(s) of glycolysis or PEP-mediated signaling or generation or pathways induced, e.g., in response to glucose-depravation.
The metabolic pathways and events and reactions may include one or more of a lipid metabolism pathway or step or reaction thereof, for example, lipid synthesis, lipid oxidation, and/or lipid storage. For example, it may include fatty acid uptake, fatty acid synthesis (FAS), and/or fatty acid oxidation (FAO). In some embodiments, the metabolic pathway, event, step or reaction is or comprises triacylglyceride (TAG) synthesis, TAG storage, glycerol phosphate pathway, glycerophospholipid synthesis and/or glycerol uptake.
In some embodiments, the metabolic pathway or event is or comprises lipid metabolism, oxidative phosphorylation (OXPHOS), a reactive oxygen species (ROS)-induced signal, glycolysis, or mitochondrial biogenesis. In some aspects, the metabolic pathway or event is or comprises generation of energy or ATP in a process that occurs within the mitochondria or via mitochondrial proteins, or occurs independently of glucose or via a pathway other than glycolysis. In some embodiments, the metabolic pathway, step, reaction, or event is or comprises a glycolysis pathway, a reaction thereof, or a reaction or pathway that metabolizes a metabolite of the glycolysis pathway, such as under glucose-low, hypoxic, or nutrient-deprived conditions. In some aspects, the metabolic pathway or event is or comprises the generation of ATP in a glucose-low environment, in a nutrient-poor environment, or in a hypoxic environment. In some embodiments, the metabolic pathway or event comprises glutaminolysis, TCA cycle, glucose metabolism, amino acid or nucleotide metabolism, or beta-oxidation.
In some embodiments, the metabolic pathway or event, reaction or step comprises oxidative phosphorylation (OXPHOS), generation or accumulation of reactive oxygen species (ROS), cellular respiration, spare respiratory capacity (SPC) or mitochondrial respiratory capacity.
In some embodiments, the molecule is capable of promoting the metabolic pathway or step or reaction thereof or a metabolic event, directly or indirectly. In some aspects, the molecule is capable of inhibiting the metabolic pathway. In some embodiments, the molecule promotes a catabolic molecular profile. In some aspects, the molecule promotes an anabolic molecular profile. In some embodiments, the molecule is not directly involved in the pathway but is capable of associating with a component thereof.
In some embodiments, the molecule is involved in or promotes a pathway resulting in the generation of energy or ATP, which may be independent of the presence of glucose or independent of glycolysis. In some embodiments, the molecule does not promote or enhance glycolysis, does not promote or enhance glycolysis in T cells, or does not promote or enhance glycolysis under conditions under which the molecule promotes or enhances FAS, FAO, OXPHOS, ROS accumulation or generation, cellular respiration, or respiratory capacity. In some embodiments, the molecule is capable of binding to or interacting with an OXPHOS complex protein or is capable of binding to or interacting with a mitochondrial membrane protein. In some aspects, the molecule promotes, acts upstream of, or is required for, such as in lymphocytes or a population of T cells, the translation or insertion of one or more OXPHOS proteins into a mitochondrial membrane. In some embodiments, the molecule is capable of promoting or enhancing the metabolic pathway, reaction or step under conditions of hypoxia, low glucose, poor vascularization, or ROS. In some embodiments, the molecule is capable of promoting generation of a glycolysis metabolite, such as PEP.
In some embodiments, the molecule is or comprises an enzyme. In some aspects, the molecule is or comprises an adapter. In some embodiments, the molecule is involved in fatty acid synthesis, fatty acid storage or fatty acid uptake. In some embodiments, the molecule promotes or enhances FAS or FAO. In some embodiments, the molecule is involved in or is capable of promoting glycerol transport, is or comprises a glycerol transporter, a TAG synthase molecule, a glycerol kinase, or an acyltransferase.
In some embodiments, the molecule comprises a mitochondrial protein. In some embodiments, the molecule is or comprises a Glycerol kinase (GYK), a Glycerol-3-phosphate acetyltransferase mitochondrial (GPAT), a Monoacylglycerol O-acetyltransferase (MOGAT), a DAG O-acetyltransferase (DGAT), an acylglycerolphosphate acyltransferase (AGPAT), or a Lipin. In some aspects, the molecule is any described in Takeuchi et al. Am J Physiol Endocrinol Metab. 2009 June; 296(6): E1195-E1209. In some embodiments, the molecule is or comprises a glycerol transporter or functional portion thereof. In some embodiments, the molecule is or comprises a palmitoyltransferase, such as carnitine palmitoyltransferase (CPT), such as CPTI. In some embodiments, the molecule is or comprises NADH ubiquinone oxidoreductase chain (ND), such as ND chain 1 (ND1), a ubiquinol cytochrome c oxidoreductase chain (UQRC), e.g., chain 2 (UQRC2), or a cytochrome c oxidoreductase (COX), such as COX1.
In some embodiments, molecule is capable of interacting with or associating with, directly or indirectly, a CR6 interacting factor (CRIF), a LEM or a 39S subunit, such as MRPL23. In some embodiments, the molecule in the metabolic pathway is or comprises lymphocyte expansion molecule (LEM), glycerol kinase (Gyk), diacylglycerol O-acetyltransferase 1 (DGAT1) or 2 (DGAT2), glycerol-3-phosphate acetyltransferase mitochondrial (GPAM), monoacylglycerol O-acetyltransferase 1 (MOGAT1) or 2 (MOGAT2), hypoxia-inducible factor alpha (HIF1α), PCK1, AGPAT1, AGPAT2, AGPAT3, AGPAT 6 (LPA-MAG PA), GPAT1, GPAT2, AQP9, LPIN1, or CRIF1.
II. Recombinant Receptors
In some embodiments, the provided cells express and/or are engineered to express receptors, such as recombinant receptors, including those containing ligand-binding domains or binding fragments thereof, and T cell receptors (TCRs) and components thereof, and/or functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). In some embodiments, the recombinant receptor contains an extracellular ligand-binding domain that specifically binds to an antigen. In some embodiments, the recombinant receptor is a CAR that contains an extracellular antigen-recognition domain that specifically binds to an antigen. In some embodiments, the ligand, such as an antigen, is a protein expressed on the surface of cells. In some embodiments, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which, like a TCR, is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule.
Exemplary recombinant receptors, including CARs and recombinant TCRs, as well as methods for engineering and introducing the receptors into cells, include those described, for example, in international patent application publication numbers WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 Mar. 18(2): 160-75. In some aspects, the genetically engineered antigen receptors include a CAR as described in U.S. Pat. No. 7,446,190, and those described in International Patent Application Publication No.: WO/2014055668 A1.
The recombinant receptor, such as a CAR, generally include an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some aspects via linkers and/or transmembrane domain(s). Such molecules typically mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone.
A. Ligand-Binding Domain
In some embodiments, the recombinant receptor includes a ligand-binding domain that binds, such as specifically binds, to an antigen (or a ligand). Among the antigens targeted by the recombinant receptors are those expressed in the context of a disease, condition, or cell type to be targeted via the adoptive cell therapy. In some embodiments, the disease or condition is a tumor, such as a solid tumor, lymphoma, leukemia, blood tumor, metastatic tumor, or other cancer or tumor type. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and/or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas.
In some embodiments, the disease or condition is an infectious disease or condition, such as, but not limited to, viral, retroviral, bacterial, and protozoal infections, immunodeficiency, Cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus. In some embodiments, the disease or condition is an autoimmune or inflammatory disease or condition, such as arthritis, e.g., rheumatoid arthritis (RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Grave's disease, Crohn's disease, multiple sclerosis, asthma, and/or a disease or condition associated with transplant.
In some embodiments, the antigen (or a ligand) is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen (or a ligand) is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on the engineered cells.
In some embodiments, the antigen associated with the disease or disorder is selected from the group consisting of orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, BCMA, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, fetal acethycholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1), a cyclin, such as cyclin A1 (CCNA1), and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens.
In some embodiments, the antigen is a pathogen-specific antigen. In some embodiments, the antigen is a viral antigen (such as a viral antigen from HIV, HCV, HBV, etc.), bacterial antigens, and/or parasitic antigens.
1. Antigen Receptor
In some embodiments, the recombinant receptor includes a CAR. In some embodiments, the CAR is constructed with a specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type to be targeted by adoptive therapy, e.g., a cancer marker, and/or an antigen intended to induce a dampening response, such as an antigen expressed on a normal or non-diseased cell type. Thus, the CAR typically includes in its extracellular portion one or more antigen binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable domains, and/or antibody molecules. In some embodiments, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).
The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab′)2 fragments, Fab′ fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
In some embodiments, the antigen-binding proteins, antibodies and antigen binding fragments thereof specifically recognize an antigen of a full-length antibody. In some embodiments, the heavy and light chains of an antibody can be full-length or can be an antigen-binding portion (a Fab, F(ab′)2, Fv or a single chain Fv fragment (scFv)). In other embodiments, the antibody heavy chain constant region is chosen from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly chosen from, e.g., IgG1, IgG2, IgG3, and IgG4, more particularly, IgG1 (e.g., human IgG1). In another embodiment, the antibody light chain constant region is chosen from, e.g., kappa or lambda, particularly kappa.
Among the provided antibodies are antibody fragments. An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; variable heavy chain (VH) regions, single-chain antibody molecules such as scFvs and single-domain VH single antibodies; and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody. In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds the antigen, such as a cancer marker or cell surface antigen of a cell or disease to be targeted, such as a tumor cell or a cancer cell, such as any of the target antigens described herein or known in the art.
Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some aspects, the antibody fragments are scFvs.
A “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
In some embodiments, the CAR contains an antibody or an antigen-binding fragment (e.g. scFv) that specifically recognizes an antigen, such as an intact antigen, expressed on the surface of a cell.
In some embodiments, the CAR contains a TCR-like antibody, such as an antibody or an antigen-binding fragment (e.g. scFv) that specifically recognizes an intracellular antigen, such as a tumor-associated antigen, presented on the cell surface as a MHC-peptide complex. In some embodiments, an antibody or antigen-binding portion thereof that recognizes an MHC-peptide complex can be expressed on cells as part of a recombinant receptor, such as an antigen receptor. Among the antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Generally, a CAR containing an antibody or antigen-binding fragment that exhibits TCR-like specificity directed against peptide-MHC complexes also may be referred to as a TCR-like CAR.
Reference to “Major histocompatibility complex” (MHC) refers to a protein, generally a glycoprotein, that contains a polymorphic peptide binding site or binding groove that can, in some cases, complex with peptide antigens of polypeptides, including peptide antigens processed by the cell machinery. In some cases, MHC molecules can be displayed or expressed on the cell surface, including as a complex with peptide, i.e. MHC-peptide complex, for presentation of an antigen in a conformation recognizable by an antigen receptor on T cells, such as a TCRs or TCR-like antibody. Generally, MHC class I molecules are heterodimers having a membrane spanning a chain, in some cases with three a domains, and a non-covalently associated (32 microglobulin. Generally, MHC class II molecules are composed of two transmembrane glycoproteins, a and (3, both of which typically span the membrane. An MHC molecule can include an effective portion of an MHC that contains an antigen binding site or sites for binding a peptide and the sequences necessary for recognition by the appropriate antigen receptor. In some embodiments, MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where a MHC-peptide complex is recognized by T cells, such as generally CD8+ T cells, but in some cases CD4+ T cells. In some embodiments, MHC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are typically recognized by CD4+ T cells. Generally, MHC molecules are encoded by a group of linked loci, which are collectively termed H-2 in the mouse and human leukocyte antigen (HLA) in humans. Hence, typically human MHC can also be referred to as human leukocyte antigen (HLA).
The term “MHC-peptide complex” or “peptide-MHC complex” or variations thereof, refers to a complex or association of a peptide antigen and an MHC molecule, such as, generally, by non-covalent interactions of the peptide in the binding groove or cleft of the MHC molecule. In some embodiments, the MHC-peptide complex is present or displayed on the surface of cells. In some embodiments, the MHC-peptide complex can be specifically recognized by an antigen receptor, such as a TCR, TCR-like CAR or antigen-binding portions thereof.
The term “peptide antigen” or “peptide epitope” refers to a peptide of a polypeptide that can associate with an MHC molecule, such as for recognition by an antigen receptor. Generally, the peptide is derived from or based on a fragment of a longer biological molecule, such as a polypeptide or protein. In some embodiments, the peptide typically is about 8 to about 24 amino acids in length. In some embodiments, a peptide has a length of from or from about 9 to 22 amino acids for recognition in the MHC Class II complex. In some embodiments, a peptide has a length of from or from about 8 to 13 amino acids for recognition in the MHC Class I complex. In some embodiments, upon recognition of the peptide in the context of an MHC molecule, such as MHC-peptide complex, the antigen receptor, such as TCR or TCR-like CAR, produces or triggers an activation signal to the T cell that induces a T cell response, such as T cell proliferation, cytokine production, a cytotoxic T cell response or other response.
In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to a MHC-peptide complex, can be produced by immunizing a host with an effective amount of an immunogen containing a specific MHC-peptide complex. In some cases, the peptide of the MHC-peptide complex is an epitope of antigen capable of binding to the MHC, such as a tumor antigen, for example a universal tumor antigen, myeloma antigen or other antigen as described below. In some embodiments, an effective amount of the immunogen is then administered to a host for eliciting an immune response, wherein the immunogen retains a three-dimensional form thereof for a period of time sufficient to elicit an immune response against the three-dimensional presentation of the peptide in the binding groove of the MHC molecule. Serum collected from the host is then assayed to determine if desired antibodies that recognize a three-dimensional presentation of the peptide in the binding groove of the MHC molecule is being produced. In some embodiments, the produced antibodies can be assessed to confirm that the antibody can differentiate the MHC-peptide complex from the MHC molecule alone, the peptide of interest alone, and a complex of MHC and irrelevant peptide. The desired antibodies can then be isolated.
In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to an MHC-peptide complex can be produced by employing antibody library display methods, such as phage antibody libraries. In some embodiments, phage display libraries of mutant Fab, scFV or other antibody forms can be generated, for example, in which members of the library are mutated at one or more residues of a CDR or CDRs. Exemplary of such methods are known in the art (see e.g. US published application No. US20020150914, US2014/0294841; and Cohen C J. et al. (2003) J Mol. Recogn. 16:324-332).
2. TCR
In some embodiments, the recombinant receptors include recombinant T cell receptors (TCRs) and/or TCRs cloned from naturally occurring T cells.
A “T cell receptor” or “TCR” refers to a molecule that contains a variable α and β chains (also known as TCRα and TCRβ, respectively) or a variable γ and δ chains (also known as TCRγ and TCRδ, respectively) and that is capable of specifically binding to an antigen peptide bound to a MHC receptor. In some embodiments, the TCR is in the αβ form. Typically, TCRs that exist in αβ and γδ forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions. A TCR can be found on the surface of a cell or in soluble form. Generally, a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, a TCR also can contain a constant domain, a transmembrane domain and/or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997). For example, in some aspects, each chain of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end. In some embodiments, a TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. Unless otherwise stated, the term “TCR” should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full-length TCRs, including TCRs in the αβ form or γδ form.
Thus, for purposes herein, reference to a TCR includes any TCR or functional fragment, such as an antigen-binding portion of a TCR that binds to a specific antigenic peptide bound in an MHC molecule, i.e. MHC-peptide complex. An “antigen-binding portion” or antigen-binding fragment” of a TCR, which can be used interchangeably, refers to a molecule that contains a portion of the structural domains of a TCR, but that binds the antigen (e.g. MHC-peptide complex) to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable α chain and variable β chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex, such as generally where each chain contains three complementarity determining regions.
In some embodiments, the variable domains of the TCR chains associate to form loops, or complementarity determining regions (CDRs) analogous to immunoglobulins, which confer antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule and determine peptide specificity. Typically, like immunoglobulins, the CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the main CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the beta chain interacts with the C-terminal part of the peptide. CDR2 is thought to recognize the MHC molecule. In some embodiments, the variable region of the β-chain can contain a further hypervariability (HV4) region.
In some embodiments, the TCR chains contain a constant domain. For example, like immunoglobulins, the extracellular portion of TCR chains (e.g., α-chain, β-chain) can contain two immunoglobulin domains, a variable domain (e.g., Vα, or Vβ; typically amino acids 1 to 116 based on Kabat numbering Kabat et al., “Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.) at the N-terminus, and one constant domain (e.g., α-chain constant domain or Cα, typically amino acids 117 to 259 based on Kabat, β-chain constant domain or Cβ, typically amino acids 117 to 295 based on Kabat) adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains, and two membrane-distal variable domains containing CDRs. The constant domain of the TCR domain contains short connecting sequences in which a cysteine residue forms a disulfide bond, making a link between the two chains. In some embodiments, a TCR may have an additional cysteine residue in each of the α and β chains such that the TCR contains two disulfide bonds in the constant domains.
In some embodiments, the TCR chains can contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chains contain a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules like CD3. For example, a TCR containing constant domains with a transmembrane region can anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex.
Generally, CD3 is a multi-protein complex that can possess three distinct chains (γ, δ, and ε) in mammals and the ζ-chain. For example, in mammals the complex can contain a CD3γ chain, a CD3δ chain, two CD3ε chains, and a homodimer of CD3ζ chains. The CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T cell receptor chains. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, whereas each CD3ζ chain has three. Generally, ITAMs are involved in the signaling capacity of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in propagating the signal from the TCR into the cell. The CD3- and ζ-chains, together with the TCR, form what is known as the T cell receptor complex.
In some embodiments, the TCR may be a heterodimer of two chains α and β (or optionally γ and δ) or it may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (α and β chains or γ and δ chains) that are linked, such as by a disulfide bond or disulfide bonds.
In some embodiments, a TCR for a target antigen (e.g., a cancer antigen) is identified and introduced into the cells. In some embodiments, nucleic acid encoding the TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological source, such as from cells such as from a T cell (e.g. cytotoxic T cell), T-cell hybridomas or other publicly available source. In some embodiments, the T-cells can be obtained from in vivo isolated cells. In some embodiments, a high-affinity T cell clone can be isolated from a patient, and the TCR isolated. In some embodiments, the T-cells can be a cultured T-cell hybridoma or clone. In some embodiments, the TCR clone for a target antigen has been generated in transgenic mice engineered with human immune system genes (e.g., the human leukocyte antigen system, or HLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al. (2009) Clin Cancer Res. 15:169-180 and Cohen et al. (2005) J Immunol. 175:5799-5808. In some embodiments, phage display is used to isolate TCRs against a target antigen (see, e.g., Varela-Rohena et al. (2008) Nat Med. 14:1390-1395 and Li (2005) Nat Biotechnol. 23:349-354. In some embodiments, the TCR or antigen-binding portion thereof can be synthetically generated from knowledge of the sequence of the TCR.
In some embodiments, after the T-cell clone is obtained, the TCR alpha and beta chains are isolated and cloned into a gene expression vector. In some embodiments, the TCR alpha and beta genes are linked via a picornavirus 2A ribosomal skip peptide so that both chains are coexpressed. In some embodiments, genetic transfer of the TCR is accomplished via retroviral or lentiviral vectors, or via transposons (see, e.g., Baum et al. (2006) Molecular Therapy: The Journal of the American Society of Gene Therapy. 13:1050-1063; Frecha et al. (2010) Molecular Therapy: The Journal of the American Society of Gene Therapy. 18:1748-1757; an Hackett et al. (2010) Molecular Therapy: The Journal of the American Society of Gene Therapy. 18:674-683.
B. Intracellular Signaling Domain
In some aspects, the ligand-binding domain, such as an antigen-specific binding, or recognition component is linked to one or more transmembrane and intracellular signaling domains.
In some embodiments, the recombinant receptor, such as a CAR, such as the antibody portion thereof, further includes a spacer, which may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region, e.g., an IgG4 hinge region, and/or a CH1/CL and/or Fc region. In some aspects, the portion of the constant region serves as a spacer region between the ligand-binding domain, such as the antigen-recognition component, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. In some examples, the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, international patent application publication number WO2014031687, U.S. Pat. No. 8,822,647 or published app. No. US2014/0271635.
In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgG1. In some embodiments, the spacer has the sequence ESKYGPPCPPCP (set forth in SEQ ID NO: 49), and is encoded by the sequence set forth in SEQ ID NO: 50. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 51. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 52. In some embodiments, the constant region or portion is of IgD. In some embodiments, the spacer has the sequence set forth in SEQ ID NO:53. In some embodiments, the spacer has a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 49, 51, 52 or 53.
The ligand-binding domain, such as antigen recognition domain, generally is linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and/or signal via another cell surface receptor. Thus, in some embodiments, the ligand-binding domain (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154, and/or transmembrane regions containing functional variants thereof such as those retaining a substantial portion of the structural, e.g., transmembrane, properties thereof. In some embodiments, the transmembrane domain is a transmembrane domain derived from CD4, CD28, or CD8, e.g., CD8alpha, or functional variant thereof. In some embodiments the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and/or transmembrane domain(s).
Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
The receptor, e.g., the CAR, generally includes at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the CD19-binding antibody is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25 or CD16.
In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling domain of the CAR activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functional capability.
In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.
T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.
In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD8, CD22, CD79a, CD79b, and CD66d. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.
In some embodiments, the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, or ICOS, or CD27. In some aspects, the same CAR includes both the activating and costimulatory components.
In some embodiments, the activating domain (e.g. CD3 zeta) is included within one CAR, whereas the costimulatory component (e.g. CD28 or 4-1BB) is provided by another CAR recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see WO2014/055668). In some aspects, the CD19-targeting CAR is the stimulatory or activating CAR; in other aspects, it is the costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than CD19, whereby an activating signal delivered through the CD19-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.
In some embodiments, the intracellular signaling component of the recombinant receptor, such as CAR, comprises a CD3 zeta intracellular domain and a costimulatory signaling region. In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and/or CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.
In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.
In some embodiments, the CAR or other antigen receptor, or the engineered cell expressing the same, further includes a marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor, such as a truncated version of a cell surface receptor, such as truncated EGFR (tEGFR). In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor (e.g., tEGFR) or a functional variant thereof. In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. For example, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence. An exemplary polypeptide for a truncated EGFR (e.g. tEGFR) comprises the sequence of amino acids set forth in SEQ ID NO: 55 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 55, or the mature form thereof set forth in SEQ ID NO: 83 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 83. An exemplary T2A linker sequence comprises the sequence of amino acids set forth in SEQ ID NO: 54, and is encoded by the sequence set forth in SEQ ID NO: 71, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 54, or the sequence of amino acids set forth in SEQ ID NO: 64 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 64. In some embodiments, the linker sequence comprise P2A, E2A or F2A cleavable linker sequence or other linkers, such as those set forth in SEQ ID NOS: 65-68 and 82, or the sequence of amino acids set forth in SEQ ID NO: 64 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 65-68 and 82.
In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.
In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self” by the immune system of the host into which the cells will be adoptively transferred.
In some embodiments, the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive transfer and encounter with ligand.
In some cases, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR in some aspects is one that includes multiple costimulatory domains of different costimulatory receptors.
In some embodiments, the receptor, e.g., chimeric antigen receptor, includes an extracellular portion containing the antibody or fragment described herein. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment includes an scFv and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain includes a signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some aspects, the transmembrane domain contains a transmembrane portion of CD28. The extracellular domain and transmembrane domain can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein. In some embodiments, the receptor contains extracellular portion of the molecule from which the transmembrane domain is derived, such as a CD28 extracellular portion. In some embodiments, the chimeric antigen receptor contains an intracellular domain derived from a T cell costimulatory molecule or a functional variant thereof, such as between the transmembrane domain and intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.
For example, in some embodiments, the CAR contains an antibody, e.g., an antibody fragment, as provided herein, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some embodiments, the CAR contains an antibody, e.g., antibody fragment, as provided herein, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4-1BB or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some such embodiments, the receptor further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4 hinge, such as a hinge-only spacer.
In some embodiments, the transmembrane domain of the recombinant receptor, e.g., the CAR, is or includes a transmembrane domain of human CD28 (e.g. Accession No. P01747.1) or variant thereof, such as a transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 56 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 56; in some embodiments, the transmembrane-domain containing portion of the recombinant receptor comprises the sequence of amino acids set forth in SEQ ID NO: 57 or a sequence of amino acids having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
In some embodiments, the intracellular signaling component(s) of the recombinant receptor, e.g. the CAR, contains an intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as a domain with an LL to GG substitution at positions 186-187 of a native CD28 protein. For example, the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 58 or 59 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 58 or 59. In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of 4-1BB (e.g. (Accession No. Q07011.1) or functional variant or portion thereof, such as the sequence of amino acids set forth in SEQ ID NO: 60 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 60.
In some embodiments, the intracellular signaling domain of the recombinant receptor, e.g. the CAR, comprises a human CD3 zeta stimulatory signaling domain or functional variant thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human CD3ζ (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. No. 7,446,190 or 8,911,993. For example, in some embodiments, the intracellular signaling domain comprises the sequence of amino acids set forth in SEQ ID NO: 61, 62 or 63 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 61, 62 or 63.
In some aspects, the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgG1, such as the hinge only spacer set forth in SEQ ID NO: 49. In other embodiments, the spacer is or contains an Ig hinge, e.g., an IgG4-derived hinge, optionally linked to a CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains, such as set forth in SEQ ID NO: 52. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a CH3 domain only, such as set forth in SEQ ID NO: 51. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers, such as set forth in SEQ ID NO:82.
For example, in some embodiments, the CAR includes an anti-CD19 antibody such as an anti-CD19 antibody fragment, such as any of the provided human anti-CD19 antibodies, e.g., single-chain antibodies including scFvs, described herein, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR includes an anti-CD19 antibody or fragment, such as any of the human anti-CD19 antibodies, including scFvs described herein, a spacer such as any of the Ig-hinge containing spacers, a CD28-derived transmembrane domain, a 4-1BB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.
In some embodiments, such CAR constructs further includes a T2A ribosomal skip element and/or a tEGFR sequence, e.g., downstream of the CAR, such as set forth in SEQ ID NO: 54 or 64 and/or 55 or 83, respectively, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 54 or 64 and/or 55 or 83.
III. Molecules Involved in Metabolic Pathway
In some embodiments, the molecule, e.g., recombinant molecule, which can be exogenous or heterologous, or a functional and/or catalytically-active portion or variant thereof, is involved in or capable of modulating a metabolic pathway. In some embodiments, provided are engineered cells, such as recombinant receptor-expressing engineered cells, that are modified by recombinant, engineered and/or ectopic expression of one or more metabolic pathway-modulating molecule, such as those described herein. In some embodiments, expression of the metabolic pathway-modulating molecule is under the control of a heterologous promoter or enhancer, such as is under the control of an inducible promoter. In some embodiments, a nucleic acid molecule, e.g. vector, encoding the metabolic pathway-modulating molecule is introduced into the cell, which introduction can occur simultaneously or sequentially with introduction of the nucleic acid encoding the transgenic receptor, such as the CAR. Exemplary nucleic acid constructs for engineering cells are provided.
In some embodiments, the metabolic pathway or event is or comprises lipid metabolism, oxidative phosphorylation (OXPHOS), a reactive oxygen species (ROS)-induced signal, glycolysis, or mitochondrial biogenesis. In some aspects, the metabolic pathway or event is or comprises generation of energy or ATP in a process that occurs within the mitochondria or via mitochondrial proteins, or occurs independently of glucose or via a pathway other than glycolysis. In some embodiments, the metabolic pathway, step, reaction, or event is or comprises a glycolysis pathway, a reaction thereof, or a reaction or pathway that metabolizes a metabolite of the glycolysis pathway, such as under glucose-low, hypoxic, or nutrient-deprived conditions. In some aspects, the metabolic pathway or event is or comprises the generation of ATP in a glucose-low environment, in a nutrient-poor environment, or in a hypoxic environment. In some embodiments, the metabolic pathway or event comprises glutaminolysis, TCA cycle, glucose metabolism, amino acid or nucleotide metabolism, or beta-oxidation.
In some embodiments, the metabolic pathway or event, reaction or step comprises fatty acid uptake, fatty acid synthesis (FAS), and/or fatty acid oxidation (FAO). In some embodiments, the metabolic pathway, event, step or reaction is or comprises triacylglyceride (TAG) synthesis, TAG storage, glycerol phosphate pathway, glycerophospholipid synthesis and/or glycerol uptake. In some aspects, the metabolic pathway or event comprises oxidative phosphorylation (OXPHOS), generation or accumulation of reactive oxygen species (ROS), cellular respiration, spare respiratory capacity (SPC) or mitochondrial respiratory capacity.
In some embodiments, the molecule is capable of promoting the metabolic pathway or step or reaction thereof or a metabolic event, directly or indirectly. In some aspects, the molecule is capable of inhibiting the metabolic pathway. In some embodiments, the molecule promotes a catabolic molecular profile. In some aspects, the molecule promotes an anabolic molecular profile.
In some embodiments, the molecule is involved in or promotes a pathway resulting in the generation of energy or ATP, which may be independent of the presence of glucose or independent of glycolysis. In some embodiments, the molecule does not promote or enhance glycolysis, does not promote or enhance glycolysis in T cells, or does not promote or enhance glycolysis under conditions under which the molecule promotes or enhances FAS, FAO, OXPHOS, ROS accumulation or generation, cellular respiration, or respiratory capacity. In some embodiments, the molecule is capable of binding to or interacting with an OXPHOS complex protein or is capable of binding to or interacting with a mitochondrial membrane protein. In some aspects, the molecule promotes, acts upstream of, or is required for, such as in lymphocytes or a population of T cells, the translation or insertion of one or more OXPHOS proteins into a mitochondrial membrane. In some embodiments, the molecule is capable of promoting or enhancing the metabolic pathway, reaction or step under conditions of hypoxia, low glucose, poor vascularization, or ROS. In some embodiments, the molecule is capable of promoting generation of a glycolysis metabolite, such as PEP.
In some embodiments, the molecule is or comprises an enzyme. In some aspects, the molecule is or comprises an adapter. In some embodiments, the molecule is involved in fatty acid synthesis, fatty acid storage or fatty acid uptake. In some embodiments, the molecule promotes or enhances FAS or FAO. In some embodiments, the molecule is involved in or is capable of promoting glycerol transport, is or comprises a glycerol transporter, a TAG synthase molecule, a glycerol kinase, or an acyltransferase.
In some embodiments, the molecule comprises a mitochondrial protein. In some embodiments, the molecule is or comprises a Glycerol kinase (GYK), a Glycerol-3-phosphate acetyltransferase mitochondrial (GPAT), a Monoacylglycerol O-acetyltransferase (MOGAT), a DAG O-acetyltransferase (DGAT), an acylglycerolphosphate acyltransferase (AGPAT), or a Lipin. In some aspects, the molecule is any described in Takeuchi et al. Am J Physiol Endocrinol Metab. 2009 June; 296(6): E1195-E1209. In some embodiments, the molecule is or comprises a glycerol transporter or functional portion thereof. In some embodiments, the molecule is or comprises a palmitoyltransferase, such as carnitine palmitoyltransferase (CPT), such as CPTI. In some embodiments, the molecule is or comprises NADH ubiquinone oxidoreductase chain (ND), such as ND chain 1 (ND1), a ubiquinol cytochrome c oxidoreductase chain (UQRC), e.g., chain 2 (UQRC2), or a cytochrome c oxidoreductase (COX), such as COX1.
In some embodiments, molecule is capable of interacting with or associating with, directly or indirectly, a CR6 interacting factor (CRIF), a LEM or a 39S subunit, such as MRPL23. In some embodiments, the molecule in the metabolic pathway is or comprises lymphocyte expansion molecule (LEM), glycerol kinase (Gyk), diacylglycerol 0-acetyltransferase 1 (DGAT1) or 2 (DGAT2), glycerol-3-phosphate acetyltransferase mitochondrial (GPAM), monoacylglycerol O-acetyltransferase 1 (MOGAT1) or 2 (MOGAT2), hypoxia-inducible factor alpha (HIF1α), PCK1, AGPAT1, AGPAT2, AGPAT3, AGPAT 6 (LPA-MAG PA), GPAT1, GPAT2, AQP9, LPIN1, or CRIF1.
In some embodiments, the molecule involved in the metabolic pathway is or comprises lymphocyte expansion molecule (LEM) or a functional fragment or functional variant thereof, such as a functional fragment or functional variant that is catalytically active and/or that exhibits an activity to modulate a metabolic pathway. In some embodiments, LEM comprises the sequence of amino acids set forth in any of SEQ ID NOs: 1-3, 27 or 69, or is a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 1-3, 27 or 69, or is a functional fragment thereof. In some embodiments, LEM lacks or does not contain a prodomain and/or is a mature or catalytically active protein. In some embodiments, LEM is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in any of SEQ ID NOs: 28, 29, 48 or 70, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 28, 29, 48 or 70, and that encodes a functional protein, variant, or fragment thereof. In some embodiments, the LEM-encoding nucleic acid molecule comprises a signal sequence. In some embodiments, LEM is a human protein. In some embodiments, LEM comprises a sequence of human LEM (huLEM or C1ORF177) set forth in NCBI Reference Sequence: NP 689820.2, NCBI Reference Sequence: NP_001104003.1, or UniProt accession number Q3ZCV2, is encoded by a sequence or coding portion of a sequence set forth in NCBI Reference Sequence: NM_152607.2, NCBI Reference Sequence: NM_001110533.1 or is described in Okoye et al. Science. 2015 May 29; 348(6238):995-1001. In some embodiments, LEM is a mouse LEM set forth in SEQ ID NO:27, GenBank: AKD95359.1 or a functional protein, variant, or fragment thereof, or is encoded by the sequence of nucleotides set forth in SEQ ID NO:48, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 48, and that encodes a functional protein, variant, or fragment thereof.
In some embodiments, the molecule involved in the metabolic pathway is or comprises glycerol kinase (Gyk) or a functional fragment or functional variant thereof, such as a functional fragment or functional variant that is catalytically active and/or that exhibits an activity to modulate a metabolic pathway. In some embodiments, Gyk comprises the sequence of amino acids set forth in any of SEQ ID NOs: 4-6, or is a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 4-6, or is a functional fragment thereof. In some embodiments, Gyk lacks or does not contain a prodomain and/or is a mature or catalytically active protein. In some embodiments, Gyk is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in SEQ ID NO: 30 or 77, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 30 or 77, and that encodes a functional protein, variant, or fragment thereof. In some embodiments, the Gyk-encoding nucleic acid molecule comprises a signal sequence. In some embodiments, Gyk is a human protein. In some embodiments, Gyk comprises a sequence set forth in UniProt accession number Q14410, GenBank: CAA55365.1, or GenBank: CAA55364.1, or is encoded by a sequence or coding portion of a sequence set forth in NG_008178.1 RefSeqGene.
In some embodiments, the molecule involved in the metabolic pathway is or comprises Diacylglycerol O-acetyltransferase 1 (DGAT1) or a functional fragment or functional variant thereof, such as a functional fragment or functional variant that is catalytically active and/or that exhibits an activity to modulate a metabolic pathway. In some embodiments, DGAT1 comprises the sequence of amino acids set forth in SEQ ID NO: 7 or 11, or a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7 or 11, or is a functional fragment thereof. In some embodiments, DGAT1 lacks or does not contain a prodomain and/or is a mature or catalytically active protein. In some embodiments, DGAT1 is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in SEQ ID NO: 31 or 76, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 31 or 76, and that encodes a functional protein, variant, or fragment thereof. In some embodiments, the DGAT1-encoding nucleic acid molecule comprises a signal sequence. In some embodiments, DGAT1 is a human protein. In some embodiments, DGAT1 comprises a sequence set forth in UniProt accession number 075907 or NCBI Reference Sequence: NP_036211.2, or is encoded by a sequence or coding portion of a sequence set forth in NCBI Reference Sequence: NM_012079.5.
In some embodiments, the molecule involved in the metabolic pathway is or comprises Glycerol-3-phosphate acetyltransferase mitochondrial (GPAM) or a functional fragment or functional variant thereof, such as a functional fragment or functional variant that is catalytically active and/or that exhibits an activity to modulate a metabolic pathway. In some embodiments, GPAM comprises the sequence of amino acids set forth in SEQ ID NO: 8, or is a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8, or is a functional fragment thereof. In some embodiments, GPAM lacks or does not contain a prodomain and/or is a mature or catalytically active protein. In some embodiments, GPAM is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in SEQ ID NO: 32 or 78, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 32 or 78, and that encodes a functional protein, variant, or fragment thereof. In some embodiments, the GPAM-encoding nucleic acid molecule comprises a signal sequence. In some embodiments, GPAM is a human protein. In some embodiments, GPAM comprises a sequence set forth in UniProt accession number Q9HCL2 or NCBI Reference Sequence: NP_001231878.1, or is encoded by a sequence or coding portion of a sequence set forth in NCBI Reference Sequence: NM_001244949.1.
In some embodiments, the molecule involved in the metabolic pathway is or comprises Monoacylglycerol O-acetyltransferase (MOGAT1) or a functional fragment or functional variant thereof, such as a functional fragment or functional variant that is catalytically active and/or that exhibits an activity to modulate a metabolic pathway. In some embodiments, MOGAT1 comprises the sequence of amino acids set forth in SEQ ID NO: 9, or a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 9, or is a functional fragment thereof. In some embodiments, MOGAT1 lacks or does not contain a prodomain and/or is a mature or catalytically active protein. In some embodiments, MOGAT1 is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in SEQ ID NO: 33 or 80, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 33 or 80, and that encodes a functional protein, variant, or fragment thereof. In some embodiments, the MOGAT1-encoding nucleic acid molecule comprises a signal sequence. In some embodiments, MOGAT1 is a human protein. In some embodiments, MOGAT1 comprises a sequence set forth in UniProt accession number Q96PD6 or NCBI Reference Sequence: NP_477513.2, or is encoded by a sequence or coding portion of a sequence set forth in NCBI Reference Sequence: NM_058165.2.
In some embodiments, the molecule involved in the metabolic pathway is or comprises hypoxia-inducible factor alpha (HIF1α) or a functional fragment or functional variant thereof, such as a functional fragment or functional variant that is catalytically active and/or that exhibits an activity to modulate a metabolic pathway. In some embodiments, HIF1α comprises the sequence of amino acids set forth in SEQ ID NO: 10, or a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10, or is a functional fragment thereof. In some embodiments, HIF1α lacks or does not contain a prodomain and/or is a mature or catalytically active protein. In some embodiments, HIF1α is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in SEQ ID NO: 34 or 79, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 34 or 79, and that encodes a functional protein, variant, or fragment thereof. In some embodiments, the HIF1α-encoding nucleic acid molecule comprises a signal sequence. In some embodiments, HIF1α is a human protein. In some embodiments, HIF1α comprises a sequence set forth in UniProt accession number Q16665 or NCBI Reference Sequence: NP_036211.2, is encoded by a sequence or coding portion of a sequence set forth in NCBI Reference Sequence: NM_001530.3, or is described in Doedens et al. Nat Immunol. 2013 November; 14(11):1173-82.
In some embodiments, the molecule involved in the metabolic pathway is or comprises PCK1 or a functional fragment or functional variant thereof, such as a functional fragment or functional variant that is catalytically active and/or that exhibits an activity to modulate a metabolic pathway. In some embodiments, PCK1 comprises the sequence of amino acids set forth in SEQ ID NO: 12, or a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12, or is a functional fragment thereof. In some embodiments, PCK1 lacks or does not contain a prodomain and/or is a mature or catalytically active protein. In some embodiments, PCK1 is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in SEQ ID NO: 35 or 81, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 35 or 81, and that encodes a functional protein, variant, or fragment thereof. In some embodiments, the PCK1-encoding nucleic acid molecule comprises a signal sequence. In some embodiments, PCK1 is a human protein. In some embodiments, PCK1 comprises a sequence set forth in UniProt accession number P35558 or NCBI Reference Sequence: NP_002582.3, is encoded by a sequence or coding portion of a sequence set forth in NCBI Reference Sequence: NM_002591.3, or is described in Ho et al. Cell. 2015 Sep. 10; 162(6):1217-28.
In some embodiments, the molecule involved in the metabolic pathway is or comprises AGPAT1 or a functional fragment or functional variant thereof, such as a functional fragment or functional variant that is catalytically active and/or that exhibits an activity to modulate a metabolic pathway. In some embodiments, AGPAT1 comprises the sequence of amino acids set forth in SEQ ID NO: 13, or a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13, or is a functional fragment thereof. In some embodiments, AGPAT1 lacks or does not contain a prodomain and/or is a mature or catalytically active protein. In some embodiments, AGPAT1 is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in SEQ ID NO: 36, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 36, and that encodes a functional protein, variant, or fragment thereof. In some embodiments, the AGPAT1-encoding nucleic acid molecule comprises a signal sequence. In some embodiments, AGPAT1 is a human protein. In some embodiments, AGPAT1 comprises a sequence set forth in amino acids 27-283 of UniProt accession number Q99943 or NCBI Reference Sequence: XP_011546679.1, or is encoded by a sequence or coding portion of a sequence set forth in NCBI Reference Sequence: NM_006411.3.
In some embodiments, the molecule involved in the metabolic pathway is or comprises AGPAT2 or a functional fragment or functional variant thereof, such as a functional fragment or functional variant that is catalytically active and/or that exhibits an activity to modulate a metabolic pathway. In some embodiments, AGPAT2 comprises the sequence of amino acids set forth in SEQ ID NO: 14 or 15, or a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14 or 15, or is a functional fragment thereof. In some embodiments, AGPAT2 lacks or does not contain a prodomain and/or is a mature or catalytically active protein. In some embodiments, AGPAT2 is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in SEQ ID NO: 37 or 38, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 37 or 38, and that encodes a functional protein, variant, or fragment thereof. In some embodiments, the AGPAT2-encoding nucleic acid molecule comprises a signal sequence. In some embodiments, AGPAT2 is a human protein. In some embodiments, AGPAT2 comprises a sequence or portion of a sequence set forth in UniProt accession number 015120, NCBI Reference Sequence: NP_006403.2, or NCBI Reference Sequence: NP_001012745.1, or is encoded by a sequence or coding portion of a sequence set forth in NCBI Reference Sequence: NM_006412.3 or NCBI Reference Sequence: NM_001012727.1.
In some embodiments, the molecule involved in the metabolic pathway is or comprises AGPAT3 or a functional fragment or functional variant thereof, such as a functional fragment or functional variant that is catalytically active and/or that exhibits an activity to modulate a metabolic pathway. In some embodiments, AGPAT3 comprises the sequence of amino acids set forth in SEQ ID NO: 16 or 17, or a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 16 or 17, or is a functional fragment thereof. In some embodiments, AGPAT3 lacks or does not contain a prodomain and/or is a mature or catalytically active protein. In some embodiments, AGPAT3 is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in SEQ ID NO: 39, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 39, and that encodes a functional protein, variant, or fragment thereof. In some embodiments, the AGPAT3-encoding nucleic acid molecule comprises a signal sequence. In some embodiments, AGPAT3 is a human protein. In some embodiments, AGPAT3 comprises a sequence or portion of a sequence set forth in NCBI Reference Sequence: NP_001012745.1, UniProt accession number Q9NRZ7, or NCBI Reference Sequence: NP_001032642.1, or is encoded by a sequence or coding portion of a sequence set forth in NCBI Reference Sequence: NM_006412.3.
In some embodiments, the molecule involved in the metabolic pathway is or comprises AGPAT6 (LPA-MAG PA) or a functional fragment or functional variant thereof, such as a functional fragment or functional variant that is catalytically active and/or that exhibits an activity to modulate a metabolic pathway. In some embodiments, AGPAT6 comprises the sequence of amino acids set forth in SEQ ID NO: 18, or a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 18, or is a functional fragment thereof. In some embodiments, AGPAT6 lacks or does not contain a prodomain and/or is a mature or catalytically active protein. In some embodiments, AGPAT6 is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in SEQ ID NO: 40, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 40, and that encodes a functional protein, variant, or fragment thereof. In some embodiments, the AGPAT6-encoding nucleic acid molecule comprises a signal sequence. In some embodiments, AGPAT6 is a human protein. In some embodiments, AGPAT6 comprises a sequence or portion of a sequence set forth in UniProt accession number Q86UL3, or is encoded by a sequence or coding portion of a sequence set forth in NCBI Reference Sequence: NM_178819.3.
In some embodiments, the molecule involved in the metabolic pathway is or comprises GPAT2 or a functional fragment or functional variant thereof, such as a functional fragment or functional variant that is catalytically active and/or that exhibits an activity to modulate a metabolic pathway. In some embodiments, GPAT2 comprises the sequence of amino acids set forth in SEQ ID NO: 19, or a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 19, or is a functional fragment thereof. In some embodiments, GPAT2 lacks or does not contain a prodomain and/or is a mature or catalytically active protein. In some embodiments, GPAT2 is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in SEQ ID NO: 41, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 41, and that encodes a functional protein, variant, or fragment thereof. In some embodiments, the GPAT2-encoding nucleic acid molecule comprises a signal sequence. In some embodiments, GPAT2 is a human protein. In some embodiments, GPAT2 comprises a sequence or portion of a sequence set forth in UniProt accession number Q86UL3, or is encoded by a sequence or coding portion of a sequence set forth in NCBI Reference Sequence: NM_178819.3.
In some embodiments, the molecule involved in the metabolic pathway is or comprises MOGAT2 or a functional fragment or functional variant thereof, such as a functional fragment or functional variant that is catalytically active and/or that exhibits an activity to modulate a metabolic pathway. In some embodiments, MOGAT2 comprises the sequence of amino acids set forth in SEQ ID NO: 20 or 21, or a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 20 or 21, or is a functional fragment thereof. In some embodiments, MOGAT2 lacks or does not contain a prodomain and/or is a mature or catalytically active protein. In some embodiments, MOGAT2 is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in SEQ ID NO: 42, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 42, and that encodes a functional protein, variant, or fragment thereof. In some embodiments, the MOGAT2-encoding nucleic acid molecule comprises a signal sequence. In some embodiments, MOGAT2 is a human protein. In some embodiments, MOGAT2 comprises a sequence or portion of a sequence set forth in UniProt accession number Q3SYC2 or GenBank: AA103879.1, or is encoded by a sequence or coding portion of a sequence set forth in NCBI Reference Sequence: NM_025098.2.
In some embodiments, the molecule involved in the metabolic pathway is or comprises DGAT2 or a functional fragment or functional variant thereof, such as a functional fragment or functional variant that is catalytically active and/or that exhibits an activity to modulate a metabolic pathway. In some embodiments, DGAT2 comprises the sequence of amino acids set forth in SEQ ID NO: 22, or a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 22, or is a functional fragment thereof. In some embodiments, DGAT2 lacks or does not contain a prodomain and/or is a mature or catalytically active protein. In some embodiments, DGAT2 is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in SEQ ID NO: 43, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 43, and that encodes a functional protein, variant, or fragment thereof. In some embodiments, the DGAT2-encoding nucleic acid molecule comprises a signal sequence. In some embodiments, DGAT2 is a human protein. In some embodiments, DGAT2 comprises a sequence or portion of a sequence set forth in UniProt accession number Q96PD7 or GenBank: AAQ88896.1, or is encoded by a sequence or coding portion of a sequence set forth in NCBI Reference Sequence: NM_032564.4.
In some embodiments, the molecule involved in the metabolic pathway is or comprises AQP9 or a functional fragment or functional variant thereof, such as a functional fragment or functional variant that is catalytically active and/or that exhibits an activity to modulate a metabolic pathway. In some embodiments, AQP9 comprises the sequence of amino acids set forth in SEQ ID NO: 23, or a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 23, or is a functional fragment thereof. In some embodiments, AQP9 lacks or does not contain a prodomain and/or is a mature or catalytically active protein. In some embodiments, AQP9 is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in SEQ ID NO: 44, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 44, and that encodes a functional protein, variant, or fragment thereof. In some embodiments, the AQP9-encoding nucleic acid molecule comprises a signal sequence. In some embodiments, AQP9 is a human protein. In some embodiments, AQP9 comprises a sequence or portion of a sequence set forth in UniProt accession number 043315 or GenBank: AAH26258.1, is encoded by a sequence or coding portion of a sequence set forth in NCBI Reference Sequence: NM_020980.3, or is described in Cui et al. Cell. 2015 May 7; 161(4):750-61.
In some embodiments, the molecule involved in the metabolic pathway is or comprises LPIN1 or a functional fragment or functional variant thereof, such as a functional fragment or functional variant that is catalytically active and/or that exhibits an activity to modulate a metabolic pathway. In some embodiments, LPIN1 comprises the sequence of amino acids set forth in SEQ ID NO: 24, or a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 24, or is a functional fragment thereof. In some embodiments, LPIN1 lacks or does not contain a prodomain and/or is a mature or catalytically active protein. In some embodiments, LPIN1 is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in SEQ ID NO: 45, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 45, and that encodes a functional protein, variant, or fragment thereof. In some embodiments, the LPIN1-encoding nucleic acid molecule comprises a signal sequence. In some embodiments, LPIN1 is a human protein. In some embodiments, LPIN1 comprises a sequence or portion of a sequence set forth in UniProt accession number 043315 or GenBank: AAH26258.1, or is encoded by a sequence or coding portion of a sequence set forth in NCBI Reference Sequence: NM_145693.2.
In some embodiments, the molecule involved in the metabolic pathway is or comprises CRIF1 or a functional fragment or functional variant thereof, such as a functional fragment or functional variant that is catalytically active and/or that exhibits an activity to modulate a metabolic pathway. In some embodiments, CRIF1 comprises the sequence of amino acids set forth in SEQ ID NO: 25, or a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 25, or is a functional fragment thereof. In some embodiments, CRIF1 lacks or does not contain a prodomain and/or is a mature or catalytically active protein. In some embodiments, CRIF1 is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in SEQ ID NO: 46, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 46, and that encodes a functional protein, variant, or fragment thereof. In some embodiments, the CRIF1-encoding nucleic acid molecule comprises a signal sequence. In some embodiments, CRIF1 is a human protein. In some embodiments, CRIF1 comprises a sequence or portion of a sequence set forth in UniProt accession number Q8TAE8, or is encoded by a sequence or coding portion of a sequence set forth in NCBI Reference Sequence: NM_052850.3.
In some embodiments, the molecule is selected from among huLEM, DGAT1, GYK, GPAM, HIF1α, MOGAT1 and PCK1, In some embodiments, the molecule comprises a sequence of amino acids set forth in any of SEQ ID NOS: 4, 7-10, 12 and 69, or a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 4, 7-10, 12 and 69, or is a functional fragment thereof. In some embodiments, the molecule is encoded by a sequence of nucleotides that comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 70 and 76-81, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 70 and 76-81, and that encodes a functional protein, variant, or fragment thereof.
IV. Nucleic Acids and Engineered Cells
Provided are methods, nucleic acids, compositions, and kits for producing the genetically engineered cells. The genetic engineering generally involves introduction of a nucleic acid encoding the recombinant receptor into a composition containing the cultured cells, such as by retroviral transduction, transfection, or transformation.
Also provided are cells such as cells that contain an engineered recombinant receptor, such as described herein. Also provided are populations of such cells, compositions containing such cells and/or enriched for such cells, such as in which cells expressing the recombinant receptor make up at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or more percent of the total cells in the composition or cells of a certain type such as T cells or CD8+ or CD4+ cells. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.
Thus also provided are genetically engineered cells expressing the recombinant receptors e.g., cells containing the CARs. The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, as described herein, and re-introducing them into the same patient, before or after cryopreservation.
Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naïve T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
In some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.
A. Preparation of Cells for Engineering
In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for introduction of the recombinant receptor, e.g., CAR, may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, or pig.
In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.
In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++/Mg++ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.
In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques.
For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (markerhigh) on the positively or negatively selected cells, respectively.
In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.
In embodiments, memory T cells are present in both CD62L+ and CD62L− subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L−CD8+ and/or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.
In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or subpopulation, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA or ROR1, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.
CD4+ T helper cells are sorted into naïve, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L− and CD45RO−.
In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In vitro and In vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, N.J.).
In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.
In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.
The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.
In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.
In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.
In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.
In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Number WO2009/072003, or US 20110003380 A1.
In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.
In some aspects, the separation and/or other steps is carried out using CliniMACS system (Miltenyi Biotec), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.
The CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.
In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood may be automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.
In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.
In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to −80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.
In some embodiments, the provided methods include cultivation, incubation, culture, and/or genetic engineering steps. For example, in some embodiments, provided are methods for incubating and/or engineering the depleted cell populations and culture-initiating compositions.
Thus, in some embodiments, the cell populations are incubated in a culture-initiating composition. The incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.
In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agents include IL-2 and/or IL-15, for example, an IL-2 concentration of at least about 10 units/mL.
In some aspects, incubation is carried out in accordance with techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, the T cells are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.
In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.
In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigen-specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.
B. Nucleic Acids, Vectors and Methods for Genetic Engineering
Also provided are nucleic acids and vectors encoding the receptors and/or molecules of any of the provided embodiments, and combinations thereof, as well as cells and compositions containing the same. The vectors may be viral or non-viral, such as gammaretroviral, lentiviral, and/or use transposon-based systems such as Sleeping Beauty or PiggyBac systems. Various methods for the introduction of genetically engineered components, e.g., antigen receptors, e.g., CARs or TCRs, are well known and may be used with the provided methods and compositions. Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation. Transfer of genetic material in the vectors may be achieved by a number of known methods, via transduction, transformation, electroporation, and/or any known method.
1. Nucleic Acids and Promoters
In some embodiments, a nucleic acid encodes a genetically engineered receptor that specifically binds to a ligand, such as a recombinant, e.g., chimeric, receptor, and a molecule involved in a metabolic pathway, or functional portion thereof. In some embodiments, the molecule involved in a metabolic pathway is a recombinant molecule, including an exogenous or heterologous molecule. In some embodiments, the molecule involved in a metabolic pathway is a protein, including a recombinant protein, including an exogenous or heterologous protein. In some embodiments, the genetically engineered receptor and the molecule involved in a metabolic pathway are encoded by one nucleic acid. In some embodiments, the genetically engineered receptor and the molecule involved in a metabolic pathway are encoded by two or more different nucleic acids. In some embodiments, a first nucleic acid encodes the genetically engineered receptor that specifically binds to a ligand and a second nucleic acid encodes the molecule involved in a metabolic pathway. In some embodiments, the nucleic acid or acids encoding the genetically engineered receptor that specifically binds to a ligand and the molecule involved in a metabolic pathway are expressed in the same or different vectors. The vectors can be any of the vectors described herein, including viral vectors and expression vectors.
In some embodiments, the nucleic acid is multicistronic, such as bicistronic or otherwise permits the coexpression of multiple, separate peptide chains, such as two or more, from the same promoter. The transcript in some embodiments has the potential to code for more than one final product, such as two final products. In some embodiments, at least one of the nucleic acids contains an internal ribosome binding site (IRES) separating the encoded molecules such that the genetically engineered receptor and the molecule involved in a metabolic pathway are expressed under the control of the same promoter. As used herein, an “internal ribosome entry site” (IRES) refers to a nucleotide sequence that allows for translation initiation in the middle of a messenger RNA (mRNA) sequence as part of protein synthesis. In some embodiments, the nucleic acid includes one or more ribosomal skip sequences, such as picornavirus 2A ribosomal skip peptide, so that the two or more peptide chains or other products may be expressed in operable linkage with the same promoter, but produced as separate chains.
In some embodiments, a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF), two or three genes (e.g. encoding the molecule involved in modulating a metabolic pathway and encoding the recombinant receptor) separated from one another by sequences encoding a self-cleavage peptide (e.g., 2A sequences) or a protease recognition site (e.g., furin). The ORF thus encodes a single polypeptide, which, either during (in the case of 2A) or after translation, is processed into the individual proteins. In some cases, the peptide, such as T2A, can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream (see, for example, de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and deFelipe et al. Traffic 5:616-626 (2004)). Many 2A elements are known in the art. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein, without limitation, 2A sequences from the foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 68), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 67), Thosea asigna virus (T2A, e.g., SEQ ID NO: 54 or 64), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 65 or 66) as described in U.S. Patent Publication No. 20070116690.
In some embodiments, expression or activity of the genetically engineered or recombinant receptor and/or of the recombinant or engineered molecule involved in a metabolic pathway is constitutive; in some embodiments, one or more of such expression or activity is engineered to be conditional, for example, induced or repressed by one or more natural or non-natural events or molecules.
In some embodiments, the genetically engineered receptor and the molecule involved in a metabolic pathway are operably linked to the same promoter or other inducing or repressing element, such as one or more enhancer(s), and/or transactivator(s) or repressors or other sequences or molecules for control of expression, e.g., via control of transcription or translation. In other embodiments, they are operably linked to different promoters and/or such other elements, for example, such that expression thereof is controlled via different mechanisms or events.
In some embodiments, expression of the receptor and/or the molecule is under the control of a constitutive promoter, enhancer, or transactivator. In some embodiments, the expression is under the control of a conditional promoter, enhancer or transactivator. In some embodiments, expression of the genetically engineered receptor is under the control of a constitutive promoter and expression of the molecule involved in a metabolic pathway is under the control of a conditional promoter. In some embodiments, expression of the genetically engineered receptor is under the control of a conditional promoter and expression of the molecule involved in a metabolic pathway is under the control of a constitutive promoter.
In some embodiments, the expression is under the control of a conditional promoter, enhancer or transactivator. In some embodiments, the conditional promoter, enhancer or transactivator is an inducible promoter, enhancer or transactivator, a repressible promoter, enhancer or transactivator, or a tissue-specific promoter, enhancer or transactivator.
Exemplary tissue specific promoters include, but are not limited to, those that are active in heart, lung, esophagus, muscle, intestine, breast, prostate, stomach, bladder, liver, spleen, pancreas, kidney, neurons, myocytes, leukocytes, immortalized cells, neoplastic cells, tumor cells, cancer cells, duodenum, jejunum, ileum, cecum, colon, rectum, salivary glands, gall bladder, urinary bladder, trachea, larynx, pharynx, aorta, arteries capillaries, veins, thymus, mandibular lymph nodes, mesenteric lymph node, bone marrow, pituitary gland, thyroid gland, parathyroid glands, adrenal glands, brain, cerebrum, cerebellum, medulla, pons, spinal cord, sciatic nerve, skeletal muscle, smooth muscle, bone, testes, epididymis, prostate, seminal vesicles, penis, ovaries, uterus, mammary glands, vagina, skin, eyes or optic nerve.
Exemplary cell specific promoters include T cells, such as the CD4 mini-promoter/enhancer described in Zhao-Emonet, et al. (2000) J. Gene. Med., 2: 416-425.
In some embodiments, the expression of the molecule or receptor, generally the molecule, is conditional upon (e.g., is induced or repressed by, such as via an inducible promoter or other element) by one or more specific conditions, events, or molecules found or found at relatively higher levels in particular, regions of the body, disease, activation state, or tissues. For example, the in some embodiments the promoter can be inducible or suppressible by hypoxia, glucose-poor or other nutrient-poor conditions, deficiencies in metabolites such as amino acids or nucleic acids or lipids, elements of the tumor microenvironment, or other elements of metabolic pathways or metabolites or levels thereof. See, e.g. Cao, et al. (2001) Gene Ther., 8: 1357-1362 and Dachs, et al. (2000) Eur. J. Cancer, 36:1649-1660, and Greco et al., (2002) Gene Ther., 9:1403-1411. In some embodiments, expression is conditioned upon activation signals or pathways, or signaling via a particular receptor, such as a cytokine or antigen receptor. In some embodiments, expression is regulated by activation or proliferative events. Exemplary inducible systems are those activatable by NFκB, NFAT or Nur77.
In some embodiments, expression of any of the peptides or nucleic acids described herein may be externally controlled by treating the cell with a modulating factor, such as doxycycline, tetracycline or analogues thereof. Analogues of tetracycline include, for example, chlortetracycline, oxytetracycline, demethylchloro-tetracycline, methacycline, doxycycline and minocycline.
In some embodiments, inducible transcription and/or expression can be implemented using a transactivator together with a transactivator induced promoter. In some embodiments, such a transactivator induced promoter comprises control elements for the enhancement or repression of transcription of the transgene or nucleic acid of interest. Control elements include, without limitation, operators, enhancers and promoters. In some embodiments, a transactivator inducible promoter is transcriptionally active when bound to a transactivator, which in turn is activated under a specific set of conditions, for example, in the presence or in the absence of a particular combination of chemical signals, for example, by a modulating factor selected for example from the previous list.
The transactivator induced promoter may be any promoter herein mentioned which has been modified to incorporate transactivator binding sequences, such as several tet-operator sequences, for example 3, 4, 5, 6, 7, 8, 9, or 10 tet-operator sequences. In some embodiments, the tet-operator sequences are in tandem. Such sequences can for example replace the functional recognition sites for Staf and Oct-1 in the distal sequence element (DSE) of the U6 promoter, including the human U6 promoter.
Specific examples of transcription modulator domains that induce expression in the presence of modulating factor include, but are not limited to, the transcription modulator domains found in the following transcription modulators: the Tet-On transcription modulator; and the Tet-On Advanced transcription modulator and the Tet-On 3G transcription modulator; all of which are available from Clontech Laboratories, Mountain View, Calif. Specific examples of transcription modulator domains that induce expression in the absence of modulating factor include, but are not limited to, the transcription modulator domains found in the following transcription modulators: the Tet-off transcription modulator and the Tet-Off Advanced transcription modulator, both of which are available from Clontech Laboratories, Mountain View, Calif. These systems can be adapted and used according to procedures that are well known in the art and that will be familiar to the ordinarily skilled artisan.
In some embodiments, the transactivator induced promoter comprises a plurality of transactivator binding sequences operatively linked to the inhibitory nucleic acid molecule.
The transactivator may be provided by a nucleic acid sequence, in the same expression vector or in a different expression vector, comprising a modulating factor-dependent promoter operatively linked to a sequence encoding the transactivator. The term “different expression vector” is intended to include any vehicle for delivery of a nucleic acid, for example, a virus, plasmid, cosmid or transposon. Suitable promoters for use in said nucleic acid sequence include, for example, constitutive, regulated, tissue-specific or ubiquitous promoters, which may be of cellular, viral or synthetic origin, such as CMV, RSV, PGK, EF1α, NSE, synapsin, β-actin, GFAP.
An exemplary transactivator according to some embodiments is the rtTA-Oct.2 transactivator composed of the DNA binding domain of rtTA2-M2 and of the Oct-2Q(Q→A) activation domain. Another exemplary transactivator according to some embodiments is the rtTA-Oct.3 transactivator composed of the DNA binding domain of the Tet-repressor protein (E. coli) and of the Oct-2Q(Q→A) activation domain. Both are described in patent application WO 2007/004062.
In some embodiments, the molecule involved in a metabolic pathway is an inhibitory nucleic acid that inhibits expression of a molecule involved in a metabolic pathway. In some embodiments, expression is reduced or eliminated using small-hairpin RNAs (shRNAs) that target nucleic acids encoding, molecule involved in a metabolic pathway. In some embodiments, shRNA and siRNA segments may further comprise stop and/or polyadenylation sequences. Suitable shRNA sequences for the knock down of a given target gene or nucleic acid sequence are well known in the art or can readily be determined by a person skilled in the art.
Expression of shRNAs in mammalian cells, such as T cells, can be achieved using any conventional expression system, e.g., a lentiviral expression system. In some embodiments, the RNA can be a component of a viral vector, or a shRNA encoded by a viral vector. In some embodiments, the viral vector comprises an oligonucleotide that inhibits expression of a molecule involved in a metabolic pathway, or encodes a shRNA having such capability. In some embodiments, the viral vector is a lentivirus vector. In some embodiments, the lentivirus vector is an integrating lentivirus vector.
In some embodiments, suitable promoters include, for example, RNA polymerase (pol) III promoters including, but not limited to, the (human and murine) U6 promoters, the (human and murine) H1 promoters, and the (human and murine) 7SK promoters, including conditional variants thereof. In some embodiments, a hybrid promoter also can be prepared that contains elements derived from, for example, distinct types of RNA polymerase (pol) III promoters. In some embodiments, modified promoters that contain sequence elements derived from two or more naturally occurring promoter sequences can be combined by the skilled person to effect transcription under a desired set of conditions or in a specific context. For example, the human and murine U6 RNA polymerase (pol) III and H1 RNA pol III promoters are well characterized. One skilled in the art will be able to select and/or modify the promoter that is most effective for the desired application and cell type so as to optimize modulation of the expression of one or more genes. In some embodiments, the promoter sequence can be one that does not occur in nature, so long as it functions in a eukaryotic cell, such as, for example, a mammalian cell.
2. Vectors and Introduction
Introduction of the molecules encoding the receptor and/or molecule involved with metabolism may be carried out using any of a number of known vectors. Such vectors include viral and non-viral systems, including lentiviral and gammaretroviral systems, as well as transposon-based systems such as PiggyBac or Sleeping Beauty-based gene transfer systems.
In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.
In some contexts, overexpression of a stimulatory factor (for example, a lymphokine or a cytokine) may be toxic to a subject. Thus, in some contexts, the engineered cells include gene segments that cause the cells to be susceptible to negative selection in vivo, such as upon administration in adoptive immunotherapy. For example in some aspects, the cells are engineered so that they can be eliminated as a result of a change in the in vivo condition of the patient to which they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell 2:223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).
In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557.
In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
Methods of lentiviral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.
In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).
Other approaches and vectors for transfer of the nucleic acids encoding the recombinant products are those described, e.g., in international patent application, Publication No.: WO2014055668, and U.S. Pat. No. 7,446,190.
In some embodiments, the cells, e.g., T cells, may be transfected either during or after expansion e.g. with a T cell receptor (TCR) or a chimeric antigen receptor (CAR) and/or a metabolic pathway-modulating molecule. This transfection for the introduction of the gene of the desired receptor can be carried out with any suitable retroviral vector, for example. The genetically modified cell population can then be liberated from the initial stimulus (the CD3/CD28 stimulus, for example) and subsequently be stimulated with a second type of stimulus e.g. via a de novo introduced receptor). This second type of stimulus may include an antigenic stimulus in form of a peptide/MHC molecule, the cognate (cross-linking) ligand of the genetically introduced receptor (e.g. natural ligand of a CAR) or any ligand (such as an antibody) that directly binds within the framework of the new receptor (e.g. by recognizing constant regions within the receptor). See, for example, Cheadle et al, “Chimeric antigen receptors for T-cell based therapy” Methods Mol Biol. 2012; 907:645-66 or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol. 65: 333-347 (2014).
Among additional nucleic acids, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.
In some embodiments, introduction of the receptor and/or molecule involved with metabolism into a population of cells are carried out separately, such as by co-transfection or transduction of a vector encoding each. Such introduction can be performed simulataneously or sequentially in any order. In some embodiments, the introduction of the receptor and/or molecule involved with metabolism into a population of cells are carried out together, such as by introduction of a vector encoding both the receptor and the molecule involved with metabolism.
In some embodiments, in the nucleic acid molecule is a single polynucleotide encoding a plurality of different polypeptide chains, such as the genetically engineered receptor and the molecule involved in a metabolic pathway. For example, in some embodiments, the polynucleotide contains at least one promoter operatively linked to control expression of the recombinant receptor and the metabolic pathway-modulating molecule. In some embodiments, the coding sequences encoding each of the different polypeptide chains can be operatively linked to a promoter, which can be the same or different. In some embodiments, the nucleic acid molecule can contain a promoter that drives the expression of the recombinant receptor and the metabolic pathway-modulating molecule. In some embodiments, such nucleic acid molecules can be multicistronic (bicistronic or tricistronic) For example, in some embodiments, the sequence of nucleotides encoding the genetically engineered receptor and the sequence of nucleotides encoding the metabolic pathway-modulating molecule are separated by an internal ribosome entry site (IRES), a self-cleaving peptide or a peptide that causes ribosome skipping, optionally a T2A, F2A, E2A or P2A peptide, e.g., any ribosome skipping peptides described herein, and the first and second chimeric receptor are expressed under the control of the same promoter. In some embodiments, two, three or more separate polypeptide chains can be encoded in one nucleic acid molecule, separated from one another by sequences encoding a self-cleavage peptide (e.g., 2A sequences) or a protease recognition site (e.g., furin). For example, in some embodiments, one nucleic acid molecule can encode multiple polypeptide chains: the genetically engineered receptor, the sequence of nucleotides encoding the metabolic pathway-modulating molecule and one or more surface markers. In some embodiments, the nucleotide sequences encoding the polypeptide chains can be operatively linked to one or more separate promoter, such as any of the promoters described herein, and/or can be separated by any IRES, self-cleaving peptide, ribosome skipping peptide and/or protease sites, such as any described herein.
In some embodiments, nucleic acid molecules encoding the recombinant receptor, e.g., CAR and/or the metabolic pathway-modulating molecules provided herein can be engineered using the same method or different methods. In some embodiments, the recombinant receptor, e.g., CAR and the metabolic pathway-modulating molecule provided herein can be introduced as two separate nucleic acid molecules. In some embodiments, one nucleic acid molecule, e.g., a viral vector, can contain nucleic acids encoding the recombinant receptor, e.g., CAR, and the metabolic pathway-modulating molecule. In some examples, the recombinant receptor and metabolic pathway-modulating molecule are encoded by one nucleic acid molecule, and are transcribed into one transcript, as described above. In other examples, the recombinant receptor and metabolic pathway-modulating molecule provided herein are encoded by one nucleic acid molecule, and are transcribed into two or more transcripts. In some embodiments, the recombinant receptor and metabolic pathway-modulating molecule are encoded by one nucleic acid molecule, and are transcribed into one transcript, which is translated into one polypeptide, then are separated, e.g., cleaved, in a post-translational manner.
In other embodiments, the recombinant receptor and metabolic pathway-modulating molecule are encoded by two or more different nucleic acid molecules, e.g., viral vectors. For example, recombinant receptor, e.g. CAR is encoded by one nucleic acid molecule, and the metabolic pathway-modulating molecule can be encoded by a separate nucleic acid molecule. In some embodiments, one or more of the nucleic acid molecules can further contain nucleotide sequences encoding a cell surface marker, e.g., any of those described herein, that can be used to confirm introduction of particular nucleic acid molecules into cells and/or transduction and/or engineering of cells.
In some embodiments, the recombinant receptor and metabolic pathway-modulating molecule are encoded by same types of vectors. In other embodiments, the recombinant receptor and metabolic pathway-modulating molecule are encoded by different types of vectors. In some embodiments, the recombinant receptor and metabolic pathway-modulating molecule are engineered into a cell using the same methods, e.g., viral transduction. In other embodiments, the recombinant receptor and metabolic pathway-modulating molecule are engineered into a cell using different methods.
3. Exemplary Constructs
Provided herein are expression vectors, such as viral vectors, e.g. lentiviral vectors encoding a metabolic pathway modulating molecule. In some embodiments, the vector comprises a nucleotide sequence encoding any of the metabolic pathway modulators as described in Section III. In some embodiments, the vector comprises a nucleotide sequence encoding a molecule selected from among huLEM, DGAT1, GYK, GPAM, HIF1α, MOGAT1 and PCK1. In some embodiments, the nucleotide sequence encodes a molecule comprising a sequence of amino acids set forth in any of SEQ ID NOS: 4, 7-10, 12 and 69, or a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 4, 7-10, 12 and 69, or is a functional fragment thereof. In some embodiments, the nucleotide sequence comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 70 and 76-81, or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 70 and 76-81, and that encodes a functional protein, variant, or fragment thereof.
In some embodiments, such expression vectors can further encode a recombinant receptor, such as a CAR, e.g. any as described. In some embodiments, CAR contains a sequence of nucleotides encoding a genetically engineered receptor that specifically binds to a ligand, a marker, a transmembrane portion of a costimulatory receptor, a signal peptide, a activating domain (e.g. CD3 zeta), and a 2A element. Exemplary CD3 zeta sequences are set forth in SEQ ID NOs: 61-63. Exemplary signaling domain and/or transmembrane portion of a costimulatory receptor include CD28, 4-1BB, OX40, DAP10, ICOS, or CD27.
In some aspects, the nucleic acid molecule can be modified for use in the constructs described herein. In some cases, the sequences can be designed to contain terminal restriction site sequences for purposes of cloning into vectors. In some cases, the sequences can be modified by codon optimization. Codon optimization involves balancing the percentages of codons selected with the published abundance of human transfer RNAs so that none is overloaded or limiting. This may be necessary in some cases because most amino acids are encoded by more than one codon, and codon usage varies from organism to organism. Differences in codon usage between transfected genes and host cells can have effects on protein expression and immunogenicity of a nucleic acid construct. In general, for codon optimization, codons are chosen to select for those codons that are in balance with human usage frequency. Typically, the redundancy of the codons for amino acids is such that different codons code for one amino acid. In some embodiments, in selecting a codon for replacement, it may be desired that the resulting mutation is a silent mutation such that the codon change does not affect the amino acid sequence. Generally, the last nucleotide of the codon can remain unchanged without affecting the amino acid sequence.
In some embodiments, the nucleic acid(s) that encodes a genetically engineered receptor and/or and a molecule involved in a metabolic pathway, or functional portion thereof further comprises a marker, such as a cell surface marker, that can be used to confirm introduction of the nucleic acid(s) into the cell. Exemplary markers, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor and/or the molecule involved in a metabolic pathway, include truncated and/or modified versions of a cell surface receptor, such as all or part (e.g., truncated form) of CD34, a NGFR, epidermal growth factor receptor (e.g., tEGFR), ErbB2/HER2/neu (e.g., modified Her2t) or a functional variant thereof. In some specific embodiments, exemplary markers include tEGFR (sequences set forth in SEQ ID NO:55 or 83) and modified Her2t (sequence set forth in SEQ ID NO:75 and encoded by the nucleic acid sequences set forth in SEQ ID NO:74). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence. For example, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2014031687.
Exemplary markers include tEGFR sequences set forth in SEQ ID NO:55 or 83, and modified Her2t sequence set forth in SEQ ID NO:75 and encoded by the nucleic acid sequences set forth in SEQ ID NO:74, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 55, 83 or 75, or encoded by a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 74 and encodes a functional protein, variant, or fragment.
In some embodiments, a nucleic acids encoding one or more cell surface markers is operatively linked to a promoter, such as any described herein, to control expression of the cell surface marker. In some embodiments, a nucleic acids encoding one or more cell surface markers is linked to nucleic acids encoding the genetically engineered receptor and/or the molecule involved in a metabolic pathway, separated from one another by sequences encoding a ribosome-skipping peptide or a self-cleavage peptide, such as any described herein. Exemplary 2A elements include F2A, E2A, T2A, and P2A (set forth in SEQ ID NOs: 54 and 64-68).
In some cases, the sequence of nucleotides encoding the genetically engineered receptor, the recombinant, engineered or ectopically expressed molecule and/or the surface marker contains a signal sequence that encodes a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from the native cell surface molecule. In other aspects, the signal sequence may encode a heterologous or non-native signal peptide, such as the exemplary signal peptide of the GMCSFR alpha chain set forth in SEQ ID NO: 72 and encoded by the nucleotide sequence set forth in SEQ ID NO:73. In some cases, the nucleic acid sequence encoding the chimeric antigen receptor (CAR) and/or a cell surface marker contains a signal sequence that encodes a signal peptide. Non-limiting exemplary examples of signal peptides include, for example, the GMCSFR alpha chain signal peptide set forth in SEQ ID NO:72.
In some embodiments, exemplary provided nucleic acid molecule include those that encode an amino acid sequence selected from among: any of SEQ ID NO: 85, 87, 89, 91, 93, 95 and 97; and a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 85, 87, 89, 91, 93, 95 and 97. In some embodiments, the nucleic acid molecule contains the nucleotide sequence is selected from among: any of SEQ ID NO: 84, 86, 88, 90, 92, 94 and 96; and a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 84, 86, 88, 90, 92, 94 and 96 that encodes a functional protein, variant, or fragment thereof. In some embodiments, such nucleic acid molecules encoding a metabolic pathway molecule can be co-expressed in a cell with a recombinant receptor, e.g., CAR. In some embodiments, such nucleic acid molecules can further comprise a sequence encoding a recombinant receptor, e.g. CAR.
Also provided are vectors encoding any of the provided nucleic acid constructs. The vectors can include any as described above, including lentiviral vectors, e.g. retroviral or gammaretroviral vectors. Also provided are cells encoding any of the provided nucleic acid constructs. The cells include any of the cells as described above. In some embodiments, the cells are T cell, such as CD4+ or CD8+ T cells. In some embodiments, the T cells are primary T cells obtained from a subject.
V. Compositions, Formulations and Methods of Administration
Also provided are compositions containing the recombinant receptor, such as CAR or TCR, and compositions containing the engineered cells, including pharmaceutical compositions and formulations. Also provided are methods of using and uses of the compositions, such as in the treatment of diseases, conditions, and disorders in which the antigen is expressed, or in detection, diagnostic, and prognostic methods.
A. Compositions/Formulations
The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
In some aspects, the choice of carrier is determined in part by the particular cell and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).
Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cell, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
The pharmaceutical composition in some embodiments contains cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
The cells may be administered using standard administration techniques, formulations, and/or devices. Provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. Administration of the cells can be autologous or heterologous. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cell populations are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyoi (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.
Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
B. Methods of Administration
Provided are methods of administering the cells, populations, and compositions, and uses of such cells, populations, and compositions to treat or prevent diseases, conditions, and disorders, including cancers. In some embodiments, the cells, populations, and compositions are administered to a subject or patient having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, cells and compositions prepared by the provided methods, such as engineered compositions and end-of-production compositions following incubation and/or other processing steps, are administered to a subject, such as a subject having or at risk for the disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptom of, the disease or condition, such as by lessening tumor burden in a cancer expressing an antigen recognized by an engineered T cell.
Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.
As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human. In some embodiments, the subject, e.g., patient, to whom the cells, cell populations, or compositions are administered is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.
As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.
As used herein, “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.
“Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided cells and compositions are used to delay development of a disease or to slow the progression of a disease.
As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, cells that suppress tumor growth reduce the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the cells.
An “effective amount” of an agent, e.g., a pharmaceutical formulation, cells, or composition, in the context of administration, refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.
A “therapeutically effective amount” of an agent, e.g., a pharmaceutical formulation or cells, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered. In some embodiments, the provided methods involve administering the cells and/or compositions at effective amounts, e.g., therapeutically effective amounts.
A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
The disease or condition that is treated can be any in which expression of an antigen is associated with and/or involved in the etiology of a disease condition or disorder, e.g. causes, exacerbates or otherwise is involved in such disease, condition, or disorder. Exemplary diseases and conditions can include diseases or conditions associated with malignancy or transformation of cells (e.g. cancer), autoimmune or inflammatory disease, or an infectious disease, e.g. caused by a bacterial, viral or other pathogen. Exemplary antigens, which include antigens associated with various diseases and conditions that can be treated, are described above. In particular embodiments, the chimeric antigen receptor or transgenic TCR specifically binds to an antigen associated with the disease or condition.
Thus, the provided methods and uses include methods and uses for adoptive cell therapy. In some embodiments, the methods include administration of the cells or a composition containing the cells to a subject, tissue, or cell, such as one having, at risk for, or suspected of having the disease, condition or disorder. In some embodiments, the cells, populations, and compositions are administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells or compositions are administered to the subject, such as a subject having or at risk for the disease or condition, ameliorate one or more symptom of the disease or condition.
In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.
In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject. The cells can be administered by any suitable means. Dosing and administration may depend in part on whether the administration is brief or chronic. Various dosing schedules include but are not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion.
In certain embodiments, the cells, or individual populations of sub-types of cells, are administered to the subject at a range of about one million to about 100 billion cells and/or that amount of cells per kilogram of body weight, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges and/or per kilogram of body weight. Again, dosages may vary depending on attributes particular to the disease or disorder and/or patient and/or other treatments. In some embodiments, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents includes a cytokine, such as IL-2, for example, to enhance persistence. In some embodiments, the methods comprise administration of a chemotherapeutic agent.
Following administration of the cells, the biological activity of the engineered cell populations in some embodiments is measured, e.g., by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying expression and/or secretion of one or more cytokines, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
In certain embodiments, the engineered cells are further modified in any number of ways, such that their therapeutic or prophylactic efficacy is increased. For example, the engineered CAR or TCR expressed by the population can be conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating compounds, e.g., the CAR or TCR, to targeting moieties is known in the art. See, for instance, Wadwa et al., J. Drug Targeting 3: 1 1 1 (1995), and U.S. Pat. No. 5,087,616.
VI. Exemplary Embodiments
Among the embodiments provided herein are:
1. An engineered immune cell, comprising:
(a) a genetically engineered receptor that specifically binds to a ligand; and
(b) a recombinant, engineered and/or ectopically expressed molecule or a functional and/or catalytically-active portion or variant thereof, which recombinant molecule is involved in or capable of modulating a metabolic pathway.
2. The engineered immune cell of embodiment 1, wherein the recombinant molecule is capable of promoting said metabolic pathway or step or reaction thereof or a metabolic event, directly or indirectly.
3. The engineered immune cell of embodiment 1, wherein the recombinant molecule is capable of inhibiting said metabolic pathway.
4. The engineered immune cell of any of embodiments 1-3, wherein the recombinant molecule is or comprises an enzyme; and/or wherein the recombinant molecule is or comprises an adapter.
5. The engineered immune cell of any of embodiments 1-4,
wherein the metabolic pathway or event comprises lipid metabolism;
wherein the metabolic pathway or event comprises oxidative phosphorylation (OXPHOS);
wherein the metabolic pathway or event comprises a reactive oxygen species (ROS)-induced signal, event or pathway; and/or
wherein the metabolic pathway or event comprises glycolysis,
wherein the metabolic pathway or event comprises mitochondrial biogenesis; and/or
wherein the metabolic pathway or event comprises generation of energy or ATP in a process that occurs within the mitochondria or via mitochondrial proteins, or occurs independently of glucose, or occurs via a pathway other than glycolysis;
wherein the metabolic pathway or event comprises the generation of ATP in a glucose-low environment, in a nutrient-poor environment, in a hypoxic environment,
wherein the molecule promotes a catabolic molecular profile, optionally in the engineered cell, as compared to a reference cell substantially similar to the engineered cell but not comprising the recombinant molecule; and/or
wherein the molecule promotes an anabolic molecular profile, optionally in the engineered cell, as compared to a reference cell substantially similar to the engineered cell but not comprising the recombinant molecule.
6. The engineered immune cell of any of embodiments 1-5, wherein the metabolic pathway or event comprises glutaminolysis, TCA cycle, glucose metabolism, amino acid or nucleotide metabolism, or beta-oxidation.
7. The engineered immune cell of any of embodiments 1-6, wherein
the molecule is involved in fatty acid synthesis, fatty acid storage and/or fatty acid uptake; and/or
the metabolic pathway or event or reaction or step comprises fatty acid uptake, fatty acid synthesis (FAS), and/or fatty acid oxidation (FAO).
8. The engineered immune cell of embodiment 7, wherein the molecule promotes or enhances FAS or FAO.
9. The engineered immune cell of embodiment 7 or embodiment 8, wherein the metabolic pathway, event or step or reaction is or comprises triacylglyceride (TAG) synthesis, TAG storage, glycerol phosphate pathway, glycerophospholipid synthesis and/or glycerol uptake.
10. The engineered immune cell of any of embodiments 7-9, wherein the recombinant molecule is involved in and/or is capable of promoting glycerol transport, is or comprises a glycerol transporter, is a TAG synthase molecule, is or comprises a glycerol kinase, or is or comprises an acyltransferase.
11. The engineered immune cell of any of embodiments 1-10, wherein the molecule is or comprises a Glycerol kinase (GYK), a Glycerol-3-phosphate acetyltransferase mitochondrial (GPAT), a Monoacylglycerol O-acetyltransferase (MOGAT), a DAG O-acetyltransferase (DGAT), an acylglycerolphosphate acyltransferase (AGPAT), or a Lipin.
12. The engineered immune cell of embodiment 11, wherein the molecule is or comprises a GYK, a GPAT1, a MOGAT1, or a DGAT1.
13. The engineered immune cell of embodiment 11 or embodiment 12, wherein the molecule comprises an amino acid sequence selected from among:
(i) any of SEQ ID NO: 4, 7, 8 and 9; and
(ii) a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 4, 7, 8 and 9.
14. The engineered immune cell of any of embodiments 11-13, wherein the molecule comprises an amino acid sequence encoded by a nucleotide sequence selected from among:
(i) any of SEQ ID NO: 76, 77, 78 and 80; and
(ii) a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 76, 77, 78 and 80 that encodes a functional protein, variant, or fragment thereof.
15. The engineered immune cell of any of embodiments 1-14, wherein the molecule is or comprises a glycerol transporter or functional portion thereof.
16. The engineered immune cell of embodiment 15, wherein the molecule is or comprises an AQP9.
17. The engineered immune cell of any of embodiments 1-16, wherein the recombinant molecule is or comprises a palmitoyltransferase, which optionally is carnitine palmitoyltransferase (CPT), which optionally is CPTI.
18. The engineered immune cell of any of embodiments 1-17, wherein the metabolic pathway or event comprises oxidative phosphorylation (OXPHOS), generation or accumulation of reactive oxygen species (ROS), cellular respiration, spare respiratory capacity (SPC) and/or mitochondrial respiratory capacity.
19. The engineered immune cell of any of embodiments 1-18,
wherein the recombinant molecule does not promote or enhance glycolysis;
wherein the recombinant molecule does not promote or enhance glycolysis in T cells;
wherein the recombinant molecule does not promote or enhance and/or does not promote or enhance glycolysis under conditions under which the molecule promotes or enhances FAS, FAO, OXPHOS, ROS accumulation or generation, cellular respiration, or respiratory capacity.
20. The engineered immune cell of any of embodiments 1-19, wherein the recombinant molecule is capable of binding to or interacting with an OXPHOS complex protein, is capable of binding to or interacting with a mitochondrial membrane protein, promotes, acts upstream of, or is required for, optionally in lymphocytes, optionally in a population of T cells, the translation and/or insertion of one or more OXPHOS proteins into a mitochondrial membrane.
21. The engineered immune cell of any of embodiments 1-20, wherein the recombinant molecule comprises a mitochondrial protein.
22. The engineered immune cell of any of embodiments 1-21, wherein the recombinant molecule is capable of interacting with or associating with, directly or indirectly, a CR6 interacting factor (CRIF), optionally CRIF1, a LEM or a 39S subunit, optionally MRPL23.
23. The engineered immune cell of any of embodiments 1-22, wherein the recombinant molecule is or comprises a lymphocyte enhancer molecule (LEM).
24. The engineered immune cell of embodiment 22 or embodiment 23, wherein the molecule comprises an amino acid sequence set forth in SEQ ID NO:69 or a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:69.
25. The engineered immune cell of any of embodiments 22-24, wherein the molecule comprises an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NO:70 or a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:70 that encodes a functional protein, variant, or fragment thereof.
26. The engineered immune cell of any of embodiments 1-25, wherein the recombinant molecule is or comprises CRIF, e.g., CRIF1 optionally, CRIF1.
27. The engineered immune cell of any of embodiments 1-26, wherein:
the recombinant molecule is capable of promoting or enhancing the metabolic pathway, reaction or step under conditions of hypoxia, low glucose, poor vascularization, and/or ROS;
the recombinant molecule is capable of promoting or enhancing the metabolic pathway, reaction or step in the engineered cell under conditions of hypoxia, low glucose, poor vascularization, and/or ROS, at a level increased as compared to a reference cell, substantially identical to the engineered cell but not comprising the molecule.
28. The engineered immune cell of any of embodiments 1-27, wherein the recombinant molecule is involved in or promotes a pathway resulting in the generation of energy or ATP, which optionally is independent of the presence of glucose or independent of glycolysis.
29. The engineered immune cell of any of embodiments 1-28, wherein the metabolic pathway, step, reaction, or event is or comprises a glycolysis pathway, a reaction thereof, and/or a reaction or pathway that metabolizes a metabolite of the glycolysis pathway, optionally under glucose-low, hypoxic, or nutrient-deprived conditions.
30. The engineered immune cell of any of embodiments 1-29, wherein the recombinant molecule is a molecule that is upregulated or activated in response to antigen-receptor signaling, IL-17-mediated signaling, IL-15-mediated signaling, TRAF-mediated signaling, TRAF6-mediated signaling, IL-7-mediated signaling, IL-21-mediated signaling, low-oxygen conditions, succinate, release of reactive oxygen species (ROS), mTOR-induced signaling, or a functional variant thereof, and/or is differentially expressed or activated under nutrient-rich versus nutrient-poor conditions, or under hypoxic vs. normoxic conditions, or in effector or vs naïve or central memory T cells and/or in exhausted vs non-exhausted T cells, and/or in terminally differentiated T cells vs. non-terminally differentiated T cells.
31. The engineered immune cell of any of embodiments 1-30, wherein the recombinant molecule comprises a hypoxia-induced factor (HIF).
32. The engineered immune cell of embodiment 31, wherein the recombinant molecule comprises a HIF1-alpha.
33. The engineered immune cell of embodiment 31 or embodiment 32, wherein the molecule comprises an amino acid sequence set forth in SEQ ID NO:10 or a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:10.
34. The engineered immune cell of any of embodiments 31-33, wherein the molecule comprises an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NO:79 or a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:79 that encodes a functional protein, variant, or fragment thereof.
35. The engineered immune cell of any of embodiments 1-34, wherein:
the recombinant molecule is capable of promoting generation of a glycolysis metabolite, which optionally is PEP, and/or
the engineered cells exhibit increased generation of said glycolysis metabolite compared to reference cells substantially identical to the engineered cells but not expressing the recombinant molecule, under the same conditions.
36. The engineered immune cell of any of embodiments 1-35, wherein the recombinant molecule comprises a phosphoenolpyruvate carboxykinase 1 (PCK1).
37. The engineered immune cell of embodiment 35 or embodiment 36, wherein the molecule comprises an amino acid sequence set forth in SEQ ID NO:12 or a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:12.
38. The engineered immune cell of any of embodiments 35-37, wherein the molecule comprises an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NO:81 or a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:81 that encodes a functional protein, variant, or fragment thereof.
39. The engineered immune cell of any of embodiments 1-24, wherein the recombinant molecule is or interacts with GLUT4.
40. The engineered immune cell of any of embodiments 1-24, wherein the recombinant molecule is or interacts with SGK1.
41. The engineered cell of any of embodiments 1-40,
wherein the molecule is capable of promoting said metabolic pathway, event, or step or reaction;
wherein the molecule is capable of inhibiting said metabolic pathway, event, or step or reaction;
wherein an outcome of the metabolic pathway, event, or step or reaction is enhanced in the engineered cell compared to a reference cell substantially identical to the engineered cell, but not expressing the recombinant molecule;
wherein the molecule is capable of inhibiting said metabolic pathway, event, or step or reaction; and/or
wherein an outcome of the metabolic pathway, event, or step is inhibited or reduced in the engineered cell compared to a reference cell substantially identical to the engineered cell, but not expressing the recombinant molecule.
42. The engineered cell of any of embodiments 1-40, wherein the recombinant molecule is a nucleic acid or protein capable of interfering with expression, activity, or stability of a negative regulator of the metabolic pathway or event or step or reaction, or a molecule that stabilizes the expression or longevity of a molecule that promotes said pathway, event, step or reaction.
43. The engineered cell of embodiment 42, wherein the nucleic acid or protein is an RNAi, siRNA, or shRNA molecule.
44. The engineered immune cell of any of embodiments 1-43, further comprising a disruption in expression or function of an immune checkpoint molecule, wherein the disruption or expression thereby promotes activation, proliferation, expansion, or reduced exhaustion, of the immune cell and/or is capable of reducing generation of or longevity of memory T cells or central memory T cells.
45. The engineered immune cell of embodiment 44, wherein the checkpoint molecule comprises a PD-1, PD-L1, TIM3, CTLA4 or an adenosine receptor.
46. The engineered cell of any of embodiments 1-45, wherein persistence of the engineered cell and/or of reprograming in favor of memory T cell, central memory T cell, Tscm, and/or undifferentiated phenotype cells, and/or reduction in exhaustion phenotype, and/or reduction in regulatory T cells, is enhanced or increased in the cell as compared to a cell substantially the same as the engineered cell without the recombinant molecule, under the same conditions.
47. The engineered cell of embodiment 46, wherein the conditions comprise activation via an antigen receptor, a TCR, an ITAM-containing signaling molecule, cytokine signaling, TNFR signaling, and/or adoptive transfer to a subject containing cells expressing the ligand.
48. The engineered cell of any of embodiments 1-47, wherein the cell is a T cell.
49. The engineered cell of embodiment 48, wherein the T cell is a CD8+ T cell.
50. The engineered cell of embodiment 48, wherein the T cell is a CD4+ T cell.
51. The engineered cell of any of embodiments 1-47, wherein the cell is a natural killer (NK) cell.
52. The engineered cell of any of embodiments 1-47, wherein the cell is an iPS-derived cell.
53. The engineered cell of any of embodiments 1-52, wherein the genetically engineered receptor that specifically binds to a ligand is a functional non-T cell receptor.
54. The engineered cell of any of embodiments 1-53, wherein the genetically engineered receptor that specifically binds to a ligand is a chimeric antigen receptor (CAR).
55. The engineered cell of embodiment 54, wherein the CAR comprises an extracellular antigen-recognition domain that specifically binds to the antigen and an intracellular signaling domain comprising an ITAM.
56. The engineered cell of embodiment 55, wherein the intracellular signaling domain comprises an intracellular domain of a CD3-zeta (CD3ζ) chain.
57. The engineered cell of embodiment 55 or embodiment 56, wherein the CAR further comprises a costimulatory signaling region.
58. The engineered cell of embodiment 57, wherein the costimulatory signaling region comprises a signaling domain of CD28 or 4-1BB.
59. The engineered cell of embodiment 57 or embodiment 58, wherein the costimulatory domain is CD28.
60. The engineered cell of any of embodiments 1-52, wherein the genetically engineered receptor that specifically binds to a ligand is a recombinant receptor.
61. The engineered cell of any of embodiments 1-52, wherein the genetically engineered receptor that specifically binds to a ligand is a transgenic T cell receptor (TCR).
62. The engineered cell of any of embodiments 1-61, wherein expression of the molecule in the cell is under the control of a conditional promoter or enhancer or transactivator.
63. The engineered cell of embodiment 62, wherein the conditional promoter or enhancer or transactivator is an inducible promoter, enhancer, or transactivator or a repressible promoter, enhancer, or transactivator.
64. The engineered cell of embodiment 62 or embodiment 63, wherein the promoter is selected from among an RNA pol I, pol II or pol III promoter.
65. The engineered cell of embodiment 64, wherein the promoter is selected from: a pol III promoter that is a U6 or H1 promoter; or a pol II promoter that is a CMV, SV40 early region or adenovirus major late promoter.
66. The engineered cell of any of embodiments 62-65, wherein the promoter is an inducible promoter.
67. The engineered cell of embodiment 66, wherein the promoter comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence or a doxycycline operator sequence, or is an analog thereof.
68. The engineered cell of any of embodiments 62-65, wherein the promoter is a repressible promoter.
69. The engineered cell of embodiment 68, wherein the promoter comprises a Lac repressor or a tetracycline repressor, or is an analog thereof.
70. The engineered immune cell of any of embodiments 1-69, further comprising a recombinant truncated cell surface receptor.
71. The engineered immune cell of embodiment 70, wherein the cell surface receptor is selected from among a modified and/or truncated form of EGFR (tEGFR), ErbB2/HER2/neu (Her2t), CD34 and/or NGFR.
72. The engineered immune cell of embodiment 70 or embodiment 71, wherein the cell surface receptor comprises an amino acid sequence selected from among:
(i) any of SEQ ID NO: 55, 74 and 83; and
(ii) a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 55, 74 and 83.
73. The engineered immune cell of any of embodiments 70-72, wherein the cell surface marker comprises an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NO:74 or a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:74 that encodes a functional protein, variant, or fragment thereof.
74. A nucleic acid molecule, comprising the nucleotide sequence set forth in any of SEQ ID NOS: 85, 87, 89, 91, 93, 95 and 97 or a sequence of nucleotides that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to 85, 87, 89, 91, 93, 95 and 97 that encodes a functional protein, variant, or fragment thereof.
75. A nucleic acid molecule(s), comprising:
a nucleotide sequence encoding a genetically engineered receptor that specifically binds to a ligand; and
a nucleotide sequence encoding a molecule that is involved in or capable of modulating a metabolic pathway or a functional and/or catalytically-active portion or variant thereof.
76. The nucleic acid molecule of embodiment 75, wherein the encoded genetically engineered receptor and encoded molecule involved in or capable of modulating a metabolic pathway are any as present in cells as forth in embodiments 1-58.
77. The nucleic acid molecule of embodiment 75 or embodiment 76 that is a single polynucleotide.
78. The nucleic acid molecule of any of embodiments 75-77, further comprising at least one promoter operatively linked to control expression of the genetically engineered receptor and the molecule involved in or capable of modulating a metabolic pathway.
79. The nucleic acid molecule of any of embodiments 75-78, wherein the nucleotide sequence encoding the genetically engineered receptor is operatively linked to a first promoter and the nucleotide sequence encoding the molecule involved in or capable of modulating a metabolic pathway is operatively linked to a second promoter, which first and second promoter can be the same or different.
80. The nucleic acid molecule of embodiment 78 or embodiment 79, wherein the nucleotide sequence encoding the genetically engineered receptor and the nucleotide sequence encoding the molecule involved in or capable of modulating a metabolic pathway are separated by an internal ribosome entry site (IRES), a self-cleaving peptide or a peptide that causes ribosome skipping, optionally a T2A, P2A, E2A and/or F2A peptide, and the first and second chimeric receptor are expressed under the control of the same promoter.
81. The nucleic acid molecule of embodiment 80, wherein the peptide that causes ribosome skipping comprises an amino acid sequence selected from among:
(i) any of SEQ ID NO: 54 and 64-68; and
(ii) a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 54 and 64-68.
82. The nucleic acid molecule of embodiment 80 or embodiment 81, wherein nucleotide sequence encoding the peptide that causes ribosome skipping is selected from among a nucleotide sequence set forth in SEQ ID NO:71 and a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:71 that encodes a functional protein, variant, or fragment thereof.
83. The nucleic acid molecule of any of embodiments 75-82, wherein the encoded molecule that is involved in or capable of modulating a metabolic pathway comprises an amino acid sequence selected from among:
(i) any of SEQ ID NO: 4, 7-10, 12 and 69; and
(ii) a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 4, 7-10, 12 and 69.
84. The nucleic acid molecule of any of embodiments 75-83, wherein the nucleotide sequence encoding the molecule that is involved in or capable of modulating a metabolic pathway is selected from among:
(i) any of SEQ ID NO: 70 and 76-81; and
(ii) a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 70 and 76-81 that encodes a functional protein, variant, or fragment thereof.
85. The nucleic acid molecule of any of embodiments 75-84, further comprising a nucleotide sequence encoding a recombinant truncated cell surface receptor.
86. The nucleic acid molecule of embodiment 85, wherein the cell surface receptor is selected from among a modified and/or truncated form of EGFR (tEGFR), ErbB2/HER2/neu (Her2t), CD34 and/or NGFR.
87. The nucleic acid molecule of embodiment 85 or embodiment 86, wherein the cell surface receptor comprises an amino acid sequence selected from among:
(i) any of SEQ ID NO: 55, 74 and 83; and
(ii) a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 55, 74 and 83.
88. The nucleic acid molecule of any of embodiments 85-87, wherein the nucleotide sequence encoding the cell surface receptor is set forth in SEQ ID NO:74 or is a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:74 that encodes a functional protein, variant, or fragment thereof.
89. The nucleic acid molecule of any of embodiments 75-88, wherein the nucleotide sequence encodes an amino acid sequence selected from among:
(i) any of SEQ ID NO: 85, 87, 89, 91, 93, 95 and 97; and
(ii) a functional variant comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 85, 87, 89, 91, 93, 95 and 97.
90. The nucleic acid molecule of any of embodiments 75-89, wherein the nucleotide sequence is selected from among:
(i) any of SEQ ID NO: 84, 86, 88, 90, 92, 94 and 96; and
(ii) a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NO: 84, 86, 88, 90, 92, 94 and 96 that encodes a functional protein, variant, or fragment thereof.
91. A vector, comprising the nucleic acid molecule of any of embodiments 74-90.
92. The vector of embodiment 91 that is a viral vector.
93. The vector of embodiment 91 or embodiment 92 that is a retroviral vector.
94. The vector of any of embodiments 91-93 that is a lentiviral vector or a gammaretroviral vector.
95. An engineered cell, comprising the nucleic acid molecules(s) of any of embodiments 74-90 or the vector of any of embodiments 91-94.
96. The engineered cell of embodiment 95, wherein the cell is a T cell.
97. The engineered cell of embodiment 96, wherein the T cell is a CD8+ T cell.
98. The engineered cell of embodiment 96, wherein the T cell is a CD4+ T cell.
99. The engineered cell of embodiment 95, wherein the cell is a natural killer (NK) cell.
100. The engineered cell of embodiment 95, wherein the cell is an iPS-derived cell.
101. The engineered cell of any of embodiments 95-98, wherein the genetically engineered receptor that specifically binds to a ligand is a functional non-T cell receptor.
102. The engineered cell of any of embodiments 95-99, wherein the genetically engineered receptor that specifically binds to a ligand is a chimeric antigen receptor (CAR).
103. The engineered cell of embodiment 102, wherein the CAR comprises an extracellular antigen-recognition domain that specifically binds to the antigen and an intracellular signaling domain comprising an ITAM.
104. The engineered cell of embodiment 103, wherein the intracellular signaling domain comprises an intracellular domain of a CD3-zeta (CD3ζ) chain.
105. The engineered cell of embodiment 103 or embodiment 104, wherein the CAR further comprises a costimulatory signaling region.
106. The engineered cell of embodiment 105, wherein the costimulatory signaling region comprises a signaling domain of CD28 or 4-1BB.
107. The engineered cell of embodiment 105 or embodiment 106, wherein the costimulatory domain is CD28.
108. The engineered cell of any of embodiments 95-107, wherein the genetically engineered receptor that specifically binds to a ligand is a recombinant receptor.
109. The engineered cell of any of embodiments 95-108, wherein the genetically engineered receptor that specifically binds to a ligand is a transgenic T cell receptor (TCR).
110. The engineered cell of any of embodiments 1-73 and 95-109, wherein the recombinant molecule is inducible upon activation, stress, hypoxia, decreased vasculature, alterations in tumor microenvironment, checkpoint molecule upregulation, proliferation, expression of an activation marker, or exposure to low glucose conditions.
111. A composition comprising the engineered cell of any of embodiments 1-73 and 95-110.
112. The composition of embodiment 111, wherein the cells are CD4+ or CD8+ cells.
113. The composition of embodiment 112, wherein the cells are CD4+ cells and the composition further comprises CD8+ cells that are genetically engineered with the ligand-binding receptor but do not express the recombinant molecule.
114. The composition of embodiment 112, wherein the cells are CD4+ cells and the composition further comprises CD8+ cells that are genetically engineered with the ligand-binding receptor and express the recombinant molecule.
115. The composition of embodiment 112, wherein the cells are CD8+ cells and the composition further comprises CD4+ cells that are genetically engineered with the ligand-binding receptor but do not express the recombinant molecule.
116. The composition of embodiment 112, wherein the cells are CD8+ cells and the composition further comprises CD4+ cells that are genetically engineered with the ligand-binding receptor and express the recombinant molecule.
117. The composition of any of embodiments 112-116, wherein the CD4+ cells express a different ligand-binding receptor than the CD8+ cells.
118. The composition of embodiment 117, wherein the only difference in the ligand-binding receptor expressed in the CD4+ cell compared to the CD8+ cell is the different costimulatory signaling domain.
119. The composition of embodiment 118, wherein the different costimulatory signaling domain is or comprises a cytoplasmic signaling domain of a CD28, a 4-1BB, or an ICOS molecule, or is a functional variant of a signaling portion thereof.
120. A method of treatment, comprising administering the engineered cell of any of embodiments 1-73 and 95-110 or the composition of any of embodiments 111-119 to a subject having a disease or condition.
121. The method of embodiment 120, wherein the ligand-binding receptor specifically binds to a ligand or antigen associated with the disease or condition.
122. The method of embodiment 120 or embodiment 121, wherein the disease or condition is a cancer, a tumor, an autoimmune disease or disorder, or an infectious disease.
123. The method of any of embodiments 120-122, wherein the engineered cells exhibit increased or longer expansion and/or persistence in the subject than in a subject administered the same or about the same dosage amount of a reference cell composition.
124. The method of any of embodiments 120-123, wherein there is an increase or greater number of memory T cells or a memory T cell subset and/or an increased or longer persistence of memory T cells or a memory T cell subset in the subject derived from the administered the engineered cells compared to the number or persistence of the memory T cells or memory T cell subset in a subject derived from a reference cell composition administered at the same or about the same dosage.
125. The method of embodiment 124, wherein the memory T cells or memory T cell subset are CD62L+.
126. The method of embodiment 124 or embodiment 125, wherein the memory T cells or memory T cell subset are central memory T cells (Tcm), long-lived memory T cells or T memory stem cells (Tscm).
127. The method of any of embodiments 124-126, wherein the memory T cells or memory T cell subset further comprises a phenotype comprising:
a) CD127+; and/or
b) any one or more of CD45RA+, CD45RO−, CCR7+ and CD27+ and any one or more of t-betlow, IL-7Ra+, CD95+, IL-2Rβ+, CXCR3+ and LFA-1+.
128. The method of any of embodiments 120-127, wherein the memory T cells or memory T cell subset are CD8+.
129. The method of any of embodiments 120-128, wherein the number of memory T cells or a memory T cell subset derived from the administered genetically engineered cells comprises an increase or greater percentage of central memory T cells (Tcm), long-lived memory T cells or T memory stem cells (Tscm) compared to the number of such cells derived from a reference cell composition administered at the same or about the same dosage.
130. The method of any of embodiments 120-129, wherein there is an increase or greater number of non-terminally differentiated T cells in the subject derived from the administered genetically engineered T cells compared to the number of the non-terminally differentiated cells in a subject derived from a reference cell composition administered at the same or about the same dosage amount.
131. The method of any of embodiments 120-130, wherein the genetically engineered cells in the subject derived from the administered genetically engineered cells exhibit an increase in activation or proliferation upon restimulation ex vivo in the presence of a stimulatory agent or agent compared to the activation or proliferation of genetically engineered cells in a subject derived from a reference cell composition administered at the same or about the same dosage when restimulated ex vivo in the presence of the same stimulatory agent or agents.
132. The method of embodiment 131, wherein the stimulatory agent or agents comprise an antigen, an anti-CD3/anti-CD28 antibody or comprises an IL-2, IL-15 and/or IL-7 cytokine.
133. The method of any of embodiments 123-132, wherein the increase is at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, or 5-fold.
134. The method of any of embodiments 120-133, wherein there is a decreased or reduced expression of an exhaustion marker genetically engineered cells in the subject derived from the administered genetically engineered T cells compared to the expression of the exhaustion marker in genetically engineered cells in a subject administered the same or about the same dosage amount of a reference cell composition.
135. The method of embodiment 134, wherein the exhaustion marker is selected from among CD244, CD160 and PD-1.
136. The method of embodiment 134 or embodiment 135, wherein the expression is decreased or reduced 1.2-fold, 1.5-fold, 2.0-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more.
137. The method of any of embodiments 120-136, wherein the increase or decrease is observed or is present within a month, within two months, within six months or within one year of administering the cells.
138. The composition of any of embodiments 111-119 or the method of any of embodiments 120-137, wherein the reference cell composition contains genetically engineered cells that are substantially the same except the expressed chimeric receptor comprises a different costimulatory molecule and/or comprises a costimulatory domain of CD28, 4-1BB or ICOS.
139. A composition of any of embodiments 111-119 for use in treating a disease or condition in a subject having a disease or condition.
140. Use of a composition of any of embodiments 111-119 for treating a disease or condition in a subject having a disease or condition.
141. Use of a composition of any of embodiments 111-119 for the manufacture of a medicament for treating a disease or condition in a subject having a disease or condition.
142. The composition for use of embodiment 139 or the use of embodiment 140 or embodiment 141, wherein the ligand-binding receptor specifically binds to a ligand or antigen associated with the disease or condition.
143. The composition for use of embodiment 139 or the use of embodiment 140 or embodiment 141 or the composition for use or the use of embodiment 142, wherein the disease or condition is a cancer, a tumor, an autoimmune disease or disorder, or an infectious disease.
VII. Definitions
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include “consisting” and/or “consisting essentially of” aspects and variations.
Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.
The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.
As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
As used herein, “enriching” when referring to one or more particular cell type or cell population, refers to increasing the number or percentage of the cell type or population, e.g., compared to the total number of cells in or volume of the composition, or relative to other cell types, such as by positive selection based on markers expressed by the population or cell, or by negative selection based on a marker not present on the cell population or cell to be depleted. The term does not require complete removal of other cells, cell type, or populations from the composition and does not require that the cells so enriched be present at or even near 100% in the enriched composition.
As used herein, a statement that a cell or population of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.
As used herein, a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.
The term “expression”, as used herein, refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.
As used herein, a subject includes any living organism, such as humans and other mammals. Mammals include, but are not limited to, humans, and non-human animals, including farm animals, sport animals, rodents and pets.
As used herein, a control refers to a sample that is substantially identical to the test sample, except that it is not treated with a test parameter, or, if it is a plasma sample, it can be from a normal volunteer not affected with the condition of interest. A control also can be an internal control.
As used herein, “operably linked” or “operatively linked” refers to the association of components, such as a DNA sequence, e.g. a heterologous nucleic acid and a regulatory sequence(s), in such a way as to permit gene expression when the appropriate molecules (e.g. transcriptional activator proteins) are bound to the regulatory sequence. Hence, it means that the components described are in a relationship permitting them to function in their intended manner and/or in which there is a functional linkage of at least two sequences. For example, operably linked includes linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Operably associated includes linkage between an inducing or repressing element and a promoter, wherein the inducing or repressing element acts as a transcriptional activator of the promoter.
As used herein, “percent (%) sequence identity” and “percent identity” when used with respect to a nucleotide sequence (reference nucleotide sequence) or amino acid sequence (reference amino acid sequence) is defined as the percentage of nucleotide residues or amino acid residues, respectively, in a candidate sequence that are identical with the residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Among the vectors are viral vectors, such as lentiviral vectors.
VIII. Examples
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1: Generation of Nucleic Acid Constructs Encoding Metabolic Pathway-Modulating Molecules
Various exemplary nucleic acid molecule constructs were generated encoding a metabolic pathway-modulating molecule that included either: (1) nucleotides set forth in SEQ ID NO:70 encoding lymphocyte expansion molecule (LEM) isoform 2 set forth in SEQ ID NO:69; (2) nucleotides set forth in SEQ ID NO: 76 encoding DAG O-acetyltransferase 1 (DGAT1) set forth in SEQ ID NO:7; (3) nucleotides set forth in SEQ ID NO: 77 encoding Glycerol kinase (Gyk) set forth in SEQ ID NO: 4; (4) nucleotides set forth in SEQ ID NO: 78 encoding Glycerol-3-phosphate acetyltransferase mitochondrial (GPAM) set forth in SEQ ID NO:8; (5) nucleotides set forth in SEQ ID NO: 79 encoding HIF1α set forth in SEQ ID NO: 10; (6) nucleotides set forth in SEQ ID NO: 80 encoding MOGAT1 set forth in SEQ ID NO:9; (7) nucleotides set forth in SEQ ID NO: 81 encoding PCK1 set forth in SEQ ID NO: 12.
Each nucleic acid construct also included a nucleic acid molecule encoding a truncated Her2 (Her2t, SEQ ID NO: 75, nucleotides set forth in SEQ ID NO: 74) for use as a transduction marker separated from the nucleic acid molecule encoding the metabolic pathway-modulating molecule by a self-cleaving T2A peptide (SEQ ID NO: 54, nucleotides set forth in SEQ ID NO: 71). The nucleic acid molecule was also designed to encode an N-terminal signal sequence derived from GMCSFR (SEQ ID NO: 72, nucleotides set forth in SEQ ID NO: 73).
Nucleic acid sequences of exemplary constructs are set forth in SEQ ID NOS: 84, 86, 88, 90, 92, 94 and 96 and encode the sequences set forth in SEQ ID NOS: 85, 87, 89, 91, 93, 95 and 97, respectively. The nucleic acid molecules were cloned into a lentiviral vector for transduction of primary T cells.
Example 2: Assessment of Proliferation, Anti-Tumor Efficacy, and Expansion of Engineered T Cells Co-Expressing a CAR and a Metabolic Pathway-Modulating Molecule
T cells are isolated by immunoaffinity-based enrichment from human donor subjects and cells from each donor are activated and co-transduced with a lentiviral vector encoding a chimeric antigen receptor (CAR) and a lentiviral vector encoding a metabolic pathway-modulating molecule as described in Example 1. An exemplary CAR contains an anti-CD19 scFv, an Ig-derived spacer, a human CD28-derived transmembrane domain, a human 4-1BB-derived intracellular signaling domain and a human CD3 zeta-derived signaling domain. An alternative exemplary CAR may be substantially similar to the aforementioned CAR, but may comprise a human CD28-derived intracellular signaling domain. As a control, T cells also were generated by transduction with the CAR only, with the metabolic pathway-modulating molecule only or with an empty vector.
The genetically engineered T cells, including CAR-expressing cells and/or cells ectopically expressing a metabolic pathway-modulating molecule, produced as described above, are assessed for various responses following co-culture with CD19-expressing cells. In vitro assays to assess CAR-T cell activity were conducted using K562 cells transduced with CD19 (K562-CD19) as target cells.
To assess cell expansion and proliferation K562-CD19 target cells are incubated with the various genetically engineered T cells (at various effector:target ratios). The fold change in total number of cells at Day 0 prior to stimulation compared to Day 5 post-stimulation is determined for T cells co-expressing a CAR and a metabolic pathway-modulating molecule and compared to the fold change of T cells expressing only the CAR. In some cases, proliferation of the various genetically engineered cells following incubation with CD19-expressing target cells is assessed by flow cytometry. Genetically engineered T cells are labeled with 0.2 μM carboxyfluorescein succinmidyl ester (CFSE). Cells are washed and incubated for 72 hours in triplicate with target cells (K562-CD19) in serum-containing medium without exogenous cytokines. Division of live T cells is indicated by CFSE dilution, as assessed by flow cytometry.
To assess T cell killing activity, CD19-expressing K562-CD19 target cells are co-cultured with the various genetically engineered T cells at various E:T ratios (e.g. from 1:1 to 9:1). Cell lysis is monitored in real-time over a 0 to about 110-hour time course by adding an IncuCyte™ fluorescent Caspase 3/7 Reagent to the co-cultures to detect apoptotic cells. Target cell death is quantitated by automated image analysis over time. The area under the curve (AUC) of fluorescent signal over time for each concentration is determined and a killing index is determined using the formula: 1/AUC. The cell killing achieved with T cells expressing both a CAR and a metabolic pathway-modulating molecule is compared with control T cells.
Cytokine release is assessed from supernatants following incubation of the various genetically engineered cells with antigen-expressing K562-CD19 target cells at various E:T ratios (e.g. from 1:1 to 9:1). The presence of cytokines, such as TNF-α, IFNγ, GM-CSF and IL-2, in culture supernatants is assessed using an ELISA or other immunoassay method. The concentration of various cytokines in the supernatants from co-cultures incubated with T cells expressing both a CAR and a metabolic pathway-modulating molecule is compared with control T cells.
The ability of cells to expand ex vivo following repeated stimulations in some aspects can indicate capacity of CAR-T cells to persist (e.g. following initial activation) and/or is indicative of function in vivo (Zhao et al. (2015) Cancer Cell, 28:415-28). To assess expansion following restimulation in a serial stimulation assay, CAR-T cells are plated on 96 well Poly-D-Lysine coated plates to which irradiated target cells (K562-CD19) are added at various effector to target ratios (e.g. from 1:1 to 9:1). Cells are stimulated, harvested every 3-4 days and counted, and restimulated with new target cells using the same culture conditions after resetting cell number to initial seeding density for each round. Multiple rounds of stimulation during a 14 to 28 day culture period are carried out. For each round of stimulation, the number of doublings of the cell population is determined for T cells expressing both a CAR and a metabolic pathway-modulating molecule and compared to control T cells.
The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.
SEQUENCES
SEQ
ID
NO.
SEQUENCE
DESCRIPTION
1
MRESQDAAGAHGWNRVGSTATKWFTGAPFGVQSHREDISAVYPNWKKFSTFTEAP
C1ORF177 isoform
YSTRYSTQVSHIGPGTYSSKETCFSKKKLMKEVDTGWAKAQEATRLTQLPHFQYQ
1(huLEM)
AIMKEKRLKEQKLGPGSYNLKDFLEQLREKPCSTRGLLSSGEVRFRGLTGNYYPG
Homo sapiens
PGNYGEKGNPYTKLEENAWNRSHSEGLMCRMSNKPHPRPHQGSGLGPGTYFEKSD
NCCBI Reference
LETYVARSVGTRGPYDTFSGDRSKPLPYGHYSMQKKKPRELMNEKSFVEELNSHH
Sequence:
NKKHGVESKLPRNPKTPTERIYWANLSQCPRTLATSGPSEWLPQEKKCKPVNQPP
NP_689820.2
FLLTSKGSGAKACQMIMGSWNPVGVGRYLNTWLMETKDRRQRYRSLELSGSKRYL
SDLARDMLMQIRLFCWKGLEVINFGLSPM
2
MRESQDAAGAHGWNRVGSTATKWFTGAPFGVQSHREDISAVYPNWKKFSTFTEAP
C1ORF177
YSTRYSTQVSHIGPGTYSSKETCFSKKKLMKEVDTGWAKAQEATRLTQLPHFQYQ
isoform2 (huLEM)
AIMKEKRLKEQKLGPGSYNLKDFLEQLREKPCSTRGLLSSGEVRFRGLTGNYYPG
Homo sapiens
PGNYGEKGNPYTKLEENAWNRSHSEGLMCRMSNKPHPRPYQGSGLGPGTYFEKSD
UniProt accession
LETYVARSVGTRGPYDTFSGDRSKPLPYGHYSMQKKKPRELMNEKSFVEELNSHH
number Q3ZCV2
NKKHGVESKLPRNPKTPTERIYWANLSQCPRTLATSGPSEWLPQEKKCKPVNQPP
FLLTSKGSGAKACQMIMGSWNPVGVGRYLNTWLMETKDRRQRYRSLELSGSKRYL
SDLARDMLMQERITPFTKGKCPPTVDYNSDPTP
3
MRESQDAAGAHGWNRVGSTATKWFTGAPFGVQSHREDISAVYPNWKKFSTFTEAP
C1ORF177 isoform
YSTRYSTQVSHIGPGTYSSKETCFSKKKLMKEVDTGWAKAQEATRLTQLPHFQYQ
1(huLEM)
AIMKEKRLKEQKLGPGSYNLKDFLEQLREKPCSTRGLLSSGEVRFRGLTGNYYPG
Homo sapiens
PGNYGEKGNPYTKLEENAWNRSHSEGLMCRMSNKPHPRPHQGSGLGPGTYFEKSD
LETYVARSVGTRGPYDTFSGDRSKPLPYGHYSMQKKKPRELMNEKSFVEELNSHH
NKKHGVESKLPRNPKTPTERIYWANLSQCPRTLATSGPSEWLPQEKKCKPVNQPP
FLLTSKGSGAKACQMIMGSWNPVGVGRYLNTWLMETKDRRQRYRSLELSGSKRYL
SDLARDMLMQ
4
MAASKKAVLGPLVGAVDQGTSSTRELVENSKTAELLSHHQVEIKQEFPREGWVEQ
Glycerol kinase
DPKEILHSVYECIEKTCEKLGQLNIDISNIKAIGVSNQRETTVVWDKITGEPLYN
(Gyk)
AVVWLDLRTQSTVESLSKRIPGNNNEVKSKTGLPLSTYFSAVKLRWLLDNVRKVQ
Homo sapiens
KAVEEKRALFGTIDSWLIWSLTGGVNGGVHCTDVTNASRTMLFNIHSLEWDKQLC
UniProt accession
EFFGIPMEILPNVRSSSEIYGLMKISHSVKAGALEGVPISGCLGDQSAALVGQMC
number Q14410
FQIGQAKNTYGTGCELLCNTGHKCVESDHGLLTTVAYKLGRDKPVYYALEGSVAI
AGAVIRWLRDNLGIIKTSEEIEKLAKEVGTSYGCYFVPAFSGLYAPYWEPSARGI
ICGLTQFTNKCHIAFAALEAVCFQTREILDAMNRDCGIPLSHLQVDGGMTSNKIL
MQLQADILYIPVVKPSMPETTALGAAMAAGAAEGVGVWSLEPEDLSAVTMERFEP
QINAEESEIRYSTWKKAVMKSMGWVTTQSPESGDPSIFCSLPLGFFIVSSMVMLI
GARYISGIP
5
MAAPKTAAVGPLVGAVVQGTNSTRELVENSKTAELLSHHKVELTQEFPKEGWVEQ
Glycerol kinase
DPKEILQSVYECIARTCEKLDELNIDISNIKAVGVSNQRETTVIWDKLTGEPLYN
(Gyk)
AVVWLDLRTQTTVEDLSKKIPGNSNEVKSKTGLPLSTYFSAVKLRWMLDNVRNVQ
Homo sapiens
KAVEEGRALFGTIDSWLIWSLTGGVNGGVHCTDVTNASRTMLFNIHSLEWDKELC
GenBank:
DFFEIPMDLLPNVFSSSEIYGLIKTGALEGVPISGCLGDQCAALVGQMCFQEGQA
CAA55365.1
KNTYGTGCELLCNTGRKCVESEHGLLTTVAYKLGREKPAYYALEGSVAIAGAVIR
WLRDNLGIIETSGDIERLAKEVGTSYGCYFVPAFSGLYAPYWEPSARGILCGLTQ
FTNKCHIAFAALEAVCFQTREILEAMNRDCGIPLRHLQVDGGMTNNKVLMQLQAD
ILHIPVIKPFMPETTALGAAMAAGAAEGVSVWSLEPQALSVLRMERFEPQIQATE
SEIRYATWKKAVMKSMGWVTSQSPEGGDPSIFCSLPLGFFIVSSMVMLIGARYIS
GVP
6
MAASKKAVLGPLVGAVDQGTSSTRELVENSRTAELLSHHQVEIKQEFPREGWVEQ
Glycerol kinase
DPKEILHSVYECIEKTCEKLGQLNIGISNIKAIGVSNQRETTVAWDKITGEPLYN
(Gyk)
AVVWLDLRTQSTVESLSKRIPGNNNEVKSKTGLPLSTYFSAVKLRWLLDNVRKVQ
Homo sapiens
KAVEEKRALFGTIDSWLIWSLTGGVNGGVHCTDVTNASRTMLFNIHSLEWDKQLC
GenBank:
EFFGIPMEILPHVRSSSEIYGLMKAGALEGVPISGCLGDQSAALVGQMCFQIGQA
CAA55364.1
KNTYGTGCELLCNTGHKCVESDHGLLTTVAYKLGRDKPVYYALEGSVAIAGAVIR
WLRDNLGIIKTSEEIEKLAKEVGTSYGCYFVPAFSGLYAPYWEPSARGIICGLTQ
FTNKCHIAFAALEAVCFQTREILDAMNRDCGIPLSHLQVDGGMTSNKILMQLQAD
ILYIPVVKPLMPETTALGAAMAAGAAEGVDVWSLEPEDLSAVTMERFEPQINAEE
SEIRYSTWKKAVMKSMGWVTTQSPEGGDPSVFCSLPLGEFIVSSMAMLIGARYIS
GIP
7
MGDRGSSRRRRTGSRPSSHGGGGPAAAEEEVRDAAAGPDVGAAGDAPAPAPNKDG
DAG O-
DAGVGSGHWELRCHRLQDSLFSSDSGFSNYRGILNWCVVMLILSNARLFLENLIK
acetyltransferase 1
YGILVDPIQVVSLFLKDPYSWPAPCLVIAANVFAVAAFQVEKRLAVGALTEQAGL
(DGAT1)
LLHVANLATILCFPAAVVLLVESITPVGSLLALMAHTILELKLESYRDVNSWCRR
Homo sapiens
ARAKAASAGKKASSAAAPHTVSYPDNLTYRDLYYFLFAPTLCYELNFPRSPRIRK
UniProt accession
RFLLRRILEMLFFTQLQVGLIQQWMVPTIQNSMKPFKDMDYSRIIERLLKLAVPN
number 075907
HLIWLIFFYWLFHSCLNAVAELMQFGDREFYRDWWNSESVTYFWQNWNIPVHKWC
NCBI Reference
IRHFYKPMLRRGSSKWMARTGVFLASAFFHEYLVSVPLRMFRLWAFTGMMAQIPL
Sequence:
AWFVGRFFQGNYGNAAVWLSLIIGQPIAVLMYVHDYYVLNYEAPAAEA
NP_036211.2
8
MDESALTLGTIDVSYLPHSSEYSVGRCKHTSEEWGECGFRPTIFRSATLKWKESL
Glycerol-3-
MSRKRPFVGRCCYSCTPQSWDKFFNPSIPSLGLRNVIYINETHTRHRGWLARRLS
phosphate
YVLFIQERDVHKGMFATNVTENVLNSSRVQEAIAEVAAELNPDGSAQQQSKAVNK
acetyltransferase
VKKKAKRILQEMVATVSPAMIRLTGWVLLKLENSFEWNIQIHKGQLEMVKAATET
mitochondrial
NLPLLFLPVHRSHIDYLLLTFILFCHNIKAPYIASGNNLNIPIFSTLIHKLGGFF
(GPAM)
IRRRLDETPDGRKDVLYRALLHGHIVELLRQQQFLEIFLEGTRSRSGKTSCARAG
Homo sapiens
LLSVVVDTLSTNVIPDILIIPVGISYDRIIEGHYNGEQLGKPKKNESLWSVARGV
UniProt accession
IRMLRKNYGCVRVDFAQPFSLKEYLESQSQKPVSALLSLEQALLPAILPSRPSDA
number Q9HCL2
ADEGRDTSINESRNATDESLRRRLIANLAEHILFTASKSCAIMSTHIVACLLLYR
NCBI Reference
HRQGIDLSTLVEDFFVMKEEVLARDFDLGFSGNSEDVVMHAIQLLGNCVTITHTS
Sequence:
RNDEFFITPSTTVPSVFELNEYSNGVLHVFIMEAIIACSLYAVLNKRGLGGPTST
NP_001231878.1
PPNLISQEQLVRKAASLCYLLSNEGTISLPCQTFYQVCHETVGKFIQYGILTVAE
HDDQEDISPSLAEQQWDKKLPEPLSWRSDEEDEDSDFGEEQRDCYLKVSQSKEHQ
QFITFLQRLLGPLLEAYSSAAIFVHNFSGPVPEPEYLQKLHKYLITRTERNVAVY
AESATYCLVKNAVKMFKDIGVFKETKQKRVSVLELSSTFLPQCNRQKLLEYILSF
VVL
9
MKVEFAPLNIQLARRLQTVAVLQWVLKYLLLGPMSIGITVMLIIHNYLFLYIPYL
Monoacylglycerol
MWLYEDWHTPERGGRRSSWIKNWTLWKHEKDYFPIHLIKTQDLDPSHNYIFGFHP
O-acetyltransferase
HGIMAVGAFGNESVNYSDEKDLFPGFTSYLHVLPLWFWCPVEREYVMSVGLVSVS
(MOGAT1)
KKSVSYMVSKEGGGNISVIVLGGAKESLDAHPGKFTLFIRQRKGFVKIALTHGAS
Homo sapiens
LVPVVSFGENELFKQTDNPEGSWIRTVQNKLQKIMGFALPLFHARGVFQYNEGLM
UniProt accession
TYRKAIHTVVGRPIPVRQTLNPTQEQIEELHQTYMEELRKLFEEHKGKYGIPEHE
number Q96PD6
TLVLK
NCBI Reference
Sequence:
NP_477513.2
10
MEGAGGANDKKKISSERRKEKSRDAARSRRSKESEVFYELAHQLPLPHNVSSHLD
HIF1α
KASVMRLTISYLRVRKLLDAGDLDIEDDMKAQMNCFYLKALDGFVMVLTDDGDMI
Homo sapiens
YISDNVNKYMGLTQFELTGHSVEDFTHPCDHEEMREMLTHRNGLVKKGKEQNTQR
UniProt accession
SFFLRMKCTLTSRGRTMNIKSATWKVLHCTGHIHVYDTNSNQPQCGYKKPPMTCL
number Q16665
VLICEPIPHPSNIEIPLDSKTFLSRHSLDMKFSYCDERITELMGYEPEELLGRSI
YEYYHALDSDHLTKTHHDMFTKGQVTTGQYRMLAKRGGYVWVETQATVIYNTKNS
QPQCIVCVNYVVSGIIQHDLIFSLQQTECVLKPVESSDMKMTQLFTKVESEDTSS
LFDKLKKEPDALTLLAPAAGDTIISLDFGSNDTETDDQQLEEVPLYNDVMLPSPN
EKLQNINLAMSPLPTAETPKPLRSSADPALNQEVALKLEPNPESLELSFTMPQIQ
DQTPSPSDGSTRQSSPEPNSPSEYCFYVDSDMVNEFKLELVEKLFAEDTEAKNPF
STQDTDLDLEMLAPYIPMDDDEQLRSEDQLSPLESSSASPESASPQSTVTVFQQT
QIQEPTANATTTTATTDELKTVTKDRMEDIKILIASPSPTHIHKETTSATSSPYR
DTQSRTASPNRAGKGVIEQTEKSHPRSPNVLSVALSQRTTVPEEELNPKILALQN
AQRKRKMEHDGSLFQAVGIGTLLQQPDDHAATTSLSWKRVKGCKSSEQNGMEQKT
IILIPSDLACRLLGQSMDESGLPQLTSYDCEVNAPIQGSRNLLQGEELLRALDQV
N
11
MGDRGSSRRRRTGSRPSSHGGGGPAAAEEEVRDAAAGPDVGAAGDAPAPAPNKDG
DAG O-
DAGVGSGHWELRCHRLQDSLFSSDSGFSNYRGILNWCVVMLILSNARLFLENLIK
acetyltransferase 1
YGILVDPIQVVSLFLKDPYSWPAPCLVIAANVFAVAAFQVEKRLAVGALTEQAGL
(DGAT1)
LLHVANLATILCFPAAVVLLVESITPVGSLLALMAHTILFLKLFSYRDVNSWCRR
Homo sapiens
ARAKAASAGKKASSAAAPHTVSYPDNLTYRDLYYFLFAPTLCYELNFPRSPRIRK
NCBI Reference
RFLLRRILEMLFFTQLQVGLIQQWMVPTIQNSMKPFKDMDYSRIIERLLKLAVPN
Sequence:
HLIWLIFFYWLFHSCLNAVAELMQFGDREFYRDWWNSESVTYFWQNWNIPVHKWC
NP_036211.2
IRHFYKPMLRRGSSKWMARTGVFLASAFFHEYLVSVPLRMFRLWAFTGMMAQIPL
AWFVGRFFQGNYGNAAVWLSLIIGQPIAVLMYVHDYYVLNYEAPAAEA
12
MPPQLQNGLNLSAKVVQGSLDSLPQAVREFLENNAELCQPDHIHICDGSEEENGR
PCK1
LLGQMEEEGILRRLKKYDNCWLALTDPRDVARIESKTVIVTQEQRDTVPIPKTGL
Homo sapiens
SQLGRWMSEEDFEKAFNARFPGCMKGRTMYVIPFSMGPLGSPLSKIGIELTDSPY
UniProt accession
VVASMRIMTRMGTPVLEAVGDGEFVKCLHSVGCPLPLQKPLVNNWPCNPELTLIA
number P35558
HLPDRREIISFGSGYGGNSLLGKKCFALRMASRLAKEEGWLAEHMLILGITNPEG
NCBI Reference
EKKYLAAAFPSACGKTNLAMMNPSLPGWKVECVGDDIAWMKFDAQGHLRAINPEN
Sequence:
GFFGVAPGTSVKTNPNAIKTIQKNTIFTNVAETSDGGVYWEGIDEPLASGVTITS
NP_002582.3
WKNKEWSSEDGEPCAHPNSRFCTPASQCPIIDAAWESPEGVPIEGIIFGGRRPAG
VPLVYEALSWQHGVFVGAAMRSEATAAAEHKGKIIMHDPFAMRPFFGYNFGKYLA
HWLSMAQHPAAKLPKIFHVNWFRKDKEGKFLWPGFGENSRVLEWMFNRIDGKAST
KLTPIGYIPKEDALNLKGLGHINMMELFSISKEFWEKEVEDIEKYLEDQVNADLP
CEIEREILALKQRISQM
13
FCSPSAKYFFKMAFYNGWILFLAVLAIPVCAVRGRNVENMKILRLMLLHIKYLYG
AGPAT1
IRVEVRGAHHFPPSQPYVVVSNHQSSLDLLGMMEVLPGRCVPIAKRELLWAGSAG
Homo sapiens
LACWLAGVIFIDRKRTGDAISVMSEVAQTLLTQDVRVWVFPEGTRNHNGSMLPFK
Amino acids 27-
RGAFHLAVQAQVPIVPIVMSSYQDFYCKKERRFTSGQCQVRVLPPVPTEGLTPDD
283 of UniProt
VPALADRVRHSMLTVFREISTDGRGGGDYLKKPGGGG
accession number
Q99943 and NCBI
Reference
Sequence:
XP_011546679.1
14
AEFYAKVALYCALCFTVSAVASLVCLLRHGGRTVENMSIIGWFVRSFKYFYGLRF
AGPAT2 isoform α
EVRDPRRLQEARPCVIVSNHQSILDMMGLMEVLPERCVQIAKRELLFLGPVGLIM
precursor
YLGGVFFINRQRSSTAMTVMADLGERMVRENLKVWIYPEGTRNDNGDLLPFKKGA
Homo sapiens
FYLAVQAQVPIVPVVYSSFSSFYNTKKKFFTSGTVTVQVLEAIPTSGLTAADVPA
Amino acids 24-
LVDTCHRAMRTTFLHISKTPQENGATAGSGVQPAQ
278 of UniProt
accession number
O15120 and NCBI
Reference
Sequence:
NP_006403.2
15
AEFYAKVALYCALCFTVSAVASLVCLLRHGGRTVENMSIIGWFVRSFKYFYGLRF
AGPAT2 isoform β
EVRDPRRLQEARPCVIVSNHQSILDMMGLMEVLPERCVQIAKRELLFLGPVGLIM
precursor
YLGGVFFINRQRSSTAMTVMADLGERMVRENVPIVPVVYSSFSSFYNTKKKFFTS
Amino acids 24-
GTVTVQVLEAIPTSGLTAADVPALVDTCHRAMRTTFLHISKTPQENGATAGSGVQ
246 of NCBI
PAQ
Reference
Sequence:
NP_001012745.1
Homo sapiens
16
MGLLAFLKTQFVLHLLVGFVFVVSGLVINFVQLCTLALWPVSKQLYRRLNCRLAY
AGPAT3
SLWSQLVMLLEWWSCTECTLFTDQATVERFGKEHAVIILNHNFEIDFLCGWTMCE
Homo sapiens
RFGVLGSSKVLAKKELLYVPLIGWTWYFLEIVFCKRKWEEDRDTVVEGLRRLSDY
UniProt accession
PEYMWFLLYCEGTRFTETKHRVSMEVAAAKGLPVLKYHLLPRTKGFTTAVKCLRG
number Q9NRZ7
TVAAVYDVTLNFRGNKNPSLLGILYGKKYEADMCVRRFPLEDIPLDEKEAAQWLH
NCBI Reference
KLYQEKDALQEIYNQKGMFPGEQFKPARRPWTLLNFLSWATILLSPLFSFVLGVF
Sequence:
ASGSPLLILTFLGFVGAASFGVRRLIGVTEIEKGSSYGNQEFKKKE
NP_001032642.1
17
MCLLAFLKTQFVLHLLVGFVFVVSGLVINFVQLCTLALWPVSKQLYRRLNCRLAY
AGPAT3
SLWSQLVMLLEWWSCTECTLFTDQATVERFGKEHAVIILNHNFEIDFLCGWTMCE
Homo sapiens
RFGVLGSSKVLAKKELLYVPLIGWTWYFLEIVFCKRKWEEDRDTVVEGLRRLSDY
GenBank:
PEYMWFLLYCEGTRFTETKHRVSMEVAAAKGLPVLKYHLLPRTKGFTTAVKCLRG
AAQ89067.1
TVAAVYDVTLNFRGNKNPSLLGILYGKKYEADMCVRRFPLEDIPLDEKEAAQWLH
KLYQEKDALQEIYNQKGMFPGEQFKPARRPWTLLNFLSWATILLSPLFSFVLGVF
ASGSPLLILTFLGFVGAASFGVRRLIGESLEPGRWRLQ
18
VSFGIRKLYMKSLLKIFAWATLRMERGAKEKNHQLYKPYTNGIIAKDPTSLEEEI
AGPAT6 (LPA-
KEIRRSGSSKALDNTPEFELSDIFYFCRKGMETIMDDEVTKRFSAEELESWNLLS
MAG PA)
RTNYNFQYISLRLTVLWGLGVLIRYCFLLPLRIALAFTGISLLVVGTTVVGYLPN
Homo sapiens
GRFKEFMSKHVHLMCYRICVRALTAIITYHDRENRPRNGGICVANHTSPIDVIIL
Amino acids 38-
ASDGYYAMVGQVHGGLMGVIQRAMVKACPHVWFERSEVKDRHLVAKRLTEHVQDK
456 of UniProt
SKLPILIFPEGTCINNTSVMMFKKGSFEIGATVYPVAIKYDPQFGDAFWNSSKYG
accession number
MVTYLLRMMTSWAIVCSVWYLPPMTREADEDAVQFANRVKSAIARQGGLVDLLWD
Q86UL3
GGLKREKVKDTFKEEQQKLYSKMIVGNHKDRSRS
19
MATMLEGRCQTQPRSSPSCREASLWSSGFGMKLEAVTPFLGKYRPFVGRCCQTCT
GPAT2
PKSWESLFHRSITDLGFCNVILVKEENTRFRGWLVRRLCYFLWSLEQHIPPCQDV
Homo sapiens
PQKIMESTGVQNLLSGRVPGGTGEGQVPDLVKKEVQRILGHIQAPPRPFLVRLFS
UniProt accession
WALLRFLNCLFLNVQLHKGQMKMVQKAAQAGLPLVLLSTHKTLLDGILLPFMLLS
number Q6NUI2
QGLGVLRVAWDSRACSPALRALLRKLGGLFLPPEASLSLDSSEGLLARAVVQAVI
NCBI Reference
EQLLVSGQPLLIFLEEPPGALGPRLSALGQAWVGFVVQAVQVGIVPDALLVPVAV
Sequence:
TYDLVPDAPCDIDHASAPLGLWTGALAVLRSLWSRWGCSHRICSRVHLAQPFSLQ
NP_997211.2
EYIVSARSCWGGRQTLEQLLQPIVLGQCTAVPDTEKEQEWTPITGPLLALKEEDQ
LLVRRLSCHVLSASVGSSAVMSTAIMATLLLFKHQKLLGEFSWLTEEILLRGFDV
GFSGQLRSLLQHSLSLLRAHVALLRIRQGDLLVVPQPGPGLTHLAQLSAELLPVF
LSEAVGACAVRGLLAGRVPPQGPWELQGILLLSQNELYRQILLLMHLLPQDLLLL
KPCQSSYCYCQEVLDRLIQCGLLVAEETPGSRPACDTGRQRLSRKLLWKPSGDFT
DSDSDDFGEADGRYFRLSQQSHCPDFFLFLCRLLSPLLKAFAQAAAFLRQGQLPD
TELGYTEQLFQFLQATAQEEGIFECADPKLAISAVWTFRDLGVLQQTPSPAGPRL
HLSPTFASLDNQEKLEQFIRQFICS
20
MVEFAPLFMPWERRLQTLAVLQFVFSFLALAEICTVGFIALLFTRFWLLTVLYAA
MOGAT2
WWYLDRDKPRQGGRHIQAIRCWTIWKYMKDYFPISLVKTAELDPSRNYIAGFHPH
Homo sapiens
GVLAVGAFANLCTESTGFSSIFPGIRPHLMMLTLWFRAPFFRDYIMSAGLVTSEK
UniProt accession
ESAAHILNRKGGGNLLGIIVGGAQEALDARPGSFTLLLRNRKGFVRLALTHGAPL
number Q3SYC2
VPIFSFGENDLFDQIPNSSGSWLRYIQNRLQKIMGISLPLFHGRGVFQYSFGLIP
YRRPITTVVGKPIEVQKTLHPSEEEVNQLHQRYIKELCNLFEAHKLKFNIPADQH
LEFC
21
MVEFAPLFMPWERRLQTLAVLQFVFSFLALAEICTVGFIALLFTRFWLLTVLYAA
MOGAT2
WWYLDRDKPRQGGRHIQAIRCWTIWKYMKDYFPISLVKTAELDPSRNYIAGFHPH
Homo sapiens
GVLAVGAFANLCTESTGFSSIFPGIRPHLMMLTLWFRAPFFRDYIMSAGLVTSEK
GenBank:
ESAAHILNRKGGGNLLGIIVGGAQEALDARPGSFTLLLRNRKGFVRLALTHGYQA
AAI03879.1
SGKSTLGSVGNWQGFYFGGKMAETNADSILVEIFSPFTIKIIFWCLMPKYLEKFP
QRRLSDLRN
22
MKTLIAAYSGVLRGERQAEADRSQRSHGGPALSREGSGRWGTGSSILSALQDLFS
DGAT2
VTWLNRSKVEKQLQVISVLQWVLSFLVLGVACSAILMYIFCTDCWLIAVLYFTWL
Homo sapiens
VFDWNTPKKGGRRSQWVRNWAVWRYFRDYFPIQLVKTHNLLTTRNYIFGYHPHGI
UniProt accession
MGLGAFCNFSTEATEVSKKFPGIRPYLATLAGNFRMPVLREYLMSGGICPVSRDT
number Q96PD7
IDYLLSKNGSGNAIIIVVGGAAESLSSMPGKNAVTLRNRKGFVKLALRHGADLVP
GenBank:
IYSFGENEVYKQVIFEEGSWGRWVQKKFQKYIGFAPCIFHGRGLFSSDTWGLVPY
AAQ88896.1
SKPITTVVGEPITIPKLEHPTQQDIDLYHTMYMEALVKLFDKHKTKFGLPETEVL
EVN
23
MQPECAEKGKSFKQRLVLKSSLAKETLSEFLGTFILIVLGCCCVAQAILSRGRFC
AQP9
GVITINVGFSMAVAMAIYVAGGVSGGHINPAVSLAMCLFGRMKWFKLPFYVGAQF
Homo sapiens
LGAFVGAATVFGIYYDGLMSFAGGKLLIVGENATAHIFATYPAPYLSLANAFADQ
UniProt accession
VVATMILLIIVFAIFDSRNLGAPRGLEPIAIGLLIIVIASSLGLNSGCAMNPARD
number 043315
LSPRLFTALAGWGFEVFRAGNNFWWIPVVGPLVGAVIGGLIYVLVIEIHHPEPDS
GenBank:
VFKTEQSEDKPEKYELSVIM
AAH26258.1
24
MNYVGQLAGQVFVTVKELYKGLNPATLSGCIDIIVIRQPNGNLQCSPFHVRFGKM
LPIN1 (Lipin 1)
GVLRSREKVVDIEINGESVDLHMKLGDNGEAFFVQETDNDQEVIPMHLATSPILS
Homo sapiens
EGASRMECQLKRGSVDRMRGLDPSTPAQVIAPSETPSSSSVVKKRRKRRRKSQLD
UniProt accession
SLKRDDNMNTSEDEDMFPIEMSSDEAMELLESSRTLPNDIPPFQDDIPEENLSLA
number Q14693
VIYPQSASYPNSDREWSPTPSPSGSRPSTPKSDSELVSKSTERTGQKNPEMLWLW
GELPQAAKSSSPHKMKESSPLSSRKICDKSHFQAIHSESSDTFSDQSPTLVGGAL
LDQNKPQTEMQFVNEEDLETLGAAAPLLPMIEELKPPSASVVQTANKTDSPSRKR
DKRSRHLGADGVYLDDLTDMDPEVAALYFPKNGDPSGLAKHASDNGARSANQSPQ
SVGSSGVDSGVESTSDGLRDLPSIAISLCGGLSDHREITKDAFLEQAVSYQQFVD
NPAIIDDPNLVVKIGSKYYNWTTAAPLLLAMQAFQKPLPKATVESIMRDKMPKKG
GRWWFSWRGRNTTIKEESKPEQCLAGKAHSTGEQPPQLSLATRVKHESSSSDEER
AAAKPSNAGHLPLLPNVSYKKTLRLTSEQLKSLKLKNGPNDVVFSVTTQYQGTCR
CEGTIYLWNWDDKVIISDIDGTITRSDTLGHILPTLGKDWTHQGIAKLYHKVSQN
GYKFLYCSARAIGMADMTRGYLHWVNERGTVLPQGPLLLSPSSLFSALHREVIEK
KPEKFKVQCLTDIKNLFFPNTEPFYAAFGNRPADVYSYKQVGVSLNRIFTVNPKG
ELVQEHAKTNISSYVRLCEVVDHVFPLLKRSHSSDFPCSDTFSNFTFWREPLPPF
ENQDIHSASA
25
MAASVRQARSLLGVAATLAPGSRGYRARPPPRRRPGPRWPDPEDLLTPRWQLGPR
CRIF1
YAAKQFARYGAASGVVPGSLWPSPEQLRELEAEEREWYPSLATMQESLRVKQLAE
Homo sapiens
EQKRREREQHIAECMAKMPQMIVNWQQQQRENWEKAQADKERRARLQAEAQELLG
UniProt accession
YQVDPRSARFQELLQDLEKKERKRLKEEKQKRKKEARAAALAAAVAQDPAASGAP
number Q8TAE8
SS
26
PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSN4
PD1
TDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAP
Homo sapiens
KAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLV
Amino acids 21-
WVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVP
288 of UniProt
CVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
accession number
Q15116
27
MRPRTHGAPPRNIMSTIPKWFKGAPFGVQSHRFDVSAVYPNQKKFSTFTEAPYSR
Lymphocyte
HHSVELSHIGPGTYNSKDTCFSKKFLEQKLGSGWSQAHEATRLTQLPHFHYQAIK
expansion molecule
KEKEQQVHKRGPGSYNIKDFITELQKKPQSKRGLLSSGETRFRGFIGNYYPGPGN
(LEM)
YGEKGNPYTQLEEKAWNRSHSDGLMCRVSNKPPLFHQGSGLGPGTYTIKSDLETF
Mus musculus
VKKSTGNRGPYDIFSGERSSPLPYGHYSVQKMKPKELTDYKSFLDEMNSQHKKKQ
GenBank:
GVFSKYPRDPKHPTERIFWTTLSQCPKNMDIAGPGSWLPHETEQKHVNRPPFLLA
AKD95359.1
SKRCGLKAYQMILGTWNPVGVGRYLNTTLMESIDRRQRYRSLYMSEPKRYLQDLT
RDRLMQKRITPITKGKCRPTVDYNSDPTP
28
ATGAGGGAAAGCCAGGATGCCGCCGGAGCTCATGGCTGGAACCGCGTCGGCTCCA
C1ORF177 1
CGGCCACCAAGTGGTTCACCGGGGCGCCCTTCGGGGTGCAGAGCCACAGGTTTGA
(huLEM)
CATCTCTGCTGTTTATCCCAACTGGAAGAAGTTCAGCACCTTCACTGAGGCCCCA
Homo sapiens
TACTCCACGCGTTATTCTACCCAAGTGTCCCACATAGGCCCTGGGACTTACAGCT
Coding sequence
CCAAAGAGACCTGCTTCAGCAAGAAGAAGCTGATGAAGGAGGTGGACACAGGCTG
NCBI Reference
GGCCAAGGCCCAGGAAGCCACGCGGCTGACCCAGCTACCCCACTTCCAGTACCAG
Sequence:
GCCATCATGAAAGAGAAGCGGCTGAAGGAGCAAAAGCTGGGCCCCGGCTCCTACA
NM_152607.2
ACCTCAAAGACTTCTTAGAACAGCTGCGGGAGAAACCATGTAGCACCCGGGGGCT
GCTCAGCTCTGGGGAGGTTCGCTTCCGAGGACTCACTGGGAACTACTATCCAGGC
CCTGGAAATTATGGGGAGAAGGGTAACCCATACACCAAGCTGGAGGAGAATGCCT
GGAACCGGTCTCATTCCGAGGGCCTCATGTGCAGGATGAGCAACAAGCCACACCC
CCGGCCTCATCAGGGGAGTGGTCTGGGACCCGGCACCTACTTCTTCAAAAGCGAC
CTTGAGACATATGTGGCACGATCCGTCGGCACCCGCGGCCCCTATGACACTTTCT
CTGGTGATCGGAGCAAGCCACTGCCTTATGGGCACTACTCCATGCAGAAAAAAAA
GCCCAGGGAACTGATGAATTTCAAGAGCTTTGTAGAAGAACTTAACTCACATCAC
AATAAGAAGCATGGGGTTTTTTCTAAACTTCCCCGAAACCCGAAAACCCCTACAG
AGAGGATTTACTGGGCCAACCTCAGCCAGTGCCCCCGCACACTGGCCACATCTGG
CCCCAGTTTCTGGCTTCCACAAGAGAAGAAATGCAAACCCGTCAACCAGCCCCCA
TTCCTGTTGACCTCCAAGGGGTCAGGTGCAAAGGCCTGCCAGATGATTATGGGAA
GCTGGAACCCAGTAGGTGTGGGCCGCTACCTCAACACCTGGCTGATGGAGACAAA
GGACAGGCGGCAGCGATATCGATCCCTATTCCTGAGTGGATCCAAACGCTACCTC
TCAGACCTGGCCCGGGACATGCTCATGCAAATCAGGCTGTTTTGTTGGAAAGGCC
TAGAAGTCATCAATTTTGGCCTCTCACCAATGTAG
29
ATGAGGGAAAGCCAGGATGCCGCCGGAGCTCATGGCTGGAACCGCGTCGGCTCCA
C1ORF177 2
CGGCCACCAAGTGGTTCACCGGGGCGCCCTTCGGGGTGCAGAGCCACAGGTTTGA
(huLEM)
CATCTCTGCTGTTTATCCCAACTGGAAGAAGTTCAGCACCTTCACTGAGGCCCCA
Homo sapiens
TACTCCACGCGTTATTCTACCCAAGTGTCCCACATAGGCCCTGGGACTTACAGCT
Coding sequence of
CCAAAGAGACCTGCTTCAGCAAGAAGAAGCTGATGAAGGAGGTGGACACAGGCTG
NCBI Reference
GGCCAAGGCCCAGGAAGCCACGCGGCTGACCCAGCTACCCCACTTCCAGTACCAG
Sequence:
GCCATCATGAAAGAGAAGCGGCTGAAGGAGCAAAAGCTGGGCCCCGGCTCCTACA
NM_001110533.1
ACCTCAAAGACTTCTTAGAACAGCTGCGGGAGAAACCATGTAGCACCCGGGGGCT
GCTCAGCTCTGGGGAGGTTCGCTTCCGAGGACTCACTGGGAACTACTATCCAGGC
CCTGGAAATTATGGGGAGAAGGGTAACCCATACACCAAGCTGGAGGAGAATGCCT
GGAACCGGTCTCATTCCGAGGGCCTCATGTGCAGGATGAGCAACAAGCCACACCC
CCGGCCTCATCAGGGGAGTGGTCTGGGACCCGGCACCTACTTCTTCAAAAGCGAC
CTTGAGACATATGTGGCACGATCCGTCGGCACCCGCGGCCCCTATGACACTTTCT
CTGGTGATCGGAGCAAGCCACTGCCTTATGGGCACTACTCCATGCAGAAAAAAAA
GCCCAGGGAACTGATGAATTTCAAGAGCTTTGTAGAAGAACTTAACTCACATCAC
AATAAGAAGCATGGGGTTTTTTCTAAACTTCCCCGAAACCCGAAAACCCCTACAG
AGAGGATTTACTGGGCCAACCTCAGCCAGTGCCCCCGCACACTGGCCACATCTGG
CCCCAGTTTCTGGCTTCCACAAGAGAAGAAATGCAAACCCGTCAACCAGCCCCCA
TTCCTGTTGACCTCCAAGGGGTCAGGTGCAAAGGCCTGCCAGATGATTATGGGAA
GCTGGAACCCAGTAGGTGTGGGCCGCTACCTCAACACCTGGCTGATGGAGACAAA
GGACAGGCGGCAGCGATATCGATCCCTATTCCTGAGTGGATCCAAACGCTACCTC
TCAGACCTGGCCCGGGACATGCTCATGCAGGAAAGGATCACACCATTTACTAAGG
GAAAGTGCCCTCCAACTGTGGATTACAATTCAGATCCTACTCCTTAA
30
ATGGCAGCCTCAAAGAAGGCAGTTTTGGGGCCATTGGTGGGGGCGGTGGACCAGG
Glycerol kinase
GCACCAGTTCGACGCGCTTTTTGGTTTTCAATTCAAAAACAGCTGAACTACTTAG
(Gyk)
TCATCATCAAGTAGAAATAAAACAAGAGTTCCCAAGAGAAGGATGGGTGGAACAG
Homo sapiens
GACCCTAAGGAAATTCTACATTCTGTCTATGAGTGTATAGAGAAAACATGTGAGA
NG_008178.1
AACTTGGACAGCTCAATATTGATATTTCCAACATAAAAGCTATTGGTGTCAGCAA
RefSeqGene
CCAGAGGGAAACCACTGTAGTCTGGGACAAGATAACTGGAGAGCCTCTCTACAAT
GCTGTGGTGTGGCTTGATCTAAGAACCCAGTCTACCGTTGAGAGTCTTAGTAAAA
GAATTCCAGGAAATAATAACTTTGTCAAGTCCAAGACAGGCCTTCCACTTAGCAC
TTACTTCAGTGCAGTGAAACTTCGTTGGCTCCTTGACAATGTGAGAAAAGTTCAA
AAGGCCGTTGAAGAAAAACGAGCTCTTTTTGGGACTATTGATTCATGGCTTATTT
GGAGTTTGACAGGAGGAGTCAATGGAGGTGTCCACTGTACAGATGTAACAAATGC
AAGTAGGACTATGCTTTTCAACATTCATTCTTTGGAATGGGATAAACAACTCTGC
GAATTTTTTGGAATTCCAATGGAAATTCTTCCAAATGTCCGGAGTTCTTCTGAGA
TCTATGGCCTAATGAAAATCTCTCATAGCGTGAAAGCTGGGGCCTTGGAAGGTGT
GCCAATATCTGGGTGTTTAGGGGACCAGTCTGCTGCATTGGTGGGACAAATGTGC
TTCCAGATTGGACAAGCCAAAAATACGTATGGAACAGGATGTTTCTTACTATGTA
ATACAGGCCATAAGTGTGTATTTTCTGATCATGGCCTTCTCACCACAGTGGCTTA
CAAACTTGGCAGAGACAAACCAGTATATTATGCTTTGGAAGGTTCTGTAGCTATA
GCTGGTGCTGTTATTCGCTGGCTAAGAGACAATCTTGGAATTATAAAGACCTCAG
AAGAAATTGAAAAACTTGCTAAAGAAGTAGGTACTTCTTATGGCTGCTACTTCGT
CCCAGCATTTTCGGGGTTATATGCACCTTATTGGGAGCCCAGCGCAAGAGGGATA
ATCTGTGGACTCACTCAGTTCACCAATAAATGCCATATTGCTTTTGCTGCATTAG
AAGCTGTTTGTTTCCAAACTCGAGAGATTTTGGATGCCATGAATCGAGACTGTGG
AATTCCACTCAGTCATTTGCAGGTAGATGGAGGAATGACCAGCAACAAAATTCTT
ATGCAGCTACAAGCAGACATTCTGTATATACCAGTAGTGAAGCCCTCAATGCCCG
AAACCACTGCACTGGGTGCGGCTATGGCGGCAGGGGCTGCAGAAGGAGTCGGCGT
ATGGAGTCTCGAACCCGAGGATTTGTCTGCCGTCACGATGGAGCGGTTTGAACCT
CAGATTAATGCGGAGGAAAGTGAAATTCGTTATTCTACATGGAAGAAAGCTGTGA
TGAAGTCAATGGGTTGGGTTACAACTCAATCTCCAGAAAGTGGTATTCCATAA
31
ATGGGCGACCGCGGCAGCTCCCGGCGCCGGAGGACAGGGTCGCGGCCCTCGAGCC
DAG O-
ACGGCGGCGGCGGGCCTGCGGCGGCGGAAGAGGAGGTGCGGGACGCCGCTGCGGG
acetyltransferase 1
CCCCGACGTGGGAGCCGCGGGGGACGCGCCAGCCCCGGCCCCCAACAAGGACGGA
(DGAT1)
GACGCCGGCGTGGGCAGCGGCCACTGGGAGCTGAGGTGCCATCGCCTGCAGGATT
Homo sapiens
CTTTATTCAGCTCTGACAGTGGCTTCAGCAACTACCGTGGCATCCTGAACTGGTG
Coding sequence of
TGTGGTGATGCTGATCTTGAGCAATGCCCGGTTATTTCTGGAGAACCTCATCAAG
NCBI Reference
TATGGCATCCTGGTGGACCCCATCCAGGTGGTTTCTCTGTTCCTGAAGGATCCCT
Sequence:
ATAGCTGGCCCGCCCCATGCCTGGTTATTGCGGCCAATGTCTTTGCTGTGGCTGC
NM_012079.5
ATTCCAGGTTGAGAAGCGCCTGGCGGTGGGTGCCCTGACGGAGCAGGCGGGACTG
CTGCTGCACGTGGCCAACCTGGCCACCATTCTGTGTTTCCCAGCGGCTGTGGTCT
TACTGGTTGAGTCTATCACTCCAGTGGGCTCCCTGCTGGCGCTGATGGCGCACAC
CATCCTCTTCCTCAAGCTCTTCTCCTACCGCGACGTCAACTCATGGTGCCGCAGG
GCCAGGGCCAAGGCTGCCTCTGCAGGGAAGAAGGCCAGCAGTGCTGCTGCCCCGC
ACACCGTGAGCTACCCGGACAATCTGACCTACCGCGATCTCTACTACTTCCTCTT
CGCCCCCACCTTGTGCTACGAGCTCAACTTTCCCCGCTCTCCCCGCATCCGGAAG
CGCTTTCTGCTGCGACGGATCCTTGAGATGCTGTTCTTCACCCAGCTCCAGGTGG
GGCTGATCCAGCAGTGGATGGTCCCCACCATCCAGAACTCCATGAAGCCCTTCAA
GGACATGGACTACTCACGCATCATCGAGCGCCTCCTGAAGCTGGCGGTCCCCAAT
CACCTCATCTGGCTCATCTTCTTCTACTGGCTCTTCCACTCCTGCCTGAATGCCG
TGGCTGAGCTCATGCAGTTTGGAGACCGGGAGTTCTACCGGGACTGGTGGAACTC
CGAGTCTGTCACCTACTTCTGGCAGAACTGGAACATCCCTGTGCACAAGTGGTGC
ATCAGACACTTCTACAAGCCCATGCTTCGACGGGGCAGCAGCAAGTGGATGGCCA
GGACAGGGGTGTTCCTGGCCTCGGCCTTCTTCCACGAGTACCTGGTGAGCGTCCC
TCTGCGAATGTTCCGCCTCTGGGCGTTCACGGGCATGATGGCTCAGATCCCACTG
GCCTGGTTCGTGGGCCGCTTTTTCCAGGGCAACTATGGCAACGCAGCTGTGTGGC
TGTCGCTCATCATCGGACAGCCAATAGCCGTCCTCATGTACGTCCACGACTACTA
CGTGCTCAACTATGAGGCCCCAGCGGCAGAGGCCTGA
32
ATGGATGAATCTGCACTGACCCTTGGTACAATAGATGTTTCTTATCTGCCACATT
Glycerol-3-
CATCAGAATACAGTGTTGGTCGATGTAAGCACACAAGTGAGGAATGGGGTGAGTG
phosphate
TGGCTTTAGACCCACCATCTTCAGATCTGCAACTTTAAAATGGAAAGAAAGCCTA
acetyltransferase
ATGAGTCGGAAAAGGCCATTTGTTGGAAGATGTTGTTACTCCTGCACTCCCCAGA
mitochondrial
GCTGGGACAAATTTTTCAACCCCAGTATCCCGTCTTTGGGTTTGCGGAATGTTAT
(GPAM)
TTATATCAATGAAACTCACACAAGACACCGCGGATGGCTTGCAAGACGCCTTTCT
Homo sapiens
TACGTTCTTTTTATTCAAGAGCGAGATGTGCATAAGGGCATGTTTGCCACCAATG
Coding sequence of
TGACTGAAAATGTGCTGAACAGCAGTAGAGTACAAGAGGCAATTGCAGAAGTGGC
NCBI Reference
TGCTGAATTAAACCCTGATGGTTCTGCCCAGCAGCAATCAAAAGCCGTTAACAAA
Sequence:
GTGAAAAAGAAAGCTAAAAGGATTCTTCAAGAAATGGTTGCCACTGTCTCACCGG
NM_001244949.1
CAATGATCAGACTGACTGGGTGGGTGCTGCTAAAACTGTTCAACAGCTTCTTTTG
GAACATTCAAATTCACAAAGGTCAACTTGAGATGGTTAAAGCTGCAACTGAGACG
AATTTGCCGCTTCTGTTTCTACCAGTTCATAGATCCCATATTGACTATCTGCTGC
TCACTTTCATTCTCTTCTGCCATAACATCAAAGCACCATACATTGCTTCAGGCAA
TAATCTCAACATCCCAATCTTCAGTACCTTGATCCATAAGCTTGGGGGCTTCTTC
ATACGACGAAGGCTCGATGAAACACCAGATGGACGGAAAGATGTTCTCTATAGAG
CTTTGCTCCATGGGCATATAGTTGAATTACTTCGACAGCAGCAATTCTTGGAGAT
CTTCCTGGAAGGCACACGTTCTAGGAGTGGAAAAACCTCTTGTGCTCGGGCAGGA
CTTTTGTCAGTTGTGGTAGATACTCTGTCTACCAATGTCATCCCAGACATCTTGA
TAATACCTGTTGGAATCTCCTATGATCGCATTATCGAAGGTCACTACAATGGTGA
ACAACTGGGCAAACCTAAGAAGAATGAGAGCCTGTGGAGTGTAGCAAGAGGTGTT
ATTAGAATGTTACGAAAAAACTATGGTTGTGTCCGAGTGGATTTTGCACAGCCAT
TTTCCTTAAAGGAATATTTAGAAAGCCAAAGTCAGAAACCGGTGTCTGCTCTACT
TTCCCTGGAGCAAGCGTTGTTACCAGCTATACTTCCTTCAAGACCCAGTGATGCT
GCTGATGAAGGTAGAGACACGTCCATTAATGAGTCCAGAAATGCAACAGATGAAT
CCCTACGAAGGAGGTTGATTGCAAATCTGGCTGAGCATATTCTATTCACTGCTAG
CAAGTCCTGTGCCATTATGTCCACACACATTGTGGCTTGCCTGCTCCTCTACAGA
CACAGGCAGGGAATTGATCTCTCCACATTGGTCGAAGACTTCTTTGTGATGAAAG
AGGAAGTCCTGGCTCGTGATTTTGACCTGGGGTTCTCAGGAAATTCAGAAGATGT
AGTAATGCATGCCATACAGCTGCTGGGAAATTGTGTCACAATCACCCACACTAGC
AGGAACGATGAGTTTTTTATCACCCCCAGCACAACTGTCCCATCAGTCTTCGAAC
TCAACTTCTACAGCAATGGGGTACTTCATGTCTTTATCATGGAGGCCATCATAGC
TTGCAGCCTTTATGCAGTTCTGAACAAGAGGGGACTGGGGGGTCCCACTAGCACC
CCACCTAACCTGATCAGCCAGGAGCAGCTGGTGCGGAAGGCGGCCAGCCTGTGCT
ACCTTCTCTCCAATGAAGGCACCATCTCACTGCCTTGCCAGACATTTTACCAAGT
CTGCCATGAAACAGTAGGAAAGTTTATCCAGTATGGCATTCTTACAGTGGCAGAG
CACGATGACCAGGAAGATATCAGTCCTAGTCTTGCTGAGCAGCAGTGGGACAAGA
AGCTTCCAGAACCTTTGTCTTGGAGAAGTGATGAAGAAGATGAAGACAGTGACTT
TGGGGAGGAACAGCGAGATTGCTACCTGAAGGTGAGCCAATCCAAGGAGCACCAG
CAGTTTATCACCTTCTTACAGAGACTCCTTGGGCCTTTGCTGGAGGCCTACAGCT
CTGCTGCCATCTTTGTTCACAACTTCAGTGGTCCTGTTCCAGAACCTGAGTATCT
GCAAAAGTTGCACAAATACCTAATAACCAGAACAGAAAGAAATGTTGCAGTATAT
GCTGAGAGTGCCACATATTGTCTTGTGAAGAATGCTGTGAAAATGTTTAAGGATA
TTGGGGTTTTCAAGGAGACCAAACAAAAGAGAGTGTCTGTTTTAGAACTGAGCAG
CACTTTTCTACCTCAATGCAACCGACAAAAACTTCTAGAATATATTCTGAGTTTT
GTGGTGCTGTAG
33
ATGAAGGTAGAGTTTGCACCGCTCAACATCCAGCTGGCGCGGCGGCTGCAGACGG
Monoacylglycerol
TGGCCGTGCTGCAGTGGGTCCTGAAATACCTGCTGCTCGGGCCGATGTCCATTGG
O-acetyltransferase
AATCACTGTGATGCTGATCATACACAACTATTTGTTCCTTTACATCCCTTATTTG
(MOGAT1)
ATGTGGCTTTACTTTGACTGGCATACCCCAGAGCGAGGAGGCAGGAGATCCAGCT
Homo sapiens
GGATCAAAAATTGGACTCTTTGGAAACACTTTAAGGACTATTTTCCAATTCATCT
Coding sequence of
TATCAAAACTCAAGATTTGGATCCAAGTCACAACTATATATTTGGGTTTCACCCC
NCBI Reference
CATGGAATAATGGCAGTTGGAGCCTTTGGGAATTTTTCTGTAAATTATTCTGACT
Sequence:
TCAAGGACCTGTTTCCTGGCTTTACTTCATATCTTCACGTGCTGCCACTTTGGTT
NM_058165.2
CTGGTGTCCTGTCTTTCGAGAATATGTGATGAGTGTTGGGCTGGTTTCAGTTTCC
AAGAAAAGTGTGTCCTACATGGTAAGCAAGGAGGGAGGTGGAAACATCTCTGTCA
TTGTCCTTGGGGGTGCAAAAGAATCACTGGATGCTCATCCTGGAAAGTTCACTCT
GTTCATCCGCCAGCGGAAAGGATTTGTTAAAATTGCTTTGACCCATGGCGCCTCT
CTGGTCCCAGTGGTTTCTTTTGGTGAAAATGAACTGTTTAAACAAACTGACAACC
CTGAAGGATCATGGATTAGAACTGTTCAGAATAAACTGCAGAAGATCATGGGGTT
TGCTTTGCCCCTGTTTCATGCCAGGGGAGTTTTTCAGTACAATTTTGGCCTAATG
ACCTATAGGAAAGCCATCCACACTGTTGTTGGCCGCCCGATCCCTGTTCGTCAGA
CTCTGAACCCGACCCAGGAGCAGATTGAGGAGTTACATCAGACCTATATGGAGGA
ACTTAGGAAATTGTTTGAGGAACACAAAGGAAAGTATGGCATTCCAGAGCACGAG
ACTCTTGTTTTAAAATGA
34
ATGGAGGGCGCCGGCGGCGCGAACGACAAGAAAAAGATAAGTTCTGAACGTCGAA
HIF1α
AAGAAAAGTCTCGAGATGCAGCCAGATCTCGGCGAAGTAAAGAATCTGAAGTTTT
Homo sapiens
TTATGAGCTTGCTCATCAGTTGCCACTTCCACATAATGTGAGTTCGCATCTTGAT
Coding sequence of
AAGGCCTCTGTGATGAGGCTTACCATCAGCTATTTGCGTGTGAGGAAACTTCTGG
NCBI Reference
ATGCTGGTGATTTGGATATTGAAGATGACATGAAAGCACAGATGAATTGCTTTTA
Sequence:
TTTGAAAGCCTTGGATGGTTTTGTTATGGTTCTCACAGATGATGGTGACATGATT
NM_001530.3
TACATTTCTGATAATGTGAACAAATACATGGGATTAACTCAGTTTGAACTAACTG
GACACAGTGTGTTTGATTTTACTCATCCATGTGACCATGAGGAAATGAGAGAAAT
GCTTACACACAGAAATGGCCTTGTGAAAAAGGGTAAAGAACAAAACACACAGCGA
AGCTTTTTTCTCAGAATGAAGTGTACCCTAACTAGCCGAGGAAGAACTATGAACA
TAAAGTCTGCAACATGGAAGGTATTGCACTGCACAGGCCACATTCACGTATATGA
TACCAACAGTAACCAACCTCAGTGTGGGTATAAGAAACCACCTATGACCTGCTTG
GTGCTGATTTGTGAACCCATTCCTCACCCATCAAATATTGAAATTCCTTTAGATA
GCAAGACTTTCCTCAGTCGACACAGCCTGGATATGAAATTTTCTTATTGTGATGA
AAGAATTACCGAATTGATGGGATATGAGCCAGAAGAACTTTTAGGCCGCTCAATT
TATGAATATTATCATGCTTTGGACTCTGATCATCTGACCAAAACTCATCATGATA
TGTTTACTAAAGGACAAGTCACCACAGGACAGTACAGGATGCTTGCCAAAAGAGG
TGGATATGTCTGGGTTGAAACTCAAGCAACTGTCATATATAACACCAAGAATTCT
CAACCACAGTGCATTGTATGTGTGAATTACGTTGTGAGTGGTATTATTCAGCACG
ACTTGATTTTCTCCCTTCAACAAACAGAATGTGTCCTTAAACCGGTTGAATCTTC
AGATATGAAAATGACTCAGCTATTCACCAAAGTTGAATCAGAAGATACAAGTAGC
CTCTTTGACAAACTTAAGAAGGAACCTGATGCTTTAACTTTGCTGGCCCCAGCCG
CTGGAGACACAATCATATCTTTAGATTTTGGCAGCAACGACACAGAAACTGATGA
CCAGCAACTTGAGGAAGTACCATTATATAATGATGTAATGCTCCCCTCACCCAAC
GAAAAATTACAGAATATAAATTTGGCAATGTCTCCATTACCCACCGCTGAAACGC
CAAAGCCACTTCGAAGTAGTGCTGACCCTGCACTCAATCAAGAAGTTGCATTAAA
ATTAGAACCAAATCCAGAGTCACTGGAACTTTCTTTTACCATGCCCCAGATTCAG
GATCAGACACCTAGTCCTTCCGATGGAAGCACTAGACAAAGTTCACCTGAGCCTA
ATAGTCCCAGTGAATATTGTTTTTATGTGGATAGTGATATGGTCAATGAATTCAA
GTTGGAATTGGTAGAAAAACTTTTTGCTGAAGACACAGAAGCAAAGAACCCATTT
TCTACTCAGGACACAGATTTAGACTTGGAGATGTTAGCTCCCTATATCCCAATGG
ATGATGACTTCCAGTTACGTTCCTTCGATCAGTTGTCACCATTAGAAAGCAGTTC
CGCAAGCCCTGAAAGCGCAAGTCCTCAAAGCACAGTTACAGTATTCCAGCAGACT
CAAATACAAGAACCTACTGCTAATGCCACCACTACCACTGCCACCACTGATGAAT
TAAAAACAGTGACAAAAGACCGTATGGAAGACATTAAAATATTGATTGCATCTCC
ATCTCCTACCCACATACATAAAGAAACTACTAGTGCCACATCATCACCATATAGA
GATACTCAAAGTCGGACAGCCTCACCAAACAGAGCAGGAAAAGGAGTCATAGAAC
AGACAGAAAAATCTCATCCAAGAAGCCCTAACGTGTTATCTGTCGCTTTGAGTCA
AAGAACTACAGTTCCTGAGGAAGAACTAAATCCAAAGATACTAGCTTTGCAGAAT
GCTCAGAGAAAGCGAAAAATGGAACATGATGGTTCACTTTTTCAAGCAGTAGGAA
TTGGAACATTATTACAGCAGCCAGACGATCATGCAGCTACTACATCACTTTCTTG
GAAACGTGTAAAAGGATGCAAATCTAGTGAACAGAATGGAATGGAGCAAAAGACA
ATTATTTTAATACCCTCTGATTTAGCATGTAGACTGCTGGGGCAATCAATGGATG
AAAGTGGATTACCACAGCTGACCAGTTATGATTGTGAAGTTAATGCTCCTATACA
AGGCAGCAGAAACCTACTGCAGGGTGAAGAATTACTCAGAGCTTTGGATCAAGTT
AACTGA
35
ATGCCTCCTCAGCTGCAAAACGGCCTGAACCTCTCGGCCAAAGTTGTCCAGGGAA
PCK1
GCCTGGACAGCCTACCCCAGGCAGTGAGGGAGTTTCTCGAGAATAACGCTGAGCT
Homo sapiens
GTGTCAGCCTGATCACATCCACATCTGTGACGGCTCTGAGGAGGAGAATGGGCGG
Coding sequence of
CTTCTGGGCCAGATGGAGGAAGAGGGCATCCTCAGGCGGCTGAAGAAGTATGACA
NCBI Reference
ACTGCTGGTTGGCTCTCACTGACCCCAGGGATGTGGCCAGGATCGAAAGCAAGAC
Sequence:
GGTTATCGTCACCCAAGAGCAAAGAGACACAGTGCCCATCCCCAAAACAGGCCTC
NM_002591.3
AGCCAGCTCGGTCGCTGGATGTCAGAGGAGGATTTTGAGAAAGCGTTCAATGCCA
GGTTCCCAGGGTGCATGAAAGGTCGCACCATGTACGTCATCCCATTCAGCATGGG
GCCGCTGGGCTCGCCTCTGTCAAAGATCGGCATCGAGCTGACGGATTCACCCTAC
GTGGTGGCCAGCATGCGGATCATGACGCGGATGGGCACGCCCGTCCTGGAAGCAG
TGGGCGATGGGGAGTTTGTCAAATGCCTCCATTCTGTGGGGTGCCCTCTGCCTTT
ACAAAAGCCTTTGGTCAACAACTGGCCCTGCAACCCGGAGCTGACGCTCATCGCC
CACCTGCCTGACCGCAGAGAGATCATCTCCTTTGGCAGTGGGTACGGCGGGAACT
CGCTGCTCGGGAAGAAGTGCTTTGCTCTCAGGATGGCCAGCCGGCTGGCCAAGGA
GGAAGGGTGGCTGGCAGAGCACATGCTGATTCTGGGTATAACCAACCCTGAGGGT
GAGAAGAAGTACCTGGCGGCCGCATTTCCCAGCGCCTGCGGGAAGACCAACCTGG
CCATGATGAACCCCAGCCTCCCCGGGTGGAAGGTTGAGTGCGTCGGGGATGACAT
TGCCTGGATGAAGTTTGACGCACAAGGTCATTTAAGGGCCATCAACCCAGAAAAT
GGCTTTTTCGGTGTCGCTCCTGGGACTTCAGTGAAGACCAACCCCAATGCCATCA
AGACCATCCAGAAGAACACAATCTTTACCAATGTGGCCGAGACCAGCGACGGGGG
CGTTTACTGGGAAGGCATTGATGAGCCGCTAGCTTCAGGTGTCACCATCACGTCC
TGGAAGAATAAGGAGTGGAGCTCAGAGGATGGGGAACCTTGTGCCCACCCCAACT
CGAGGTTCTGCACCCCTGCCAGCCAGTGCCCCATCATTGATGCTGCCTGGGAGTC
TCCGGAAGGTGTTCCCATTGAAGGCATTATCTTTGGAGGCCGTAGACCTGCTGGT
GTCCCTCTAGTCTATGAAGCTCTCAGCTGGCAACATGGAGTCTTTGTGGGGGCGG
CCATGAGATCAGAGGCCACAGCGGCTGCAGAACATAAAGGCAAAATCATCATGCA
TGACCCCTTTGCCATGCGGCCCTTCTTTGGCTACAACTTCGGCAAATACCTGGCC
CACTGGCTTAGCATGGCCCAGCACCCAGCAGCCAAACTGCCCAAGATCTTCCATG
TCAACTGGTTCCGGAAGGACAAGGAAGGCAAATTCCTCTGGCCAGGCTTTGGAGA
GAACTCCAGGGTGCTGGAGTGGATGTTCAACCGGATCGATGGAAAAGCCAGCACC
AAGCTCACGCCCATAGGCTACATCCCCAAGGAGGATGCCCTGAACCTGAAAGGCC
TGGGGCACATCAACATGATGGAGCTTTTCAGCATCTCCAAGGAATTCTGGGAGAA
GGAGGTGGAAGACATCGAGAAGTATCTGGAGGATCAAGTCAATGCCGACCTCCCC
TGTGAAATCGAGAGAGAGATCCTTGCCTTGAAGCAAAGAATAAGCCAGATGTAA
36
ATGGATTTGTGGCCAGGGGCATGGATGCTGCTGCTGCTGCTCTTCCTGCTGCTGC
AGPAT1
TCTTCCTGCTGCCCACCCTGTGGTTCTGCAGCCCCAGTGCCAAGTACTTCTTCAA
Homo sapiens
GATGGCCTTCTACAATGGCTGGATCCTCTTCCTGGCTGTGCTCGCCATCCCTGTG
Coding sequence of
TGTGCCGTGCGAGGACGCAACGTCGAGAACATGAAGATCTTGCGTCTAATGCTGC
NCBI Reference
TCCACATCAAATACCTGTACGGGATCCGAGTGGAGGTGCGAGGGGCTCACCACTT
Sequence:
CCCTCCCTCGCAGCCCTATGTTGTTGTCTCCAACCACCAGAGCTCTCTCGATCTG
NM_006411.3
CTTGGGATGATGGAGGTACTGCCAGGCCGCTGTGTGCCCATTGCCAAGCGCGAGC
TACTGTGGGCTGGCTCTGCCGGGCTGGCCTGCTGGCTGGCAGGAGTCATCTTCAT
CGACCGGAAGCGCACGGGGGATGCCATCAGTGTCATGTCTGAGGTCGCCCAGACC
CTGCTCACCCAGGACGTGAGGGTCTGGGTGTTTCCTGAGGGAACGAGAAACCACA
ATGGCTCCATGCTGCCCTTCAAACGTGGCGCCTTCCATCTTGCAGTGCAGGCCCA
GGTTCCCATTGTCCCCATAGTCATGTCCTCCTACCAAGACTTCTACTGCAAGAAG
GAGCGTCGCTTCACCTCGGGACAATGTCAGGTGCGGGTGCTGCCCCCAGTGCCCA
CGGAAGGGCTGACACCAGATGACGTCCCAGCTCTGGCTGACAGAGTCCGGCACTC
CATGCTCACTGTTTTCCGGGAAATCTCCACTGATGGCCGGGGTGGTGGTGACTAT
CTGAAGAAGCCTGGGGGCGGTGGGTGA
37
ATGGAGCTGTGGCCGTGTCTGGCCGCGGCGCTGCTGTTGCTGCTGCTGCTGGTGC
AGPAT2 isoform α
AGCTGAGCCGCGCGGCCGAGTTCTACGCCAAGGTCGCCCTGTACTGCGCGCTGTG
precursor
CTTCACGGTGTCCGCCGTGGCCTCGCTCGTCTGCCTGCTGCGCCACGGCGGCCGG
Homo sapiens
ACGGTGGAGAACATGAGCATCATCGGCTGGTTCGTGCGAAGCTTCAAGTACTTTT
Coding sequence of
ACGGGCTCCGCTTCGAGGTGCGGGACCCGCGCAGGCTGCAGGAGGCCCGTCCCTG
NCBI Reference
TGTCATCGTCTCCAACCACCAGAGCATCCTGGACATGATGGGCCTCATGGAGGTC
Sequence:
CTTCCGGAGCGCTGCGTGCAGATCGCCAAGCGGGAGCTGCTCTTCCTGGGGCCCG
NM_006412.3
TGGGCCTCATCATGTACCTCGGGGGCGTCTTCTTCATCAACCGGCAGCGCTCTAG
CACTGCCATGACAGTGATGGCCGACCTGGGCGAGCGCATGGTCAGGGAGAACCTC
AAAGTGTGGATCTATCCCGAGGGTACTCGCAACGACAATGGGGACCTGCTGCCTT
TTAAGAAGGGCGCCTTCTACCTGGCAGTCCAGGCACAGGTGCCCATCGTCCCCGT
GGTGTACTCTTCCTTCTCCTCCTTCTACAACACCAAGAAGAAGTTCTTCACTTCA
GGAACAGTCACAGTGCAGGTGCTGGAAGCCATCCCCACCAGCGGCCTCACTGCGG
CGGACGTCCCTGCGCTCGTGGACACCTGCCACCGGGCCATGAGGACCACCTTCCT
CCACATCTCCAAGACCCCCCAGGAGAACGGGGCCACTGCGGGGTCTGGCGTGCAG
CCGGCCCAGTAG
38
ATGGAGCTGTGGCCGTGTCTGGCCGCGGCGCTGCTGTTGCTGcTGcTGcTGGTGC
AGPAT2 isoform β
AGCTGAGCCGCGCGGCCGAGTTCTACGCCAAGGTCGCCCTGTACTGCGCGCTGTG
precursor
CTTCACGGTGTCCGCCGTGGCCTCGCTCGTCTGCCTGCTGCGCCACGGCGGCCGG
Homo sapiens
ACGGTGGAGAACATGAGCATCATCGGCTGGTTCGTGCGAAGCTTCAAGTACTTTT
Coding sequence of
ACGGGCTCCGCTTCGAGGTGCGGGACCCGCGCAGGCTGCAGGAGGCCCGTCCCTG
NCBI Reference
TGTCATCGTCTCCAACCACCAGAGCATCCTGGACATGATGGGCCTCATGGAGGTC
Sequence:
CTTCCGGAGCGCTGCGTGCAGATCGCCAAGCGGGAGCTGCTCTTCCTGGGGCCCG
NM_001012727.1
TGGGCCTCATCATGTACCTCGGGGGCGTCTTCTTCATCAACCGGCAGCGCTCTAG
CACTGCCATGACAGTGATGGCCGACCTGGGCGAGCGCATGGTCAGGGAGAACGTG
CCCATCGTCCCCGTGGTGTACTCTTCCTTCTCCTCCTTCTACAACACCAAGAAGA
AGTTCTTCACTTCAGGAACAGTCACAGTGCAGGTGCTGGAAGCCATCCCCACCAG
CGGCCTCACTGCGGCGGACGTCCCTGCGCTCGTGGACACCTGCCACCGGGCCATG
AGGACCACCTTCCTCCACATCTCCAAGACCCCCCAGGAGAACGGGGCCACTGCGG
GGTCTGGCGTGCAGCCGGCCCAGTAG
39
ATGGGCCTGCTGGCCTTCCTGAAGACCCAGTTCGTGCTGCACCTGCTGGTCGGCT
AGPAT3
TTGTCTTCGTGGTGAGTGGTCTGGTCATCAACTTCGTCCAGCTGTGCACGCTGGC
Homo sapiens
GCTCTGGCCGGTCAGCAAGCAGCTCTACCGCCGCCTCAACTGCCGCCTCGCCTAC
Coding sequence of
TCACTCTGGAGCCAACTGGTCATGCTGCTGGAGTGGTGGTCCTGCACGGAGTGTA
NCBI Reference
CACTGTTCACGGACCAGGCCACGGTAGAGCGCTTTGGGAAGGAGCACGCAGTCAT
Sequence:
CATCCTCAACCACAACTTCGAGATCGACTTCCTCTGTGGGTGGACCATGTGTGAG
NM_001037553.1
CGCTTCGGAGTGCTGGGGAGCTCCAAGGTCCTCGCTAAGAAGGAGCTGCTCTACG
or NM_020132.4
TGCCCCTCATCGGCTGGACGTGGTACTTTCTGGAGATTGTGTTCTGCAAGCGGAA
GTGGGAGGAGGACCGGGACACCGTGGTCGAAGGGCTGAGGCGCCTGTCGGACTAC
CCCGAGTACATGTGGTTTCTCCTGTACTGCGAGGGGACGCGCTTCACGGAGACCA
AGCACCGCGTTAGCATGGAGGTGGCGGCTGCTAAGGGGCTTCCTGTCCTCAAGTA
CCACCTGCTGCCGCGGACCAAGGGCTTCACCACCGCAGTCAAGTGCCTCCGGGGG
ACAGTCGCAGCTGTCTATGATGTAACCCTGAACTTCAGAGGAAACAAGAACCCGT
CCCTGCTGGGGATCCTCTACGGGAAGAAGTACGAGGCGGACATGTGCGTGAGGAG
ATTTCCTCTGGAAGACATCCCGCTGGATGAAAAGGAAGCAGCTCAGTGGCTTCAT
AAACTGTACCAGGAGAAGGACGCGCTCCAGGAGATATATAATCAGAAGGGCATGT
TTCCAGGGGAGCAGTTTAAGCCTGCCCGGAGGCCGTGGACCCTCCTGAACTTCCT
GTCCTGGGCCACCATTCTCCTGTCTCCCCTCTTCAGTTTTGTCTTGGGCGTCTTT
GCCAGCGGATCACCTCTCCTGATCCTGACTTTCTTGGGGTTTGTGGGAGCAGCTT
CCTTTGGAGTTCGCAGACTGATAGGAGTAACTGAGATAGAAAAAGGCTCCAGCTA
CGGAAACCAAGAGTTTAAGAAAAAGGAATAA
40
ATGTTCCTGTTGCTGCCTTTTGATAGCCTGATTGTCAACCTTCTGGGCATCTCCC
AGPAT6 (LPA-
TGACTGTCCTCTTCACCCTCCTTCTCGTTTTCATCATAGTGCCAGCCATTTTTGG
MAG PA)
AGTCTCCTTTGGTATCCGCAAACTCTACATGAAAAGTCTGTTAAAAATCTTTGCG
Homo sapiens
TGGGCTACCTTGAGAATGGAGCGAGGAGCCAAGGAGAAGAACCACCAGCTTTACA
Coding sequence of
AGCCCTACACCAACGGAATCATTGCAAAGGATCCCACTTCACTAGAAGAAGAGAT
NCBI Reference
CAAAGAGATTCGTCGAAGTGGTAGTAGTAAGGCTCTGGACAACACTCCAGAGTTC
Sequence:
GAGCTCTCTGACATTTTCTACTTTTGCCGGAAAGGAATGGAGACCATTATGGATG
NM_178819.3
ATGAGGTGACAAAGAGATTCTCAGCAGAAGAACTGGAGTCCTGGAACCTGCTGAG
CAGAACCAATTATAACTTCCAGTACATCAGCCTTCGGCTCACGGTCCTGTGGGGG
TTAGGAGTGCTGATTCGGTACTGCTTTCTGCTGCCGCTCAGGATAGCACTGGCTT
TCACAGGGATTAGCCTTCTGGTGGTGGGCACAACTGTGGTGGGATACTTGCCAAA
TGGGAGGTTTAAGGAGTTCATGAGTAAACATGTTCACTTAATGTGTTACCGGATC
TGCGTGCGAGCGCTGACAGCCATCATCACCTACCATGACAGGGAAAACAGACCAA
GAAATGGTGGCATCTGTGTGGCCAATCATACCTCACCGATCGATGTGATCATCTT
GGCCAGCGATGGCTATTATGCCATGGTGGGTCAAGTGCACGGGGGACTCATGGGT
GTGATTCAGAGAGCCATGGTGAAGGCCTGCCCACACGTCTGGTTTGAGCGCTCGG
AAGTGAAGGATCGCCACCTGGTGGCTAAGAGACTGACTGAACATGTGCAAGATAA
AAGCAAGCTGCCTATCCTCATCTTCCCAGAAGGAACCTGCATCAATAATACATCG
GTGATGATGTTCAAAAAGGGAAGTTTTGAAATTGGAGCCACAGTTTACCCTGTTG
CTATCAAGTATGACCCTCAATTTGGCGATGCCTTCTGGAACAGCAGCAAATACGG
GATGGTGACGTACCTGCTGCGAATGATGACCAGCTGGGCCATTGTCTGCAGCGTG
TGGTACCTGCCTCCCATGACTAGAGAGGCAGATGAAGATGCTGTCCAGTTTGCGA
ATAGGGTGAAATCTGCCATTGCCAGGCAGGGAGGACTTGTGGACCTGCTGTGGGA
TGGGGGCCTGAAGAGGGAGAAGGTGAAGGACACGTTCAAGGAGGAGCAGCAGAAG
CTGTACAGCAAGATGATCGTGGGGAACCACAAGGACAGGAGCCGCTCCTGA
41
ATGGCCACCATGTTGGAAGGCAGATGCCAAACTCAGCCAAGGAGCAGCCCCAGTG
GPAT2
GCCGAGAGGCTAGCCTGTGGTCGTCAGGCTTTGGGATGAAGCTGGAGGCTGTCAC
Homo sapiens
TCCATTCCTGGGGAAGTATCGCCCCTTTGTGGGTCGCTGTTGCCAGACCTGCACC
Coding sequence of
CCCAAGAGCTGGGAGTCCCTCTTCCACAGAAGCATAACGGACCTAGGCTTCTGCA
NCBI Reference
ATGTGATCCTGGTGAAGGAGGAGAACACAAGGTTTCGGGGCTGGCTGGTTCGGAG
Sequence:
GCTCTGCTATTTCCTGTGGTCCCTGGAGCAGCACATCCCCCCCTGCCAGGATGTC
NM_207328.2
CCACAGAAGATCATGGAAAGCACCGGGGTGCAGAACCTCCTCTCAGGGAGGGTCC
CAGGAGGCACTGGGGAAGGCCAGGTGCCTGACCTTGTGAAGAAGGAGGTACAGCG
CATCCTGGGTCACATCCAGGCCCCACCCCGTCCCTTCCTGGTCAGGCTGTTCAGC
TGGGCGCTGCTGAGGTTCCTGAACTGCCTGTTCCTGAATGTGCAGCTCCACAAGG
GTCAGATGAAGATGGTCCAGAAGGCCGCCCAGGCAGGCTTGCCGCTTGTCCTCCT
CTCTACTCACAAAACCCTCCTGGATGGGATCCTGCTGCCCTTTATGCTGCTCTCC
CAGGGCCTGGGTGTGCTTCGTGTGGCCTGGGACTCCCGCGCCTGCTCCCCTGCCC
TCAGAGCTCTGCTGAGGAAGCTTGGGGGGCTTTTCCTGCCCCCAGAGGCCAGCCT
CTCCCTGGACAGCTCTGAGGGGCTCCTTGCCAGGGCTGTGGTCCAGGCGGTCATA
GAGCAGCTGCTGGTTAGTGGGCAGCCCCTGCTCATCTTCCTGGAGGAACCTCCTG
GGGCTCTGGGGCCACGGCTGTCAGCCCTGGGCCAGGCTTGGGTGGGGTTTGTGGT
GCAGGCAGTCCAGGTGGGCATCGTCCCAGATGCTCTGCTGGTACCAGTGGCCGTC
ACCTATGACCTGGTTCCGGATGCACCGTGTGACATAGACCATGCCTCGGCCCCCC
TGGGGCTGTGGACAGGAGCTCTGGCTGTCCTACGTAGCTTGTGGAGCCGCTGGGG
CTGCAGCCACCGGATCTGCTCCCGGGTGCACCTAGCTCAGCCCTTTTCCCTGCAG
GAATACATCGTCAGTGCCAGAAGCTGCTGGGGCGGCAGACAGACCCTGGAGCAGC
TACTGCAGCCCATCGTGCTGGGCCAATGTACTGCTGTCCCAGACACTGAGAAGGA
GCAGGAGTGGACCCCCATAACTGGGCCTCTCCTGGCCCTCAAGGAAGAGGACCAG
CTCCTGGTCAGGAGACTGAGCTGTCATGTCCTGAGTGCCAGTGTAGGGAGCTCTG
CGGTGATGAGCACGGCCATTATGGCAACGCTGCTGCTCTTCAAGCATCAGAAGCT
CCTGGGGGAGTTCTCCTGGCTGACGGAGGAGATACTGTTGCGTGGCTTTGATGTA
GGCTTCTCTGGGCAGCTGCGGAGCCTGCTGCAGCACTCACTGAGCCTGCTGCGGG
CGCACGTGGCCCTGCTGCGCATCCGTCAGGGTGACTTGCTGGTGGTGCCGCAGCC
TGGCCCAGGCCTCACACACCTGGCACAACTGAGTGCTGAGCTGCTGCCCGTCTTC
CTGAGCGAGGCTGTGGGCGCCTGTGCAGTGCGGGGGCTGCTGGCAGGCAGAGTGC
CGCCCCAGGGGCCCTGGGAGCTGCAGGGCATATTGCTGCTGAGCCAGAATGAGCT
GTACCGCCAGATCCTGCTGCTGATGCACCTGCTGCCGCAAGACCTGCTGCTGCTA
AAGCCCTGCCAGTCTTCCTACTGCTACTGTCAGGAGGTGCTGGACCGGCTCATCC
AATGCGGGCTCCTGGTTGCTGAGGAGACCCCAGGCTCCCGGCCAGCCTGTGACAC
AGGGCGACAGCGATTGAGCAGAAAGCTGCTGTGGAAACCGAGTGGGGACTTTACT
GATAGTGACAGTGATGACTTCGGAGAGGCTGACGGCCGGTACTTCAGGCTCAGCC
AGCAGTCACACTGCCCAGATTTCTTTCTTTTCCTCTGCCGCCTGCTCAGCCCGCT
GCTCAAGGCCTTTGCACAGGCTGCCGCCTTCCTCCGCCAGGGCCAGCTGCCCGAT
ACTGAGTTGGGCTACACAGAGCAGCTGTTCCAGTTCCTGCAGGCCACCGCCCAGG
AAGAAGGGATCTTCGAGTGTGCGGACCCAAAGCTCGCCATCAGTGCTGTCTGGAC
CTTCAGAGACCTAGGGGTTCTGCAGCAGACGCCGAGCCCTGCAGGCCCCAGGCTC
CACCTGTCCCCTACTTTTGCCAGCCTGGACAATCAGGAAAAACTAGAACAGTTCA
TCCGGCAGTTCATTTGTAGCTAG
42
ATGGTAGAGTTCGCGCCCTTGTTTATGCCGTGGGAGCGCAGGCTGCAGACACTTG
MOGAT2
CTGTCCTACAGTTTGTCTTCTCCTTCTTGGCACTGGCCGAGATCTGCACTGTGGG
Homo sapiens
CTTCATAGCCCTCCTGTTTACAAGATTCTGGCTCCTCACTGTCCTGTATGCGGCC
Coding sequence of
TGGTGGTATCTGGACCGAGACAAGCCACGGCAGGGGGGCCGGCACATCCAGGCCA
NCBI Reference
TCAGGTGCTGGACTATATGGAAGTACATGAAGGACTATTTCCCCATCTCGCTGGT
Sequence:
CAAGACTGCTGAGCTGGACCCCTCTCGGAACTACATTGCGGGCTTCCACCCCCAT
NM_025098.2
GGAGTCCTGGCAGTCGGAGCCTTTGCCAACCTGTGCACTGAGAGCACAGGCTTCT
CTTCGATCTTCCCCGGTATCCGCCCCCATCTGATGATGCTGACCTTGTGGTTCCG
GGCCCCCTTCTTCAGAGATTACATCATGTCTGCAGGGTTGGTCACATCAGAAAAG
GAGAGTGCTGCTCACATTCTGAACAGGAAGGGTGGCGGAAACTTGCTGGGCATCA
TTGTAGGGGGTGCCCAGGAGGCCCTGGATGCCAGGCCTGGATCCTTCACGCTGTT
ACTGCGGAACCGAAAGGGCTTCGTCAGGCTCGCCCTGACACACGGGGCACCCCTG
GTGCCAATCTTCTCCTTCGGGGAGAATGACCTATTTGACCAGATTCCCAACTCTT
CTGGCTCCTGGTTACGCTATATCCAGAATCGGTTGCAGAAGATCATGGGCATCTC
CCTCCCACTCTTTCATGGCCGTGGTGTCTTCCAGTACAGCTTTGGTTTAATACCC
TACCGCCGGCCCATCACCACTGTGGTGGGGAAGCCCATCGAGGTACAGAAGACGC
TGCATCCCTCGGAGGAGGAGGTGAACCAGCTGCACCAGCGTTATATCAAAGAGCT
GTGCAACCTCTTCGAGGCCCACAAACTTAAGTTCAACATCCCTGCTGACCAGCAC
TTGGAGTTCTGCTGA
43
ATGAAGACCCTCATAGCCGCCTACTCCGGGGTCCTGCGCGGCGAGCGTCAGGCCG
DGAT2
AGGCTGACCGGAGCCAGCGCTCTCACGGAGGACCTGCGCTGTCGCGCGAGGGGTC
Homo sapiens
TGGGAGATGGGGCACTGGATCCAGCATCCTCTCCGCCCTCCAGGACCTCTTCTCT
Coding sequence of
GTCACCTGGCTCAATAGGTCCAAGGTGGAAAAGCAGCTACAGGTCATCTCAGTGC
NCBI Reference
TCCAGTGGGTCCTGTCCTTCCTTGTACTGGGAGTGGCCTGCAGTGCCATCCTCAT
Sequence:
GTACATATTCTGCACTGATTGCTGGCTCATCGCTGTGCTCTACTTCACTTGGCTG
NM_032564.4
GTGTTTGACTGGAACACACCCAAGAAAGGTGGCAGGAGGTCACAGTGGGTCCGAA
ACTGGGCTGTGTGGCGCTACTTTCGAGACTACTTTCCCATCCAGCTGGTGAAGAC
ACACAACCTGCTGACCACCAGGAACTATATCTTTGGATACCACCCCCATGGTATC
ATGGGCCTGGGTGCCTTCTGCAACTTCAGCACAGAGGCCACAGAAGTGAGCAAGA
AGTTCCCAGGCATACGGCCTTACCTGGCTACACTGGCAGGCAACTTCCGAATGCC
TGTGTTGAGGGAGTACCTGATGTCTGGAGGTATCTGCCCTGTCAGCCGGGACACC
ATAGACTATTTGCTTTCAAAGAATGGGAGTGGCAATGCTATCATCATCGTGGTCG
GGGGTGCGGCTGAGTCTCTGAGCTCCATGCCTGGCAAGAATGCAGTCACCCTGCG
GAACCGCAAGGGCTTTGTGAAACTGGCCCTGCGTCATGGAGCTGACCTGGTTCCC
ATCTACTCCTTTGGAGAGAATGAAGTGTACAAGCAGGTGATCTTCGAGGAGGGCT
CCTGGGGCCGATGGGTCCAGAAGAAGTTCCAGAAATACATTGGTTTCGCCCCATG
CATCTTCCATGGTCGAGGCCTCTTCTCCTCCGACACCTGGGGGCTGGTGCCCTAC
TCCAAGCCCATCACCACTGTTGTGGGAGAGCCCATCACCATCCCCAAGCTGGAGC
ACCCAACCCAGCAAGACATCGACCTGTACCACACCATGTACATGGAGGCCCTGGT
GAAGCTCTTCGACAAGCACAAGACCAAGTTCGGCCTCCCGGAGACTGAGGTCCTG
GAGGTGAACTGA
44
ATGCAGCCTGAGGGAGCAGAAAAGGGAAAAAGCTTCAAGCAGAGACTGGTCTTGA
AQP9
AGAGCAGCTTAGCGAAAGAAACCCTCTCTGAGTTCTTGGGCACGTTCATCTTGAT
Homo sapiens
TGTCCTTGGATGTGGCTGTGTTGCCCAAGCTATTCTCAGTCGAGGACGTTTTGGA
Coding sequence of
GGGGTCATCACTATCAATGTTGGATTTTCAATGGCAGTTGCAATGGCCATTTATG
NCBI Reference
TGGCTGGCGGTGTCTCTGGTGGTCACATCAACCCAGCTGTGTCTTTAGCAATGTG
Sequence:
TCTCTTTGGACGGATGAAATGGTTCAAATTGCCATTTTATGTGGGAGCCCAGTTC
NM_020980.3
TTGGGAGCCTTTGTGGGGGCTGCAACCGTCTTTGGCATTTACTATGATGGACTTA
TGTCCTTTGCTGGTGGAAAACTGCTGATCGTGGGAGAAAATGCAACAGCACACAT
TTTTGCAACATACCCAGCTCCGTATCTATCTCTGGCGAACGCATTTGCAGATCAA
GTGGTGGCCACCATGATACTCCTCATAATCGTCTTTGCCATCTTTGACTCCAGAA
ACTTGGGAGCCCCCAGAGGCCTAGAGCCCATTGCCATCGGCCTCCTGATTATTGT
CATTGCTTCCTCCCTGGGACTGAACAGTGGCTGTGCCATGAACCCAGCTCGAGAC
CTGAGTCCCAGACTTTTCACTGCCTTGGCAGGCTGGGGGTTTGAAGTCTTCAGAG
CTGGAAACAACTTCTGGTGGATTCCTGTAGTGGGCCCTTTGGTTGGTGCTGTCAT
TGGAGGCCTCATCTATGTTCTTGTCATTGAAATCCACCATCCAGAGCCTGACTCA
GTCTTTAAGACAGAACAATCTGAGGACAAACCAGAGAAATATGAACTCAGTGTCA
TCATGTAG
45
ATGAATTACGTGGGGCAGTTAGCCGGCCAGGTGTTTGTCACCGTGAAGGAGCTCT
LPIN1 (Lipin 1)
ACAAGGGGCTGAATCCCGCCACACTCTCAGGGTGCATTGACATCATTGTCATCCG
Homo sapiens
CCAGCCCAATGGAAACCTCCAATGCTCCCCTTTCCACGTCCGCTTTGGGAAGATG
Coding sequence of
GGGGTCCTGCGCTCCCGAGAGAAAGTGGTTGACATAGAAATCAATGGGGAATCTG
NCBI Reference
TGGATTTGCATATGAAATTGGGAGATAATGGAGAAGCATTTTTTGTTCAAGAAAC
Sequence:
AGATAATGATCAGGAAGTTATCCCTATGCACCTGGCCACCTCCCCCATCCTGTCA
NM_145693.2
GAAGGAGCTTCGAGAATGGAATGCCAGCTGAAAAGGGGCTCTGTGGACAGGATGA
GAGGCCTGGACCCCAGCACGCCAGCCCAAGTGATCGCTCCCAGCGAGACGCCGTC
AAGCAGCTCTGTAGTAAAGAAGAGAAGAAAAAGGAGGAGAAAGTCACAGCTGGAC
AGCCTGAAGAGAGATGACAACATGAACACATCTGAGGATGAGGACATGTTCCCCA
TCGAGATGAGCTCGGATGAGGCCATGGAGCTGCTGGAGAGCAGCAGAACTCTTCC
TAATGATATACCTCCATTCCAAGATGATATTCCTGAGGAAAACCTCTCCCTGGCT
GTGATTTACCCTCAGTCAGCCTCATACCCTAATTCGGATAGAGAGTGGTCACCCA
CTCCCAGTCCTTCCGGTTCCCGACCTTCAACACCTAAAAGTGATTCAGAATTGGT
CAGCAAGTCCACGGAAAGGACAGGGCAGAAGAACCCAGAAATGCTTTGGCTGTGG
GGAGAGCTGCCGCAGGCTGCTAAGTCTTCTTCTCCACACAAGATGAAAGAGTCCA
GCCCATTGAGCAGTAGAAAAATTTGTGATAAAAGTCACTTTCAGGCCATTCACAG
CGAATCTTCAGACACTTTTAGTGACCAATCGCCAACTCTGGTCGGTGGGGCACTT
TTGGACCAGAACAAGCCTCAGACAGAAATGCAGTTTGTGAATGAAGAAGACCTGG
AGACCTTAGGAGCAGCAGCGCCACTCTTGCCCATGATCGAGGAGCTCAAACCCCC
CTCTGCCAGTGTAGTCCAGACAGCAAACAAGACGGATTCTCCTTCCAGGAAAAGA
GATAAACGAAGCCGACATCTTGGTGCTGACGGCGTCTACTTGGATGACCTCACAG
ACATGGATCCTGAAGTGGCGGCCCTGTATTTTCCCAAAAACGGAGATCCTTCCGG
ACTCGCAAAACATGCAAGCGACAACGGAGCCCGGTCAGCCAACCAGTCCCCGCAG
TCGGTGGGCAGCTCGGGCGTGGACAGTGGCGTGGAGAGCACCTCGGACGGGCTGA
GGGACCTCCCTTCCATCGCCATCTCCCTCTGCGGGGGCCTCAGCGACCACCGGGA
GATCACGAAAGATGCATTCCTGGAGCAAGCTGTGTCATATCAACAGTTTGTGGAC
AACCCCGCTATTATCGATGACCCCAATCTCGTGGTAAAGATTGGGAGTAAATATT
ATAACTGGACAACAGCAGCACCCCTCCTCCTGGCAATGCAGGCCTTCCAGAAACC
TTTGCCAAAGGCCACTGTGGAATCTATCATGAGGGATAAAATGCCCAAAAAGGGA
GGAAGATGGTGGTTTTCATGGAGGGGAAGAAACACCACAATCAAGGAGGAAAGTA
AGCCAGAGCAGTGCTTGGCTGGCAAGGCCCATAGCACCGGAGAGCAACCGCCGCA
GCTCAGCTTGGCCACCAGGGTAAAGCATGAATCATCCTCCAGTGATGAGGAGCGC
GCAGCTGCCAAGCCATCAAACGCAGGCCACCTCCCTCTTCTGCCTAATGTCAGCT
ACAAGAAGACTCTCCGGCTGACTTCCGAGCAGCTTAAAAGCTTGAAGTTGAAGAA
TGGCCCCAACGACGTGGTTTTCAGTGTCACCACGCAGTACCAAGGCACGTGCCGC
TGTGAGGGCACCATCTATCTGTGGAACTGGGATGATAAAGTCATCATTTCTGATA
TTGATGGGACAATTACCAGATCAGATACTCTTGGCCACATTTTGCCCACCCTTGG
GAAGGATTGGACCCATCAGGGCATCGCTAAGCTGTACCATAAAGTGAGCCAGAAT
GGATATAAATTTCTCTACTGTTCTGCCCGTGCCATCGGGATGGCGGACATGACGC
GGGGCTACCTGCACTGGGTCAACGAGAGGGGCACGGTGCTGCCCCAGGGGCCCCT
GCTGCTGAGTCCCAGCAGCCTCTTCTCTGCCCTGCACAGAGAAGTGATTGAAAAG
AAGCCAGAAAAGTTTAAAGTCCAGTGTTTGACAGACATCAAAAACCTGTTTTTCC
CCAACACAGAACCCTTTTATGCTGCTTTTGGAAACCGACCAGCTGATGTGTATTC
ATACAAGCAAGTAGGAGTGTCTTTGAATAGAATATTTACCGTCAACCCTAAAGGA
GAGCTGGTACAGGAACATGCAAAGACCAACATCTCTTCGTATGTGAGACTCTGTG
AAGTAGTCGACCACGTTTTCCCGTTGCTGAAAAGAAGCCATTCTTCAGACTTTCC
CTGTTCGGATACCTTCAGTAACTTCACCTTTTGGAGAGAGCCACTGCCACCTTTT
GAAAACCAGGACATTCATTCTGCCTCAGCGTAA
46
ATGGCGGCGTCCGTGCGACAGGCACGCAGCCTACTAGGTGTGGCGGCGACCCTGG
CRIF1
CCCCGGGTTCCCGTGGCTACCGGGCGCGGCCGCCCCCGCGCCGCAGGCCGGGACC
Homo sapiens
CCGGTGGCCAGACCCCGAGGACCTCCTGACCCCGCGGTGGCAGCTGGGACCGCGC
Coding sequence of
TACGCGGCTAAGCAGTTCGCGCGTTACGGCGCCGCCTCCGGGGTGGTCCCCGGTT
NCBI Reference
CGTTATGGCCGTCGCCGGAGCAGCTGCGGGAGCTGGAGGCCGAAGAACGCGAATG
Sequence:
GTACCCGAGCCTGGCGACCATGCAGGAGTCGCTGCGGGTGAAGCAGCTGGCCGAA
NM_052850.3
GAGCAGAAGCGTCGGGAGAGGGAGCAGCACATCGCAGAGTGCATGGCCAAGATGC
CACAGATGATTGTGAACTGGCAGCAGCAGCAGCGGGAGAACTGGGAGAAGGCCCA
GGCTGACAAGGAGAGGAGGGCCCGACTGCAGGCTGAGGCCCAGGAGCTCCTGGGC
TACCAGGTGGACCCAAGGAGTGCCCGCTTCCAGGAGCTGCTCCAGGACCTAGAGA
AGAAGGAGCGCAAGCGCCTCAAGGAGGAAAAACAGAAACGGAAGAAGGAGGCGCG
AGCTGCTGCATTGGCTGCAGCTGTGGCTCAAGACCCAGCAGCCTCTGGGGCACCC
AGCTCCTGA
47
ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCT
PD1
GGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTT
Homo sapiens
CTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGC
Coding sequence of
TTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCA
GenBank:
ACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGA
KJ865859.1
CTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTG
GTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGG
CCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAG
AAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAG
TTCCAAACCCTGGTGGTTGGTGTCGTGGGCGGCCTGCTGGGCAGCCTGGTGCTGC
TAGTCTGGGTCCTGGCCGTCATCTGCTCCCGGGCCGCACGAGGGACAATAGGAGC
CAGGCGCACCGGCCAGCCCCTGAAGGAGGACCCCTCAGCCGTGCCTGTGTTCTCT
GTGGACTATGGGGAGCTGGATTTCCAGTGGCGAGAGAAGACCCCGGAGCCCCCCG
TGCCCTGTGTCCCTGAGCAGACGGAGTATGCCACCATTGTCTTTCCTAGCGGAAT
GGGCACCTCATCCCCCGCCCGCAGGGGCTCAGCTGACGGCCCTCGGAGTGCCCAG
CCACTGAGGCCTGAGGATGGACACTGCTCTTGGCCCCTCTGA
48
ATGAGACCTCGCACCCACGGCGCCCCCCCGCGCAACATCATGTCCACCATCCCCA
Lymphocyte
AGTGGTTCAAAGGGGCGCCCTTTGGGGTGCAGAGCCACAGGTTTGATGTCTCCGC
expansion molecule
TGTCTATCCCAACCAGAAGAAATTCAGCACCTTCACAGAGGCCCCATACTCCAGA
(LEM)
CATCATTCGGTGGAACTGTCCCACATAGGACCTGGGACCTATAACTCCAAGGATA
Mus musculus
CCTGCTTCAGCAAGAAGTTCCTGGAACAGAAGTTGGGCTCAGGATGGTCCCAGGC
Coding sequence of
CCACGAAGCCACTCGGCTGACCCAGCTACCCCACTTCCACTACCAGGCCATCAAG
GenBank:
AAGGAAAAAGAGCAGCAGGTGCACAAGCGTGGCCCTGGCTCCTACAACATCAAAG
KP939367.1
ACTTCATAACTGAGCTGCAGAAGAAACCACAGAGCAAACGGGGGCTGCTCAGCTC
TGGGGAGACACGTTTCCGAGGTTTTATTGGGAATTATTATCCTGGCCCTGGAAAT
TATGGGGAGAAGGGGAACCCGTACACACAGCTGGAGGAGAAGGCCTGGAACCGCT
CACATTCTGACGGCCTGATGTGTAGAGTGTCTAACAAGCCACCCTTGTTTCATCA
GGGCAGTGGCCTGGGACCTGGTACCTACACCATCAAAAGCGATCTTGAGACCTTT
GTGAAAAAGTCCACTGGTAATCGTGGCCCCTATGACATTTTCTCTGGTGAACGGA
GCAGTCCTTTGCCCTATGGACATTACTCTGTGCAGAAAATGAAGCCCAAGGAACT
GACAGATTACAAGAGCTTTCTGGACGAAATGAACTCACAACACAAGAAGAAACAA
GGGGTTTTCTCGAAATATCCCCGAGATCCGAAACACCCCACAGAGAGAATTTTCT
GGACAACCCTTAGTCAGTGCCCCAAAAATATGGATATAGCTGGCCCTGGTTCTTG
GCTTCCTCATGAGACGGAACAGAAACATGTCAACCGGCCACCGTTCCTCCTGGCC
TCCAAACGGTGCGGCCTAAAGGCCTACCAGATGATTTTGGGAACCTGGAACCCAG
TTGGCGTAGGCCGCTATCTCAACACCACGCTGATGGAGTCCATAGACCGAAGGCA
GCGATACCGTTCTCTGTACATGAGTGAGCCCAAGCGATACCTGCAAGACCTAACC
CGAGACAGACTCATGCAGAAACGGATTACACCTATTACGAAGGGCAAGTGCCGTC
CAACTGTGGACTACAATTCAGATCCTACTCCTTAA
49
ESKYGPPCPPCP
spacer (IgG4hinge)
(aa)
Homo sapiens
50
GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT
spacer (IgG4hinge)
(nt)
Homo sapiens
51
ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
Hinge-CH3 spacer
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ
Homo sapiens
KSLSLSLGK
52
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV
Hinge-CH2-CH3
QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL
spacer
PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
Homo sapiens
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ
KSLSLSLGK
53
RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEE
IgD-hinge-Fc
RETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAG
Homo sapiens
KVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMA
LREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTS
GFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLE
VSYVTDH
54
LEGGGEGRGSLLTCGDVEENPGPR
T2A
artificial
55
MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSIS
tEGFR
GDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFE
artificial
NLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTIN
WKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSR
GRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYI
DGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGP
KIPSIATGMVGALLLLLVVALGIGLFM
56
FWVLVVVGGVLACYSLLVTVAFIIFWV
CD28 (amino acids
153-179 of
Accession No.
P10747)
Homo sapiens
57
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSL
CD28 (amino acids
LVTVAFIIFWV
114-179 of
Accession No.
P10747)
Homo sapiens
58
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
CD28 (amino acids
180-220 of P10747)
Homo sapiens
59
RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
CD28 (LL to GG)
Homo sapiens
60
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
4-1BB (amino acids
214-255 of
Q07011.1)
Homo sapiens
61
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ
CD3 zeta
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP
Homo sapiens
PR
62
RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ
CD3 zeta
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP
Homo sapiens
PR
63
RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ
CD3 zeta
EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP
Homo sapiens
PR
64
EGRGSLLTCGDVEENPGP
T2A
65
GSGATNFSLLKQAGDVEENPGP
P2A
66
ATNFSLLKQAGDVEENPGP
P2A
67
QCTNYALLKLAGDVESNPGP
E2A
68
VKQTLNFDLLKLAGDVESNPGP
F2A
69
MRESQDAAGAHGWNRVGSTATKWFTGAPFGVQSHREDISAVYPNWKKFSTFTEAP
lymphocyte
YSTRYSTQVSHIGPGTYSSKETCFSKKKLMKEVDTGWAKAQEATRLTQLPHFQYQ
expansion molecule
AIMKEKRLKEQKLGPGSYNLKDFLEQLREKPCSTRGLLSSGEVRFRGLTGNYYPG
(LEM) isoform 2
PGNYGEKGNPYTKLEENAWNRSHSEGLMCRMSNKPHPRPHQGSGLGPGTYFEKSD
Homo sapiens
LETYVARSVGTRGPYDTFSGDRSKPLPYGHYSMQKKKPRELMNEKSFVEELNSHH
NCBI Reference
NKKHGVESKLPRNPKTPTERIYWANLSQCPRTLATSGPSEWLPQEKKCKPVNQPP
Sequence:
FLLTSKGSGAKACQMIMGSWNPVGVGRYLNTWLMETKDRRQRYRSLELSGSKRYL
NP_001104003.1
SDLARDMLMQERITPFTKGKCPPTVDYNSDPTP
70
ATGAGGGAAAGCCAGGATGCCGCCGGAGCTCATGGCTGGAACCGCGTCGGCTCCA
lymphocyte
CGGCCACCAAGTGGTTCACCGGGGCGCCCTTCGGGGTGCAGAGCCACAGGTTTGA
expansion molecule
CATCTCTGCTGTTTATCCCAACTGGAAGAAGTTCAGCACCTTCACTGAGGCCCCA
(LEM) isoform 2
TACTCCACGCGTTATTCTACCCAAGTGTCCCACATAGGCCCTGGGACTTACAGCT
Homo sapiens
CCAAAGAGACCTGCTTCAGCAAGAAGAAGCTGATGAAGGAGGTGGACACAGGCTG
NCBI Reference
GGCCAAGGCCCAGGAAGCCACGCGGCTGACCCAGCTACCCCACTTCCAGTACCAG
Sequence:
GCCATCATGAAAGAGAAGCGGCTGAAGGAGCAAAAGCTGGGCCCCGGCTCCTACA
NM_001110533.1
ACCTCAAAGACTTCTTAGAACAGCTGCGGGAGAAACCATGTAGCACCCGGGGGCT
GCTCAGCTCTGGGGAGGTTCGCTTCCGAGGACTCACTGGGAACTACTATCCAGGC
CCTGGAAATTATGGGGAGAAGGGTAACCCATACACCAAGCTGGAGGAGAATGCCT
GGAACCGGTCTCATTCCGAGGGCCTCATGTGCAGGATGAGCAACAAGCCACACCC
CCGGCCTCATCAGGGGAGTGGTCTGGGACCCGGCACCTACTTCTTCAAAAGCGAC
CTTGAGACATATGTGGCACGATCCGTCGGCACCCGCGGCCCCTATGACACTTTCT
CTGGTGATCGGAGCAAGCCACTGCCTTATGGGCACTACTCCATGCAGAAAAAAAA
GCCCAGGGAACTGATGAATTTCAAGAGCTTTGTAGAAGAACTTAACTCACATCAC
AATAAGAAGCATGGGGTTTTTTCTAAACTTCCCCGAAACCCGAAAACCCCTACAG
AGAGGATTTACTGGGCCAACCTCAGCCAGTGCCCCCGCACACTGGCCACATCTGG
CCCCAGTTTCTGGCTTCCACAAGAGAAGAAATGCAAACCCGTCAACCAGCCCCCA
TTCCTGTTGACCTCCAAGGGGTCAGGTGCAAAGGCCTGCCAGATGATTATGGGAA
GCTGGAACCCAGTAGGTGTGGGCCGCTACCTCAACACCTGGCTGATGGAGACAAA
GGACAGGCGGCAGCGATATCGATCCCTATTCCTGAGTGGATCCAAACGCTACCTC
TCAGACCTGGCCCGGGACATGCTCATGCAGGAAAGGATCACACCATTTACTAAGG
GAAAGTGCCCTCCAACTGTGGATTACAATTCAGATCCTACTCCT
71
CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGG
T2A (nt)
AGAATCCCGGCCCTAGG
72
MLLLVTSLLLCELPHPAFLLIP
GMCSFR alpha
chain signal
sequence
Homo sapiens
UniProt No.
P15509
73
ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCC
GMCSFR alpha
TCCTGATCCCA
chain signal
sequence DNA
Homo sapiens
74
TGCCACCCTGAGTGTCAGCCCCAGAATGGCTCAGTGACCTGTTTTGGACCGGAGG
Modified Her2t (nt)
CTGACCAGTGTGTGGCCTGTGCCCACTATAAGGACCCTCCCTTCTGCGTGGCCCG
CTGCCCCAGCGGTGTGAAACCTGACCTCTCCTACATGCCCATCTGGAAGTTTCCA
GATGAGGAGGGCGCATGCCAGCCTTGCCCCATCAACTGCACCCACTCCTGTGTGG
ACCTGGATGACAAGGGCTGCCCCGCCGAGCAGAGAGCCAGCCCTCTGACGGGTGG
AGGAAGCGGAGGTGGCAGCTCCATCATCTCTGCGGTGGTTGGCATTCTGCTGGTC
GTGGTCTTGGGGGTGGTCTTTGGGATCCTCATC
75
CHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFP
Modified Her2t (aa)
DEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTGGGSGGGSSIISAVVGILLV
VVLGVVFGILI
76
ATGGGCGATCGCGGCAGCTCCCGGAGAAGGCGCACCGGCAGCCGGCCCTCTAGCC
DAG O-
ACGGCGGCGGCGGCCCTGCTGCCGCCGAGGAGGAGGTGCGCGACGCCGCCGCCGG
acetyltransferase 1
CCCTGATGTGGGAGCAGCAGGCGACGCACCAGCACCTGCCCCAAACAAGGACGGC
(DGAT1) (nt)
GATGCAGGAGTGGGAAGCGGACACTGGGAGCTGAGATGCCACAGGCTGCAGGATT
CCCTGTTCTCCTCTGACAGCGGCTTTTCCAACTACAGAGGCATCCTGAATTGGTG
CGTGGTCATGCTGATCCTGTCCAACGCCAGGCTGTTCCTGGAGAATCTGATCAAG
TACGGCATCCTGGTGGATCCTATCCAGGTGGTGAGCCTGTTTCTGAAGGACCCAT
ATTCCTGGCCAGCACCTTGCCTGGTCATCGCAGCAAACGTGTTCGCAGTGGCAGC
CTTTCAGGTGGAGAAGCGGCTGGCCGTGGGCGCCCTGACCGAGCAGGCAGGCCTG
CTGCTGCACGTGGCCAATCTGGCCACAATCCTGTGCTTCCCAGCAGCAGTGGTGC
TGCTGGTGGAGTCTATCACCCCTGTGGGAAGCCTGCTGGCCCTGATGGCACACAC
AATCCTGTTCCTGAAGCTGTTTTCCTACAGAGACGTGAATTCTTGGTGTAGGAGA
GCAAGGGCAAAGGCAGCCTCTGCCGGCAAGAAGGCCAGCAGCGCCGCCGCCCCTC
ACACCGTGAGCTACCCAGATAACCTGACATATAGAGACCTGTACTATTTCCTGTT
TGCCCCCACCCTGTGCTATGAGCTGAATTTCCCAAGGTCCCCCAGGATCCGCAAG
CGGTTTCTGCTGAGGCGCATCCTGGAGATGCTGTTCTTTACCCAGCTGCAAGTGG
GCCTGATCCAGCAGTGGATGGTGCCAACAATCCAGAACTCCATGAAGCCCTTCAA
GGACATGGATTACTCTAGAATCATCGAGAGGCTGCTGAAGCTGGCCGTGCCCAAC
CACCTGATCTGGCTGATCTTCTTTTATTGGCTGTTTCACTCTTGCCTGAATGCCG
TGGCCGAGCTGATGCAGTTCGGCGATCGCGAGTTTTACCGGGACTGGTGGAATTC
CGAGTCTGTGACATATTTCTGGCAGAACTGGAATATCCCAGTGCACAAGTGGTGT
ATCCGCCACTTTTACAAGCCCATGCTGCGGAGAGGCTCTAGCAAGTGGATGGCCA
GAACCGGCGTGTTCCTGGCCTCTGCCTTCTTTCACGAGTATCTGGTGAGCGTGCC
TCTGCGCATGTTCCGGCTGTGGGCCTTTACAGGCATGATGGCCCAGATCCCACTG
GCCTGGTTTGTGGGCCGGTTCTTTCAGGGCAACTACGGCAATGCCGCCGTGTGGC
TGAGCCTGATCATCGGCCAGCCCATCGCCGTGCTGATGTACGTGCACGATTACTA
TGTGCTGAACTATGAGGCCCCTGCCGCCGAGGCC
77
ATGGCAGCCAGCAAGAAGGCCGTGCTGGGCCCACTGGTGGGAGCAGTGGACCAGG
Glycerol kinase
GCACCAGCTCCACAAGGTTCCTGGTGTTTAATAGCAAGACCGCAGAGCTGCTGTC
(Gyk) (nt)
CCACCACCAGGTGGAGATCAAGCAGGAGTTTCCAAGGGAGGGATGGGTGGAGCAG
GACCCAAAGGAGATCCTGCACTCCGTGTACGAGTGCATCGAGAAGACCTGTGAGA
AGCTGGGCCAGCTGAATATCGACATCAGCAACATCAAGGCCATCGGCGTGTCCAA
TCAGCGGGAGACCACAGTGGTGTGGGACAAGATCACAGGCGAGCCCCTGTATAAC
GCCGTGGTGTGGCTGGATCTGAGGACCCAGAGCACAGTGGAGTCCCTGTCTAAGC
GCATCCCTGGCAACAATAACTTTGTGAAGTCCAAGACCGGCCTGCCACTGTCCAC
ATATTTCTCTGCCGTGAAGCTGAGGTGGCTGCTGGACAATGTGCGCAAGGTGCAG
AAGGCCGTGGAGGAGAAGAGGGCCCTGTTTGGCACCATCGATTCTTGGCTGATCT
GGAGCCTGACAGGAGGAGTGAACGGAGGCGTGCACTGCACCGACGTGACAAATGC
CTCTCGGACCATGCTGTTCAACATCCACAGCCTGGAGTGGGATAAGCAGCTGTGC
GAGTTCTTTGGCATCCCTATGGAGATCCTGCCAAACGTGAGATCTAGCTCCGAGA
TCTATGGCCTGATGAAGATCAGCCACTCCGTGAAGGCAGGCGCCCTGGAGGGAGT
GCCTATCTCTGGATGCCTGGGCGACCAGAGCGCCGCCCTGGTGGGACAGATGTGC
TTCCAGATCGGCCAGGCCAAGAATACCTACGGCACAGGCTGCTTTCTGCTGTGCA
ACACCGGCCACAAGTGCGTGTTCAGCGACCACGGCCTGCTGACCACAGTGGCCTA
TAAGCTGGGCAGGGATAAGCCCGTGTACTATGCACTGGAGGGATCTGTGGCAATC
GCAGGAGCCGTGATCAGGTGGCTGAGAGATAATCTGGGCATCATCAAGACCAGCG
AGGAGATCGAGAAGCTGGCCAAGGAAGTGGGCACATCCTACGGCTGTTATTTCGT
GCCTGCCTTTTCTGGCCTGTACGCACCATATTGGGAGCCAAGCGCCAGGGGAATC
ATCTGCGGCCTGACCCAGTTCACAAACAAGTGTCACATCGCCTTTGCCGCCCTGG
AGGCCGTGTGCTTCCAGACCCGGGAGATCCTGGACGCCATGAATAGAGATTGTGG
CATCCCTCTGTCCCACCTGCAGGTGGACGGAGGCATGACATCTAACAAGATCCTG
ATGCAGCTGCAGGCCGACATCCTGTATATCCCAGTGGTGAAGCCCTCCATGCCTG
AGACCACAGCCCTGGGAGCAGCAATGGCAGCAGGAGCAGCCGAGGGCGTGGGCGT
GTGGTCCCTGGAGCCAGAGGACCTGTCTGCCGTGACCATGGAGCGGTTTGAGCCT
CAGATCAATGCCGAGGAGTCCGAGATCAGATACTCTACATGGAAGAAGGCCGTGA
TGAAGTCCATGGGCTGGGTGACCACACAGTCTCCCGAGAGCGGCGATCCTAGCAT
CTTCTGCTCCCTGCCACTGGGCTTCTTTATCGTGTCTAGCATGGTCATGCTGATC
GGCGCCCGGTATATCTCTGGCATCCCC
78
ATGGACGAGTCCGCCCTGACACTGGGCACCATCGACGTGAGCTACCTGCCACACA
Glycerol-3-
GCTCCGAGTATTCTGTGGGCAGGTGCAAGCACACAAGCGAGGAGTGGGGAGAGTG
phosphate
TGGCTTCCGGCCAACAATCTTTAGATCCGCCACCCTGAAGTGGAAGGAGAGCCTG
acetyltransferase
ATGTCCCGGAAGAGACCATTCGTGGGCCGGTGCTGTTACTCCTGCACCCCCCAGT
mitochondrial
CTTGGGACAAGTTCTTTAACCCTTCTATCCCAAGCCTGGGCCTGAGAAACGTGAT
(GPAM) (nt)
CTACATCAATGAGACCCACACAAGGCACAGGGGATGGCTGGCCCGGAGACTGAGC
TATGTGCTGTTCATCCAGGAGAGGGACGTGCACAAGGGCATGTTTGCCACAAATG
TGACCGAGAACGTGCTGAATTCTAGCCGCGTGCAGGAGGCAATCGCAGAGGTGGC
AGCAGAGCTGAACCCTGATGGAAGCGCCCAGCAGCAGTCCAAGGCAGTGAATAAG
GTGAAGAAGAAGGCCAAGCGGATCCTGCAGGAGATGGTGGCCACAGTGTCCCCAG
CCATGATCAGACTGACCGGCTGGGTGCTGCTGAAGCTGTTCAACTCTTTCTTTTG
GAATATCCAGATCCACAAGGGCCAGCTGGAGATGGTGAAGGCCGCCACCGAGACA
AACCTGCCACTGCTGTTTCTGCCCGTGCACCGCAGCCACATCGATTACCTGCTGC
TGACCTTCATCCTGTTTTGTCACAACATCAAGGCCCCTTATATCGCCAGCGGCAA
CAATCTGAATATCCCAATCTTCTCCACACTGATCCACAAGCTGGGCGGCTTCTTT
ATCAGGCGCCGGCTGGATGAGACCCCTGACGGCAGGAAGGATGTGCTGTACCGCG
CCCTGCTGCACGGACACATCGTGGAGCTGCTGAGGCAGCAGCAGTTCCTGGAGAT
CTTTCTGGAGGGCACACGGTCTAGAAGCGGCAAGACCTCCTGCGCAAGGGCAGGA
CTGCTGTCCGTGGTGGTGGACACACTGTCTACCAACGTGATCCCCGACATCCTGA
TCATCCCTGTGGGCATCTCTTACGACCGGATCATCGAGGGCCACTATAACGGCGA
GCAGCTGGGCAAGCCCAAGAAGAATGAGTCCCTGTGGTCTGTGGCCAGGGGCGTG
ATCCGGATGCTGAGAAAGAATTACGGATGCGTGCGGGTGGATTTCGCACAGCCTT
TTTCCCTGAAGGAGTATCTGGAGTCCCAGTCTCAGAAGCCCGTGAGCGCCCTGCT
GTCCCTGGAGCAGGCCCTGCTGCCTGCAATCCTGCCAAGCAGACCTTCCGATGCA
GCAGACGAGGGAAGGGACACATCTATCAACGAGAGCAGAAATGCCACCGATGAGA
GCCTGAGAAGGCGCCTGATCGCCAACCTGGCCGAGCACATCCTGTTCACAGCCAG
CAAGTCCTGCGCCATCATGAGCACCCACATCGTGGCCTGTCTGCTGCTGTACCGG
CACAGGCAGGGAATCGACCTGTCCACACTGGTGGAGGATTTCTTTGTGATGAAGG
AGGAGGTGCTGGCCAGGGACTTCGATCTGGGCTTTTCTGGCAATAGCGAGGACGT
GGTCATGCACGCCATCCAGCTGCTGGGCAACTGCGTGACCATCACACACACCTCC
CGCAATGATGAGTTCTTTATCACCCCTTCTACCACAGTGCCAAGCGTGTTCGAGC
TGAACTTTTACTCTAATGGCGTGCTGCACGTGTTTATCATGGAGGCCATCATCGC
CTGCAGCCTGTATGCCGTGCTGAACAAGAGGGGACTGGGCGGCCCAACAAGCACC
CCCCCTAATCTGATCTCCCAGGAGCAGCTGGTGAGAAAGGCCGCCTCCCTGTGCT
ATCTGCTGTCTAACGAGGGCACAATCAGCCTGCCCTGCCAGACCTTCTACCAGGT
GTGCCACGAGACAGTGGGCAAGTTTATCCAGTATGGCATCCTGACCGTGGCCGAG
CACGACGATCAGGAGGACATCTCTCCTAGCCTGGCCGAGCAGCAGTGGGATAAGA
AGCTGCCAGAGCCCCTGTCCTGGAGGTCTGACGAGGAGGACGAGGATAGCGACTT
CGGCGAGGAGCAGCGCGATTGTTACCTGAAGGTGTCCCAGTCTAAGGAGCACCAG
CAGTTCATCACCTTTCTGCAGCGGCTGCTGGGCCCACTGCTGGAGGCCTATTCCT
CTGCCGCCATCTTCGTGCACAACTTTTCCGGCCCTGTGCCAGAGCCAGAGTACCT
GCAGAAGCTGCACAAGTATCTGATCACAAGGACCGAGAGGAACGTGGCCGTGTAC
GCAGAGAGCGCCACCTATTGCCTGGTGAAGAATGCCGTGAAGATGTTCAAGGACA
TCGGCGTGTTTAAGGAGACAAAGCAGAAGCGGGTGTCTGTGCTGGAGCTGAGCTC
CACCTTCCTGCCCCAGTGTAATAGACAGAAGCTGCTGGAGTACATCCTGAGCTTT
GTGGTGCTG
79
ATGGAGGGCGCCGGCGGCGCCAACGATAAGAAGAAGATCAGCTCCGAGCGGAGAA
HIF1α (nt)
AGGAGAAGAGCAGGGACGCAGCACGCTCTAGGCGCAGCAAGGAGTCCGAGGTGTT
CTACGAGCTGGCCCACCAGCTGCCACTGCCACACAACGTGTCTAGCCACCTGGAT
AAGGCCAGCGTGATGCGGCTGACCATCTCCTATCTGCGGGTGAGAAAGCTGCTGG
ACGCCGGCGATCTGGACATCGAGGACGATATGAAGGCCCAGATGAATTGCTTCTA
CCTGAAGGCCCTGGACGGCTTTGTGATGGTGCTGACCGACGATGGCGACATGATC
TACATCTCCGATAACGTGAATAAGTATATGGGCCTGACCCAGTTTGAGCTGACAG
GCCACAGCGTGTTCGACTTTACCCACCCCTGCGATCACGAGGAGATGAGGGAGAT
GCTGACACACCGCAACGGCCTGGTGAAGAAGGGCAAGGAGCAGAATACCCAGCGG
TCTTTCTTTCTGAGAATGAAGTGTACCCTGACAAGCAGGGGCCGCACCATGAACA
TCAAGTCCGCCACATGGAAGGTGCTGCACTGCACCGGCCACATCCACGTGTACGA
TACCAACTCCAATCAGCCACAGTGTGGCTATAAGAAGCCCCCTATGACATGCCTG
GTGCTGATCTGTGAGCCTATCCCACACCCCTCTAATATCGAGATCCCCCTGGACA
GCAAGACCTTCCTGTCTCGGCACAGCCTGGACATGAAGTTTAGCTACTGCGATGA
GAGAATCACAGAGCTGATGGGCTATGAGCCTGAGGAGCTGCTGGGCAGATCTATC
TACGAGTACTATCACGCCCTGGATAGCGACCACCTGACCAAGACACACCACGACA
TGTTCACCAAGGGCCAGGTGACCACAGGCCAGTACAGGATGCTGGCCAAGAGGGG
AGGATACGTGTGGGTGGAGACCCAGGCCACAGTGATCTATAACACCAAGAATAGC
CAGCCCCAGTGCATCGTGTGCGTGAACTACGTGGTGTCCGGCATCATCCAGCACG
ATCTGATCTTTTCTCTGCAGCAGACCGAGTGCGTGCTGAAGCCTGTGGAGTCCTC
TGACATGAAGATGACCCAGCTGTTCACAAAGGTGGAGTCCGAGGACACAAGCTCC
CTGTTTGATAAGCTGAAGAAGGAGCCAGACGCACTGACCCTGCTGGCCCCAGCAG
CAGGCGATACAATCATCTCTCTGGACTTCGGCAGCAATGATACCGAGACAGACGA
TCAGCAGCTGGAGGAGGTGCCTCTGTATAACGATGTGATGCTGCCTTCTCCAAAT
GAGAAGCTGCAGAACATCAATCTGGCAATGAGCCCACTGCCTACCGCAGAGACAC
CAAAGCCACTGAGGTCTAGCGCCGACCCAGCCCTGAACCAGGAGGTGGCCCTGAA
GCTGGAGCCTAATCCAGAGTCCCTGGAGCTGTCTTTTACAATGCCACAGATCCAG
GACCAGACCCCATCCCCTTCTGATGGCAGCACACGCCAGTCCTCTCCAGAGCCCA
ACAGCCCTTCCGAGTACTGCTTCTATGTGGATTCCGACATGGTGAATGAGTTCAA
GCTGGAGCTGGTGGAGAAGCTGTTTGCCGAGGATACCGAGGCCAAGAACCCCTTC
AGCACCCAGGATACAGACCTGGATCTGGAGATGCTGGCCCCCTATATCCCTATGG
ACGATGACTTCCAGCTGCGGTCCTTTGACCAGCTGTCTCCTCTGGAGAGCTCCTC
TGCCTCCCCTGAGTCTGCCAGCCCACAGTCTACCGTGACAGTGTTCCAGCAGACC
CAGATCCAGGAGCCAACAGCCAATGCCACCACAACCACAGCCACCACAGATGAGC
TGAAGACCGTGACAAAGGACCGGATGGAGGACATCAAGATCCTGATCGCCTCCCC
TTCTCCAACCCACATCCACAAGGAGACCACATCCGCCACAAGCTCCCCTTACCGG
GACACCCAGAGCAGAACAGCCTCCCCAAACAGAGCCGGCAAGGGCGTGATCGAGC
AGACCGAGAAGTCTCACCCAAGGAGCCCCAATGTGCTGTCCGTGGCCCTGTCTCA
GCGCACCACAGTGCCCGAGGAGGAGCTGAACCCTAAGATCCTGGCCCTGCAGAAT
GCCCAGCGGAAGAGAAAGATGGAGCACGATGGAAGCCTGTTCCAGGCAGTGGGAA
TCGGCACCCTGCTGCAGCAGCCAGATGACCACGCCGCCACCACAAGCCTGTCCTG
GAAGAGGGTGAAGGGCTGTAAGTCTAGCGAGCAGAACGGCATGGAGCAGAAGACC
ATCATCCTGATCCCATCCGACCTGGCATGCAGGCTGCTGGGCCAGAGCATGGATG
AGTCCGGCCTGCCCCAGCTGACAAGCTACGACTGTGAGGTGAACGCCCCTATCCA
GGGCTCCCGGAATCTGCTGCAGGGCGAGGAGCTGCTGAGAGCCCTGGACCAGGTG
AAT
80
ATGAAGGTGGAGTTCGCCCCTCTGAACATCCAGCTGGCCCGGAGACTGCAGACCG
MOGAT1 (nt)
TGGCCGTGCTGCAGTGGGTGCTGAAGTACCTGCTGCTGGGCCCAATGTCCATCGG
CATCACAGTGATGCTGATCATCCACAATTACCTGTTCCTGTATATCCCCTATCTG
ATGTGGCTGTATTTTGACTGGCACACCCCTGAGAGGGGCGGCAGGCGCAGCTCCT
GGATCAAGAACTGGACACTGTGGAAGCACTTCAAGGATTACTTTCCAATCCACCT
GATCAAGACCCAGGACCTGGATCCTTCTCACAATTATATCTTCGGCTTTCACCCA
CACGGAATCATGGCAGTGGGAGCCTTCGGCAACTTTAGCGTGAATTACTCCGACT
TCAAGGATCTGTTCCCCGGCTTTACCAGCTATCTGCACGTGCTGCCACTGTGGTT
CTGGTGCCCCGTGTTTAGAGAGTACGTGATGTCCGTGGGCCTGGTGTCTGTGAGC
AAGAAGTCCGTGTCTTATATGGTGTCCAAGGAGGGCGGCGGCAACATCTCTGTGA
TCGTGCTGGGAGGAGCAAAGGAGTCTCTGGACGCCCACCCTGGCAAGTTCACCCT
GTTTATCCGGCAGAGAAAGGGCTTTGTGAAGATCGCCCTGACACACGGAGCCTCT
CTGGTGCCAGTGGTGAGCTTCGGCGAGAACGAGCTGTTTAAGCAGACCGATAATC
CCGAGGGCAGCTGGATCAGGACAGTGCAGAACAAGCTGCAGAAGATCATGGGCTT
CGCACTGCCACTGTTTCACGCAAGGGGCGTGTTCCAGTACAATTTTGGCCTGATG
ACCTATAGAAAGGCCATCCACACAGTGGTGGGCAGGCCCATCCCTGTGCGCCAGA
CCCTGAATCCCACACAGGAGCAGATCGAGGAGCTGCACCAGACCTACATGGAGGA
GCTGCGCAAGCTGTTCGAGGAGCACAAGGGCAAGTATGGCATCCCTGAGCACGAG
ACACTGGTGCTGAAG
81
ATGCCCCCTCAGCTGCAGAACGGCCTGAATCTGTCCGCCAAGGTGGTGCAGGGCT
PCK1 (nt)
CCCTGGACTCTCTGCCTCAGGCCGTGAGGGAGTTTCTGGAGAACAATGCCGAGCT
GTGCCAGCCAGACCACATCCACATCTGTGATGGCTCTGAGGAGGAGAACGGCCGC
CTGCTGGGACAGATGGAGGAGGAGGGCATCCTGCGGAGACTGAAGAAGTACGATA
ATTGCTGGCTGGCCCTGACCGACCCAAGGGATGTGGCACGCATCGAGAGCAAGAC
CGTGATCGTGACACAGGAGCAGAGGGACACCGTGCCAATCCCCAAGACAGGCCTG
TCTCAGCTGGGCCGCTGGATGAGCGAGGAGGATTTCGAGAAGGCCTTTAACGCCC
GGTTCCCTGGCTGTATGAAGGGCAGAACCATGTACGTGATCCCCTTCAGCATGGG
ACCTCTGGGAAGCCCACTGTCCAAGATCGGCATCGAGCTGACAGACTCCCCATAT
GTGGTGGCCTCTATGCGGATCATGACCAGAATGGGAACACCCGTGCTGGAGGCAG
TGGGCGATGGCGAGTTCGTGAAGTGCCTGCACTCCGTGGGCTGTCCTCTGCCACT
GCAGAAGCCCCTGGTGAACAATTGGCCCTGCAACCCTGAGCTGACCCTGATCGCA
CACCTGCCTGACAGGAGGGAGATCATCTCTTTTGGCAGCGGCTACGGCGGCAATA
GCCTGCTGGGCAAGAAGTGTTTCGCACTGAGGATGGCCTCCCGCCTGGCCAAGGA
GGAGGGATGGCTGGCCGAGCACATGCTGATCCTGGGCATCACCAATCCCGAGGGC
GAGAAGAAGTATCTGGCTGCCGCCTTTCCTTCTGCCTGCGGCAAGACAAACCTGG
CCATGATGAATCCAAGCCTGCCAGGATGGAAGGTGGAGTGCGTGGGCGACGACAT
CGCCTGGATGAAGTTCGATGCACAGGGACACCTGAGGGCCATCAACCCAGAGAAT
GGCTTCTTTGGCGTGGCCCCAGGCACCTCTGTGAAGACAAACCCCAATGCCATCA
AGACCATCCAGAAGAACACCATCTTTACAAATGTGGCCGAGACAAGCGACGGAGG
CGTGTACTGGGAGGGAATCGATGAGCCCCTGGCCAGCGGCGTGACCATCACATCC
TGGAAGAACAAGGAGTGGAGCTCCGAGGACGGAGAGCCATGCGCACACCCTAATT
CCAGATTCTGCACCCCCGCCTCTCAGTGTCCTATCATCGATGCAGCATGGGAGTC
TCCAGAGGGAGTGCCAATCGAGGGCATCATCTTTGGCGGCCGGAGACCTGCAGGA
GTGCCACTGGTGTATGAGGCCCTGTCCTGGCAGCACGGCGTGTTCGTGGGAGCAG
CAATGCGGTCTGAGGCAACAGCTGCCGCCGAGCACAAGGGCAAGATCATCATGCA
CGACCCATTTGCCATGAGACCCTTCTTTGGCTACAACTTCGGCAAGTATCTGGCA
CACTGGCTGTCCATGGCACAGCACCCTGCAGCAAAGCTGCCAAAGATCTTTCACG
TGAATTGGTTCAGGAAGGATAAGGAGGGCAAGTTTCTGTGGCCTGGCTTCGGCGA
GAACAGCAGGGTGCTGGAGTGGATGTTCAATCGCATCGACGGCAAGGCCTCCACC
AAGCTGACACCCATCGGCTACATCCCTAAGGAGGATGCCCTGAACCTGAAGGGCC
TGGGCCACATCAATATGATGGAGCTGTTTTCTATCAGCAAGGAGTTCTGGGAGAA
GGAGGTGGAGGACATCGAGAAGTATCTGGAGGACCAGGTGAACGCCGATCTGCCC
TGTGAGATCGAGCGGGAGATCCTGGCCCTGAAGCAGAGAATCTCCCAGATG
82
GGGSGGGS
Linker
83
RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLD
tEGFR
PQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSL
artificial
NITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENS
CKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENS
ECIQCHPECLPQAMNITCTGRGPDNCTQCAHYIDGPHCVKTCPAGVMGENNTLVW
KYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALG
IGLFM
84
ATGAGGGAAAGCCAGGATGCCGCCGGAGCTCATGGCTGGAACCGCGTCGGCTCCA
huLEM-T2A-Her2t
CGGCCACCAAGTGGTTCACCGGGGCGCCCTTCGGGGTGCAGAGCCACAGGTTTGA
(nt)
CATCTCTGCTGTTTATCCCAACTGGAAGAAGTTCAGCACCTTCACTGAGGCCCCA
TACTCCACGCGTTATTCTACCCAAGTGTCCCACATAGGCCCTGGGACTTACAGCT
CCAAAGAGACCTGCTTCAGCAAGAAGAAGCTGATGAAGGAGGTGGACACAGGCTG
GGCCAAGGCCCAGGAAGCCACGCGGCTGACCCAGCTACCCCACTTCCAGTACCAG
GCCATCATGAAAGAGAAGCGGCTGAAGGAGCAAAAGCTGGGCCCCGGCTCCTACA
ACCTCAAAGACTTCTTAGAACAGCTGCGGGAGAAACCATGTAGCACCCGGGGGCT
GCTCAGCTCTGGGGAGGTTCGCTTCCGAGGACTCACTGGGAACTACTATCCAGGC
CCTGGAAATTATGGGGAGAAGGGTAACCCATACACCAAGCTGGAGGAGAATGCCT
GGAACCGGTCTCATTCCGAGGGCCTCATGTGCAGGATGAGCAACAAGCCACACCC
CCGGCCTCATCAGGGGAGTGGTCTGGGACCCGGCACCTACTTCTTCAAAAGCGAC
CTTGAGACATATGTGGCACGATCCGTCGGCACCCGCGGCCCCTATGACACTTTCT
CTGGTGATCGGAGCAAGCCACTGCCTTATGGGCACTACTCCATGCAGAAAAAAAA
GCCCAGGGAACTGATGAATTTCAAGAGCTTTGTAGAAGAACTTAACTCACATCAC
AATAAGAAGCATGGGGTTTTTTCTAAACTTCCCCGAAACCCGAAAACCCCTACAG
AGAGGATTTACTGGGCCAACCTCAGCCAGTGCCCCCGCACACTGGCCACATCTGG
CCCCAGTTTCTGGCTTCCACAAGAGAAGAAATGCAAACCCGTCAACCAGCCCCCA
TTCCTGTTGACCTCCAAGGGGTCAGGTGCAAAGGCCTGCCAGATGATTATGGGAA
GCTGGAACCCAGTAGGTGTGGGCCGCTACCTCAACACCTGGCTGATGGAGACAAA
GGACAGGCGGCAGCGATATCGATCCCTATTCCTGAGTGGATCCAAACGCTACCTC
TCAGACCTGGCCCGGGACATGCTCATGCAGGAAAGGATCACACCATTTACTAAGG
GAAAGTGCCCTCCAACTGTGGATTACAATTCAGATCCTACTCCTCTCGAGGGCGG
CGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGC
CCTAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAG
CATTCCTCCTGATCCCATGCCACCCTGAGTGTCAGCCCCAGAATGGCTCAGTGAC
CTGTTTTGGACCGGAGGCTGACCAGTGTGTGGCCTGTGCCCACTATAAGGACCCT
CCCTTCTGCGTGGCCCGCTGCCCCAGCGGTGTGAAACCTGACCTCTCCTACATGC
CCATCTGGAAGTTTCCAGATGAGGAGGGCGCATGCCAGCCTTGCCCCATCAACTG
CACCCACTCCTGTGTGGACCTGGATGACAAGGGCTGCCCCGCCGAGCAGAGAGCC
AGCCCTCTGACGGGTGGAGGAAGCGGAGGTGGCAGCTCCATCATCTCTGCGGTGG
TTGGCATTCTGCTGGTCGTGGTCTTGGGGGTGGTCTTTGGGATCCTCATC
85
MRESNAAGAHGWNRVGSTATKWFTGAPFGVQSHRFDISAVYPNWKKFSTFTEAP
huLEM-T2A-Her2t
YSTRYSTQVSHIGPGTYSSKETCFSKKKLMKEVDTGWAKAQEATRLTQLPHFQYQ
(aa)
AIMKEKRLKEQKLGPGSYNLKDFLEQLREKPCSTRGLLSSGEVRFRGLTGNYYPG
PGNYGEKGNPYTKLEENAWNRSHSEGLMCRMSNKPHPRPHQGSGLGPGTYFFKSD
LETYVARSVGTRGPYDTFSGDRSKPLPYGHYSMQKKKPRELMNFKSFVEELNSHH
NKKHGVFSKLPRNPKTPTERIYWANLSQCPRTLATSGPSFWLPQEKKCKPVNQPP
FLLTSKGSGAKACQMIMGSWNPVGVGRYLNTWLMETKDRRQRYRSLFLSGSKRYL
SDLARDMLMQERITPFTKGKCPPTVDYNSDPTPLEGGGEGRGSLLTCGDVEENPG
PRMLLLVTSLLLCELPHPAFLLIPCHPECQPQNGSVTCFGPEADQCVACAHYKDP
PFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRA
SPLTGGGSGGGSSIISAVVGILLVVVLGVVFGILI
86
ATGGGCGATCGCGGCAGCTCCCGGAGAAGGCGCACCGGCAGCCGGCCCTCTAGCC
DGAT1-T2A-Her2t
ACGGCGGCGGCGGCCCTGCTGCCGCCGAGGAGGAGGTGCGCGACGCCGCCGCCGG
(nt)
CCCTGATGTGGGAGCAGCAGGCGACGCACCAGCACCTGCCCCAAACAAGGACGGC
GATGCAGGAGTGGGAAGCGGACACTGGGAGCTGAGATGCCACAGGCTGCAGGATT
CCCTGTTCTCCTCTGACAGCGGCTTTTCCAACTACAGAGGCATCCTGAATTGGTG
CGTGGTCATGCTGATCCTGTCCAACGCCAGGCTGTTCCTGGAGAATCTGATCAAG
TACGGCATCCTGGTGGATCCTATCCAGGTGGTGAGCCTGTTTCTGAAGGACCCAT
ATTCCTGGCCAGCACCTTGCCTGGTCATCGCAGCAAACGTGTTCGCAGTGGCAGC
CTTTCAGGTGGAGAAGCGGCTGGCCGTGGGCGCCCTGACCGAGCAGGCAGGCCTG
CTGCTGCACGTGGCCAATCTGGCCACAATCCTGTGCTTCCCAGCAGCAGTGGTGC
TGCTGGTGGAGTCTATCACCCCTGTGGGAAGCCTGCTGGCCCTGATGGCACACAC
AATCCTGTTCCTGAAGCTGTTTTCCTACAGAGACGTGAATTCTTGGTGTAGGAGA
GCAAGGGCAAAGGCAGCCTCTGCCGGCAAGAAGGCCAGCAGCGCCGCCGCCCCTC
ACACCGTGAGCTACCCAGATAACCTGACATATAGAGACCTGTACTATTTCCTGTT
TGCCCCCACCCTGTGCTATGAGCTGAATTTCCCAAGGTCCCCCAGGATCCGCAAG
CGGTTTCTGCTGAGGCGCATCCTGGAGATGCTGTTCTTTACCCAGCTGCAAGTGG
GCCTGATCCAGCAGTGGATGGTGCCAACAATCCAGAACTCCATGAAGCCCTTCAA
GGACATGGATTACTCTAGAATCATCGAGAGGCTGCTGAAGCTGGCCGTGCCCAAC
CACCTGATCTGGCTGATCTTCTTTTATTGGCTGTTTCACTCTTGCCTGAATGCCG
TGGCCGAGCTGATGCAGTTCGGCGATCGCGAGTTTTACCGGGACTGGTGGAATTC
CGAGTCTGTGACATATTTCTGGCAGAACTGGAATATCCCAGTGCACAAGTGGTGT
ATCCGCCACTTTTACAAGCCCATGCTGCGGAGAGGCTCTAGCAAGTGGATGGCCA
GAACCGGCGTGTTCCTGGCCTCTGCCTTCTTTCACGAGTATCTGGTGAGCGTGCC
TCTGCGCATGTTCCGGCTGTGGGCCTTTACAGGCATGATGGCCCAGATCCCACTG
GCCTGGTTTGTGGGCCGGTTCTTTCAGGGCAACTACGGCAATGCCGCCGTGTGGC
TGAGCCTGATCATCGGCCAGCCCATCGCCGTGCTGATGTACGTGCACGATTACTA
TGTGCTGAACTATGAGGCCCCTGCCGCCGAGGCCCTCGAGGGCGGCGGAGAGGGC
AGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGATGC
TTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCT
GATCCCATGCCACCCTGAGTGTCAGCCCCAGAATGGCTCAGTGACCTGTTTTGGA
CCGGAGGCTGACCAGTGTGTGGCCTGTGCCCACTATAAGGACCCTCCCTTCTGCG
TGGCCCGCTGCCCCAGCGGTGTGAAACCTGACCTCTCCTACATGCCCATCTGGAA
GTTTCCAGATGAGGAGGGCGCATGCCAGCCTTGCCCCATCAACTGCACCCACTCC
TGTGTGGACCTGGATGACAAGGGCTGCCCCGCCGAGCAGAGAGCCAGCCCTCTGA
CGGGTGGAGGAAGCGGAGGTGGCAGCTCCATCATCTCTGCGGTGGTTGGCATTCT
GCTGGTCGTGGTCTTGGGGGTGGTCTTTGGGATCCTCATC
87
MGDRGSSRRRRTGSRPSSHGGGGPAAAEEEVRDAAAGPDVGAAGDAPAPAPNKDG
DGAT1-T2A-Her2t
DAGVGSGHWELRCHRLQDSLFSSDSGFSNYRGILNWCVVMLILSNARLFLENLIK
(aa)
YGILVDPIQVVSLFLKDPYSWPAPCLVIAANVFAVAAFQVEKRLAVGALTEQAGL
LLHVANLATILCFPAAVVLLVESITPVGSLLALMAHTILFLKLFSYRDVNSWCRR
ARAKAASAGKKASSAAAPHTVSYPDNLTYRDLYYFLFAPTLCYELNFPRSPRIRK
RFLLRRILEMLFFTQLQVGLIQQWMVPTIQNSMKPFKDMDYSRIIERLLKLAVPN
HLIWLIFFYWLFHSCLNAVAELMQFGDREFYRDWWNSESVTYFWQNWNIPVHKWC
IRHFYKPMLRRGSSKWMARTGVFLASAFFHEYLVSVPLRMFRLWAFTGMMAQIPL
AWFVGRFFQGNYGNAAVWLSLIIGQPIAVLMYVHDYYVLNYEAPAAEALEGGGEG
RGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPCHPECQPQNGSVTCFG
PEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHS
CVDLDDKGCPAEQRASPLTGGGSGGGSSIISAVVGILLVVVLGVVFGILI
88
ATGGCAGCCAGCAAGAAGGCCGTGCTGGGCCCACTGGTGGGAGCAGTGGACCAGG
GYK-T2A-Her2t
GCACCAGCTCCACAAGGTTCCTGGTGTTTAATAGCAAGACCGCAGAGCTGCTGTC
(nt)
CCACCACCAGGTGGAGATCAAGCAGGAGTTTCCAAGGGAGGGATGGGTGGAGCAG
GACCCAAAGGAGATCCTGCACTCCGTGTACGAGTGCATCGAGAAGACCTGTGAGA
AGCTGGGCCAGCTGAATATCGACATCAGCAACATCAAGGCCATCGGCGTGTCCAA
TCAGCGGGAGACCACAGTGGTGTGGGACAAGATCACAGGCGAGCCCCTGTATAAC
GCCGTGGTGTGGCTGGATCTGAGGACCCAGAGCACAGTGGAGTCCCTGTCTAAGC
GCATCCCTGGCAACAATAACTTTGTGAAGTCCAAGACCGGCCTGCCACTGTCCAC
ATATTTCTCTGCCGTGAAGCTGAGGTGGCTGCTGGACAATGTGCGCAAGGTGCAG
AAGGCCGTGGAGGAGAAGAGGGCCCTGTTTGGCACCATCGATTCTTGGCTGATCT
GGAGCCTGACAGGAGGAGTGAACGGAGGCGTGCACTGCACCGACGTGACAAATGC
CTCTCGGACCATGCTGTTCAACATCCACAGCCTGGAGTGGGATAAGCAGCTGTGC
GAGTTCTTTGGCATCCCTATGGAGATCCTGCCAAACGTGAGATCTAGCTCCGAGA
TCTATGGCCTGATGAAGATCAGCCACTCCGTGAAGGCAGGCGCCCTGGAGGGAGT
GCCTATCTCTGGATGCCTGGGCGACCAGAGCGCCGCCCTGGTGGGACAGATGTGC
TTCCAGATCGGCCAGGCCAAGAATACCTACGGCACAGGCTGCTTTCTGCTGTGCA
ACACCGGCCACAAGTGCGTGTTCAGCGACCACGGCCTGCTGACCACAGTGGCCTA
TAAGCTGGGCAGGGATAAGCCCGTGTACTATGCACTGGAGGGATCTGTGGCAATC
GCAGGAGCCGTGATCAGGTGGCTGAGAGATAATCTGGGCATCATCAAGACCAGCG
AGGAGATCGAGAAGCTGGCCAAGGAAGTGGGCACATCCTACGGCTGTTATTTCGT
GCCTGCCTTTTCTGGCCTGTACGCACCATATTGGGAGCCAAGCGCCAGGGGAATC
ATCTGCGGCCTGACCCAGTTCACAAACAAGTGTCACATCGCCTTTGCCGCCCTGG
AGGCCGTGTGCTTCCAGACCCGGGAGATCCTGGACGCCATGAATAGAGATTGTGG
CATCCCTCTGTCCCACCTGCAGGTGGACGGAGGCATGACATCTAACAAGATCCTG
ATGCAGCTGCAGGCCGACATCCTGTATATCCCAGTGGTGAAGCCCTCCATGCCTG
AGACCACAGCCCTGGGAGCAGCAATGGCAGCAGGAGCAGCCGAGGGCGTGGGCGT
GTGGTCCCTGGAGCCAGAGGACCTGTCTGCCGTGACCATGGAGCGGTTTGAGCCT
CAGATCAATGCCGAGGAGTCCGAGATCAGATACTCTACATGGAAGAAGGCCGTGA
TGAAGTCCATGGGCTGGGTGACCACACAGTCTCCCGAGAGCGGCGATCCTAGCAT
CTTCTGCTCCCTGCCACTGGGCTTCTTTATCGTGTCTAGCATGGTCATGCTGATC
GGCGCCCGGTATATCTCTGGCATCCCCCTCGAGGGCGGCGGAGAGGGCAGAGGAA
GTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGATGCTTCTCCT
GGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCA
TGCCACCCTGAGTGTCAGCCCCAGAATGGCTCAGTGACCTGTTTTGGACCGGAGG
CTGACCAGTGTGTGGCCTGTGCCCACTATAAGGACCCTCCCTTCTGCGTGGCCCG
CTGCCCCAGCGGTGTGAAACCTGACCTCTCCTACATGCCCATCTGGAAGTTTCCA
GATGAGGAGGGCGCATGCCAGCCTTGCCCCATCAACTGCACCCACTCCTGTGTGG
ACCTGGATGACAAGGGCTGCCCCGCCGAGCAGAGAGCCAGCCCTCTGACGGGTGG
AGGAAGCGGAGGTGGCAGCTCCATCATCTCTGCGGTGGTTGGCATTCTGCTGGTC
GTGGTCTTGGGGGTGGTCTTTGGGATCCTCATC
89
MAASKKAVLGPLVGAVDQGTSSTRFLVFNSKTAELLSHHQVEIKQEFPREGWVE4
GYK-T2A-Her2t
DPKEILHSVYECIEKTCEKLGQLNIDISNIKAIGVSNQRETTVVWDKITGEPLYN
(aa)
AVVWLDLRTQSTVESLSKRIPGNNNFVKSKTGLPLSTYFSAVKLRWLLDNVRKVQ
KAVEEKRALFGTIDSWLIWSLTGGVNGGVHCTDVTNASRTMLFNIHSLEWDKQLC
EFFGIPMEILPNVRSSSEIYGLMKISHSVKAGALEGVPISGCLGDQSAALVGQMC
FQIGQAKNTYGTGCFLLCNTGHKCVFSDHGLLTTVAYKLGRDKPVYYALEGSVAI
AGAVIRWLRDNLGIIKTSEEIEKLAKEVGTSYGCYFVPAFSGLYAPYWEPSARGI
ICGLTQFTNKCHIAFAALEAVCFQTREILDAMNRDCGIPLSHLQVDGGMTSNKIL
MQLQADILYIPVVKPSMPETTALGAAMAAGAAEGVGVWSLEPEDLSAVTMERFEP
QINAEESEIRYSTWKKAVMKSMGWVTTQSPESGDPSIFCSLPLGFFIVSSMVMLI
GARYISGIPLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIP
CHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFP
DEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTGGGSGGGSSIISAVVGILLV
VVLGVVFGILI
90
ATGGACGAGTCCGCCCTGACACTGGGCACCATCGACGTGAGCTACCTGCCACACA
GPAM-T2A-Her2t
GCTCCGAGTATTCTGTGGGCAGGTGCAAGCACACAAGCGAGGAGTGGGGAGAGTG
(nt)
TGGCTTCCGGCCAACAATCTTTAGATCCGCCACCCTGAAGTGGAAGGAGAGCCTG
ATGTCCCGGAAGAGACCATTCGTGGGCCGGTGCTGTTACTCCTGCACCCCCCAGT
CTTGGGACAAGTTCTTTAACCCTTCTATCCCAAGCCTGGGCCTGAGAAACGTGAT
CTACATCAATGAGACCCACACAAGGCACAGGGGATGGCTGGCCCGGAGACTGAGC
TATGTGCTGTTCATCCAGGAGAGGGACGTGCACAAGGGCATGTTTGCCACAAATG
TGACCGAGAACGTGCTGAATTCTAGCCGCGTGCAGGAGGCAATCGCAGAGGTGGC
AGCAGAGCTGAACCCTGATGGAAGCGCCCAGCAGCAGTCCAAGGCAGTGAATAAG
GTGAAGAAGAAGGCCAAGCGGATCCTGCAGGAGATGGTGGCCACAGTGTCCCCAG
CCATGATCAGACTGACCGGCTGGGTGCTGCTGAAGCTGTTCAACTCTTTCTTTTG
GAATATCCAGATCCACAAGGGCCAGCTGGAGATGGTGAAGGCCGCCACCGAGACA
AACCTGCCACTGCTGTTTCTGCCCGTGCACCGCAGCCACATCGATTACCTGCTGC
TGACCTTCATCCTGTTTTGTCACAACATCAAGGCCCCTTATATCGCCAGCGGCAA
CAATCTGAATATCCCAATCTTCTCCACACTGATCCACAAGCTGGGCGGCTTCTTT
ATCAGGCGCCGGCTGGATGAGACCCCTGACGGCAGGAAGGATGTGCTGTACCGCG
CCCTGCTGCACGGACACATCGTGGAGCTGCTGAGGCAGCAGCAGTTCCTGGAGAT
CTTTCTGGAGGGCACACGGTCTAGAAGCGGCAAGACCTCCTGCGCAAGGGCAGGA
CTGCTGTCCGTGGTGGTGGACACACTGTCTACCAACGTGATCCCCGACATCCTGA
TCATCCCTGTGGGCATCTCTTACGACCGGATCATCGAGGGCCACTATAACGGCGA
GCAGCTGGGCAAGCCCAAGAAGAATGAGTCCCTGTGGTCTGTGGCCAGGGGCGTG
ATCCGGATGCTGAGAAAGAATTACGGATGCGTGCGGGTGGATTTCGCACAGCCTT
TTTCCCTGAAGGAGTATCTGGAGTCCCAGTCTCAGAAGCCCGTGAGCGCCCTGCT
GTCCCTGGAGCAGGCCCTGCTGCCTGCAATCCTGCCAAGCAGACCTTCCGATGCA
GCAGACGAGGGAAGGGACACATCTATCAACGAGAGCAGAAATGCCACCGATGAGA
GCCTGAGAAGGCGCCTGATCGCCAACCTGGCCGAGCACATCCTGTTCACAGCCAG
CAAGTCCTGCGCCATCATGAGCACCCACATCGTGGCCTGTCTGCTGCTGTACCGG
CACAGGCAGGGAATCGACCTGTCCACACTGGTGGAGGATTTCTTTGTGATGAAGG
AGGAGGTGCTGGCCAGGGACTTCGATCTGGGCTTTTCTGGCAATAGCGAGGACGT
GGTCATGCACGCCATCCAGCTGCTGGGCAACTGCGTGACCATCACACACACCTCC
CGCAATGATGAGTTCTTTATCACCCCTTCTACCACAGTGCCAAGCGTGTTCGAGC
TGAACTTTTACTCTAATGGCGTGCTGCACGTGTTTATCATGGAGGCCATCATCGC
CTGCAGCCTGTATGCCGTGCTGAACAAGAGGGGACTGGGCGGCCCAACAAGCACC
CCCCCTAATCTGATCTCCCAGGAGCAGCTGGTGAGAAAGGCCGCCTCCCTGTGCT
ATCTGCTGTCTAACGAGGGCACAATCAGCCTGCCCTGCCAGACCTTCTACCAGGT
GTGCCACGAGACAGTGGGCAAGTTTATCCAGTATGGCATCCTGACCGTGGCCGAG
CACGACGATCAGGAGGACATCTCTCCTAGCCTGGCCGAGCAGCAGTGGGATAAGA
AGCTGCCAGAGCCCCTGTCCTGGAGGTCTGACGAGGAGGACGAGGATAGCGACTT
CGGCGAGGAGCAGCGCGATTGTTACCTGAAGGTGTCCCAGTCTAAGGAGCACCAG
CAGTTCATCACCTTTCTGCAGCGGCTGCTGGGCCCACTGCTGGAGGCCTATTCCT
CTGCCGCCATCTTCGTGCACAACTTTTCCGGCCCTGTGCCAGAGCCAGAGTACCT
GCAGAAGCTGCACAAGTATCTGATCACAAGGACCGAGAGGAACGTGGCCGTGTAC
GCAGAGAGCGCCACCTATTGCCTGGTGAAGAATGCCGTGAAGATGTTCAAGGACA
TCGGCGTGTTTAAGGAGACAAAGCAGAAGCGGGTGTCTGTGCTGGAGCTGAGCTC
CACCTTCCTGCCCCAGTGTAATAGACAGAAGCTGCTGGAGTACATCCTGAGCTTT
GTGGTGCTGCTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTG
ACGTGGAGGAGAATCCCGGCCCTAGGATGCTTCTCCTGGTGACAAGCCTTCTGCT
CTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCATGCCACCCTGAGTGTCAG
CCCCAGAATGGCTCAGTGACCTGTTTTGGACCGGAGGCTGACCAGTGTGTGGCCT
GTGCCCACTATAAGGACCCTCCCTTCTGCGTGGCCCGCTGCCCCAGCGGTGTGAA
ACCTGACCTCTCCTACATGCCCATCTGGAAGTTTCCAGATGAGGAGGGCGCATGC
CAGCCTTGCCCCATCAACTGCACCCACTCCTGTGTGGACCTGGATGACAAGGGCT
GCCCCGCCGAGCAGAGAGCCAGCCCTCTGACGGGTGGAGGAAGCGGAGGTGGCAG
CTCCATCATCTCTGCGGTGGTTGGCATTCTGCTGGTCGTGGTCTTGGGGGTGGTC
TTTGGGATCCTCATC
91
MDESALTLGTIDVSYLPHSSEYSVGRCKHTSEEWGECGFRPTIFRSATLKWKESL
GPAM-T2A-Her2t
MSRKRPFVGRCCYSCTPQSWDKFFNPSIPSLGLRNVIYINETHTRHRGWLARRLS
(aa)
YVLFIQERDVHKGMFATNVTENVLNSSRVQEAIAEVAAELNPDGSAQQQSKAVNK
VKKKAKRILQEMVATVSPAMIRLTGWVLLKLFNSFFWNIQIHKGQLEMVKAATET
NLPLLFLPVHRSHIDYLLLTFILFCHNIKAPYIASGNNLNIPIFSTLIHKLGGFF
IRRRLDETPDGRKDVLYRALLHGHIVELLRQQQFLEIFLEGTRSRSGKTSCARAG
LLSVVVDTLSTNVIPDILIIPVGISYDRIIEGHYNGEQLGKPKKNESLWSVARGV
IRMLRKNYGCVRVDFAQPFSLKEYLESQSQKPVSALLSLEQALLPAILPSRPSDA
ADEGRDTSINESRNATDESLRRRLIANLAEHILFTASKSCAIMSTHIVACLLLYR
HRQGIDLSTLVEDFFVMKEEVLARDFDLGFSGNSEDVVMHAIQLLGNCVTITHTS
RNDEFFITPSTTVPSVFELNFYSNGVLHVFIMEAIIACSLYAVLNKRGLGGPTST
PPNLISQEQLVRKAASLCYLLSNEGTISLPCQTFYQVCHETVGKFIQYGILTVAE
HDDQEDISPSLAEQQWDKKLPEPLSWRSDEEDEDSDFGEEQRDCYLKVSQSKEHQ
QFITFLQRLLGPLLEAYSSAAIFVHNFSGPVPEPEYLQKLHKYLITRTERNVAVY
AESATYCLVKNAVKMFKDIGVFKETKQKRVSVLELSSTFLPQCNRQKLLEYILSF
VVLLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPCHPECQ
PQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGAC
QPCPINCTHSCVDLDDKGCPAEQRASPLTGGGSGGGSSIISAVVGILLVVVLGVV
FGILI
92
ATGGAGGGCGCCGGCGGCGCCAACGATAAGAAGAAGATCAGCTCCGAGCGGAGAA
HIF1α-T2A-Her2t
AGGAGAAGAGCAGGGACGCAGCACGCTCTAGGCGCAGCAAGGAGTCCGAGGTGTT
(nt)
CTACGAGCTGGCCCACCAGCTGCCACTGCCACACAACGTGTCTAGCCACCTGGAT
AAGGCCAGCGTGATGCGGCTGACCATCTCCTATCTGCGGGTGAGAAAGCTGCTGG
ACGCCGGCGATCTGGACATCGAGGACGATATGAAGGCCCAGATGAATTGCTTCTA
CCTGAAGGCCCTGGACGGCTTTGTGATGGTGCTGACCGACGATGGCGACATGATC
TACATCTCCGATAACGTGAATAAGTATATGGGCCTGACCCAGTTTGAGCTGACAG
GCCACAGCGTGTTCGACTTTACCCACCCCTGCGATCACGAGGAGATGAGGGAGAT
GCTGACACACCGCAACGGCCTGGTGAAGAAGGGCAAGGAGCAGAATACCCAGCGG
TCTTTCTTTCTGAGAATGAAGTGTACCCTGACAAGCAGGGGCCGCACCATGAACA
TCAAGTCCGCCACATGGAAGGTGCTGCACTGCACCGGCCACATCCACGTGTACGA
TACCAACTCCAATCAGCCACAGTGTGGCTATAAGAAGCCCCCTATGACATGCCTG
GTGCTGATCTGTGAGCCTATCCCACACCCCTCTAATATCGAGATCCCCCTGGACA
GCAAGACCTTCCTGTCTCGGCACAGCCTGGACATGAAGTTTAGCTACTGCGATGA
GAGAATCACAGAGCTGATGGGCTATGAGCCTGAGGAGCTGCTGGGCAGATCTATC
TACGAGTACTATCACGCCCTGGATAGCGACCACCTGACCAAGACACACCACGACA
TGTTCACCAAGGGCCAGGTGACCACAGGCCAGTACAGGATGCTGGCCAAGAGGGG
AGGATACGTGTGGGTGGAGACCCAGGCCACAGTGATCTATAACACCAAGAATAGC
CAGCCCCAGTGCATCGTGTGCGTGAACTACGTGGTGTCCGGCATCATCCAGCACG
ATCTGATCTTTTCTCTGCAGCAGACCGAGTGCGTGCTGAAGCCTGTGGAGTCCTC
TGACATGAAGATGACCCAGCTGTTCACAAAGGTGGAGTCCGAGGACACAAGCTCC
CTGTTTGATAAGCTGAAGAAGGAGCCAGACGCACTGACCCTGCTGGCCCCAGCAG
CAGGCGATACAATCATCTCTCTGGACTTCGGCAGCAATGATACCGAGACAGACGA
TCAGCAGCTGGAGGAGGTGCCTCTGTATAACGATGTGATGCTGCCTTCTCCAAAT
GAGAAGCTGCAGAACATCAATCTGGCAATGAGCCCACTGCCTACCGCAGAGACAC
CAAAGCCACTGAGGTCTAGCGCCGACCCAGCCCTGAACCAGGAGGTGGCCCTGAA
GCTGGAGCCTAATCCAGAGTCCCTGGAGCTGTCTTTTACAATGCCACAGATCCAG
GACCAGACCCCATCCCCTTCTGATGGCAGCACACGCCAGTCCTCTCCAGAGCCCA
ACAGCCCTTCCGAGTACTGCTTCTATGTGGATTCCGACATGGTGAATGAGTTCAA
GCTGGAGCTGGTGGAGAAGCTGTTTGCCGAGGATACCGAGGCCAAGAACCCCTTC
AGCACCCAGGATACAGACCTGGATCTGGAGATGCTGGCCCCCTATATCCCTATGG
ACGATGACTTCCAGCTGCGGTCCTTTGACCAGCTGTCTCCTCTGGAGAGCTCCTC
TGCCTCCCCTGAGTCTGCCAGCCCACAGTCTACCGTGACAGTGTTCCAGCAGACC
CAGATCCAGGAGCCAACAGCCAATGCCACCACAACCACAGCCACCACAGATGAGC
TGAAGACCGTGACAAAGGACCGGATGGAGGACATCAAGATCCTGATCGCCTCCCC
TTCTCCAACCCACATCCACAAGGAGACCACATCCGCCACAAGCTCCCCTTACCGG
GACACCCAGAGCAGAACAGCCTCCCCAAACAGAGCCGGCAAGGGCGTGATCGAGC
AGACCGAGAAGTCTCACCCAAGGAGCCCCAATGTGCTGTCCGTGGCCCTGTCTCA
GCGCACCACAGTGCCCGAGGAGGAGCTGAACCCTAAGATCCTGGCCCTGCAGAAT
GCCCAGCGGAAGAGAAAGATGGAGCACGATGGAAGCCTGTTCCAGGCAGTGGGAA
TCGGCACCCTGCTGCAGCAGCCAGATGACCACGCCGCCACCACAAGCCTGTCCTG
GAAGAGGGTGAAGGGCTGTAAGTCTAGCGAGCAGAACGGCATGGAGCAGAAGACC
ATCATCCTGATCCCATCCGACCTGGCATGCAGGCTGCTGGGCCAGAGCATGGATG
AGTCCGGCCTGCCCCAGCTGACAAGCTACGACTGTGAGGTGAACGCCCCTATCCA
GGGCTCCCGGAATCTGCTGCAGGGCGAGGAGCTGCTGAGAGCCCTGGACCAGGTG
AATCTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGG
AGGAGAATCCCGGCCCTAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGA
GTTACCACACCCAGCATTCCTCCTGATCCCATGCCACCCTGAGTGTCAGCCCCAG
AATGGCTCAGTGACCTGTTTTGGACCGGAGGCTGACCAGTGTGTGGCCTGTGCCC
ACTATAAGGACCCTCCCTTCTGCGTGGCCCGCTGCCCCAGCGGTGTGAAACCTGA
CCTCTCCTACATGCCCATCTGGAAGTTTCCAGATGAGGAGGGCGCATGCCAGCCT
TGCCCCATCAACTGCACCCACTCCTGTGTGGACCTGGATGACAAGGGCTGCCCCG
CCGAGCAGAGAGCCAGCCCTCTGACGGGTGGAGGAAGCGGAGGTGGCAGCTCCAT
CATCTCTGCGGTGGTTGGCATTCTGCTGGTCGTGGTCTTGGGGGTGGTCTTTGGG
ATCCTCATC
93
MEGAGGANDKKKISSERRKEKSRDAARSRRSKESEVFYELAHQLPLPHNVSSHLD
HIF1α-T2A-Her2t
KASVMRLTISYLRVRKLLDAGDLDIEDDMKAQMNCFYLKALDGFVMVLTDDGDMI
(aa)
YISDNVNKYMGLTQFELTGHSVFDFTHPCDHEEMREMLTHRNGLVKKGKEQNTQR
SFFLRMKCTLTSRGRTMNIKSATWKVLHCTGHIHVYDTNSNQPQCGYKKPPMTCL
VLICEPIPHPSNIEIPLDSKTFLSRHSLDMKFSYCDERITELMGYEPEELLGRSI
YEYYHALDSDHLTKTHHDMFTKGQVTTGQYRMLAKRGGYVWVETQATVIYNTKNS
QPQCIVCVNYVVSGIIQHDLIFSLQQTECVLKPVESSDMKMTQLFTKVESEDTSS
LFDKLKKEPDALTLLAPAAGDTIISLDFGSNDTETDDQQLEEVPLYNDVMLPSPN
EKLQNINLAMSPLPTAETPKPLRSSADPALNQEVALKLEPNPESLELSFTMPQIQ
DQTPSPSDGSTRQSSPEPNSPSEYCFYVDSDMVNEFKLELVEKLFAEDTEAKNPF
STQDTDLDLEMLAPYIPMDDDFQLRSFDQLSPLESSSASPESASPQSTVTVFQQT
QIQEPTANATTTTATTDELKTVTKDRMEDIKILIASPSPTHIHKETTSATSSPYR
DTQSRTASPNRAGKGVIEQTEKSHPRSPNVLSVALSQRTTVPEEELNPKILALQN
AQRKRKMEHDGSLFQAVGIGTLLQQPDDHAATTSLSWKRVKGCKSSEQNGMEQKT
IILIPSDLACRLLGQSMDESGLPQLTSYDCEVNAPIQGSRNLLQGEELLRALDQV
NLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPCHPECQPQ
NGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQP
CPINCTHSCVDLDDKGCPAEQRASPLTGGGSGGGSSIISAVVGILLVVVLGVVFG
ILI
94
ATGAAGGTGGAGTTCGCCCCTCTGAACATCCAGCTGGCCCGGAGACTGCAGACCG
MOGAT1-T2A-
TGGCCGTGCTGCAGTGGGTGCTGAAGTACCTGCTGCTGGGCCCAATGTCCATCGG
Her2t (nt)
CATCACAGTGATGCTGATCATCCACAATTACCTGTTCCTGTATATCCCCTATCTG
ATGTGGCTGTATTTTGACTGGCACACCCCTGAGAGGGGCGGCAGGCGCAGCTCCT
GGATCAAGAACTGGACACTGTGGAAGCACTTCAAGGATTACTTTCCAATCCACCT
GATCAAGACCCAGGACCTGGATCCTTCTCACAATTATATCTTCGGCTTTCACCCA
CACGGAATCATGGCAGTGGGAGCCTTCGGCAACTTTAGCGTGAATTACTCCGACT
TCAAGGATCTGTTCCCCGGCTTTACCAGCTATCTGCACGTGCTGCCACTGTGGTT
CTGGTGCCCCGTGTTTAGAGAGTACGTGATGTCCGTGGGCCTGGTGTCTGTGAGC
AAGAAGTCCGTGTCTTATATGGTGTCCAAGGAGGGCGGCGGCAACATCTCTGTGA
TCGTGCTGGGAGGAGCAAAGGAGTCTCTGGACGCCCACCCTGGCAAGTTCACCCT
GTTTATCCGGCAGAGAAAGGGCTTTGTGAAGATCGCCCTGACACACGGAGCCTCT
CTGGTGCCAGTGGTGAGCTTCGGCGAGAACGAGCTGTTTAAGCAGACCGATAATC
CCGAGGGCAGCTGGATCAGGACAGTGCAGAACAAGCTGCAGAAGATCATGGGCTT
CGCACTGCCACTGTTTCACGCAAGGGGCGTGTTCCAGTACAATTTTGGCCTGATG
ACCTATAGAAAGGCCATCCACACAGTGGTGGGCAGGCCCATCCCTGTGCGCCAGA
CCCTGAATCCCACACAGGAGCAGATCGAGGAGCTGCACCAGACCTACATGGAGGA
GCTGCGCAAGCTGTTCGAGGAGCACAAGGGCAAGTATGGCATCCCTGAGCACGAG
ACACTGGTGCTGAAGCTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACAT
GCGGTGACGTGGAGGAGAATCCCGGCCCTAGGATGCTTCTCCTGGTGACAAGCCT
TCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCATGCCACCCTGAG
TGTCAGCCCCAGAATGGCTCAGTGACCTGTTTTGGACCGGAGGCTGACCAGTGTG
TGGCCTGTGCCCACTATAAGGACCCTCCCTTCTGCGTGGCCCGCTGCCCCAGCGG
TGTGAAACCTGACCTCTCCTACATGCCCATCTGGAAGTTTCCAGATGAGGAGGGC
GCATGCCAGCCTTGCCCCATCAACTGCACCCACTCCTGTGTGGACCTGGATGACA
AGGGCTGCCCCGCCGAGCAGAGAGCCAGCCCTCTGACGGGTGGAGGAAGCGGAGG
TGGCAGCTCCATCATCTCTGCGGTGGTTGGCATTCTGCTGGTCGTGGTCTTGGGG
GTGGTCTTTGGGATCCTCATC
95
MKVEFAPLNIQLARRLQTVAVLQWVLKYLLLGPMSIGITVMLIIHNYLFLYIPYL
MOGAT1-T2A-
MWLYFDWHTPERGGRRSSWIKNWTLWKHFKDYFPIHLIKTQDLDPSHNYIFGFHP
Her2t (aa)
HGIMAVGAFGNFSVNYSDFKDLFPGFTSYLHVLPLWFWCPVFREYVMSVGLVSVS
KKSVSYMVSKEGGGNISVIVLGGAKESLDAHPGKFTLFIRQRKGFVKIALTHGAS
LVPVVSFGENELFKQTDNPEGSWIRTVQNKLQKIMGFALPLFHARGVFQYNFGLM
TYRKAIHTVVGRPIPVRQTLNPTQEQIEELHQTYMEELRKLFEEHKGKYGIPEHE
TLVLKLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELPHPAFLLIPCHPE
CQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEG
ACQPCPINCTHSCVDLDDKGCPAEQRASPLTGGGSGGGSSIISAVVGILLVVVLG
VVFGILI
96
ATGCCCCCTCAGCTGCAGAACGGCCTGAATCTGTCCGCCAAGGTGGTGCAGGGCT
PCK1-T2A-Her2t
CCCTGGACTCTCTGCCTCAGGCCGTGAGGGAGTTTCTGGAGAACAATGCCGAGCT
(nt)
GTGCCAGCCAGACCACATCCACATCTGTGATGGCTCTGAGGAGGAGAACGGCCGC
CTGCTGGGACAGATGGAGGAGGAGGGCATCCTGCGGAGACTGAAGAAGTACGATA
ATTGCTGGCTGGCCCTGACCGACCCAAGGGATGTGGCACGCATCGAGAGCAAGAC
CGTGATCGTGACACAGGAGCAGAGGGACACCGTGCCAATCCCCAAGACAGGCCTG
TCTCAGCTGGGCCGCTGGATGAGCGAGGAGGATTTCGAGAAGGCCTTTAACGCCC
GGTTCCCTGGCTGTATGAAGGGCAGAACCATGTACGTGATCCCCTTCAGCATGGG
ACCTCTGGGAAGCCCACTGTCCAAGATCGGCATCGAGCTGACAGACTCCCCATAT
GTGGTGGCCTCTATGCGGATCATGACCAGAATGGGAACACCCGTGCTGGAGGCAG
TGGGCGATGGCGAGTTCGTGAAGTGCCTGCACTCCGTGGGCTGTCCTCTGCCACT
GCAGAAGCCCCTGGTGAACAATTGGCCCTGCAACCCTGAGCTGACCCTGATCGCA
CACCTGCCTGACAGGAGGGAGATCATCTCTTTTGGCAGCGGCTACGGCGGCAATA
GCCTGCTGGGCAAGAAGTGTTTCGCACTGAGGATGGCCTCCCGCCTGGCCAAGGA
GGAGGGATGGCTGGCCGAGCACATGCTGATCCTGGGCATCACCAATCCCGAGGGC
GAGAAGAAGTATCTGGCTGCCGCCTTTCCTTCTGCCTGCGGCAAGACAAACCTGG
CCATGATGAATCCAAGCCTGCCAGGATGGAAGGTGGAGTGCGTGGGCGACGACAT
CGCCTGGATGAAGTTCGATGCACAGGGACACCTGAGGGCCATCAACCCAGAGAAT
GGCTTCTTTGGCGTGGCCCCAGGCACCTCTGTGAAGACAAACCCCAATGCCATCA
AGACCATCCAGAAGAACACCATCTTTACAAATGTGGCCGAGACAAGCGACGGAGG
CGTGTACTGGGAGGGAATCGATGAGCCCCTGGCCAGCGGCGTGACCATCACATCC
TGGAAGAACAAGGAGTGGAGCTCCGAGGACGGAGAGCCATGCGCACACCCTAATT
CCAGATTCTGCACCCCCGCCTCTCAGTGTCCTATCATCGATGCAGCATGGGAGTC
TCCAGAGGGAGTGCCAATCGAGGGCATCATCTTTGGCGGCCGGAGACCTGCAGGA
GTGCCACTGGTGTATGAGGCCCTGTCCTGGCAGCACGGCGTGTTCGTGGGAGCAG
CAATGCGGTCTGAGGCAACAGCTGCCGCCGAGCACAAGGGCAAGATCATCATGCA
CGACCCATTTGCCATGAGACCCTTCTTTGGCTACAACTTCGGCAAGTATCTGGCA
CACTGGCTGTCCATGGCACAGCACCCTGCAGCAAAGCTGCCAAAGATCTTTCACG
TGAATTGGTTCAGGAAGGATAAGGAGGGCAAGTTTCTGTGGCCTGGCTTCGGCGA
GAACAGCAGGGTGCTGGAGTGGATGTTCAATCGCATCGACGGCAAGGCCTCCACC
AAGCTGACACCCATCGGCTACATCCCTAAGGAGGATGCCCTGAACCTGAAGGGCC
erein the cells are CD4+ or CD8+ cells. 113. The
sition of
TGTGAGATCGAGCGGGAGATCCTGGCCCTGAAGCAGAGAATCTCCCAGATGCTCG
AGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAA
TCCCGGCCCTAGGATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCA
CACCCAGCATTCCTCCTGATCCCATGCCACCCTGAGTGTCAGCCCCAGAATGGCT
CAGTGACCTGTTTTGGACCGGAGGCTGACCAGTGTGTGGCCTGTGCCCACTATAA
GGACCCTCCCTTCTGCGTGGCCCGCTGCCCCAGCGGTGTGAAACCTGACCTCTCC
TACATGCCCATCTGGAAGTTTCCAGATGAGGAGGGCGCATGCCAGCCTTGCCCCA
TCAACTGCACCCACTCCTGTGTGGACCTGGATGACAAGGGCTGCCCCGCCGAGCA
GAGAGCCAGCCCTCTGACGGGTGGAGGAAGCGGAGGTGGCAGCTCCATCATCTCT
GCGGTGGTTGGCATTCTGCTGGTCGTGGTCTTGGGGGTGGTCTTTGGGATCCTCA
TC
97
MPPQLQNGLNLSAKVVQGSLDSLPQAVREFLENNAELCQPDHIHICDGSEEENGR
PCK1-T2A-Her2t
LLGQMEEEGILRRLKKYDNCWLALTDPRDVARIESKTVIVTQEQRDTVPIPKTGL
(aa)
SQLGRWMSEEDFEKAFNARFPGCMKGRTMYVIPFSMGPLGSPLSKIGIELTDSPY
VVASMRIMTRMGTPVLEAVGDGEFVKCLHSVGCPLPLQKPLVNNWPCNPELTLIA
HLPDRREIISFGSGYGGNSLLGKKCFALRMASRLAKEEGWLAEHMLILGITNPEG
EKKYLAAAFPSACGKTNLAMMNPSLPGWKVECVGDDIAWMKFDAQGHLRAINPEN
GFFGVAPGTSVKTNPNAIKTIQKNTIFTNVAETSDGGVYWEGIDEPLASGVTITS
WKNKEWSSEDGEPCAHPNSRFCTPASQCPIIDAAWESPEGVPIEGIIFGGRRPAG
VPLVYEALSWQHGVFVGAAMRSEATAAAEHKGKIIMHDPFAMRPFFGYNFGKYLA
HWLSMAQHPAAKLPKIFHVNWFRKDKEGKFLWPGFGENSRVLEWMFNRIDGKAST
KLTPIGYIPKEDALNLKGLGHINMMELFSISKEFWEKEVEDIEKYLEDQVNADLP
CEIEREILALKQRISQMLEGGGEGRGSLLTCGDVEENPGPRMLLLVTSLLLCELP
HPAFLLIPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLS
YMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTGGGSGGGSSIIS
AVVGILLVVVLGVVFGILI
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15773153
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juno therapeutics, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Mar 30th, 2022 06:04PM
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Mar 30th, 2022 06:04PM
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Bristol-Myers Squibb
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:bmy
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Juno Therapeutics
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Jul 27th, 2021 12:00AM
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Oct 3rd, 2017 12:00AM
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https://www.uspto.gov?id=US11072660-20210727
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HPV-specific binding molecules
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Provided are binding molecules, such as TCRs or antigen binding fragments thereof and antibodies and antigen-binding fragments thereof, such as those that recognize or bind human papilloma virus (HPV) 16, including HPV 16 E6 and HPV 16 E7. Also provided are engineered cells containing such binding molecules, compositions containing the binding molecules or engineered cells, and methods of treatment, such as administration of the binding molecules, engineered cells, or compositions.
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11072660
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1. An engineered T cell containing a heterologous TCR that binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E7 in the context of a major histocompatibility complex (MHC) molecule, wherein:
the T cell is a human T cell;
the T cell contains a genetic disruption of a T cell receptor alpha constant (TRAC) gene and/or a T cell receptor beta constant (TRBC) gene; and
the heterologous TCR comprises an alpha chain and a beta chain, wherein the Vα region of the alpha chain comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 157, 158, and 159, respectively, and the Vβ region of the beta chain comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 160, respectively.
2. The engineered T cell of claim 1, wherein the TRBC gene is one or both of a T cell receptor beta constant 1 (TRBC1) or T cell receptor beta constant 2 (TRBC2) gene.
3. The engineered T cell of claim 1, wherein the genetic disruption of the TRAC gene reduces or prevents expression of the endogenous TCR constant alpha (Cα) chain in the T cell and/or the genetic disruption of the TRBC gene reduces or prevents expression of an endogenous TCR constant beta (Cβ) chain in the T cell.
4. The engineered T cell of claim 3, wherein the endogenous TCR Cα is encoded by the TRAC gene locus of SEQ ID NO: 348, or wherein the endogenous TCR Cβ is encoded by the TRBC1 gene locus of SEQ ID NO: 349, or wherein the endogenous TCR Cβ is encoded by the TRBC2 gene locus of SEQ ID NO: 1047.
5. The engineered T cell of claim 3, wherein the reduction or prevention of expression of the endogenous TCR in the T cell increases expression of the heterologous TCR or antigen binding fragment thereof by 1.5 fold, 2-fold, 3-fold, 4-fold, 5-fold or more.
6. The engineered T cell of claim 1, wherein the heterologous TCR binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule that is or contains the sequence set forth in SEQ ID NO:236.
7. A method for producing the T cell of claim 1 comprising:
i) introducing a vector comprising a nucleic acid encoding a TCR into a human T cell in vitro or ex vivo, wherein the TCR binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E7 in the context of a major histocompatibility complex (MEW) molecule and comprises an alpha chain and a beta chain, wherein:
the Vα region of the alpha chain comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 157, 158, and 159, respectively, and the Vβ region of the beta chain comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 160, respectively; and
ii) introducing into the T cell one or more agents, wherein each of the one or more agents is independently capable of inducing a genetic disruption of the TRAC gene and/or the TRBC gene.
8. The method of claim 7, wherein the Vα region comprises the amino acid sequence set forth in SEQ ID NO: 119 or an amino acid sequence that has at least 90% sequence identity thereto, and the Vβ region comprises the amino acid sequence set forth in SEQ ID NO: 120, or an amino acid sequence that has at least 90% sequence identity thereto.
9. The method of claim 7, wherein the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 119 and 120, respectively.
10. The method of claim 7, wherein the alpha constant (Cα) region of the alpha chain and the beta constant (Cβ) region of the beta chain are human or comprise one or more amino acid replacements thereof to introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and beta chain.
11. The method of claim 7, wherein the alpha chain and beta chain comprise the amino acid sequences of SEQ ID NOs: 218 and 214, respectively.
12. The method of claim 7, wherein the alpha chain and beta chain comprise the amino acid sequences of SEQ ID NOs: 201 and 197, respectively.
13. The method of claim 7, wherein the TCR is encoded by a nucleotide sequence in which the encoded alpha chain and the encoded beta chain are separated by a peptide sequence that causes ribosome skipping.
14. The method of claim 13, wherein the peptide is a T2A or a P2A peptide.
15. A composition containing an engineered T cell of claim 1 and a pharmaceutically acceptable excipient.
16. A method of treatment, comprising administering the composition of claim 15 to a subject having a disease or disorder associated with HPV.
17. A composition of claim 15, wherein the composition comprises engineered CD8+ T cells and engineered CD4+ T cells.
18. The engineered T cell of claim 1, wherein the T cell contains a genetic disruption of a T cell receptor alpha constant (TRAC) gene, and wherein the genetic disruption of the TRAC gene reduces or prevents expression of the endogenous TCR constant alpha (Cα) chain in the T cell.
19. A composition comprising an engineered T cell of claim 18.
20. A composition of claim 19, wherein the composition comprises engineered CD8+ T cells and engineered CD4+ T cells.
21. A method of treatment, comprising administering the composition of claim 19 to a subject having a disease or disorder associated with HPV.
22. The engineered T cell of claim 18, wherein the endogenous TCR Cα is encoded by the TRAC gene locus of SEQ ID NO: 348.
23. The engineered T cell of claim 1, wherein the T cell contains a genetic disruption of a T cell receptor beta constant (TRBC) gene, and wherein the genetic disruption of the TRBC gene reduces or prevents expression of the endogenous TCR constant alpha (Cβ) chain in the cell.
24. A composition comprising an engineered T cell of claim 23.
25. A composition of claim 24, wherein the composition comprises engineered CD8+ T cells and engineered CD4+ T cells.
26. A method of treatment, comprising administering the composition of claim 24 to a subject having a disease or disorder associated with HPV.
27. The engineered T cell of claim 23, wherein the endogenous TCR Cβ is encoded by the TRBC1 gene locus of SEQ ID NO: 349 or wherein the endogenous TCR Cβ is encoded by the TRBC2 gene locus of SEQ ID NO: 1047.
28. The engineered T cell of claim 1, wherein the Vα region comprises the amino acid sequence set forth in SEQ ID NO: 119 or an amino acid sequence that has at least 90% sequence identity thereto, and the Vβ region comprises the amino acid sequence set forth in SEQ ID NO: 120, or an amino acid sequence that has at least 90% sequence identity thereto.
29. The engineered T cell of claim 1, wherein the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 119 and 120, respectively.
30. The engineered T cell of claim 1, wherein the alpha constant (Cα) region of the alpha chain and the beta constant (Cβ) region of the beta chain are human or comprise one or more amino acid replacements thereof to introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and beta chain.
31. The engineered T cell of claim 1, wherein the alpha chain and beta chain comprise the amino acid sequences of SEQ ID NOs: 218 and 214, respectively.
32. The engineered T cell of claim 1, wherein the alpha chain and beta chain comprise the amino acid sequences of SEQ ID NOs: 201 and 197, respectively.
33. The engineered T cell of claim 1, wherein the heterologous TCR is encoded by a nucleotide sequence in which the encoded alpha chain and the encoded beta chain are separated by a peptide sequence that causes ribosome skipping.
34. The engineered T cell of claim 33, wherein the peptide is a T2A or a P2A peptide.
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34
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Stage of International Application No. PCT/US2017/055005 filed Oct. 3, 2017, which claims priority from U.S. provisional application No. 62/403,661 filed Oct. 3, 2016, entitled “HPV-SPECIFIC BINDING MOLECULES,” the contents of which are incorporated by reference in their entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042003800SeqList.txt, created Mar. 29, 2019, which is 2,095,795 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.
FIELD
The present disclosure relates in some aspects to binding molecules, such as those that recognize or bind a peptide epitope of human papilloma virus (HPV) 16 E6 or E7 in the context of a major histocompatibility complex (MHC) molecule. In particular, the present disclosure relates to T cell receptors (TCRs) or antibodies, including antigen-binding fragments thereof, that bind or recognize a peptide epitope of HPV 16 E6 or E7. The present disclosure further relates to engineered cells comprising such binding molecules, e.g., TCRs or antibodies (and chimeric antigen receptors containing the antibodies), and uses thereof in adoptive cell therapy.
BACKGROUND
Human papillomavirus (HPV) is a common virus among human subjects that, in some cases, can be transmitted by skin-to-skin contact and is a common sexually transmitted virus. Certain subtypes of HPV, such as HPV 16, can lead to certain cancers, such as cervical and other cancers. In some cases, cancer can be associated with expression of the HPV oncoproteins E6 and/or E7. For example, HPV E6 and/or E7 may contribute to cancer progression by targeting tumor suppressor signaling pathways that are involved in cellular growth control. Certain therapeutic agents targeting HPV 16-expressing cells or cancers are available, but improved agents against HPV 16 are needed. Provided are embodiments that meet such needs.
SUMMARY
Provided herein is a binding molecule containing a first variable region containing a complementarity determining region 3 (CDR-3) containing an amino acid sequence set forth in any of SEQ ID NOs: 138, 144, 147, 153, 159, 163, 167, 173, 175, 301, 304, 308, 478, 493, 505, 511, 523, 539, 555, 572, 588, 600, 612, 624, 638, 650, 662, 679, 694, 712, 729, 744, 762, 776, 788, 802, 818, 832, 846, 858, 870, 882, 896, 911, 926, 940, 952, 964, 976, 988, or 1002, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 117, 119, 121, 123, 125, 127, 295, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, 676, 691, 709, 726, 741, 759, 775, 787, 799, 815, 830, 845, 857, 869, 881, 895, 908, 925, 937, 951, 963, 975, 987, or 999; and/or a second variable region containing a complementarity determining region 3 (CDR-3) containing an amino acid sequence set forth in any of SEQ ID NOs: 141, 146, 150, 156, 160, 164, 170, 174, 178, 305, 309, 486, 499, 517, 531, 548, 563, 581, 594, 606, 618, 630, 644, 656, 670, 686, 703, 721, 736, 753, 769, 782, 794, 809, 825, 840, 852, 864, 876, 888, 902, 919, 932, 946, 958, 970, 982, 994, or 1010, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 118, 120, 122, 124, 126, 128, 296, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, 685, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008. In some embodiments, the binding molecules bind or recognize a peptide epitope of HPV 16 E6 or E7.
In some embodiments, the first variable region further contains a complementarity determining region 1 (CDR-1) containing an amino acid sequence set forth in any of SEQ ID NOs: 136, 142, 151, 157, 161, 165, 171, 302, 306, 537, 570, 677, 692, 710, 727, 742, 760, 800, 816, 909, 938, or 1000, or a CDR-1 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 117, 119, 121, 123, 125, 127, 295, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, 676, 691, 709, 726, 741, 759, 775, 787, 799, 815, 830, 845, 857, 869, 881, 895, 908, 925, 937, 951, 963, 975, 987, or 999; and/or a complementarity determining region 2 (CDR-2) containing an amino acid sequence set forth in any of SEQ ID NOs: 137, 143, 152, 158, 162, 166, 172, 303, 307, 538, 571, 678, 693, 711, 728, 743, 761, 801, 817, 831, 833, 910, 939, or 1001, or a CDR-2 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 117, 119, 121, 123, 125, 127, 295, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, 676, 691, 709, 726, 741, 759, 775, 787, 799, 815, 830, 845, 857, 869, 881, 895, 908, 925, 937, 951, 963, 975, 987, or 999.
In some of any such embodiments, the second variable region contains a complementarity determining region 1 (CDR-1) containing an amino acid sequence set forth in any of SEQ ID NOs: 139, 145, 148, 154, 168, 176, 484, 546, 561, 579, 668, 701, 719, or 751 or a CDR-1 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 118, 120, 122, 124, 126, 128, 296, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, 685, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008; and/or a complementarity determining region 2 (CDR-2) containing an amino acid sequence set forth in any of SEQ ID NOs: 140, 149, 155, 169, 177, 485, 547, 562, 580, 669, 702, 720, 752, 918, or 1009, or a CDR-2 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 118, 120, 122, 124, 126, 128, 296, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, 685, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008.
In some of any such embodiments, the binding molecule is an antibody or antigen-binding fragment thereof. In some of any such embodiments, the binding molecule is a T cell receptor (TCR) or antigen-binding fragment thereof.
Provided herein is a T cell receptor (TCR) or antigen-binding fragment thereof, containing an alpha chain containing a variable alpha (Vα) region and a beta chain containing a variable beta (Vβ) region, wherein said Vα region contains the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 117, 119, 121, 123, 125, 127, 295, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, 676, 691, 709, 726, 741, 759, 775, 787, 799, 815, 830, 845, 857, 869, 881, 895, 908, 925, 937, 951, 963, 975, 987, or 999, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or said Vβ region contains the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 118, 120, 122, 124, 126, 128, 296, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, 685, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
In some embodiments, said Vα region contains a complementarity determining region 3 (CDR-3) containing the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18 (SEQ ID NO: 251), wherein X1 is A, I, or V; X2 is M, L, V, E or A; X3 is R, L, N, or S; X4 is E, V, P, T, F, I, R or A; X5 is G, I, L, A, P, R, D, or H; X6 is R, T, G, S, N or H; X7 is G, R, A, N, or null; X8 is T, G, or null; X9 is null, A or G; X10 is null or G; X11 is null or G; X12 is null or T; X13 is F, Y, A, S or null; X14 is G, Y, or N; X15 is F, G, T, N, Q, or Y; X16 is K, P, V, N or A; X17 is T, L, or F; and X18 is I, V, T, H, or N; and/or said Vβ region contains a complementarity determining region 3 (CDR-3) containing the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15 (SEQ ID NO: 261), wherein X1 is A or S; X2 is 5, I, or V; X3 is S, T, or V; X4 is H, P, L, Y, T, D, or Q; X5 is L, G, W, F, S, or R; X6 is A, G, L, S, or T; X7 is G, E, A, T, R, or null; X8 is null or G; X9 is null or G; X10 is null, F, G, T, S, or A; X11 is T, N, H, A, S, or F; X12 is G, T, Q, D, Y, or L; X13 is E, P, T, G or W; X14 is L, A, Q, Y, or K; and X15 is F, H, Y, or T.
In some of any such embodiments, said Vα region contains a complementarity determining region 1 (CDR-1) containing the amino acid sequence X1X2X3X4X5X6X7 (SEQ ID NO: 243), wherein X1 is T, D, N, or V; X2 is I or S; X3 is S, D, A, P, or M; X4 is G, Q, P, or null; X5 is T, S, I, or F; X6 is D, Y, Q, T, or S; and X7 is Y, G, N, or Q; or a complementarity determining region 2 (CDR-2) containing the amino acid sequence X1X2X3X4X5X6X7X8 (SEQ ID NO: 247), wherein X1 is G, Q, I, V, or M; X2 is L, S, Q, Y, F, T, or G; X3 is T, G, S, or F; X4 is Y, S, N, I, or null; X5 is null or D; X6 is null, E, Q, S, M, or K; X7 is S, Q, R, G, D, or N; and X8 is N, E, M, T, or K; and/or said Vβ region contains a complementarity determining region 1 (CDR-1) containing the amino acid sequence X1X2X3X4X5 (SEQ ID NO: 254), wherein X1 is S, M, or L; X2 is G, E, D, N, or Q; X3 is H or V; X4 is V, N, E, L, or T; and X5 is S, R, N, Y, A, or M; or a complementarity determining region 2 (CDR-2) containing the amino acid sequence X1X2X3X4X5X6X7 (SEQ ID NO: 257), wherein X1 is F, Y, S, or A; X2 is Q, Y, V, or N; X3 is N, D, G, F, or Q; X4 is null or G; X5 is E, V, N, K, or S; X6 is A, K, G, or E; and X7 is Q, M, T, I, or A.
In some of any such embodiments, the binding molecule or TCR or antigen-binding fragment thereof binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E6 or E7 in the context of an MHC molecule. In some aspects, the binding molecule or TCR or antigen-binding fragment thereof binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E6 in the context of an MHC molecule.
In some of any such embodiments, the peptide epitope derived from HPV16 E6 is or contains the amino acid sequence set forth in any of SEQ ID NOs: 232-234. In some embodiments, the peptide epitope derived from HPV16 E6 is or contains E6(29-38) TIHDIILECV (SEQ ID NO:233).
In some of any such embodiments, said Vα region contains a complementarity determining region 3 (CDR-3) containing the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18 (SEQ ID NO: 248), wherein X1 is A, I, or V; X2 is M, L, or V; X3 is R, L, or N; X4 is E, V, T, P, or F; X5 is G, I, L, A, or P; X6 is R, T, G, or S; X7 is G, R, or null; X8 is T, G, or null; X9 is null or A; X10 is null or G; X11 is null or G; X12 is null or T; X13 is null or S; X14 is G, Y, or N; X15 is F, G, or T; X16 is K or P; X17 is T or L; and X18 is I, V or T; and/or said Vβ region contains a complementarity determining region 3 (CDR-3) containing the amino acid sequence ASSX4X5X6X7X8X9X10X11X12X13 (SEQ ID NO: 258), wherein X4 is H, P, L, or Y; X5 is L, G, W, F, or S; X6 is A, G, or L; X7 is G, E, A, T, or null; X8 is F, G, T, or S; X9 is T, N, H, or A; X10 is G, T, Q, D, or Y; X11 is E, P, T, or G; X12 is L, A, Q, or Y; and X13 is F, H, Y, or T.
In some of any such embodiments, said Vα region contains a complementarity determining region 1 (CDR-1) containing the amino acid sequence X1X2X3X4X5X6X7 (SEQ ID NO: 240), wherein X1 is T, D, or N; X2 is I, or S; X3 is S, D, or A; X4 is G, Q, P, or null; X5 is T, S, or I; X6 is D, Y, or Q; and X7 is Y, G, N, or Q; or a complementarity determining region 2 (CDR-2) containing the amino acid sequence X1X2X3X4X5X6X7X8 (SEQ ID NO: 244), wherein X1 is G, Q, I, or V; X2 is L, S, Q, or Y; X3 is T, G, or S; X4 is Y, S, or null; X5 is null or D; X6 is null, E, Q, or S; X7 is S, Q, R, or G; and X8 is N or E; and/or said Vβ region contains a complementarity determining region 1 (CDR-1) containing the amino acid sequence X1X2HX4X5 (SEQ ID NO: 252), wherein X1 is S or M; X2 is G, E, D, or N; X4 is V, N, or E; and X5 is S, R, N, or Y; or a complementarity determining region 2 (CDR-2) containing the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO: 255), wherein X1 is F or S; X2 is Q, Y, or V; X3 is N, D, or G; X4 is E or V; X5 is A, K, or G; and X6 is Q, M, or T.
In some of any such embodiments, said Vα region contains a complementarity determining region 3 (CDR-3) containing an amino acid sequence set forth in any of SEQ ID NOs: 138, 144, 147, 163, 167 173, 304, 308, 478, 493, 505, 511, 523, 539, 555, 572, 588, 600, 612, 624, 638, 650, 662, or 679, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123 125, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, or 676; and/or a Vβ region containing a complementarity determining region 3 (CDR-3) containing an amino acid sequence set forth in any of SEQ ID NOs: 141, 146, 150, 164, 170, 174, 305, 309, 486, 499, 517, 531, 548, 563, 581, 594, 606, 618, 630, 644, 656, 670, or 686, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 122, 124 126, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, or 685. In some aspects, the Vα region further contains a complementarity determining region 1 (CDR-1) containing an amino acid sequence set forth in any of SEQ ID NOs: 136, 142, 161, 165, 171, 302, 306, 537, 570, or 677, or a CDR-1 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123, 125, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, or 676; and/or a complementarity determining region 2 (CDR-2) containing an amino acid sequence set forth in any of SEQ ID NOs: 137, 143, 162, 166, 172, 303, 307, 538, 571, or 678, or a CDR-2 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123, 125, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, or 676.
In some of any such embodiments, the Vβ region contains a complementarity determining region 1 (CDR-1) containing an amino acid sequence set forth in any of SEQ ID NOs: 139, 145, 148, 168, 484, 546, 561, 579, or 668, or a CDR-1 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 122, 124, 126, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, or 685; and/or a complementarity determining region 2 (CDR-2) containing an amino acid sequence set forth in any of SEQ ID NOs: 140, 149, 169, 485, 547, 562, 580, or 669, or a CDR-2 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 122, 124, 126, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, or 685.
In some of any such embodiments, said Vα region contains a complementarity determining region 1 (CDR-1) containing an amino acid sequence set forth in any of SEQ ID NOs: 136, 142, 161, 165, 171, 302, 306, 537, 570, or 677; a complementarity determining region 2 (CDR-2) containing an amino acid sequence set forth in any of SEQ ID NOs: 137, 143, 162, 166, 172, 303, 307, 538, 571, or 678; and/or a complementarity determining region 3 (CDR-3) containing an amino acid sequence set forth in any of SEQ ID NOs: 138, 144, 147, 163, 167, 173, 304, 308, 478, 493, 505, 511, 523, 539, 555, 572, 588, 600, 612, 624, 638, 650, 662, 679; and/or said Vβ region contains a complementarity determining region 1 (CDR-1) containing an amino acid sequence set forth in any of SEQ ID NOs: 139, 145, 148, 168, 484, 546, 561, 579, or 668; a complementarity determining region 2 (CDR-2) containing an amino acid sequence set forth in any of SEQ ID NOs: 140, 149 or 169; and/or a complementarity determining region 3 (CDR-3) containing an amino acid sequence set forth in any of SEQ ID NOs: 141, 146, 150, 164, 170, 174, 305, 309, 486, 499, 517, 531, 548, 563, 581, 594, 606, 618, 630, 644, 656, 670, or 686.
In some of any such embodiments, said Vα region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 136, 137, and 138, respectively, and said Vβ region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 139, 140, and 141, respectively; said Vα region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 142, 143, and 144, respectively, and said Vβ region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 145, 140, and 146, respectively; said Vα region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 136, 137, and 147, respectively, and said Vβ region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 148, 149, and 150, respectively; said Vα region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 161, 162, and 163, respectively, and said Vβ region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 148, 149, and 164, respectively; said Vα region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 165, 166, and 167, respectively, and said Vβ region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 168, 169, and 170, respectively; said Vα region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 171, 172, and 173, respectively, and said Vβ region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 148, 149, and 174, respectively; said Vα region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively, and said Vβ region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 139, 140, and 305, respectively; or said Vα region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 306, 307, and 308, respectively, and said Vβ region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 148, 149, and 309, respectively.
In some of any such embodiments, said Vα region contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively containing the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vα region amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123, 125, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, or 676; and/or said Vβ region contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively containing the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vβ region amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 122, 124, 126, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, or 685.
In some of any such embodiments, the Vα and Vβ regions include the amino acid sequences of SEQ ID NOs: 111 and 112, respectively; the Vα and Vβ regions include the amino acid sequences of SEQ ID NOs: 113 and 114, respectively; the Vα and Vβ regions include the amino acid sequences of SEQ ID NOs: 115 and 116, respectively; the Vα and Vβ regions include the amino acid sequences of SEQ ID NOs: 121 and 122, respectively; the Vα and Vβ regions include the amino acid sequences of SEQ ID NOs: 123 and 124, respectively; the Vα and Vβ regions include the amino acid sequences of SEQ ID NOs: 125 and 126, respectively; the Vα and Vβ regions include the amino acid sequences of SEQ ID NOs: 297 and 298, respectively; the Vα and Vβ regions include the amino acid sequences of SEQ ID NOs: 299 and 300, respectively.
In some of any such embodiments, the binding molecule or TCR or antigen-binding fragment thereof binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule.
Provided herein is a T cell receptor (TCR) or antigen-binding fragment thereof, containing an alpha chain containing a variable alpha (Vα) region and a beta chain containing a variable beta (Vβ) region, wherein the TCR or antigen-binding fragment thereof binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule. In some embodiments, the peptide epitope derived from HPV16 E7 is or contains the amino acid sequence set forth in any of SEQ ID NOs: 235-239. In some aspects, the peptide epitope derived from HPV16 E7 is or contains E7(11-19) YMLDLQPET (SEQ ID NO:236).
In some of any such embodiments, said Vα region contains a complementarity determining region 3 (CDR-3) containing the amino acid sequence X1X2SX4X5X6X7X8X9X10X11 (SEQ ID NO: 249), wherein X1 is A or V; X2 is E or V; X4 is I or R; X5 is R or D; X6 is G or N; X7 is F or Y; X8 is N or Q; X9 is V or N; X10 is L or F; and X11 is H or V; and/or said Vβ region contains a complementarity determining region 3 (CDR-3) containing the amino acid sequence AX2TX4RX6X7YX9X10X11 (SEQ ID NO: 259), wherein X2 is S or I; X4 is T or D; X6 is S or T; X7 is S or N; X9 is E or G; X10 is Q or Y; and X11 is Y or T.
In some of any such embodiments, said Vα region contains a complementarity determining region 1 (CDR-1) containing the amino acid sequence X1SX3X4X5X6 (SEQ ID NO: 241), wherein X1 is D or V; X3 is S, or P; X4 is S or F; X5 is T or S; and X6 is Y or N; or a complementarity determining region 2 (CDR-2) containing the amino acid sequence X1X2X3X4X5X6X7 (SEQ ID NO: 245), wherein X1 is I or M; X2 is F or T; X3 is S or F; X4 is N or S; X5 is M or E; X6 is D or N; and X7 is M or T; and/or said Vβ region contains a complementarity determining region 1 (CDR-1) containing the amino acid sequence set forth in SEQ ID NO: 154; or a complementarity determining region 2 (CDR-2) containing the amino acid sequence set forth in SEQ ID NO: 155.
In some of any such embodiments, said Vα region contains a complementarity determining region 3 (CDR-3) containing the amino acid sequence set forth in any of SEQ ID NOs: 153, 159, 301, 694, 712, 729, 744, 762, 776, 788, 802, 818, 832, 846, 858, 870, 882, 896, 911, 926, 940, 952, 964, 976, 988, or 1002, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 117, 119, or 295; and/or said Vβ region contains a complementarity determining region 3 (CDR-3) containing an amino acid sequence set forth in any of SEQ ID NOs: 156, 160, 703, 721, 736, 753, 769, 782, 794, 809, 825, 840, 852, 864, 876, 888, 902, 919, 932, 946, 958, 970, 982, 994, or 1010, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 118, 120, 296, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008. In some embodiments, the Vα region further contains a complementarity determining region 1 (CDR-1) containing an amino acid sequence set forth in any of SEQ ID NOs: 151, 157, 692, 710, 727, 742, 760, 800, 816, 909, 938, or 1000; and/or a complementarity determining region 2 (CDR-2) containing an amino acid sequence set forth in any of SEQ ID NOs: 152, 158, 693, 711, 728, 743, 761, 801, 817, 831, 833, 910, 939, or 1001.
In some of any such embodiments, the Vβ region contains a complementarity determining region 1 (CDR-1) containing the amino acid sequence set forth in SEQ ID NO: 154; and/or a complementarity determining region 2 (CDR-2) containing the amino acid sequence set forth in SEQ ID NO: 155.
In some of any such embodiments, said Vα region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 151, 152, and 153, respectively, and said Vβ region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 154, 155, and 156, respectively; said Vα region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 157, 158, and 159, respectively, and said Vβ region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 154, 155, and 160, respectively; or said Vα region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 151, 152, and 301, respectively, and said Vβ region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 154, 155, and 156, respectively.
In some of any such embodiments, said Vα region contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively containing the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vα region amino acid sequence set forth in any of SEQ ID NOs: 117, 119, or 295; and/or said Vβ region contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively containing the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vβ region amino acid sequence set forth in any of SEQ ID NOs: 118, 120, 296, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008.
In some of any such embodiments, the Vα and Vβ regions include the amino acid sequences of SEQ ID NOs: 117 and either 118 or 296, respectively; the Vα and Vβ regions include the amino acid sequences of SEQ ID NOs: 119 and 120, respectively; or the Vα and Vβ regions include the amino acid sequences of SEQ ID NOs: 295 and either 118 or 296, respectively.
In some of any such embodiments, the peptide epitope derived from HPV16 E7 is or contains E7(86-93) TLGIVCPI (SEQ ID NO:235).
In some of any such embodiments, said Vα region contains a complementarity determining region 3 (CDR-3) containing the amino acid sequence set forth in SEQ ID NO: 175; and/or said Vβ region contains a complementarity determining region 3 (CDR-3) containing the amino acid sequence set forth in any of SEQ ID NO: 178. In some embodiments, the Vα region contains a complementarity determining region 1 (CDR-1) containing the amino acid sequence set forth in SEQ ID NO: 142; and/or a complementarity determining region 2 (CDR-2) containing the amino acid sequence set forth in SEQ ID NO: 143.
In some embodiments, said Vβ region contains a complementarity determining region 1 (CDR-1) containing an amino acid sequence set forth in SEQ ID NOs: 176; and/or a complementarity determining region 2 (CDR-2) containing an amino acid sequence set forth in SEQ ID NOs: 177, said Vα region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 142, 143, and 175, respectively, and said Vβ region contains a CDR-1, CDR-2, and CDR-3, containing the amino acid sequences of SEQ ID NOs: 176, 177, and 178, respectively.
In some of any such embodiments, said Vα region contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively containing the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vα region amino acid sequence set forth in SEQ ID NO: 127; and/or said Vβ region contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively containing the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vβ region amino acid sequence set forth in SEQ ID NO: 128.
In some of any such embodiments, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 127 and 128, respectively.
In some of any such embodiments, the alpha chain further contains an alpha constant (Cα) region and/or the beta chain further contains a beta constant (Cβ) region. In some aspects, the Cα and Cβ regions are mouse constant regions. In some embodiments, said Cα region contains the amino acid sequence set forth in SEQ ID NO: 262, or a sequence of amino acids that has at least 90% sequence identity thereto; and/or said Cβ region contains the amino acid sequence set forth in SEQ ID NO: 263, or a sequence of amino acids that has at least 90% sequence identity thereto. In some instances, the Cα and Cβ regions are human constant regions.
In some of any such embodiments, said Cα region contains the amino acid sequence set forth in any of SEQ ID NOs: 212, 213, 215, 217, 218, 220, or 524, or a sequence of amino acids that has at least 90% sequence identity thereto; and/or said Cβ region contains the amino acid sequence set forth in any of SEQ ID NOs: 214, 216, 631, or 889, or a sequence of amino acids that has at least 90% sequence identity thereto.
In some of any such embodiments, the TCR or antigen-binding fragment thereof containing one or more modifications in the α chain and/or β chain such that when the TCR or antigen-binding fragment thereof is expressed in a cell, the frequency of mispairing between the TCR α chain and β chain and an endogenous TCR α chain and β chain is reduced, the expression of the TCR α chain and β chain is increased and/or the stability of the TCR α chain and β chain is increased. In some embodiments, the one or more modifications is a replacement, deletion, or insertion of one or more amino acids in the Cα region and/or the Cβ region. In some aspects, the one or more modifications contain replacement(s) to introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and beta chain.
In some of any such embodiments, the TCR or antigen-binding fragment thereof contains a Cα region containing a cysteine at a position corresponding to position 48 with numbering as set forth in SEQ ID NO: 212, 213, 215, 217, 218, 220, or 524, and/or a Cβ region containing a cysteine at a position corresponding to position 57 with numbering as set forth in SEQ ID NO: 214, 216, 631, or 889. In some embodiments, said Cα region contains the amino acid sequence set forth in any of SEQ ID NOs: 196, 198, 200, 201, 203, or 525, or a sequence of amino acids that has at least 90% sequence identity thereto containing one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; and/or said Cβ region contains the amino acid sequence set forth in any of SEQ ID NOs: 197, 199, 632, or 890, or a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain.
In some of any such embodiments, the TCR or antigen-binding fragment thereof is encoded by a nucleotide sequence that has been codon-optimized.
In some of any such embodiments, a) said alpha chain contains the amino acid sequence set forth in any of SEQ ID NOs: 18, 28, 38, 68, 78, 88, 287, 291, 473, 488, 500, 506, 518, 532, 550, 565, 583, 595, 607, 619, 633, 645, 657, or 672, a sequence of amino acids that has at least 90% sequence identity thereto; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 20, 30, 40, 70, 80, 90, 100, 202, 219, 389, 430, 1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045 or a nucleotide sequence that has at least 90% sequence identity thereto; and/or said beta chain contains an amino acid sequence set forth in any of SEQ ID NOs: 22, 32, 42, 72, 82, 92, 289, 293, 479, 494, 512, 526, 541, 556, 574, 589, 601, 613, 625, 639, 651, 663, or 681, a sequence of amino acids that has at least 90% sequence identity thereto; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOS: 16, 17, 24, 34, 44, 74, 84, 94, 104, 390, 431, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, or a nucleotide sequence that has at least 90% sequence identity thereto; or b) the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 18 and 22, respectively; the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 28 and 32, respectively; the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 38 and 42, respectively; the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 68 and 72, respectively; the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 78 and 82, respectively; the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 88 and 92, respectively, the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 287 and 289, respectively, or the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 291 and 293, respectively.
In some of any such embodiments, a) said alpha chain contains the amino acid sequence set forth in any of SEQ ID NOs: 19, 29, 39, 69, 89, 288, 292, 474, 489, 501, 507, 519, 533, 551, 566, 584, 596, 608, 620, 634, 646, 658, or 673, a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 10, 11, 21, 31, 41, 71, 81, 91, 101, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117, 1119, 1121, 1123, 1125, 1127, or a nucleotide sequence that has at least 90% sequence identity thereto and encodes an alpha chain that contains one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; and/or said beta chain contains an amino acid sequence set forth in any of SEQ ID NOs: 23, 33, 43, 73, 83, 93, 290, 294, 480, 495, 513, 527, 542, 557, 575, 590, 602, 614, 626, 640, 652, 664, or 682, a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 7, 8, 25, 35, 45, 75, 85, 95, 105, 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128, or a nucleotide sequence that has at least 90% sequence identity thereto and encodes a beta chain that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain; or b) the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 19 and 23, respectively; the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 29 and 33, respectively; the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 39 and 43, respectively; the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 69 and 73, respectively; the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 79 and 83, respectively; the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 89 and 93, respectively, the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 288 and 290, or the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 292 and 294.
In some of any such embodiments, a) said alpha chain contains the amino acid sequence set forth in SEQ ID NOs: 48, 58, 283, 687, 705, 722, 737, 755, 771, 783, 795, 811, 826, 841, 853, 865, 877, 891, 904, 921, 933, 947, 959, 971, 983, or 995, a sequence of amino acids that has at least 90% sequence identity thereto; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 50, 60, 183, 1049, 1051, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1225, 1226, or a nucleotide sequence that has at least 90% sequence identity thereto; and/or said beta chain contains an amino acid sequence set forth in SEQ ID NOs: 52, 62, 285, 696, 714, 731, 746, 764, 777, 789, 804, 820, 835, 847, 859, 871, 883, 897, 913, 927, 941, 953, 965, 977, 989, or 1004, a sequence of amino acids that has at least 90% sequence identity thereto; or an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NOS: 55, 64, 108, 1050, 1052, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1224, 1227, 1228, or a nucleotide sequence that has at least 90% sequence identity thereto; or b) the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 48 and either 52 or 285, respectively; the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 58 and 62, respectively; or the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 283 and either 52 or 285, respectively.
In some of any such embodiments, a) said alpha chain contains the amino acid sequence set forth in SEQ ID NOs: 49, 59, 284, 688, 706, 723, 738, 756, 772, 784, 796, 812, 827, 842, 854, 866, 878, 892, 905, 922, 934, 948, 960, 972, 984, or 996, a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 12, 51, 61, 1129, 1131, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, or a nucleotide sequence that has at least 90% sequence identity thereto and encodes an alpha chain that contains one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; and/or said beta chain contains an amino acid sequence set forth in SEQ ID NOs: 53, 63, 286, 697, 715, 732, 747, 765, 778, 790, 805, 821, 836, 848, 860, 872, 884, 898, 914, 928, 942, 954, 966, 978, 990, or 1005, a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain; or an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NOS: 9, 54, or 65, 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, or a nucleotide sequence that has at least 90% sequence identity thereto and encodes a beta chain that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain; or b) the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 49 and 53, respectively; the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 59 and 63, respectively; or the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 284 and 286, respectively.
In some of any such embodiments, a) said alpha chain contains the amino acid sequence set forth in SEQ ID NO: 98, a sequence of amino acids that has at least 90% sequence identity thereto; or an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 100, or a nucleotide sequence that has at least 90% sequence identity thereto; and/or said beta chain contains an amino acid sequence set forth in SEQ ID NO: 102, a sequence of amino acids that has at least 90% sequence identity thereto; or an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 104, or a nucleotide sequence that has at least 90% sequence identity thereto; or b) the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 98 and 102, respectively.
In some of any such embodiments, a) said alpha chain contains the amino acid sequence set forth in SEQ ID NO: 99, a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; or an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 101, or a nucleotide sequence that has at least 90% sequence identity thereto and encodes an alpha chain that contains one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; and/or said beta chain contains an amino acid sequence set forth in SEQ ID NO: 103, a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain; or an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 105, or a nucleotide sequence that has at least 90% sequence identity thereto and encodes a beta chain that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain; or b) the alpha and beta chains contain the amino acid sequences of SEQ ID NOs: 99 and 103, respectively.
In some of any such embodiments, the TCR or antigen-binding fragment thereof further contains a signal peptide. In some embodiments, the signal peptide contains the amino acid sequence set forth in any of SEQ ID NOs: 181, 184, 187, 189, 190, 192, 193, 310, 311, 182, 185, 186, 188, 191, 194, 487, 540, 549, 564, 573, 582, 671, 680, 695, 704, 713, 730, 745, 754, 763, 770, 803, 810, 819, 834, 903, 912, 920, 1003, or 1011.
In some of any such embodiments, the binding molecule or TCR or antigen-binding fragment thereof is isolated or purified or is recombinant. In some of any such embodiments, the binding molecule or TCR or antigen-binding fragment thereof is human.
In some of any such embodiments, the binding molecule or TCR or antigen-binding fragment thereof is monoclonal. In some of any such embodiments, the binding molecule or TCR or antigen-binding fragment thereof is single chain. In some of any such embodiments, the binding molecule or TCR or antigen-binding fragment thereof contains two chains.
In some of any such embodiments, the antigen-specificity is at least partially CD8-independent. In some of any such embodiments, the MHC molecule is an HLA-A2 molecule.
Provided herein is a nucleic acid molecule encoding the binding molecule or the TCR or antigen-binding fragment thereof according to any one of the embodiments described above. In some embodiments, the nucleic acid molecule containing a nucleotide sequence encoding an alpha chain and/or a nucleotide sequence encoding a beta chain, wherein said nucleotide sequence encoding an alpha chain contains the sequence selected from the group consisting of residues 61-816 of SEQ ID NO: 20, residues 58-804 of SEQ ID NO: 30, residues 61-825 of SEQ ID NO: 40, residues 64-813 of SEQ ID NO: 50, residues 64-816 of SEQ ID NO: 60, residues 58-807 of SEQ ID NO: 70, residues 61-825 of SEQ ID NO: 80, residues 67-831 of SEQ ID NO: 90, residues 58-801 of SEQ ID NO: 100, or a sequence having at least 90% sequence identity thereto; and/or said nucleotide sequence encoding a beta chain contains the sequence selected from the group consisting of residues 58-936 of SEQ ID NO: 17, residues 58-930 of SEQ ID NO: 16, residues 58-939 of SEQ ID NO: 24, residues 64-930 of SEQ ID NO: 34 or 44, residues 58-933 of SEQ ID NO: 55, residues 58-927 of SEQ ID NO: 64, residues 64-936 of SEQ ID NO: 74, residues 58-933 of SEQ ID NO: 84, residues 63-930 of SEQ ID NO: 94, residues 46-936 of SEQ ID NO: 104, residues 58-933 of SEQ ID NO: 108, or a sequence having at least 90% sequence identity thereto. In some instances, the nucleotide sequence is codon-optimized.
In some of any such embodiments, the nucleic acid molecule containing a nucleotide sequence encoding an alpha chain and/or a nucleotide sequence encoding a beta chain, wherein said nucleotide sequence encoding an alpha chain contains the sequence selected from the group consisting of residues 67-825 of SEQ ID NO: 10, residues 58-813 of SEQ ID NO: 11, residues 64-822 of SEQ ID NO: 12 residues 61-825 of SEQ ID NO: 21, residues 58-813 of SEQ ID NO: 31, residues 61-834 of SEQ ID NO: 41, residues 63-822 of SEQ ID NO: 51, residues 64-825 of SEQ ID NO: 61, residues 58-816 of SEQ ID NO: 71, residues 61-834 of SEQ ID NO: 81, residues 67-840 of SEQ ID NO: 91, residues 58-810 of SEQ ID NO: 101, or a sequence having at least 90% sequence identity thereto; and/or said nucleotide sequence encoding a beta chain contains the sequence selected from the group consisting of residues 58-939 of SEQ ID NO: 25, residues 64-930 of SEQ ID NO: 35, 45, or 95, residues 58-933 of SEQ ID NO: 54 or 85, residues 58-927 of SEQ ID NO: 65, residues 64-936 of SEQ ID NO: 75, residues 46-936 of SEQ ID NO: 105, or a sequence having at least 90% sequence identity thereto.
In some of any such embodiments, the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a nucleotide sequence encoding an internal ribosome entry site (IRES) or a peptide sequence that causes ribosome skipping. In some aspects, the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a peptide sequence that causes ribosome skipping. In some embodiments, the peptide that causes ribosome skipping is a P2A or T2A peptide and/or contains the sequence of amino acids set forth in SEQ ID NO: 204 or 211.
In some of any such embodiments, provided herein is a nucleic acid containing the nucleotide sequence set forth in any of SEQ ID NOs: 13, 14, 15, 26, 36, 46, 56, 66, 76, 86, 96, 106, 432-472, or a nucleotide sequence having at least 90% sequence identity thereto. In some of any such embodiments, the nucleic acid is synthetic. In some of any such embodiments, the nucleic acid is cDNA.
Provided herein is a vector containing the nucleic acid according to any one of the embodiments described above. In some instances, the vector is an expression vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some aspects, the lentiviral vector is derived from HIV-1.
Provided herein is an engineered cell containing the vector according to any one of the embodiments described above. Provided herein is an engineered cell containing the binding molecule or the TCR or antigen-binding fragment thereof according to any one of the embodiments described above. In some embodiments, the binding molecule or TCR or antigen-binding fragment thereof is heterologous to the cell.
Provided herein is an engineered cell containing a heterologous TCR or antigen-binding fragment thereof that binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E6 in the context of an MHC molecule, wherein the TCR or antigen-binding fragment thereof does not bind to or recognize the epitope E6(29-38) containing the amino acid sequence TIHDIILECV (SEQ ID NO: 233). In some aspects, the TCR or antigen-binding fragment thereof that binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E6 in the context of an MHC molecule is or contains the sequence set forth in SEQ ID NO: 232 or SEQ ID NO: 234.
Provided herein is an engineered cell containing a heterologous TCR or antigen-binding fragment thereof that binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule. In some embodiments, the peptide derived from HPV16 E7 is or contains the sequence set forth in any of SEQ ID NOs: 235-239. In some aspects, the peptide derived from HPV16 E7 is or contains the sequence set forth in SEQ ID NO: 236.
In some embodiments, the TCR or antigen-binding fragment thereof is a TCR or antigen-binding fragment thereof according to any one of the embodiments described above. In some aspects, the peptide derived from HPV16 E7 is or contains the sequence set forth in SEQ ID NO:235. In some embodiments, the TCR or antigen-binding fragment thereof is a TCR or antigen-binding fragment thereof according to any one of the embodiments described above.
In some of any such embodiments, the engineered cell is a T cell. In some embodiments, the T cell is CD8+. In some aspects, the T cell is CD4+.
In some embodiments, the engineered cell is a cell line. In some embodiments, the engineered cell is a primary cell obtained from a subject. In some embodiments, the subject is a mammalian subject. In some embodiments, the subject is a human.
In some embodiments, the provided engineered cells contain a genetic disruption of a T cell receptor alpha constant (TRAC) gene and/or a T cell receptor beta constant (TRBC) gene. In some embodiments, the TRBC gene is one or both of a T cell receptor beta constant 1 (TRBC1) or T cell receptor beta constant 2 (TRBC2) gene.
Also provided herein are methods for producing any of the engineered cells described herein, that includes introducing any of the vectors described herein into a cell in vitro or ex vivo. In some embodiments, the vector is a viral vector and the introducing is carried out by transduction.
In some embodiments, the methods provided herein include introducing into the cell one or more agent, wherein each of the one or more agent is independently capable of inducing a genetic disruption of a T cell receptor alpha constant (TRAC) gene and/or a T cell receptor beta constant (TRBC) gene. In some embodiments, the one or more agent capable of inducing a genetic disruption comprises a DNA binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to the target site. In some embodiments, the one or more agent capable of inducing a genetic disruption comprises (a) a fusion protein containing a DNA-targeting protein and a nuclease or (b) an RNA-guided nuclease. In some embodiments, the DNA-targeting protein or RNA-guided nuclease comprises a zinc finger protein (ZFP), a TAL protein, or a clustered regularly interspaced short palindromic nucleic acid (CRISPR)-associated nuclease (Cas) specific for a target site within the TRAC and/or TRBC gene. In some embodiments, the one or more agent comprises a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or and a CRISPR-Cas9 combination that specifically binds to, recognizes, or hybridizes to the target site. In some embodiments, the each of the one or more agent comprises a guide RNA (gRNA) having a targeting domain that is complementary to the at least one target site.
In some embodiments, the one or more agent is introduced as a ribonucleoprotein (RNP) complex containing the gRNA and a Cas9 protein. In some embodiments, the RNP is introduced via electroporation, particle gun, calcium phosphate transfection, cell compression or squeezing. In some embodiments, the RNP is introduced via electroporation.
In some embodiments, the one or more agent is introduced as one or more polynucleotide encoding the gRNA and/or a Cas9 protein.
Provided herein is a method for producing a cell according to any one of the embodiments described above, including transducing a cell in vitro or ex vivo with a vector according to any one of the embodiments described above.
Provided herein is a composition containing the binding molecule or the TCR or antigen-binding fragment thereof according to any one of the embodiments described above, or the engineered cell according to any one of the embodiments described above. Provided herein is a composition containing an engineered CD8+ cell according to any one of the embodiments described above and an engineered CD4+ cell according to any one of the embodiments described above.
In some embodiments, the TCR or antigen-binding fragment thereof binds to or recognizes a peptide epitope of HPV 16 in the context of an MHC molecule that is at least partially CD8-independent.
In some aspects, the CD8+ cell and CD4+ cell are engineered with the same TCR or antigen-binding fragment thereof and/or are each engineered with a TCR or antigen-binding fragment thereof that binds to or recognizes the same peptide epitope of HPV 16 in the context of an MHC molecule.
In some aspects, also provided are compositions according to any one of the embodiments described above, further containing a pharmaceutically acceptable excipient.
Also provided herein are methods of treatment. Provided herein is a method of treatment including administering the engineered cell according to any one of the embodiments described above to a subject having a disease or disorder associated with HPV. Provided herein is a method of treatment including administering the composition according to any one of the embodiments described above to a subject having a disease or disorder associated with HPV. In some aspect, the disease or disorder is associated with HPV16. In some instances, the disease or disorder is cancer. In some embodiments, the subject is a human.
Also provided herein are compositions, such as any of the compositions described herein, for use in treating a disease or disorder associated with HPV.
Also provided herein are uses of compositions, such as any of the compositions provided herein, for the manufacture of a medicament for treating a disease or disorder associated with HPV. In some embodiments, the disease or disorder is associated with HPV16. In some embodiments, the disease or disorder is cancer. In some embodiments, the subject is a human.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows lytic activity of monoclonal T cell lines expressing exemplary TCRs incubated with SiHa cells or Caski target cells based on the percent of caspase positive target cells at various assessed time points. Specifically, results are shown for T cell lines expressing the modified version of TCR 5 and the modified version of TCR 12.
FIG. 2A and FIG. 2B show flow cytometry results for tetramer binding by a CD4+ Jurkat-derived cell line (Neg ctrl CD4+), the CD4+ Jurkat-derived cell line expressing various E6(29-38)-specific TCRs (CD4+ TCR-E6(29)), the CD4+ Jurkat-derived cell line that also expresses exogenous CD8 (CD8), or the CD4+ Jurkat-derived cell line that also expresses exogenous CD8 and various E6(29-38)-specific TCRs (CD8+ TCR-E6(29)). Specifically, results are shown for a reference TCR, the modified version of TCR 5, the modified version of TCR 4, the modified version of TCR 3 and the modified version of TCR 8.
FIG. 3 shows flow cytometry results for tetramer binding by CD4+ Jurkat-derived cell line (Neg ctrl CD4+), the CD4+ Jurkat-derived cell line expressing various E7(11-19)-specific TCRs (CD4+ TCR-E7(11-19)), the CD4+ Jurkat-derived cell line that also expresses exogenous CD8 (CD8), or the CD4+ Jurkat-derived cell line that also expresses exogenous CD8 and various E7(11-19)-specific TCRs (CD8+ TCR-E7(11-19)). Specifically, results are shown for the modified version of TCR 7 and the modified version of TCR 12.
FIG. 4 shows flow cytometry results for tetramer binding by CD4+ Jurkat-derived cell line (Neg ctrl CD4+), the CD4+ Jurkat-derived cell line expressing various E7(86-93)-specific TCRs (CD4+ TCR-E7(86-93)), the CD4+ Jurkat-derived cell line that also expresses exogenous CD8 (CD8), or the CD4+ Jurkat-derived cell line that also expresses exogenous CD8 and various E7(86-93)-specific TCRs (CD8+ TCR-E7(86-93)). Specifically, results are shown for the modified version of TCR 11.
FIGS. 5A-5C show flow cytometry results for tetramer binding and in Jurkat-derived cell line that also expresses exogenous CD8 and various E6(29-38)-specific TCRs, in CD8+ cells. Results are shown for TCR 9, TCR13, TCR14, a reference TCR capable of binding to HLA-A2/E6(29-38) (Reference TCR) and cells that had been mock transfected (mock) (FIG. 5A); TCR 17, TCR 21, TCR 22, Reference TCR and Mock (FIG. 5B); and TCR 18, TCR 23, TCR 24 and TCR 27 (FIG. 5C).
FIGS. 5D-5F show flow cytometry results for tetramer binding and in Jurkat-derived cell line that also expresses exogenous CD8 and various E6(29-38)-specific TCRs. Results are shown for TCR 15, TCR 16, TCR 17, TCR 19, TCR 20 and TCR 21 (FIG. 5D); TCR 18, TCR 23, TCR 24, TCR 27 and TCR 28 (FIG. 5E); and TCR 25, TCR 26, TCR 29 and TCR 30 (FIG. 5F).
FIGS. 6A-6F show flow cytometry results for tetramer binding and in Jurkat-derived cell line that also expresses exogenous CD8 and various E7(11-19)-specific TCRs. Results are shown for TCR 12 and cells that had been mock transfected (mock) (FIG. 6A); TCR 31, TCR 32, TCR 33 and TCR 34 (FIG. 6B); TCR 12, TCR 49, TCR 50 and TCR 51 (FIG. 6C); TCR 35, TCR 36, TCR 37, TCR 38, TCR 53 and TCR 54 (FIG. 6D); TCR 39, TCR 40, TCR 41, TCR 42, TCR 43 and TCR 44 (FIG. 6E); and TCR 45, TCR 46, TCR 47, TCR 48, TCR 54 and TCR 55 (FIG. 6F).
DETAILED DESCRIPTION
I. T Cell Receptors and Other HPV-Specific Binding Molecules
Provided herein are binding molecules, such as those that bind or recognize a peptide epitope of human papillomavirus (HPV) 16, e.g., a peptide epitope of HPV 16 E6 or E7, in the context of an MHC molecule. Such binding molecules include T cell receptors (TCRs) and antigen-binding fragments thereof and antibodies and antigen binding fragments thereof that exhibit antigenic specificity for binding or recognizing a peptide epitope of HPV 16 E6 or HPV 16 E7. Also provided in some embodiments are nucleic acid molecules encoding the binding molecules, engineered cells containing the binding molecules, compositions and methods of treatment involving administering such binding molecules, engineered cells or compositions.
HPV is a causative organism in most cases of cervical cancer and has been implicated in anal, vaginal, vulvar, penile, and oropharyngeal cancers, and other cancers. Generally, the HPV genome contains an early region containing six open reading frames (E1, E2, E4, E5, E6 and E7), which encode proteins involved in cell transformation and replication, and a late region containing two open reading frames (L1 and L2), which encode proteins of the viral capsid. In general, E6 and E7 are oncogenes that can affect cell cycle regulation and contribute to the formation of cancers. For instance, the E6 gene product can cause p53 degradation and the E7 gene product can cause retinoblastoma (Rb) inactivation.
In some aspects, a provided HPV 16 binding molecule, including a TCR or antigen binding fragment thereof or an anti-HPV 16 antibody, e.g., antibody fragments thereof, and proteins such as chimeric molecules containing one or more of the foregoing, such as the chimeric receptors, e.g., TCR-like CARs, and/or engineered cells expressing the TCRs or CARs, bind to a peptide epitope derived from HPV16 E6 protein. In some aspects, a provided HPV 16 binding molecule, including a TCR or antigen binding fragments thereof or anti-HPV 16 antibody, e.g., antibody fragments and proteins containing the same, such as the chimeric receptors, e.g., TCR-like CARs, and/or engineered cells expressing the TCRs or CARs, binds to a peptide epitope derived from HPV16 E7 protein.
In some aspects, the binding molecule recognizes or binds HPV 16 E6 or E7 epitopes in the context of an MHC molecule, such as an MHC Class I molecule. In some aspects, the MHC Class I molecule is an HLA-A2 molecule, including any one or more subtypes thereof, e.g. HLA-A*0201, *0202, *0203, *0206, or *0207. In some cases, there can be differences in the frequency of subtypes between different populations. For example, in some embodiments, more than 95% of the HLA-A2 positive Caucasian population is HLA-A*0201, whereas in the Chinese population the frequency has been reported to be approximately 23% HLA-A*0201, 45% HLA-A*0207, 8% HLA-A*0206 and 23% HLA-A*0203. In some embodiments, the MHC molecule is HLA-A*0201.
In some embodiments, the TCR or antigen-binding fragment thereof recognizes or binds to an epitope or region of HPV16 E6 or HPV 16 E7, such as a peptide epitope containing an amino acid sequence set forth in any of SEQ ID NOs: 232-239, and as shown below in Table 1.
TABLE 1
HPV-16 Epitopes
Epitope
Epitope
SEQ ID
Description
Name
NO.
KLPQLCTEL
E6(18-26)
232
TIHDIILECV
E6(29-38)
233
FAFRDLCIV
E6(52-60)
234
TLGIVCPI
E7(86-93)
235
YMLDLQPET
E7(11-19)
236
GTLGIVCPI
E7(85-93)
237
LLMGTLGIV
E7(82-90)
238
TLHEYMLDL
E7(7-15)
239
In some embodiments, the binding molecule, e.g., TCR or antigen-binding fragment thereof or antibody or antigen-binding fragment thereof, is isolated or purified or is recombinant. In some aspects, the binding molecule, e.g., TCR or antigen-binding fragment thereof or antibody or antigen-binding fragment thereof, is human. In some embodiments, the binding molecule is monoclonal. In some aspects, the binding molecule is a single chain. In other embodiments, the binding molecule contains two chains. In some embodiments, the binding molecule, e.g., TCR or antigen-binding fragment thereof or antibody or antigen-binding fragment thereof, is expressed on the surface of a cell.
In some aspects, the provided binding molecules have one or more specified functional features, such as binding properties, including binding to particular epitopes, and/or particular binding affinities as described.
A. T Cell Receptors (TCRs)
In some embodiments, the binding molecule that recognizes or binds an epitope or region of HPV 16 is a T cell receptor (TCR) or an antigen-binding fragment thereof.
In some embodiments, a “T cell receptor” or “TCR” is a molecule that contains a variable α and β chains (also known as TCRα and TCRβ, respectively) or a variable γ and δ chains (also known as TCRγ and TCRδ, respectively), or antigen-binding portions thereof, and which is capable of specifically binding to a peptide bound to an MHC molecule. In some embodiments, the TCR is in the αβ form. Typically, TCRs that exist in αβ and γδ forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions. A TCR can be found on the surface of a cell or in soluble form. Generally, a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.
Unless otherwise stated, the term “TCR” should be understood to encompass full TCRs as well as antigen-binding portions or antigen-binding fragments thereof. In some embodiments, the TCR is an intact or full-length TCR, such as a TCR containing the α chain and β chain. In some embodiments, the TCR is an antigen-binding portion that is less than a full-length TCR but that binds to a specific peptide bound in an MHC molecule, such as binds to an MHC-peptide complex. In some cases, an antigen-binding portion or fragment of a TCR can contain only a portion of the structural domains of a full-length or intact TCR, but yet is able to bind the peptide epitope, such as MHC-peptide complex, to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable α (Vα) chain and variable β (Vβ) chain of a TCR, or antigen-binding fragments thereof sufficient to form a binding site for binding to a specific MHC-peptide complex.
In some embodiments, the variable domains of the TCR contain complementarity determining regions (CDRs), which generally are the primary contributors to antigen recognition and binding capabilities and specificity of the peptide, MHC and/or MHC-peptide complex. In some embodiments, a CDR of a TCR or combination thereof forms all or substantially all of the antigen-binding site of a given TCR molecule. The various CDRs within a variable region of a TCR chain generally are separated by framework regions (FRs), which generally display less variability among TCR molecules as compared to the CDRs (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the main CDR responsible for antigen binding or specificity, or is the most important among the three CDRs on a given TCR variable region for antigen recognition, and/or for interaction with the processed peptide portion of the peptide-MHC complex. In some contexts, the CDR1 of the alpha chain can interact with the N-terminal part of certain antigenic peptides. In some contexts, CDR1 of the beta chain can interact with the C-terminal part of the peptide. In some contexts, CDR2 contributes most strongly to or is the primary CDR responsible for the interaction with or recognition of the MHC portion of the MHC-peptide complex. In some embodiments, the variable region of the β-chain can contain a further hypervariable region (CDR4 or HVR4), which generally is involved in superantigen binding and not antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).
In some embodiments, the α-chain and/or β-chain of a TCR also can contain a constant domain, a transmembrane domain and/or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997). In some aspects, each chain (e.g. alpha or beta) of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end. In some embodiments, a TCR, for example via the cytoplasmic tail, is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. In some cases, the structure allows the TCR to associate with other molecules like CD3 and subunits thereof. For example, a TCR containing constant domains with a transmembrane region may anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex. The intracellular tails of CD3 signaling subunits (e.g. CD3γ, CD3δ, CD3ε and CD3ζ chains) contain one or more immunoreceptor tyrosine-based activation motif or ITAM and generally are involved in the signaling capacity of the TCR complex.
It is within the level of a skilled artisan to determine or identify the various domains or regions of a TCR. In some cases, the exact locus of a domain or region can vary depending on the particular structural or homology modeling or other features used to describe a particular domain. It is understood that reference to amino acids, including to a specific sequence set forth as a SEQ ID NO used to describe domain organization of a TCR are for illustrative purposes and are not meant to limit the scope of the embodiments provided. In some cases, the specific domain (e.g. variable or constant) can be several amino acids (such as one, two, three or four) longer or shorter. In some aspects, residues of a TCR are known or can be identified according to the International Immunogenetics Information System (IMGT) numbering system (see e.g. www.imgt.org; see also, Lefranc et al. (2003) Developmental and Comparative Immunology, 2&; 55-77; and The T Cell Factsbook 2nd Edition, Lefranc and LeFranc Academic Press 2001). Using this system, the CDR1 sequences within a TCR Vα chains and/or Vβ chain correspond to the amino acids present between residue numbers 27-38, inclusive, the CDR2 sequences within a TCR Vα chain and/or Vβ chain correspond to the amino acids present between residue numbers 56-65, inclusive, and the CDR3 sequences within a TCR Vα chain and/or Vβ chain correspond to the amino acids present between residue numbers 105-117, inclusive.
In some embodiments, the α chain and β chain of a TCR each further contain a constant domain. In some embodiments, the α chain constant domain (Cα) and β chain constant domain (Cβ) individually are mammalian, such as is a human or murine constant domain. In some embodiments, the constant domain is adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains, and two membrane-distal variable domains, which variable domains each contain CDRs.
In some embodiments, each of the Cα and Cβ domains is human. In some embodiments, the Cα is encoded by the TRAC gene (IMGT nomenclature) or is a variant thereof. In some embodiments, the Cα has or comprises the sequence of amino acids set forth in SEQ ID NO: 213 or 220 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 213 or 220. In some embodiments, the Cα has or comprises the sequence of amino acids set forth in SEQ ID NO: 212, 215 or 217 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 212, 215 or 217. In some embodiments, the Cα has or comprises the sequence of amino acids set forth in any of SEQ ID NOS: 212, 213, 215, 217, 220, or 524. In some embodiments, the Cβ is encoded by TRBC1 or TRBC2 genes (IMGT nomenclature) or is a variant thereof. In some embodiments, the Cβ has or comprises the sequence of amino acids set forth in SEQ ID NO:214, 216, 631, or 889 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 214, 216, 631, or 889. In some embodiments, the Cβ has or comprises the sequence of amino acids set forth in SEQ ID NO: 214, 216, 631, or 889.
In some embodiments, any of the provided TCRs or antigen-binding fragments thereof can be a human/mouse chimeric TCR. In some cases, the TCR or antigen-binding fragment thereof comprises an alpha chain and/or a beta chain comprising a mouse constant region. In some embodiments, the Cα is a mouse constant region that is or comprises the sequence of amino acids set forth in SEQ ID NO: 262, 317, 833, 1012, 1014, 1015, 1017 or 1018 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 262, 317, 833, 1012, 1014, 1015, 1017 or 1018. In some embodiments, the Cα is or comprises the sequence of amino acids set forth in SEQ ID NO: 262, 317, 833, 1012, 1014, 1015, 1017 or 1018. In some embodiments, the Cβ is a mouse constant region that is or comprises the sequence of amino acids set forth in SEQ ID NO: 263, 109, 1013 or 1016 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 263, 109, 1013 or 1016. In some embodiments, the Cβ is or comprises the sequence of amino acids set forth in SEQ ID NO: 263, 109, 1013 or 1016. In some embodiments, the Cα is or comprises the sequence of amino acids set forth in SEQ ID NO: 262 or 1014 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 262 or 1014 and/or the Cβ is or comprises the sequence of amino acids set forth in SEQ ID NO: 263 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 263. In some embodiments, the Cα and/or Cβ is or comprises any Cα and/or Cβ described in WO 2015/184228, WO 2015/009604 and WO 2015/009606.
In some embodiments, the TCR or antigen-binding fragment thereof herein comprises a variant of an alpha chain and/or a beta chain, e.g., an alpha and/or beta chain that comprises a mouse constant region. In some embodiments, the variant comprises the amino acid sequence of any of the TCRs described herein with one, two, three, or four or more amino acid substitution(s) in the constant region of the alpha or beta chain. In some embodiments, the variant comprises the amino acid sequence of any of the constant regions described herein with one, two, three, or four or more amino acid substitution(s) in the constant region. In some embodiments, the TCRs (or functional portions thereof) comprising the substituted amino acid sequence(s) advantageously provide one or more of increased recognition of HPV 16 targets, increased expression by a host cell, and increased anti-tumor activity as compared to the parent TCR comprising an unsubstituted amino acid sequence.
In some embodiments, the substituted amino acid sequences of the mouse constant regions of the TCR α and β chains, SEQ ID NOs: 1015 and 1016, respectively, correspond with all or portions of the unsubstituted mouse constant region amino acid sequences SEQ ID NOs: 1014 and 263, respectively, with SEQ ID NO: 1015 having one, two, three, or four amino acid substitution(s) when compared to SEQ ID NO: 1014 and SEQ ID NO: 1016 having one amino acid substitution when compared to SEQ ID NO: 263. In some embodiments, a variant of a TCR comprises the amino acid sequences of (a) SEQ ID NO: 1015 (constant region of alpha chain), wherein (i) X at position 48 is Thr or Cys; (ii) X at position 112 is Ser, Gly, Ala, Val, Leu, Be, Pro, Phe, Met, or Trp; (iii) X at position 114 is Met, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; and (iv) X at position 115 is Gly, Ala, Val, Leu, Be, Pro, Phe, Met, or Trp; and (b) SEQ ID NO: 1016 (constant region of beta chain), wherein X at position 56 is Ser or Cys. In some embodiments, the Cα is or comprises the sequence of amino acids set forth in SEQ ID NO: 1015 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1015 and/or the Cβ is or comprises the sequence of amino acids set forth in SEQ ID NO: 1016 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1016.
In some embodiments, the TCR may be a heterodimer of two chains α and β that are linked, such as by a disulfide bond or disulfide bonds. In some embodiments, the constant domain of the TCR may contain short connecting sequences in which a cysteine residue forms a disulfide bond, thereby linking the two chains of the TCR. In some embodiments, a TCR may have an additional cysteine residue in each of the α and β chains, such that the TCR contains two disulfide bonds in the constant domains. In some embodiments, each of the constant and variable domains contains disulfide bonds formed by cysteine residues.
In some embodiments, the TCR can contain an introduced disulfide bond or bonds. In some embodiments, the native disulfide bonds are not present. In some embodiments, the one or more of the native cysteines (e.g. in the constant domain of the α chain and β chain) that form a native interchain disulfide bond are substituted to another residue, such as to a serine or alanine. In some embodiments, an introduced disulfide bond can be formed by mutating non-cysteine residues on the alpha and beta chains, such as in the constant domain of the α chain and β chain, to cysteine. Opposing cysteines in the TCR α and β chains in provide a disulfide bond that links the constant regions of TCR α and β chains of the substituted TCR to one another and which is not present in a TCR comprising the unsubstituted human constant region or the unsubstituted mouse constant region. In some embodiments, the presence of non-native cysteine residues (e.g. resulting in one or more non-native disulfide bonds) in a recombinant TCR can favor production of the desired recombinant TCR in a cell in which it is introduced over expression of a mismatched TCR pair containing a native TCR chain.
Exemplary non-native disulfide bonds of a TCR are described in published International PCT No. WO2006/000830 and WO2006/037960. In some embodiments, cysteines can be introduced or substituted at a residue corresponding to Thr48 of the Cα chain and Ser57 of the Cβ chain, at residue Thr45 of the Cα chain and Ser77 of the Cβ chain, at residue Tyr10 of the Cα chain and Ser17 of the Cβ chain, at residue Thr45 of the Cα chain and Asp59 of the Cβ chain and/or at residue Ser15 of the Cα chain and Glu15 of the Cβ chain with reference to numbering of a Cα set forth in any of SEQ ID NOS: 212, 213, 217, or 524, or Cβ set forth in SEQ ID NO: 214 or 216. In some embodiments, the variant of the TCR is a cysteine-substituted, chimeric TCR in which one or both of the native Thr48 of SEQ ID NO: 1014 and the native Ser56 of SEQ ID NO: 263 is substituted with Cys. In some embodiments, the Cα is or comprises the sequence of amino acids set forth in SEQ ID NO: 1017 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1017 and/or the Cβ is or comprises the sequence of amino acids set forth in SEQ ID NO: 1016 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1013.
In some embodiments, any of the provided cysteine mutations can be made at a corresponding position in another sequence, for example, in the mouse Cα and Cβ sequences described above. The term “corresponding” with reference to positions of a protein, such as recitation that amino acid positions “correspond to” amino acid positions in a disclosed sequence, such as set forth in the Sequence listing, refers to amino acid positions identified upon alignment with the disclosed sequence based on structural sequence alignment or using a standard alignment algorithm, such as the GAP algorithm. For example, corresponding residues can be determined by alignment of a reference sequence with the Cα sequence set forth in any of SEQ ID NOS: 212, 213, 215, 217, 220, or 524, or the Cβ sequence set forth in SEQ ID NO: 214, 216, 631, or 889, by structural alignment methods as described herein. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides.
In some embodiments, the variant includes substitutions of one, two, or three amino acids in the transmembrane (TM) domain of the constant region of one or both of the α and β chains with a hydrophobic amino acid to provide a hydrophobic amino acid-substituted TCR. The hydrophobic amino acid substitution(s) in the TM domain of the TCR may increase the hydrophobicity of the TM domain of the TCR as compared to a TCR that lacks the hydrophobic amino acid substitution(s) in the TM domain. In some embodiments, the variant of the TCR comprises one, two, or three of the native Ser 112, Met 114, and Gly 115 of SEQ ID NO: 1014 may, independently, be substituted with Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; for example with Leu, Ile, or Val. In some embodiments, the Cα is or comprises the sequence of amino acids set forth in SEQ ID NO: 1018 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1018 and/or the Cβ is or comprises the sequence of amino acids set forth in SEQ ID NO: 263 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 263.
In some embodiments, the variant includes substitutions cysteine substitutions in the constant region of one or both of the α and β chains in combination with the substitution(s) of one, two, or three amino acids in the transmembrane (TM) domain of the constant region of one or both of the α and β chains with a hydrophobic amino acid. In some embodiments, the variant has the native Thr48 of SEQ ID NO: 1014 substituted with Cys; one, two, or three of the native Ser 112, Met 114, and Gly 115 of SEQ ID NO: 1014, independently, substituted with Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; for example with Leu, Ile, or Val; and the native Ser56 of SEQ ID NO: 19 substituted with Cys. In some embodiments, the Cα is or comprises the sequence of amino acids set forth in SEQ ID NO: 833 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 833 and/or the Cβ is or comprises the sequence of amino acids set forth in SEQ ID NO: 1013 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1013.
Exemplary sequences (e.g. CDRs, Vα and/or Vβ and constant region sequences) of provided TCRs are described below.
In some embodiments, the TCR is a full-length TCR. In some embodiments, the TCR is an antigen-binding portion. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR (sc-TCR). A TCR may be cell-bound or in soluble form. In some embodiments, the TCR is in cell-bound form expressed on the surface of a cell.
In some embodiments a dTCR contains a first polypeptide wherein a sequence corresponding to a provided TCR α chain variable region sequence is fused to the N terminus of a sequence corresponding to a TCR α chain constant region extracellular sequence, and a second polypeptide wherein a sequence corresponding to a provided TCR β chain variable region sequence is fused to the N terminus a sequence corresponding to a TCR β chain constant region extracellular sequence, the first and second polypeptides being linked by a disulfide bond. In some embodiments, the bond can correspond to the native interchain disulfide bond present in native dimeric αβ TCRs. In some embodiments, the interchain disulfide bonds are not present in a native TCR. For example, in some embodiments, one or more cysteines can be incorporated into the constant region extracellular sequences of dTCR polypeptide pair. In some cases, both a native and a non-native disulfide bond may be desirable. In some embodiments, the TCR contains a transmembrane sequence to anchor to the membrane.
In some embodiments, a dTCR contains a provided TCR α chain containing a variable α domain, a constant α domain and a first dimerization motif attached to the C-terminus of the constant α domain, and a provided TCR β chain comprising a variable β domain, a constant β domain and a first dimerization motif attached to the C-terminus of the constant β domain, wherein the first and second dimerization motifs easily interact to form a covalent bond between an amino acid in the first dimerization motif and an amino acid in the second dimerization motif linking the TCR α chain and TCR β chain together.
In some embodiments, the TCR is a scTCR, which is a single amino acid strand containing an α chain and a β chain that is able to bind to MHC-peptide complexes. Typically, a scTCR can be generated using methods known to those of skill in the art, See e.g., International published PCT Nos. WO 96/13593, WO 96/18105, WO99/18129, WO 04/033685, WO2006/037960, WO2011/044186; U.S. Pat. No. 7,569,664; and Schlueter, C. J. et al. J. Mol. Biol. 256, 859 (1996).
In some embodiments, a scTCR contains a first segment constituted by an amino acid sequence corresponding to a sequence of a provided TCR α chain variable region, a second segment constituted by an amino acid sequence corresponding to a provided TCR β chain variable region sequence fused to the N terminus of an amino acid sequence corresponding to a TCR β chain constant domain extracellular sequence, and a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.
In some embodiments, a scTCR contains a first segment constituted by an amino acid sequence corresponding to a provided TCR β chain variable region, a second segment constituted by an amino acid sequence corresponding to a provided TCR α chain variable region sequence fused to the N terminus of an amino acid sequence corresponding to a TCR α chain constant domain extracellular sequence, and a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.
In some embodiments, a scTCR contains a first segment constituted by a provided α chain variable region sequence fused to the N terminus of an α chain extracellular constant domain sequence, and a second segment constituted by a provided β chain variable region sequence fused to the N terminus of a sequence β chain extracellular constant and transmembrane sequence, and, optionally, a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.
In some embodiments, a scTCR contains a first segment constituted by a provided TCR β chain variable region sequence fused to the N terminus of a β chain extracellular constant domain sequence, and a second segment constituted by a provided α chain variable region sequence fused to the N terminus of a sequence α chain extracellular constant and transmembrane sequence, and, optionally, a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.
In some embodiments, for the scTCR to bind an MHC-peptide complex, the α and β chains must be paired so that the variable region sequences thereof are orientated for such binding. Various methods of promoting pairing of an α and β in a scTCR are well known in the art. In some embodiments, a linker sequence is included that links the α and β chains to form the single polypeptide strand. In some embodiments, the linker should have sufficient length to span the distance between the C terminus of the α chain and the N terminus of the β chain, or vice versa, while also ensuring that the linker length is not so long so that it blocks or reduces bonding of the scTCR to the target peptide-MHC complex.
In some embodiments, the linker of a scTCRs that links the first and second TCR segments can be any linker capable of forming a single polypeptide strand, while retaining TCR binding specificity. In some embodiments, the linker sequence may, for example, have the formula -P-AA-P-, wherein P is proline and AA represents an amino acid sequence wherein the amino acids are glycine and serine. In some embodiments, the first and second segments are paired so that the variable region sequences thereof are orientated for such binding. Hence, in some cases, the linker has a sufficient length to span the distance between the C terminus of the first segment and the N terminus of the second segment, or vice versa, but is not too long to block or reduces bonding of the scTCR to the target ligand. In some embodiments, the linker can contain from or from about 10 to 45 amino acids, such as 10 to 30 amino acids or 26 to 41 amino acids residues, for example 29, 30, 31 or 32 amino acids. In some embodiments, the linker has the formula -PGGG-(SGGGG)n-P-, wherein n is 5 or 6 and P is proline, G is glycine and S is serine (SEQ ID NO: 266). In some embodiments, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO: 267).
In some embodiments, a scTCR contains a disulfide bond between residues of the single amino acid strand, which, in some cases, can promote stability of the pairing between the α and β regions of the single chain molecule (see e.g. U.S. Pat. No. 7,569,664). In some embodiments, the scTCR contains a covalent disulfide bond linking a residue of the immunoglobulin region of the constant domain of the α chain to a residue of the immunoglobulin region of the constant domain of the β chain of the single chain molecule. In some embodiments, the disulfide bond corresponds to the native disulfide bond present in a native dTCR. In some embodiments, the disulfide bond in a native TCR is not present. In some embodiments, the disulfide bond is an introduced non-native disulfide bond, for example, by incorporating one or more cysteines into the constant region extracellular sequences of the first and second chain regions of the scTCR polypeptide. Exemplary cysteine mutations include any as described above. In some cases, both a native and a non-native disulfide bond may be present.
In some embodiments, a scTCR is a non-disulfide linked truncated TCR in which heterologous leucine zippers fused to the C-termini thereof facilitate chain association (see e.g. International published PCT No. WO99/60120). In some embodiments, a scTCR contain a TCRα variable domain covalently linked to a TCRβ variable domain via a peptide linker (see e.g., International published PCT No. WO99/18129).
In some embodiments, any of the provided TCRs, including a dTCR or scTCR, can be linked to signaling domains that yield an active TCR on the surface of a T cell. In some embodiments, the TCR is expressed on the surface of cells. In some embodiments, the TCR does contain a sequence corresponding to a transmembrane sequence. In some embodiments, the transmembrane domain is positively charged. In some embodiments, the transmembrane domain can be a Cα or Cβ transmembrane domain. In some embodiments, the transmembrane domain can be from a non-TCR origin, for example, a transmembrane region from CD3z, CD28 or B7.1. In some embodiments, the TCR does contain a sequence corresponding to cytoplasmic sequences. In some embodiments, the TCR contains a CD3z signaling domain. In some embodiments, the TCR is capable of forming a TCR complex with CD3.
In some embodiments, the TCR is a soluble TCR. In some embodiments, the soluble TCR has a structure as described in WO99/60120 or WO 03/020763. In some embodiments, the TCR does not contain a sequence corresponding to the transmembrane sequence, for example, to permit membrane anchoring into the cell in which it is expressed. In some embodiments, the TCR does not contain a sequence corresponding to cytoplasmic sequences.
1. Exemplary TCRs
In some embodiments, among the provided -TCRs or antigen-binding fragment thereof that bind or recognize a peptide epitope of HPV 16 in the context of an MHC (e.g. a peptide epitope of HPV 16 E6 or a peptide epitope of HPV 16 E7) are TCRs or antigen-binding fragments thereof that contain any of the alpha and/or beta chain variable (Vα or Vβ) region sequences as described, individually, or a sufficient antigen-binding portion of such chain(s). In some embodiments, the provided anti-HPV 16 TCR or antigen-binding fragment thereof (e.g. anti-HPV 16 E6 or anti-HPV 16 E7 TCRs) contains a Vα region sequence or sufficient antigen-binding portion thereof that contains a CDR-1, CDR-2 and/or CDR-3 as described. In some embodiments, the provided anti-HPV 16 TCR or antigen-binding fragment thereof (e.g., anti-HPV 16 E6 or anti-HPV 16 E7 TCRs) contains a Vβ region sequence or sufficient antigen-binding portion that contains a CDR-1, CDR-2 and/or CDR-3 as described. In some embodiments, the anti-HPV 16 TCR or antigen-binding fragment thereof (e.g. anti-HPV 16 E6 or anti-HPV 16 E7 TCRs) contains a Vα region sequence that contains a CDR-1, CDR-2 and/or CDR-3 as described and contains a Vβ region sequence that contains a CDR-1, CDR-2 and/or CDR-3 as described. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such a sequence.
In some embodiments, the TCR contains a Vα region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18 (SEQ ID NO: 251), where X1 is A, I, or V; X2 is M, L, V, E or A; X3 is R, L, N, or S; X4 is E, V, P, T, F, I, R or A; X5 is G, I, L, A, P, R, D, or H; X6 is R, T, G, S, N or H; X7 is G, R, A, N, or null; X8 is T, G, or null; X9 is null, A or G; X10 is null or G; X11 is null or G; X12 is null or T; X13 is F, Y, A, S or null; X14 is G, Y, or N; X15 is F, G, T, N, Q, or Y; X16 is K, P, V, N or A; X17 is T, L, or F; and X18 is I, V, T, H, or N.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 138, 144, 147, 153, 159, 163, 167, 173, 175, 301, 304, 308, 478, 493, 505, 511, 523, 539, 555, 572, 588, 600, 612, 624, 638, 650, 662, 679, 694, 712, 729, 744, 762, 776, 788, 802, 818, 832, 846, 858, 870, 882, 896, 911, 926, 940, 952, 964, 976, 988, or 1002, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some aspects, the TCR or antigen-binding fragment thereof contains a Vα region containing a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 117, 119, 121, 123, 125, 127, 295, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, 676, 691, 709, 726, 741, 759, 775, 787, 799, 815, 830, 845, 857, 869, 881, 895, 908, 925, 937, 951, 963, 975, 987, or 999, or a sequence at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical with such a sequence.
In some embodiments, the TCR contains a Vβ region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15 (SEQ ID NO: 261), where X1 is A or S; X2 is 5, I, or V; X3 is S, T, or V; X4 is H, P, L, Y, T, D, or Q; X5 is L, G, W, F, S, or R; X6 is A, G, L, S, or T; X7 is G, E, A, T, R, or null; X8 is null or G; X9 is null or G; X10 is null, F, G, T, S, or A; X11 is T, N, H, A, S, or F; X12 is G, T, Q, D, Y, or L; X13 is E, P, T, G or W; X14 is L, A, Q, Y, or K; and X15 is F, H, Y, or T.
In some instances, the TCR contains a Vβ region containing a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 141, 146, 150, 156, 160, 164, 170, 174, 178, 305, 309, 486, 499, 517, 531, 548, 563, 581, 594, 606, 618, 630, 644, 656, 670, 686, 703, 721, 736, 753, 769, 782, 794, 809, 825, 840, 852, 864, 876, 888, 902, 919, 932, 946, 958, 970, 982, 994, or 1010, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some embodiments, the TCR contains a Vβ region containing a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 118, 120, 122, 124, 126, 128, 296, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, 685, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008 or a sequence at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical with such a sequence.
In some aspects, the Vα region further contains a complementarity determining region 1 (CDR-1) comprising the amino acid sequence X1X2X3X4X5X6X7 (SEQ ID NO: 243), where X1 is T, D, N, or V; X2 is I or S; X3 is S, D, A, P, or M; X4 is G, Q, P, or null; X5 is T, S, I, or F; X6 is D, Y, Q, T, or S; and X7 is Y, G, N, or Q. In some embodiments, the Vα region further contains a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6X7X8 (SEQ ID NO: 247), where X1 is G, Q, I, V, or M; X2 is L, S, Q, Y, F, T, or G; X3 is T, G, S, or F; X4 is Y, S, N, I, or null; X5 is null or D; X6 is null, E, Q, S, M, or K; X7 is S, Q, R, G, D, or N; and X8 is N, E, M, T, or K.
In some embodiments, the Vα region contains a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 136, 142, 151, 157, 161, 165, 171, 302, 306, 537, 570, 677, 692, 710, 727, 742, 760, 800, 816, 909, 938, or 1000, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some aspects, the Vα region contains a CDR-1 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 117, 119, 121, 123, 125, 127, 295, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, 676, 691, 709, 726, 741, 759, 775, 787, 799, 815, 830, 845, 857, 869, 881, 895, 908, 925, 937, 951, 963, 975, 987, or 999, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some embodiments, the Vα region contains a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 137, 143, 152, 158, 162, 166, 172, 303, 307, 538, 571, 678, 693, 711, 728, 743, 761, 801, 817, 831, 833, 910, 939, or 1001, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some embodiments, the Vα region contains a CDR-2 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 117, 119, 121, 123, 125, 127, 295, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, 676, 691, 709, 726, 741, 759, 775, 787, 799, 815, 830, 845, 857, 869, 881, 895, 908, 925, 937, 951, 963, 975, 987, or 999, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some aspects, the Vβ region further contains a complementarity determining region 1 (CDR-1) comprising the amino acid sequence X1X2X3X4X5 (SEQ ID NO: 254), where X1 is S, M, or L; X2 is G, E, D, N, or Q; X3 is H or V; X4 is V, N, E, L, or T; and X5 is S, R, N, Y, A, or M. In some embodiments, the Vβ region further contains a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6X7 (SEQ ID NO: 257), where X1 is F, Y, S, or A; X2 is Q, Y, V, or N; X3 is N, D, G, F, or Q; X4 is null or G; X5 is E, V, N, K, or S; X6 is A, K, G, or E; and X7 is Q, M, T, I, or A.
In some instances, the Vβ region contains a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 139, 145, 148, 154, 168, 176, 484, 546, 561, 579, 668, 701, 719, or 751, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some aspects, the Vβ region contains a CDR-1 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 118, 120, 122, 124, 126, 128, 296, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, 685, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some embodiments, the Vβ region contains a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 140, 149, 155, 169, 177, 485, 547, 562, 580, 669, 702, 720, 752, 918, or 1009, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some embodiments, the Vβ region contains a CDR-2 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 118, 120, 122, 124, 126, 128, 296, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, 685, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the Vα region contains the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 117, 119, 121, 123, 125, 127, 295, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, 676, 691, 709, 726, 741, 759, 775, 787, 799, 815, 830, 845, 857, 869, 881, 895, 908, 925, 937, 951, 963, 975, 987, or 999, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some instances, the Vβ region contains the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 118, 120, 122, 124, 126, 128, 296, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, 685, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the TCR contains an alpha chain comprising any of such Vα chain sequences and any of such Vβ chain sequences.
In some embodiments, the alpha chain of the TCR or antigen-binding fragment thereof further contains an alpha constant (Cα) region or portion thereof. In some aspects, the beta chain further contains a beta constant (Cβ) region or portion thereof. Thus, in some embodiments, the TCR, e.g., the HPV 16 E6 or E7 TCR or antigen-binding fragment thereof, contains an alpha chain comprising a variable alpha (Vα) region and an alpha constant (Cα) region or portion thereof and/or a beta chain comprising a variable beta (Vβ) region and a beta constant region (Cβ) or portion thereof.
In some cases, the Cα and Cβ regions are mouse constant regions. In some embodiments, the Cα region contains the amino acid sequence set forth in SEQ ID NO: 262 or 317, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some cases, the Cβ region contains the amino acid sequence set forth in SEQ ID NO: 263 or 109, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the Cα and Cβ regions are human constant regions. In some such embodiments, the Cα region comprises the amino acid sequence set forth in any of SEQ ID NOs: 212, 213, 215, 217, 218, 220, or 524, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some aspects, the Cβ region contains the amino acid sequence set forth in SEQ ID NO: 214, 216, 631, or 889, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the Cα and/or Cβ regions are modified, for example, by incorporation of one or more non-native cysteine residues. In some embodiments, the constant region is a modified form of a human constant region (e.g. modified compared to a Cα region set forth in any of SEQ ID NOs: 212, 213, 215, 217, 218, 220, or 524, and/or a Cβ region set forth in SEQ ID NO:214, 216, 631, or 889. In some embodiments, the modification is by introduction of cysteine at residue Thr48 of the Cα chain and/or Ser57 of the Cβ chain, at residue Thr45 of the Cα chain and/or Ser77 of the Cβ chain, at residue Tyr10 of the Cα chain and/or Ser17 of the Cβ chain, at residue Thr45 of the Cα chain and Asp59 of the Cβ chain and/or at residue Ser15 of the Cα chain and Glu15 of the Cβ chain with reference to numbering of a Cα set forth in any of SEQ ID NOS: 212, 213, 217, 218 or 524 or Cβ set forth in SEQ ID NO: 214 or 216. Corresponding residues can be identified by aligning a reference sequence to any of SEQ ID NOS: 212, 213, 217, 218 or 524 or 214 or 216. For example, Thr48 in the Cα chain aligns with or corresponds to Thr49 in the sequence set forth in SEQ ID NO: 215 or 220 and Ser57 in the Cβ chain aligns with or corresponds to Ser58 in the sequence set forth in SEQ ID NO:631 or 889. In some such embodiments, the Cα region contains a non-native cysteine at residue 48 (or at a corresponding residue, e.g. residue 49) and comprises the amino acid sequence set forth in any of SEQ ID NOs: 196, 198, 200, 201, 203, 525, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence and that contains the introduced non-native cysteine residue or residues. In some aspects, the Cβ region contains a non-native cysteine at residue 57 (or at a corresponding residue, e.g. residue 58) and contains the amino acid sequence set forth in SEQ ID NO: 197, 199, 632, or 890, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence and that contains the non-native cysteine residue or residues.
In some embodiments, the TCR or antigen-binding fragment thereof comprises an alpha chain comprising the sequence of amino acids set forth in SEQ ID NO: 18, 28, 38, 48, 58, 68, 78, 88, 98, 287, or 291 or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence and/or a beta chain comprising the sequence of amino acids set forth in SEQ ID NO: 22, 32, 42, 52, 62, 72, 82, 92, 102, 285, 289, 293, 479, 494, 512, 526, 541, 556, 574, 589, 601, 613, 625, 639, 651, 663, 681, 696, 714, 731, 746, 764, 777, 789, 804, 820, 835, 847, 859, 871, 883, 897, 913, 927, 941, 953, 965, 977, 989, or 1004 or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the TCR or antigen-binding fragment thereof comprises an alpha chain comprising the sequence of amino acids set forth in SEQ ID NO: 19, 29, 39, 49, 59, 69, 79, 89, 99, 284, 288, 292, 474, 489, 501, 507, 519, 533, 551, 566, 584, 596, 608, 620, 634, 646, 658, 673, 688, 706, 723, 738, 756, 772, 784, 796, 812, 827, 842, 854, 866, 878, 892, 905, 922, 934, 948, 960, 972, 984, or 996, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence and/or a beta chain comprising the sequence of amino acids set forth in SEQ ID NO: 23, 33, 43, 53, 63, 73, 83, 93, 103, 286, 290, 294, 480, 495, 513, 527, 542, 557, 575, 590, 602, 614, 626, 640, 652, 664, 682, 697, 715, 732, 747, 765, 778, 790, 805, 821, 836, 848, 860, 872, 884, 898, 914, 928, 942, 954, 966, 978, 990, or 1005, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the alpha chain and/or beta chain of the TCR is encoded by a sequence of nucleotides comprising a signal peptide (also called a leader sequence). Non-limiting examples of such a signal peptide are signal peptides that have or comprise the sequence of amino acids set forth in any of SEQ ID NOS: 180-182, 184-194, 310, 311, 487, 540, 549, 564, 573, 582, 671, 680, 695, 704, 713, 730, 745, 754, 763, 770, 803, 810, 819, 834, 903, 912, 920, 1003, or 1011. In some embodiments, the TCR or antigen-binding fragment thereof is encoded by a sequence of nucleotides that encodes: a) an alpha chain comprising the sequence of amino acids set forth in SEQ ID NO: 318, 319, 322, 323, 326, 327, 330, 331, 334, 335, 338, 339, 130, 131, 134, 135, 195, 205, 222, 242, 253, 256, 313, 314, 475, 476, 490, 491, 502, 503, 508, 509, 520, 521, 534, 535, 552, 553, 567, 568, 585, 586, 597, 598, 609, 610, 621, 622, 635, 636, 647, 648, 659, 660, 674, 675, 689, 690, 707, 708, 724, 725, 739, 740, 757, 758, 773, 774, 785, 786, 797, 798, 813, 814, 828, 829, 843, 844, 855, 856, 867, 868, 879, 880, 893, 894, 906, 907, 923, 924, 935, 936, 949, 950, 961, 962, 973, 974, 985, 986, 997, 998, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence and/or b) a beta chain comprising the sequence of amino acids set forth in SEQ ID NO: 320, 321, 324, 325, 328, 329, 332, 333, 336, 337, 110, 129, 132, 133, 179, 180, 206, 221, 246, 250, 260, 312, 315, 316, 481, 482, 496, 497, 514, 515, 616, 528, 529, 543, 544, 558, 559, 576, 577, 591, 592, 603, 604, 615, 627, 628, 641, 642, 653, 654, 665, 666, 683, 684, 698, 699, 716, 717, 733, 734, 748, 749, 766, 767, 779, 780, 791, 792, 806, 807, 822, 823, 837, 838, 849, 850, 861, 862, 873, 874, 885, 886, 899, 900, 915, 916, 929, 930, 943, 944, 955, 956, 967, 968, 979, 980, 991, 992, 1006, or 1007, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some embodiments, the alpha chain and beta chain can be connected via a linker, such as any described elsewhere herein.
In some embodiments, the TCR or antigen-binding fragment thereof recognizes or binds to an epitope or region of HPV16 E6, such as a peptide epitope containing an amino acid sequence set forth in any of SEQ ID NOs: 232-234. In some cases, the TCR or antigen-binding fragment thereof does not recognize or bind the epitope E6(29-38) comprising the amino acid sequence TIHDIILECV (SEQ ID NO. 233). In some instances, the TCR or antigen-binding fragment thereof that recognizes or binds a peptide epitope derived from HPV16 E6 is or comprises the sequence set forth in SEQ ID NO: 232 or SEQ ID NO: 234.
In some aspects, the TCR or antigen-binding fragment recognizes or binds to an epitope or region of HPV16 E7 protein, such as a peptide epitope containing an amino acid sequence set forth in any of SEQ ID NOs: 235-239. In some embodiments, the TCR or antigen-binding fragment thereof does not recognize or bind the epitope E7(11-19) comprising the amino acid sequence YMLDLQPET (SEQ ID NO. 236). In some cases, the peptide derived from HPV16 E7 is or contains the sequence set forth in SEQ ID NO: 235.
a. HPV 16 E6(29-38)
In some cases, the TCR recognizes or binds a peptide epitope derived from HPV16 E6 that is or contains E6(29-38) TIHDIILECV (SEQ ID NO: 233). In some embodiments, the TCR recognizes or binds HPV 16 E6 (29-38) in the context of an MHC, such as an MHC class I, e.g. HLA-A2.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18 (SEQ ID NO: 248), where X1 is A, I, or V; X2 is M, L, or V; X3 is R, L, or N; X4 is E, V, T, P, or F; X5 is G, I, L, A, or P; X6 is R, T, G, or S; X7 is G, R, or null; X8 is T, G, or null; X9 is null or A; X10 is null or G; X11 is null or G; X12 is null or T; X13 is null or S; X14 is G, Y, or N; X15 is F, G, or T; X16 is K or P; X17 is T or L; and X18 is I, V or T.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18 (SEQ ID NO:1205), where X1 is A, I, or V; X2 is M, L, A, V, S, or E; X3 is R, L, N, S, Q, K, G, or W; X4 is E, V, P, T, F, A, G, N, D, or L; X5 is G, I, D, L, A, P, H, N, R, T, or null; X6 is G, N, R, T, M, S, P, or null; X7 is G, V, D, L, Q, T, R, N, or null; X8 is T, D, S, L, G, or null; X9 is A, G, Q, or null; X10 is G, or null; X11 is G, or null; X12 is T, or null; X13 is S, A, T, G, or null; X14 is G, Y, T, N, A, W, or null; X15 is F, G, N, T, Y, D, S, R, Q, or E; X16 is K, P, A, N, D, or Q; X17 is L, M, I, V, or T; and X18 is I, T, V, N, F, R, or Q.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18 (SEQ ID NO:1220), where X1 is A, I, or V; X2 is M, L, A, V, S, or E; X3 is R, L, N, S, Q, K, G, or W; X4 is E, V, P, T, F, A, G, N, D, or L; X5 is G, I, D, L, A, P, N, R, T, or null; X6 is G, N, R, T, M, S, P, or null; X7 is G, V, D, L, Q, T, R, or null; X8 is T, D, S, L, G, or null; X9 is A, G, Q, or null; X10 is G, or null; X11 is G, or null; X12 is T, or null; X13 is S, A, T, G, or null; X14 is G, Y, T, N, A, W, or null; X15 is F, G, N, T, Y, D, S, R, Q, or E; X16 is K, P, A, D, or Q; X17 is L, M, I, V, or T; and X18 is I, T, V, F, R, or Q.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16LT (SEQ ID NO: 1206), where X1 is A, I, or V; X2 is L, M, V, or E; X3 is L, R, N, G, or S; X4 is V, T, F, N, E, P, G, or L; X5 is I, A, P, N, G, or T; X6 is R, G, S, or T; X7 is G, R, L, V, or T; X8 is T, G, L, or null; X9 is A, G, Q, or null; X10 is G, or null; X11 is G, or null; X12 is T, or null; X13 is S, T, or G; X14 is Y, A, G, or N; X15 is G, S, N, R, or E; and X16 is K, or Q.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AMRX4X5X6X7X8X9X10X11X12X13X14X15(SEQ ID NO:1207), where X4 is E, T, A, D, or L; X5 is G, A, N, or R; X6 is R, G, R, T, M, or S; X7 is G, V, D, L, or null; X8 is T, D, or null; X9 is G, or null; X10 is S, T, G, or null; X11 is G, Y, N, A, or W; X12 is F, G, N, D, S, or Y; X13 is K, D, Q; X14 is T, L, M, or I; and X15 is I, T, R, or Q.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15KX17X18 (SEQ ID NO:1208), where X1 is I, or V; X2 is L, or V; X3 is L, N, or R; X4 is V, F, or G; X5 is I, P, G, or T; X6 is R, S, P, or G; X7 is G, R, Q, T, or V; X8 is T, G, S, or L; X9 is A, G, Q, or null; X10 is G, or null; X11 is G, or null; X12 is T, or null; X13 is G, or S; X14 is Y, or N; X15 is G, Q, or E; X17 is V, or L; and X18 is I, or T.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2RX4AX6NNDMR (SEQ ID NO:1221), where X2 is V, or M; X4 is P, or D; X6 is N, or R.
In some embodiments, the Vα region contains a complementarity determining region 1 (CDR-1) comprising the amino acid sequence X1X2X3X4X5X6X7 (SEQ ID NO: 240), where X1 is T, D, or N; X2 is I, or S; X3 is S, D, or A; X4 is G, Q, P, or null; X5 is T, S, or I; X6 is D, Y, or Q; and X7 is Y, G, N, or Q. In some embodiments, the Vα region contains a complementarity determining region 1 (CDR-1) comprising the amino acid sequence X1X2X3X4X5X6X7 (SEQ ID NO: 1209), where X1 is T, N, D, or S; X2 is 5, I, or R; X3 is D, S, M, A, Y, N, or G; X4 is Q, G, P, or null; X5 is S, T, F, I, or N; X6 is Y, D, Q, P, N, or E; and X7 is G, Y, N, S, or A.
In some examples, the Vα region contains a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6X7X8 (SEQ ID NO: 244), where X1 is G, Q, I, or V; X2 is L, S, Q, or Y; X3 is T, G, or S; X4 is Y, S, or null; X5 is null or D; X6 is null, E, Q, or S; X7 is S, Q, R, or G; and X8 is N or E. In some examples, the Vα region contains a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6X7X8 (SEQ ID NO:1210), where X1 is Q, G, I, V, Y, M, R, or N; X2 is G, L, S, Q, Y, T, N, or V; X3 is S, T, L, or K; X4 is Y, I, S, A, N, F, or null; X5 is D, A, or null; X6 is E, K, Q, S, T, G, D, or null; X7 is Q, S, N, R, G, L, or D; and X8 is N, K, E, V, or L.
In some aspects, the TCR or antigen-binding fragment thereof contains a Vβ region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4X5X6X7X8X9X10X11X12X13 (SEQ ID NO: 258), where X4 is H, P, L, or Y; X5 is L, G, W, F, or S; X6 is A, G, or L; X7 is G, E, A, T, or null; X8 is F, G, T, or S; X9 is T, N, H, or A; X10 is G, T, Q, D, or Y; X11 is E, P, T, or G; X12 is L, A, Q, or Y; and X13 is F, H, Y, or T.
In some aspects, the TCR or antigen-binding fragment thereof contains a Vβ region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15(SEQ ID NO: 1211), where X1 is A, S, or V; X2 is S, A, or V; X3 is 5, V, R, or Q; X4 is H, P, Q, L, Y, G, T, F, S, R, or E; X5 is L, G, R, W, F, S, V, T, Y, Q, or null; X6 is A, G, L, T, E, P, or null; X7 is G, T, A, R, Q, N, S, or null; X8 is G, S, or null; X9 is G, or null; X10 is F, G, A, S, T, R, Q, L, or null; X11 is T, N, F, A, R, S, G, or null; X12 is G, T, L D, Y, N, Q, S, or E; X13 is E, W, T, G, K, N, or P; X14 is L, A, K, Q, Y, or I; and X15 is F, H, Y, T, or I.
In some aspects, the TCR or antigen-binding fragment thereof contains a Vβ region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15 (SEQ ID NO: 1222), where X1 is A, S, or V; X2 is S, A, or V; X3 is S, R, or Q; X4 is H, P, Q, L, Y, G, T, F, S, R, or E; X5 is L, G, R, W, F, S, V, T, Y, Q, or null; X6 is A, G, L, E, P, or null; X7 is G, T, A, R, Q, N, S, or null; X8 is G, S, or null; X9 is G, or null; X10 is F, G, A, S, T, R, Q, L, or null; X11 is T, N, F, A, R, S, G, or null; X12 is G, T, L D, Y, N, Q, S, or E; X13 is E, W, T, G, K, N, or P; X14 is L, A, K, Q, Y, or I; and X15 is F, H, Y, T, or I.
In some aspects, the TCR or antigen-binding fragment thereof contains a Vβ region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 1212), where X4 is H, P, Q, L, Y, F, R, or E; X5 is L, G, R, W, F, S, V, T, Y, or Q; X6 is A, G, L, E P; X7 is G, T, A, R, Q, S, or null; X8 is G, S, or null; X9 is F, G, A, S, T, R, L, or null; X10 is T, N, A, F, R, S, or G; X11 is G, T, L, D, Y, Q, S, E, or N; X12 is E, W, T, G, P, K; X13 is L, A, K, Q, Y, or I; and X14 is F, H, Y, or T.
In some aspects, the TCR or antigen-binding fragment thereof contains a Vβ region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13QY (SEQ ID NO: 1213), where X1 is A, or S; X2 is 5, V, or A; X3 is S, or V; X4 is L, Y, P, or S; X5 is W, F, V, L, or Y; X6 is G, T, or A; X7 is A, R, Q, S, or null; X8 is G, or null; X9 is G, or null; X10 is S, T, R, or G; X11 is T, A, R, S, or N; X12 is D, Y, T, or G; and X13 is T, or E.
In some aspects, the TCR or antigen-binding fragment thereof contains a Vβ region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2SX4X5X6X7X8X9X10X11X12X13QY (SEQ ID NO: 1223), where X1 is A, or S; X2 is S, or A; X4 is L, Y, P, or S; X5 is W, F, V, L, or Y; X6 is G, or A; X7 is A, R, Q, S, or null; X8 is G, or null; X9 is G, or null; X10 is S, T, R, or G; X11 is T, A, R, S, or N; X12 is D, Y, T, or G; and X13 is T, or E.
In some aspects, the TCR or antigen-binding fragment thereof contains a Vβ region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASX3X4X5X6X7X8X9X10X11X12F (SEQ ID NO: 1214), where X3 is S, Q, or R; X4 is H, P, T, or E; X5 is L, G, W, or F; X6 is A, G, or null; X7 is G, N, S, R, or null; X8 is F, G, Q, L, A, or null; X9 is T, N, or A; X10 is G, T, N, or E; X11 is E, N, or K; and X12 is L, A, or Q.
In some aspects, the TCR or antigen-binding fragment thereof contains a Vβ region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4X5X6X7X8NYX11YT (SEQ ID NO: 1215), where X4 is L, or R; X5 is S, or T; X6 is G, T, or A; X7 is T, or null; X8 is G, or null; and X11 is G, or null.
In some aspects, the TCR or antigen-binding fragment thereof contains a Vβ region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4WGX7SNQPX12H (SEQ ID NO:1216), where X4 is L, F, or P; X7 is R, or Q; and X12 is Q, or L.
In some aspects, the TCR or antigen-binding fragment thereof contains a Vβ region that contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4X5X6X7X8SGNTIY (SEQ ID NO:1217), where X4 is L, or R; X5 is W, or Q; X6 is G, or P; X7 is R, or S; and X8 is S, or null.
In some instances, the Vβ region contains a complementarity determining region 1 (CDR-1) comprising the amino acid sequence X1X2HX4X5 (SEQ ID NO: 252), where X1 is S or M; X2 is G, E, D, or N; X4 is V, N, or E; and X5 is S, R, N, or Y. In some instances, the Vβ region contains a complementarity determining region 1 (CDR-1) comprising the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO: 1218), where X1 is S, M, D, or L; X2 is G, E, D, N, Q, S, or F; X3 is H, V, Y, N, or Q; X4 is A, S, F, or null; X5 is W V, N, E, T, P, Y, K, D, or L; and X6 is S, R, A, N, Y, M, or T.
In some cases, the Vβ region contains a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO: 255), where X1 is F or S; X2 is Q, Y, or V; X3 is N, D, or G; X4 is E or V; X5 is A, K, or G; and X6 is Q, M, or T. In some cases, the Vβ region contains a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6X7 (SEQ ID NO: 1219), where X1 is F, Y, S, A M; X2 is N, Q, V, T, Y, or A; X3 is N, D, E, S, G, I, F, Q, or L; X4 is G, A, N, or null; X5 is E, K, V, E, S, T, G, or N; X6 is A, E, K, G, L, D, V, or N; and X7 is Q, M, T, A, V, E, P, D, or I.
In some embodiments, the Vα region contains a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 138, 144, 147, 163, 167 173, 304, 308, 478, 493, 505, 511, 523, 539, 555, 572, 588, 600, 612, 624, 638, 650, 662, or 679, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some examples, the Vα region contains a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123 125, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, or 676, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some embodiments, the Vα region further contains a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 136, 142, 161, 165 171, 302, 306, 537, 570, or 677, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some aspects, the Vα region contains a CDR-1 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123 125, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, or 676, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some embodiments, the Vα region further contains a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 137, 143, 162, 166, 172, 303, 307, 538, 571, or 678, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some cases, the Vα region contains a CDR-2 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123 125, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, or 676, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the Vβ region contains a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 141, 146, 150, 164, 170 174, 305, 309, 486, 499, 517, 531, 548, 563, 581, 594, 606, 618, 630, 644, 656, 670, or 686, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 122, 124 126, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, or 685, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some embodiments, the Vβ region contains a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 139, 145, 148, 168, 484, 546, 561, 579, or 668, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some instances, the Vβ region contains a CDR-1 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 122, 124 126, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, or 685, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some embodiments, the Vβ region further contains a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 140, 149, 169, 485, 547, 562, 580, or 669, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some examples, the Vβ region contains a CDR-2 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 122, 124 126, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, or 685, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 136, 142, 161, 165, 171, 302, 306, 537, 570, or 677, a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 137, 143, 162, 166, 172, 303, 307, 538, 571, or 678, and/or a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 138, 144, 147, 163, 167 173, 304, 308, 478, 493, 505, 511, 523, 539, 555, 572, 588, 600, 612, 624, 638, 650, 662, or 679. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences. In some aspects, the TCR or antigen-binding fragment thereof contains a Vβ region that contains a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 139, 145, 148, 168, 484, 546, 561, 579, or 668, a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 140, 149, 169, 485, 547, 562, 580, or 669, and/or a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 141, 146, 150, 164, 170 174, 305, 309, 486, 499, 517, 531, 548, 563, 581, 594, 606, 618, 630, 644, 656, 670, or 686. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137, and 138, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 139, 140, and 141, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the Vα region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 142, 143, and 144, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 145, 140, and 146, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the Vα region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137, and 147, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 150, respectively. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the Vα region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 161, 162, and 163, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 164, respectively. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the Vα region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 165, 166, and 167, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 168, 169, and 170, respectively. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the Vα region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 171, 172, and 173, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 174, respectively. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the Vα region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 139, 140, and 305, respectively. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the Vα region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 306, 307, and 308, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 309, respectively. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137, and 478, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 484, 485, and 486, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 161, 162, and 493, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 499, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 165, 166, and 505, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 499, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 161, 162, and 511, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 517, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137, and 523, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 531, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 537, 538, and 539, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 546, 547, and 548, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137, and 555, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 561, 562, and 563, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 570, 571, and 572, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 579, 580, and 581, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137, and 588, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 594, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137, and 600, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 606, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137, and 612, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 618, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137, and 624, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 168, 169, and 630, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 142, 143, and 638, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 561, 562, and 644, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 171, 172, and 650, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 656, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137, and 662, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 668, 669, and 670, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 677, 678, and 679, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 686, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the Vα region contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vα region amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123 125, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, or 676. In some aspects, the Vβ region contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vβ region amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 122, 124 126, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, or 685. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment includes a Vα region that contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences set forth in Table 2; and aVβ region that contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences set forth in Table 2. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences. Exemplary TCRs containing such CDRs, or their modified versions as described elsewhere herein, also are set forth in the Table 2.
TABLE 2
HPV16 E6(29-38) TCR CDR SEQ ID NOs.
Exemplary
Alpha
Beta
TCR
CDR1
CDR2
CDR3
CDR1
CDR2
CDR3
TCR 3
136
137
138
139
140
141
TCR 4
142
143
144
145
140
146
TCR 5
136
137
147
148
149
150
TCR 8
161
162
163
148
149
164
TCR 9
165
166
167
168
169
170
TCR 10
171
172
173
148
149
174
TCR 13
302
303
304
139
140
305
TCR 14
306
307
308
148
149
309
TCR 15
136
137
478
484
485
486
TCR 16
161
162
493
148
149
499
TCR 17
165
166
505
148
149
499
TCR 18
161
162
511
148
149
517
TCR 19
136
137
523
148
149
531
TCR 20
537
538
539
546
547
548
TCR 21
136
137
555
561
562
563
TCR 22
570
571
572
579
580
581
TCR 23
136
137
588
148
149
594
TCR 24
136
137
600
148
149
606
TCR 25
136
137
612
148
149
618
TCR 26
136
137
624
168
169
630
TCR 27
142
143
638
561
562
644
TCR 28
171
172
650
148
149
656
TCR 29
136
137
662
668
669
670
TCR 30
677
678
679
154
155
686
In some instances, the TCR or antigen-binding fragment thereof contains Vα and Vβ regions containing the amino acid sequences of SEQ ID NOs: 111 and 112, respectively. In some embodiments, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 113 and 114, respectively. In some cases, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 115 and 116, respectively. In some embodiments, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 121 and 122, respectively. In some aspects, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 123 and 124, respectively. In some examples, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 125 and 126, respectively. In some examples, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 297 and 298, respectively. In some examples, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 299 and 300, respectively. In some embodiments, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 477 and 483, respectively. In some examples, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 492 and 498, respectively. In some cases, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 504 and 498, respectively. In some instances, the TCR or antigen-binding fragment thereof contains Vα and Vβ regions containing the amino acid sequences of SEQ ID NOs: 510 and 516, respectively. In some embodiments, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 522 and 530, respectively. In some examples, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 536 and 545, respectively. In some cases, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 554 and 560, respectively. In some instances, the TCR or antigen-binding fragment thereof contains Vα and Vβ regions containing the amino acid sequences of SEQ ID NOs: 569 and 578, respectively. In some embodiments, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 587 and 593, respectively. In some examples, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 599 and 605, respectively. In some embodiments, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 611 and 617, respectively. In some cases, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 623 and 629, respectively. In some instances, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 637 and 643, respectively. In some cases, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 649 and 655, respectively. In some examples, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 661 and 667, respectively. In some cases, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 676 and 685, respectively. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the alpha chain of the TCR or antigen-binding fragment thereof further contains a Cα region or portion thereof and/or the beta chain further contains a Cβ region or portion thereof. In some embodiments, the Cα region or portion thereof comprises the amino acid sequence set forth in any of SEQ ID NOs: 212, 213, 215, 218, or 524, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some aspects, the Cβ region contains the amino acid sequence set forth in SEQ ID NO: 214, 216, or 631, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some embodiments, the Cα and/or Cβ regions are modified, for example, by incorporation of one or more non-native cysteine residues, such as any described herein. In some embodiments, the Cα region or portion thereof contains a non-native cysteine at residue 48 and comprises the amino acid sequence set forth in any of SEQ ID NOs: 196, 198, 201, 203, or 525, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence and that contains the introduced non-native cysteine residue (e.g. Cys48). In some aspects, the Cβ region contains a non-native cysteine at residue 57 and contains the amino acid sequence set forth in SEQ ID NO: 197, 199, or 632, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the TCR or antigen-binding fragment thereof comprises an alpha chain comprising the sequence of amino acids set forth in SEQ ID NO: 18, 28, 38, 68, 78, 88, 287, 291, 473, 488, 500, 506, 518, 532, 550, 565, 583, 595, 607, 619, 633, 645, 657, or 672, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence and/or a beta chain comprising the sequence of amino acids set forth in SEQ ID NO: 22, 32, 42, 72, 82, 92, 289, 293, 479, 494, 512, 526, 541, 556, 574, 589, 601, 613, 625, 639, 651, 663, or 681, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the TCR or antigen-binding fragment thereof comprises an alpha chain comprising the sequence of amino acids set forth in SEQ ID NO: 19, 29, 39, 69, 79, 89, 288, 292, 474, 489, 501, 507, 519, 533, 551, 566, 584, 596, 608, 620, 634, 646, 658, or 673, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence and/or a beta chain comprising the sequence of amino acids set forth in SEQ ID NO: 23, 33, 43, 73, 83, 93, 290, 294, 480, 495, 513, 527, 542, 557, 575, 590, 602, 614, 626, 640, 652, 664, or 682, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the Vα and Vβ regions contain the amino acid sequences corresponding to the SEQ ID NOs. set forth in Table 3 or Table 4. In some aspects, the TCR contains constant alpha and constant beta region sequences, such as those corresponding to the SEQ ID NOs. set forth in Table 3 or Table 4. In some cases, the TCR contains a full sequence comprising the variable and constant chain, such as a sequence corresponding to the SEQ ID NOs. set forth in Tables 3 or 4(“Full”). In some embodiments, the full sequence containing the variable and constant regions also includes a signal sequence and thus comprises a sequence corresponding to the SEQ ID NOs. set forth in Table 3 or 4 (“Full+signal”). Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences. Exemplary TCRs containing such sequences, or their modified versions as described elsewhere herein, also are set forth in the Tables 3 and 4, respectively.
TABLE 3
HPV16 E6(29-38) TCR Native SEQ ID NOs.
Alpha
Beta
Exemplary
Variable
Full +
Variable
Full +
TCR
(Vα)
Constant
Full
signal
(Vβ)
Constant
Full
signal
TCR 3
111
215
18
318
112
216
22
320
TCR 4
113
213
28
322
114
214
32
324
TCR 5
115
213
38
326
116
214
42
328
TCR 8
121
213
68
338
122
216
72
110
TCR 9
123
213
78
130
124
216
82
132
TCR 10
125
212
88
134
126
214
92
179
TCR 13
297
213
287
253
298
216
289
260
TCR 14
299
218
291
313
300
214
293
315
TCR 15
477
218
473
475
483
216
479
481
TCR 16
492
213
488
490
498
214
494
496
TCR 17
504
213
500
502
498
214
494
496
TCR 18
510
213
506
508
516
214
512
514
TCR 19
522
524
518
520
530
216
526
528
TCR 20
536
218
532
534
545
216
541
543
TCR 21
554
213
550
552
560
214
556
558
TCR 22
569
524
565
567
578
214
574
576
TCR 23
587
524
583
585
593
214
589
591
TCR 24
599
524
595
597
605
216
601
603
TCR 25
611
524
607
609
617
214
613
615
TCR 26
623
213
619
621
629
631
625
627
TCR 27
637
213
633
635
643
214
639
641
TCR 28
649
213
645
647
655
214
651
653
TCR 29
661
524
657
659
667
216
663
665
TCR 30
676
213
672
674
685
214
681
683
TABLE 4
HPV16 E6(29-38) TCR Modified SEQ ID NOs.
Exemplary
modified
Alpha
Beta
version of
Variable
Full +
Variable
Full +
TCR
(Vα)
Constant
Full
signal
(Vβ)
Constant
Full
signal
TCR 3
111
198
19
319
112
199
23
321
TCR 4
113
196
29
323
114
197
33
325
TCR 5
115
196
39
327
116
197
43
329
TCR 8
121
203
69
339
122
199
73
129
TCR 9
123
203
79
131
124
199
83
133
TCR 10
125
198
89
135
126
197
93
180
TCR 13
297
203
288
256
298
199
290
312
TCR 14
299
201
292
314
300
197
294
316
TCR 15
477
201
474
476
483
199
480
482
TCR 16
492
203
489
491
498
197
495
497
TCR 17
504
203
501
503
498
197
495
497
TCR 18
510
203
507
509
516
197
513
515
TCR 19
522
525
519
521
530
199
527
529
TCR 20
536
201
533
535
545
199
542
544
TCR 21
554
203
551
553
560
197
557
559
TCR 22
569
525
566
568
578
197
575
577
TCR 23
587
525
584
586
593
197
590
592
TCR 24
599
525
596
598
605
199
602
604
TCR 25
611
525
608
610
617
197
614
616
TCR 26
623
203
620
622
629
632
626
628
TCR 27
637
203
634
636
643
197
640
642
TCR 28
649
203
646
648
655
197
652
654
TCR 29
661
525
658
660
667
199
664
666
TCR 30
676
203
673
675
685
197
682
684
b. HPV 16 E7(11-19)
In some cases, the TCR recognizes or binds a peptide epitope derived from HPV 16 E7 that is or contains E7(11-19) YMLDLQPET (SEQ ID NO: 236). In some embodiments, the TCR recognizes or binds HPV 16 E7(11-19) in the context of an MHC, such as an MHC class I, e.g., HLA-A2.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2SX4X5X6X7X8X9X10X11 (SEQ ID NO: 249), where X1 is A or V; X2 is E or V; X4 is I or R; X5 is R or D; X6 is G or N; X7 is F or Y; X8 is N or Q; X9 is V or N; X10 is L or F; and X11 is H or V.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO:1183), where X1 is V, or A; X2 is V, A, G, Q, M, or E; X3 is S, G, A, N, Y, R, T, or P; X4 is E, A, S, G, R. F, N, D, V, P, L, I, or M; X5 is R, N, H, T, D, G, S, A, P, L, Q, or F; X6 is G, H, N, A, S, L, T, or null; X7 is T, S, G, or null; X8 is G, or null; X9 is G, Y, N, S, or null; X10 is T, G, S, D, F, Y, A, N, or null; X11 is Y, F, Y, Q, N, or R; X12 is N, K, Q, or D; X13 is Y, L, T, F, M, or V; and X14 is I, T, S, V, R, or Y.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence VVX3X4X5X6X7X8GX10X11X12X13(SEQ ID NO:1184), where X3 is S, N, or T; X4 is R, or F; X5 is D, or A; X6 is N, or L; X7 is T, or null; X8 is Y, or G; X10 is Q, or F; X11 is N, or K; X12 is F, or T; and X13 is V, or I.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO:1185), where X2 is A, G, V, Q, M, or E; X3 is S, G, N, A, Y, R, or P; X4 is E, S, A, G, F, N, D, V, P, L, I, M, or R; X5 is R, N, H, T, D, G, S, P, L, Q, or F; X6 is G, H, A, S, T, or null; X7 is T, S, G, or null; X8 is G, or null; X9 is G, N, S, or null; X10 is T, G, S, D, F, Y, A, or N; X11 is Y, F, Q, R, or N; X12 is K, Q, or D; X13 is Y, L, T, M, F, or V; and X14 is I, T, S, R, Y, or V.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10KX12I (SEQ ID NO:1186), where X1 is A, or V; X2 is A, V, or E; X3 is S, N, T, R, or P; X4 is E, A, G, F, V, P, I, D, or S; X5 is R, H, T, A P, S, G, or F; X6 is G, H, L, T, S, A, or null; X7 is S, T, or null; X8 is G, or null; X9 is G, T, or null; X10 is F, Y, or N; and X12 is Y, T, or L.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9YKYI (SEQ ID NO:1187), where X2 is A, V, or E; X3 is S, N, or R; X4 is E, G, V, P, I, or D; X5 is R, T, P, S, G, or F; X6 is G, T, S, or null; X7 is S, or null; X8 is G, or null; and X9 is T, or null.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11X12X13X14(SEQ ID NO:1188), where X2 is G, V, Q, or M; X3 is G, A, Y, S, N, or R; X4 is S, G, L, I, M, or R; X5 is N, D, G, S, L, Q, or R; X6 is A, S, G, or null; X7 is G, or null; X8 is G, or null; X9 is G, N, S, or null; X10 is S, D, Y, A, N, or null; X11 is Y, Q, or R; X12 is K, or Q; X13 is L, or V; and X14 is S, T, or V.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11X12X13T (SEQ ID NO:1189), where X2 is G, V, or Q; X3 is G, Y, S, or N; X4 is S, L, or M; X5 is N, G, L, or R; X6 is A, S, G, or null; X7 is G, or null; X8 is G, or null; X9 is G, S, or null; X10 is S, Y, A, N, or null; X11 is Y, Q, or R; X12 is K, or Q; and X13 is L, or V.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7YKLS (SEQ ID NO:1190), where X2 is G, or V; X3 is A, or Y; X4 is G, S, or R; X5 is D, or S; X6 is N, or null; and X7 is D, or null.
In some embodiments, the Vα region contains a complementarity determining region 1 (CDR-1) comprising the amino acid sequence X1SX3X4X5X6 (SEQ ID NO: 241), where X1 is D or V; X3 is S, or P; X4 is S or F; X5 is T or S; and X6 is Y or N. In some embodiments, the Vα region contains a complementarity determining region 1 (CDR-1) comprising the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO:1191), where X1 is N, S, D, T, or V; X2 is S, V, R, T, or I; X3 is M, F, G, S, N, A, L, V, or P; X4 is F, S, N, A, or null; X5 is D, S, Q, Y, N, V, T, or P; and X6 is Y, S, R, N, G, or T.
In some cases, the Vα region contains a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6X7 (SEQ ID NO: 245), where X1 is I or M; X2 is F or T; X3 is S or F; X4 is N or S; X5 is M or E; X6 is D or N; and X7 is M or T. In some embodiments, the Vα region contains a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6X7X8 (SEQ ID NO:1192), where X1 is I, V, L, G, N, T, Y, or M; X2 is S, V, Y, L, P, F, I, or T; X3 is S, Y, K, L, T, or F; X4 is I, G, N, A, S, or null; X5 is S, D, or null; X6 is K, G, N, S, D, T, or E; X7 is D, E, G, A, K, L, or N; and X8 is K, V, D, P, N, T, L, or M.
In some aspects, the TCR or antigen-binding fragment thereof contains a Vβ region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2TX4RX6X7YX9X10X11 (SEQ ID NO: 259), where X2 is S or I; X4 is T or D; X6 is S or T; X7 is S or N; X9 is E or G; X10 is Q or Y; and X11 is Y or T.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vβ region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11X12X13X14(SEQ ID NO: 1193), where X2 is 5, M, I, K, or V; X3 is S, T, N, or A; X4 is R, V P, 5, T, G, L, A, I, or D; X5 is F, G, R, Y, 5, L, V, or T; X6 is L, G, D, A, S, T, V, R, or null; X7 is G, D, R, S, T, or null; X8 is S, or null; X9 is S, H, G, R, V, T, D, L, or null; X10 is T, S, A, Y, N, G, or P; X11 is D, Y, N, E, K, or G; X12 is T, E, G, or K; X13 is Q, Y, A, or L; and X14 is Y, F, T, or I.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vβ region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2TX4X5X6X7X8X9X10X11X12(SEQ ID NO: 1194), where X2 is 5, M, I, or K; X4 is P, T, G, A, S, or D; X5 is R, or S; X6 is D, G, S, T, or V; X7 is R, S, or null; X8 is T, Y, G, N, or S; X9 is Y, N, or K; X10 is E, or G; X11 is Q, A, or Y; and X12 is Y, F, or T.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vβ region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11X12X13X14(SEQ ID NO: 1195), where X2 is 5, M, I, or K; X3 is S, T, A, or N; X4 is R, V, S, P, T, G, L, or A; X5 is F, G, R, Y, S, V, or T; X6 is L, G, D, A, S, T, V, or null; X7 is G, D, R, T, or null; X8 is S, or null; X9 is S, H, G, R, V, T, L, or null; X10 is T, S, Y, A, N, G, or P; X11 is D, Y, N, K, E, or G; X12 is T, or E; X13 is Q, A, or L; and X14 is Y, or F.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vβ region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11QY (SEQ ID NO: 1196), where X2 is 5, M, I, or K; X3 is S, T, A, or N; X4 is R, P, S, G, L, A, or T; X5 is F, R, Y, V, or T; X6 is L, D, A, S, T, V, or null; X7 is G, R, or null; X8 is S, G, V, or null; X9 is T, A, G, N, S, or P; X10 is D, Y, or E; and X11 is T, or E.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vβ region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X91 YEQY (SEQ ID NO: 1197), where X2 is 5, M, I, or K; X3 is 5, T, A, or N; X4 is P, S, G, T, or A; X5 is R, or Y; X6 is D, A, S, T, or V; X7 is R, or null; X8 is G, V, or null; and X9 is S, T, A, or N.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vβ region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASTX4X5X6X7X8X9X10X11EX13X14(SEQ ID NO: 1198), where X4 is T, P, or G; X5 is R, or S; X6 is S, D, G, or V; X7 is D, or null; X8 is S, or null; X9 is S, R, or null; X10 is S, T, Y, or G; X11 is Y, N, or K; X13 is Q, or A; and X14 is Y, or F.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vβ region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8YGYT (SEQ ID NO: 1199), where X2 is S, or I; X3 is S, or T; X4 is L, A, or D; X5 is L, T, or R; X6 is L, T, or R; X7 is G, D, or null; and X8 is A, or N.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vβ region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 1200), where X2 is 5, V, or I; X3 is S, N, or A; X4 is R, V, S, L, P, G, I, or A; X5 is F, G, Y, L, V, R, T, or S; X6 is L, G, A, D, R, V, or null; X7 is G, D, R, S, T, or null; X8 is S, or null; X9 is S, H, G, V, T, D, L, or null; X10 is T, S, A, G, P, N, or Y; X11 is D, Y, E, G, or N; X12 is T, E, G, or K; X13 is Q, Y, or L; and X14 is Y, F, T, or I.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vβ region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4X5X6X7X8X9X10X11X12X13X14(SEQ ID NO: 1201), where X4 is R, V, S, L, G, or A; X5 is F, G, Y, L, V, T, or S; X6 is A, L, R, D, G, or null; X7 is G, D, T, or null; X8 is S, or null; X9 is S, H, G, T, D, L, or null; X10 is T, S, A, G, P, N, or Y; X11 is D, Y, E, G, or N; X12 is T, E, G, or T; X13 is Q, Y, or L; and X14 is Y, F, or T.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vβ region containing a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4X5X6X7X8X9X10TQY (SEQ ID NO: 1202), where X4 is R, L, or G; X5 is F, V, T, or Y; X6 is L, A, or null; X7 is G, or null; X8 is S, G, or null; X9 is T, G, P, or S; and X10 is D, or E.
In some embodiments, the Vβ region contains a complementarity determining region 1 (CDR-1) comprising the amino acid sequence SX2X3X4X5 (SEQ ID NO:1203), where X2 is G, or N; X3 is H, or D; X4 is T, L, N, or V; and X5 is A, S, Y, or T.
In some embodiments, the Vβ region contains a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO:1204), where X1 is F, or Y; X2 is Q, Y, or N; X3 is G, N, R, or Y; X4 is N, G, E, or T; X5 is S, E, A, or G; and X6 is A, E, I, or Q.
In some aspects, the Vβ region contains a complementarity determining region 1 (CDR-1) comprising the amino acid sequence set forth in SEQ ID NO: 154, 701, 719, or 751. In some embodiments, the Vβ region contains a complementarity determining region 2 (CDR-2) comprising the amino acid sequence set forth in SEQ ID NO: 155, 702, 720, 752, 918, or 1009.
In some embodiments, the Vα region contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence set forth in any of SEQ ID NOs: 153, 159, 301, 694, 712, 729, 744, 762, 776, 788, 802, 818, 832, 846, 858, 870, 882, 896, 911, 926, 940, 952, 964, 976, 988, or 1002, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 117, 119, 295, 691, 709, 726, 741, 759, 775, 787, 799, 815, 830, 845, 857, 869, 881, 895, 908, 925, 937, 951, 963, 975, 987, or 999. In some embodiments, the Vα region contains a CDR3 sequence at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the Vα region further contains a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 151, 157, 692, 710, 727, 742, 760, 800, 816, 909, 938, or 1000, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some aspects, the Vα region further contains a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 152, 158, 693, 711, 728, 743, 761, 801, 817, 831, 833, 910, 939, or 1001, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some aspects, the Vβ region contains a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 156, 160, 703, 721, 736, 753, 769, 782, 794, 809, 825, 840, 852, 864, 876, 888, 902, 919, 932, 946, 958, 970, 982, 994, or 1010, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 118, 120, 296, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008. In some embodiments, the Vβ region contains a CDR3 sequence at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences. In some embodiments, the Vβ region contains a complementarity determining region 1 (CDR-1) comprising the amino acid sequence set forth in SEQ ID NO: 154, 701, 719, or 751, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some instances, the Vβ region contains a complementarity determining region 2 (CDR-2) comprising the amino acid sequence set forth in SEQ ID NO: 155, 702, 720, 752, 918, or 1009, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 151, 152, and 153, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 156, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some aspects, the Vα region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 157, 158, and 159, respectively. In some such aspects, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 160, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the Vα region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 151, 152, and 301, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 156, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 692, 693, and 694, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 701, 702, and 703, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 710, 711, and 712, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 719, 720, and 721, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 727, 728, and 729, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 736, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 742, 743, and 744, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 751, 752, and 753, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 760, 761, and 762, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 719, 720, and 769, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 171, 172, and 776, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 782, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 742, 743, and 788, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 139, 140, and 794, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 800, 801, and 802 respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 751, 752, and 809, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 816, 817, and 818, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 825, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 816, 831, and 832, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 840, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 171, 172, and 846, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 852, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 816, 833, and 858, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 864, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 727, 728, and 870, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 876, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 570, 571, and 882, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 719, 720, and 888, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 816, 817, and 896, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 701, 702, and 902, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 909, 910, and 911, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 701, 918, and 919, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 727, 728, and 926, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 932, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 938, 939, and 940, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 946, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 727, 728, and 952, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 958, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 151, 152, and 964, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 719, 720, and 970, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 727, 728, and 976, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 982, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 710, 711, and 988, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 719, 720, and 994, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region that contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 1000, 1001, and 1002, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 139, 1009, and 1010, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some instances, the Vα region contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vα region amino acid sequence set forth in any of SEQ ID NOs: 117, 119, 295, 691, 709, 726, 741, 759, 775, 787, 799, 815, 830, 845, 857, 869, 881, 895, 908, 925, 937, 951, 963, 975, 987, or 999. In some cases, the Vβ region contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vβ region amino acid sequence set forth in any of SEQ ID NOs: 118, 120, 296, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment includes a Vα region that contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences set forth in Table Sand a Vβ region that contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences set forth in Table 5. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences. Exemplary TCRs containing such CDRs, or their modified versions as described elsewhere herein, also are set forth in the Table 5.
TABLE 5
HPV16 E7(11-19) TCR CDR SEQ ID NOs.
Exemplary
Alpha
Beta
TCR
CDR1
CDR2
CDR3
CDR1
CDR2
CDR3
TCR 6
151
152
153
154
155
156
TCR 7
157
158
159
154
155
160
TCR 12
151
152
301
154
155
156
TCR 31
692
693
694
701
702
703
TCR 32
710
711
712
719
720
721
TCR 33
727
728
729
154
155
736
TCR 34
742
743
744
751
752
753
TCR 35
760
761
762
719
720
769
TCR 36
171
172
776
154
155
782
TCR 37
742
743
788
139
140
794
TCR 38
800
801
802
751
752
809
TCR 39
816
817
818
154
155
825
TCR 40
816
831
832
154
155
840
TCR 41
171
172
846
154
155
852
TCR 42
816
833
858
154
155
864
TCR 43
727
728
870
154
155
876
TCR 44
570
571
882
719
720
888
TCR 45
816
817
896
701
702
902
TCR 46
909
910
911
701
918
919
TCR 47
727
728
926
154
155
932
TCR 48
938
939
940
154
155
946
TCR 49
727
728
952
154
155
958
TCR 50
151
152
964
719
720
970
TCR 51
727
728
976
154
155
982
TCR 52
710
711
988
719
720
994
TCR 53
1000
1001
1002
139
1009
1010
TCR 54
157
158
159
154
155
160
TCR 55
151
152
301
154
155
156
In some embodiments, the TCR or antigen-binding fragment thereof contains Vα and Vβ regions containing the amino acid sequences of SEQ ID NOs: 117 and either 118 or 296, respectively. In some aspects, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 119 and 120, respectively. In some aspects, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 295 and either 118 or 296, respectively. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences. In some cases, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 691 and 700, respectively. In some instances, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 709 and 718, respectively. In some aspects, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 726 and 735, respectively. In some embodiments, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 741 and 750, respectively. In some cases, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 759 and 768, respectively. In some aspects, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 775 and 781, respectively. In some embodiments, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 787 and 793, respectively. In some examples, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 799 and 808, respectively. In some cases, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 815 and 824, respectively. In some instances, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 830 and 839, respectively. In some embodiments, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 845 and 851, respectively. In some aspects, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 857 and 863, respectively. In some cases, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 869 and 875, respectively. In some instances, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 881 and 887, respectively. In some embodiments, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 895 and 901, respectively. In some aspects, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 908 and 917, respectively. In some cases, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 925 and 931, respectively. In some instances, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 937 and 945, respectively. In some examples, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 951 and 957, respectively. In some cases, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 963 and 969, respectively. In some instances, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 975 and 981, respectively. In some cases, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 987 and 993, respectively. In some embodiments, the Vα and Vβ regions contain the amino acid sequences of SEQ ID NOs: 999 and 1008, respectively.
In some embodiments, the alpha chain of the TCR or antigen-binding fragment thereof further contains a Cα region or portion thereof and/or the beta chain further contains a Cβ region or portion thereof. In some embodiments, the Cα region or portion thereof comprises the amino acid sequence set forth in any of SEQ ID NO: 213, 217, 218, or 524, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some aspects, the Cβ region contains the amino acid sequence set forth in SEQ ID NO: 214, 216, 631, or 889, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some embodiments, the Cα and/or Cβ regions are modified, for example, by incorporation of one or more non-native cysteine residues, such as any described herein. In some embodiments, the Cα region or portion thereof contains a non-native cysteine at residue 48 and comprises the amino acid sequence set forth in any of SEQ ID NOs: 196, 200, 201, 203, or 525, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence and that contains the introduced non-native cysteine residue (e.g., Cys48). In some aspects, the Cβ region contains a non-native cysteine at residue 57 and contains the amino acid sequence set forth in SEQ ID NO: 197, 199, or 890, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the TCR or antigen-binding fragment thereof comprises an alpha chain comprising the sequence of amino acids set forth in SEQ ID NO: 48, 58, 283, 687, 705, 722, 737, 755, 771, 783, 795, 811, 826, 841, 853, 865, 877, 891, 904, 921, 933, 947, 959, 971, 983, or 995, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence and/or a beta chain comprising the sequence of amino acids set forth in SEQ ID NO: 52, 285, 62, 696, 714, 731, 746, 764, 777, 789, 804, 820, 835, 847, 859, 871, 883, 897, 913, 927, 941, 953, 965, 977, 989, or 1004, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the TCR or antigen-binding fragment thereof comprises an alpha chain comprising the sequence of amino acids set forth in SEQ ID NO: 49, 59, 284, 688, 706, 723, 738, 756, 772, 784, 796, 812, 827, 842, 854, 866, 878, 892, 905, 922, 934, 948, 960, 972, 984, or 996, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence and/or a beta chain comprising the sequence of amino acids set forth in SEQ ID NO: 53, 63, 286, 697, 715, 732, 747, 765, 778, 790, 805, 821, 836, 848, 860, 872, 884, 898, 914, 928, 942, 954, 966, 978, 990, or 1005, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the Vα and Vβ regions contain the amino acid sequences corresponding to the SEQ ID NOs. set forth in Table 6 or Table 7. In some aspects, the TCR contains constant alpha and constant beta region sequences, such as those corresponding to the SEQ ID NOs. set forth in Table 6 or Table 7. In some cases, the TCR contains a full sequence comprising the variable and constant chain, such as a sequence corresponding to the SEQ ID NOs. set forth in Table 6 or Table 7 (“Full”). In some embodiments, the full sequence containing the variable and constant regions also includes a signal sequence and thus comprises a sequence corresponding to the SEQ ID NOs. set forth in Table 6 or Table 7 (“Full+signal”). Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences. Exemplary TCRs containing such sequences, or their modified versions as described elsewhere herein, also are set forth in the Tables 6 and 7, respectively.
TABLE 6
HPV16 E7(11-19) TCR Native SEQ ID NOs.
Alpha
Beta
Exemplary
Variable
Full +
Variable
Full +
TCR
(Vα)
Constant
Full
signal
(Vβ)
Constant
Full
signal
TCR 6
117
217
48
330
118, 296
216
52, 285
332, 246
TCR 7
119
218
58
334
120
214
62
336
TCR 12
295
213
283
222
118, 296
216
52, 285
332, 246
TCR 31
691
213
687
689
700
216
696
698
TCR 32
709
213
705
707
718
216
714
716
TCR 33
726
213
722
724
735
216
731
733
TCR 34
741
213
737
739
750
216
746
748
TCR 35
759
213
755
757
768
216
764
766
TCR 36
775
218
771
773
781
216
777
779
TCR 37
787
213
783
785
793
214
789
791
TCR 38
799
213
795
797
808
216
804
806
TCR 39
815
213
811
813
824
214
820
822
TCR 40
830
213
826
828
839
216
835
837
TCR 41
845
213
841
843
851
216
847
849
TCR 42
857
213
853
855
863
216
859
861
TCR 43
869
213
865
867
875
216
871
873
TCR 44
881
213
877
879
887
889
883
885
TCR 45
895
213
891
893
901
216
897
899
TCR 46
908
213
904
906
917
216
913
915
TCR 47
925
524
921
923
931
216
927
929
TCR 48
937
213
933
935
945
216
941
943
TCR 49
951
213
947
949
957
216
953
955
TCR 50
963
213
959
961
969
214
965
967
TCR 51
975
213
971
973
981
214
977
979
TCR 52
987
213
983
985
993
214
989
991
TCR 53
999
213
995
997
1008
216
1004
1006
TCR 54
119
218
58
334
120
214
62
336
TCR 55
295
213
283
222
118, 296
216
52, 285
332, 246
TABLE 7
HPV16 E7(11-19) TCR Modified SEQ ID NOs.
Exemplary
modified
Alpha
Beta
version of
Variable
Full +
Variable
Full +
TCR
(Vα)
Constant
Full
signal
(Vβ)
Constant
Full
signal
TCR 6
117
200
49
331
118, 296
199
53, 286
333, 250
TCR 7
119
201
59
335
120
197
63
337
TCR 12
295
196
284
242
118, 296
199
53, 286
333, 250
TCR 31
691
203
688
690
700
199
697
699
TCR 32
709
203
706
708
718
199
715
717
TCR 33
726
203
723
725
735
199
732
734
TCR 34
741
203
738
740
750
199
747
749
TCR 35
759
203
756
758
768
199
765
767
TCR 36
775
201
772
774
781
199
778
780
TCR 37
787
203
784
786
793
197
790
792
TCR 38
799
203
796
798
808
199
805
807
TCR 39
815
203
812
814
824
197
821
823
TCR 40
830
203
827
829
839
199
836
838
TCR 41
845
203
842
844
851
199
848
850
TCR 42
857
203
854
856
863
199
860
862
TCR 43
869
203
866
868
875
199
872
874
TCR 44
881
203
878
880
887
890
884
886
TCR 45
895
203
892
894
901
199
898
900
TCR 46
908
203
905
907
917
199
914
916
TCR 47
925
525
922
924
931
199
928
930
TCR 48
937
203
934
936
945
199
942
944
TCR 49
951
203
948
950
957
199
954
956
TCR 50
963
203
960
962
969
197
966
968
TCR 51
975
203
972
974
981
199
978
980
TCR 52
987
203
984
986
993
199
990
992
TCR 53
999
203
996
998
1008
199
1005
1007
TCR 54
119
201
59
335
120
197
63
337
TCR 55
295
196
284
242
118, 296
199
53, 286
333, 250
c. HPV 16 E7(86-93)
In some cases, the TCR recognizes or binds a peptide epitope derived from HPV16 E7 that is or contains E7(86-93) TLGIVCPI (SEQ ID NO: 235). In some embodiments, the TCR recognizes or binds HPV 16 E7(86-93) in the context of an MHC, such as an MHC class I, e.g. HLA-A2.
In some embodiments, the Vα region contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence set forth in SEQ ID NO: 175. In some embodiments, the Vα region contains a CDR3 sequence at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences. In some aspects, the Vα region contains a complementarity determining region 1 (CDR-1) comprising the amino acid sequence set forth in SEQ ID NO: 142, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some aspects, the Vα region comprises a complementarity determining region 2 (CDR-2) comprising the amino acid sequence set forth in SEQ ID NO: 143, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the Vβ region contains a complementarity determining region 3 (CDR-3) comprising the amino acid sequence set forth in SEQ ID NO: 178, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some cases, the Vβ region contains a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in SEQ ID NO:176, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some aspects, the Vβ region contains a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in SEQ ID NO: 177, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the Vα region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 142, 143, and 175, respectively. In some such embodiments, the Vβ region contains a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 176, 177, and 178, respectively. Also among the provided TCRs are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some aspects, the Vα region contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vα region amino acid sequence set forth in SEQ ID NO: 127. In some embodiments, the Vβ region contains a CDR-1, a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vβ region amino acid sequence set forth in SEQ ID NO: 128. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the TCR or antigen-binding fragment includes a Vα region contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences set forth in Table 8. and a Vβ region that contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences set forth in Table 8. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences. Exemplary TCRs containing such CDRs, or their modified versions as described elsewhere herein, also are set forth in the Table 8.
TABLE 8
HPV16 E7(86-93) TCR CDR SEQ ID NOs.
Exemplary
Alpha
Beta
TCR
CDR1
CDR2
CDR3
CDR1
CDR2
CDR3
TCR 11
142
143
175
176
177
178
In some embodiments, the TCR or antigen-binding fragment thereof contains Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 127 and 128, respectively. Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the alpha chain of the TCR or antigen-binding fragment thereof further contains a Cα region or portion thereof and/or the beta chain further contains a Cβ region or portion thereof. In some embodiments, the Cα region or portion thereof comprises the amino acid sequence set forth in any of SEQ ID NO: 212, 213 or 217, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some aspects, the Cβ region contains the amino acid sequence set forth in SEQ ID NO: 214, or 216, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence. In some embodiments, the Cα and/or Cβ regions are modified, for example, by incorporation of one or more non-native cysteine residues, such as any described herein. In some embodiments, the Cα region or portion thereof contains a non-native cysteine at residue 48 and comprises the amino acid sequence set forth in SEQ ID NO: 200, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence and that contains the introduced non-native cysteine residue (e.g. Cys48). In some aspects, the Cβ region contains a non-native cysteine at residue 57 and contains the amino acid sequence set forth in SEQ ID NO: 197 or 199, or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the TCR or antigen-binding fragment thereof comprises an alpha chain comprising the sequence of amino acids set forth in SEQ ID NO: 98 or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence and/or a beta chain comprising the sequence of amino acids set forth in SEQ ID NO: 102 or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the TCR or antigen-binding fragment thereof comprises an alpha chain comprising the sequence of amino acids set forth in SEQ ID NO: 99 or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence and/or a beta chain comprising the sequence of amino acids set forth in SEQ ID NO: 103 or a sequence of amino acids that has at least 90% sequence identity thereto, such as a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with such a sequence.
In some embodiments, the Vα and Vβ regions contain the amino acid sequences corresponding to the SEQ ID NOs. set forth in Table 9 or Table 10. In some aspects, the TCR contains constant alpha and constant beta region sequences, such as those corresponding to the SEQ ID NOs. set forth in Table 9 or Table 10. In some cases, the TCR contains a full sequence comprising the variable and constant chain, such as a sequence corresponding to the SEQ ID NOs. set forth in Table 9 or Table 10 (“Full”). In some embodiments, the full sequence containing the variable and constant regions also includes a signal sequence and thus comprises a sequence corresponding to the SEQ ID NOs. set forth in Table 9 or Table 10 (“Full+signal”). Also among the provided TCRs are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences. Exemplary TCRs containing such sequences, or their modified versions as described elsewhere herein, also are set forth in the Tables 9 and 10, respectively.
TABLE 9
HPV16 E7(86-93) TCR Native SEQ ID NOs.
Alpha
Beta
Exemplary
Variable
Full +
Variable
Full +
TCR
(Vα)
Constant
Full
signal
(Vβ)
Constant
Full
signal
TCR 11
127
217
98
195
128
216
102
352
TABLE 10
HPV16 E7(86-93) TCR Modified SEQ ID NOs.
Exemplary
modified
Alpha
Beta
version of
Variable
Full +
Variable
Full +
TCR
(Vα)
Constant
Full
signal
(Vβ)
Constant
Full
signal
TCR 11
127
200
99
205
128
199
103
221
2. Variants & Modifications
In some embodiments, the binding molecule, e.g., TCR or antigen-binding fragment thereof, is or has been modified. In certain embodiments, the binding molecules, e.g., TCRs or antigen-binding fragments thereof, include one or more amino acid variations, e.g., substitutions, deletions, insertions, and/or mutations, compared to the sequence of a binding molecule, e.g., TCR, described herein. Exemplary variants include those designed to improve the binding affinity and/or other biological properties of the binding molecule. Amino acid sequence variants of a binding molecule may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the binding molecule, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the binding molecule. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
In some embodiments, directed evolution methods are used to generate TCRs with altered properties, such as with higher affinity for a specific peptide in the context of an MHC molecule. In some embodiments, directed evolution is achieved by display methods including, but not limited to, yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54), or T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84). In some embodiments, display approaches involve engineering, or modifying, a known, parent or reference TCR. For example, in some cases, a reference TCR, such as any provided herein, can be used as a template for producing mutagenized TCRs in which in one or more residues of the CDRs are mutated, and mutants with an desired altered property, such as higher affinity for peptide epitope in the context of an MHC molecule, are selected.
In certain embodiments, the binding molecules, e.g., TCRs or antigen-binding fragments thereof, include one or more amino acid substitutions, e.g., as compared to a binding molecule, e.g., TCR, sequence described herein and/or compared to a sequence of a natural repertoire, e.g., human repertoire. Sites of interest for substitutional mutagenesis include the CDRs, FRs and/or constant regions. Amino acid substitutions may be introduced into a binding molecule of interest and the products screened for a desired activity, e.g., retained/improved antigen affinity or avidity, decreased immunogenicity, improved half-life, CD8-independent binding or activity, surface expression, promotion of TCR chain pairing and/or other improved properties or functions.
In some embodiments, one or more residues within a CDR of a parent binding molecule, e.g., TCR, is/are substituted. In some embodiments, the substitution is made to revert a sequence or position in the sequence to a germline sequence, such as a binding molecule sequence found in the germline (e.g., human germline), for example, to reduce the likelihood of immunogenicity, e.g., upon administration to a human subject.
In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the binding molecule, e.g., TCR or antigen-binding fragment thereof, to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. Such alterations may, for example, be outside of antigen contacting residues in the CDRs. In certain embodiments of the variable sequences provided herein, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
In some aspects, the TCR or antigen-binding fragment thereof may contain one or more modifications in the alpha chain and/or beta chain such that when the TCR or antigen-binding fragment thereof is expressed in a cell, the frequency of mis-pairing between the TCR alpha chain and beta chain and an endogenous TCR alpha chain and beta chain is reduced, the expression of the TCR alpha chain and beta chain is increased, and/or the stability of the TCR alpha chain and beta chain is increased.
In some embodiments, the TCR contains one or more non-native cysteine residues to introduce a covalent disulfide bond linking a residue of the immunoglobulin region of the constant domain of the α chain to a residue of the immunoglobulin region of the constant domain of the β chain. In some embodiments, one or more cysteines can be incorporated into the constant region extracellular sequences of the first and second segments of the TCR polypeptide. Exemplary non-limiting modifications in a TCR to introduce a non-native cysteine residues are described herein (see also, International PCT No. WO2006/000830 and WO2006037960). In some cases, both a native and a non-native disulfide bond may be desirable. In some embodiments, the TCR or antigen-binding fragment is modified such that the interchain disulfide bond in a native TCR is not present.
In some embodiments, the transmembrane domain of the constant region of the TCR can be modified to contain a greater number of hydrophobic residues (see e.g. Haga-Friedman et al. (2012) Journal of Immunology, 188:5538-5546). In some embodiments, the tranmembrane region of TCR α chain contains one or more mutations corresponding to S116L, G119V or F120L, with reference to numbering of a Cα set forth in any of SEQ ID NOS: 212, 213, 215, 217, 220, or 524.
In some embodiments, the cell expressing the TCR further includes a marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the TCR, such as a truncated version of a cell surface receptor, such as truncated EGFR (tEGFR). In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. See WO2014031687. In some embodiments, introduction of a construct encoding the TCR and EGFRt separated by a T2A, P2A or other ribosome switch can express two proteins from the same construct, such that the EGFRt can be used as a marker to detect cells expressing such construct. Exemplary of such markers that can be used are described below.
In some embodiments, the TCR or antigen-binding fragment thereof is encoded by a nucleotide sequence that is or has been codon-optimized. Exemplary codon-optimized variants are described elsewhere herein.
B. Antibodies
In some embodiments, the binding molecule is an antibody or antigen-binding fragment thereof that contains any one or more of the CDRs as described above with respect to TCRs.
In some embodiments, the antibody or antigen-binding fragment contains variable heavy and light chain containing a CDR1, CDR2 and/or CDR3 contained in the alpha chain and a CDR1, CDR2 and/or CDR3 contained in the beta chain as set forth in Table 2, Table 5, or Table 8. Also among the provided antibodies or antigen-binding fragments are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the antibody or antigen-binding fragment contains a variable region that contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vα region amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123 125, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, or 676. In some aspects, the antibody or antigen-binding fragment contains a variable region that contains a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vβ region amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 122, 124 126, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, or 685. Also among the provided antibodies or antigen-bind fragments are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the provided antibody or antibody fragment is a human antibody. In some embodiments, the provided antibody or antibody fragment contains a VH region that contains a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain V segment, a portion with at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence encoded by a germline nucleotide human heavy chain D segment, and/or a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence encoded by a germline nucleotide human heavy chain J segment; and/or contains a VL region that contains a portion with at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain V segment, and/or a portion with at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain J segment. In some embodiments, the portion of the VH region corresponds to the CDR-H1, CDR-H2 and/or CDR-H3. In some embodiments, the portion of the VH region corresponds to the framework region 1 (FR1), FR2, FR2 and/or FR4. In some embodiments, the portion of the VL region corresponds to the CDR-L1, CDR-L2 and/or CDR-L3. In some embodiments, the portion of the VL region corresponds to the FR1, FR2, FR2 and/or FR4.
In some embodiments, the antibody or antigen-binding fragment contains a framework region that contains human germline gene segment sequences. For example, in some embodiments, the antibody or antigen-binding fragment contains a VH region in which the framework region, e.g. FR1, FR2, FR3 and FR4, has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a framework region encoded by a human germline antibody segment, such as a V and/or J segment. In some embodiments, the human antibody contains a VL region in which the framework region e.g. FR1, FR2, FR3 and FR4, has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a framework region encoded by a human germline antibody segment, such as a V and/or segment. For example, in some such embodiments, the framework sequence of the VH and/or VL sequence differs by no more than 10 amino acids, such as no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid, compared to the framework region encoded by a human germline antibody segment. In some embodiments, the antibodies and antigen binding fragments thereof, e.g. TCR-like antibodies, specifically recognize a peptide epitope in the context of an MHC molecule, such as an MHC class I. In some cases, the MHC class I molecule is an HLA-A2 molecule, e.g. HLA-A2*01.
In some embodiments, the antibody or antigen-binding fragment thereof recognizes or binds to an epitope or region of HPV16 E6, such as a peptide epitope containing an amino acid sequence set forth in any of SEQ ID NOs: 232-234. In some instances, the TCR or antigen-binding fragment thereof that recognizes or binds a peptide epitope derived from HPV16 E6 is or comprises the sequence set forth in SEQ ID NO: 233.
In some aspects, the TCR or antigen-binding fragment recognizes or binds to an epitope or region of HPV16 E7 protein, such as a peptide epitope containing an amino acid sequence set forth in any of SEQ ID NOs: 235-239. In some embodiments, the TCR or antigen-binding fragment thereof does not recognize or bind the epitope E7 (11-19) comprising the amino acid sequence YMLDLQPET (SEQ ID NO. 236). In some cases, the peptide derived from HPV16 E7 is or contains the sequence set forth in SEQ ID NO: 235.
Thus, provided in some embodiments are anti-HPV antibodies, including functional antibody fragments. In some embodiments, the antibodies VH and/or VL domains, or antigen-binding site thereof, and are capable of specifically binding to a peptide epitope of HPV 16. In some embodiments, the antibodies include a variable heavy chain and a variable light chain, such as scFvs. The antibodies include antibodies that specifically bind to HPV, e.g., HPV 16 E6 or HPV 16 E7. Among the provided anti-HPV antibodies are human antibodies. The antibodies include isolated antibodies. Also provided are molecules containing such antibodies, e.g., single-chain proteins, fusion proteins, and/or recombinant receptors such as chimeric receptors, including antigen receptors.
The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab′)2 fragments, Fab′ fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
In some embodiments, the heavy and light chains of an antibody can be full-length or can be an antigen-binding portion (a Fab, F(ab′)2, Fv or a single chain Fv fragment (scFv)). In other embodiments, the antibody heavy chain constant region is chosen from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly chosen from, e.g., IgG1, IgG2, IgG3, and IgG4, more particularly, IgG1 (e.g., human IgG1). In another embodiment, the antibody light chain constant region is chosen from, e.g., kappa or lambda, particularly kappa.
Among the provided antibodies are antibody fragments. An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; variable heavy chain (VH) regions, single-chain antibody molecules such as scFvs and single-domain VH single antibodies; and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
The term “variable region” or “variable domain”, when used in reference to an antibody, such as an antibody fragment, refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody.
Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some aspects, the antibody fragments are scFvs.
Among the provided anti-HPV antibodies are human antibodies. A “human antibody” is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences, including human antibody libraries. The term excludes humanized forms of non-human antibodies comprising non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human. The term includes antigen-binding fragments of human antibodies.
A “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated. Human antibodies also may be derived from human antibody libraries, including phage display and cell-free libraries, containing antibody-encoding sequences derived from a human repertoire.
Among the provided antibodies are monoclonal antibodies, including monoclonal antibody fragments. The term “monoclonal antibody” as used herein refers to an antibody obtained from or within a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible variants containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. The term is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be made by a variety of techniques, including but not limited to generation from a hybridoma, recombinant DNA methods, phage-display and other antibody display methods.
As used herein, reference to a “corresponding form” of an antibody means that when comparing a property or activity of two antibodies, the property is compared using the same form of the antibody. For example, if it is stated that an antibody has greater activity compared to the activity of the corresponding form of a first antibody, that means that a particular form, such as a scFv of that antibody, has greater activity compared to the scFv form of the first antibody.
“Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
In some embodiments, the antibody, e.g., antibody fragment, may contain at least a portion of an immunoglobulin constant region, such as one or more constant region domain. In some embodiments, the constant regions include a light chain constant region and/or a heavy chain constant region 1 (CH1). In some embodiments, the antibody includes a CH2 and/or CH3 domain, such as an Fc region. In some embodiments, the Fc region is an Fc region of a human IgG, such as an IgG1 or IgG4.
The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.
The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).
1. Variants and Modifications
In certain embodiments, the antibodies or antigen-binding fragments thereof include one or more amino acid variations, e.g., substitutions, deletions, insertions, and/or mutations, compared to the sequence of an antibody described herein. Exemplary variants include those designed to improve the binding affinity and/or other biological properties of the antibody.
Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
In certain embodiments, the antibodies include one or more amino acid substitutions, e.g., as compared to an antibody sequence described herein and/or compared to a sequence of a natural repertoire, e.g., human repertoire. Sites of interest for substitutional mutagenesis include the CDRs and FRs. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, improved half-life, and/or improved effector function, such as the ability to promote antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).
In some embodiments, one or more residues within a CDR of a parent antibody (e.g. a humanized or human antibody) is/are substituted. In some embodiments, the substitution is made to revert a sequence or position in the sequence to a germline sequence, such as an antibody sequence found in the germline (e.g., human germline), for example, to reduce the likelihood of immunogenicity, e.g., upon administration to a human subject.
In some embodiments, alterations are made in CDR “hotspots,” residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library may then be created and screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. Such alterations may, for example, be outside of antigen contacting residues in the CDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.
In certain embodiments, the antibody or antigen-binding fragment thereof is altered to increase or decrease the extent to which the antibody is glycosylated, for example, by removing or inserting one or more glycosylation sites by altering the amino acid sequence and/or by modifying the oligosaccharide(s) attached to the glycosylation sites, e.g., using certain cell lines.
Exemplary modifications, variants, and cell lines are described, e.g., in Patent Publication Nos. US 2003/0157108, US 2004/0093621, US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107); WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.); WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
Among the modified antibodies are those having one or more amino acid modifications in the Fc region, such as those having a human Fc region sequence or other portion of a constant region (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.
Such modifications can be made, e.g., to improve half-life, alter binding to one or more types of Fc receptors, and/or alter effector functions.
Also among the variants are cysteine engineered antibodies such as “thioMAbs” and other cysteine engineered variants, in which one or more residues of an antibody are substituted with cysteine residues, in order to generate reactive thiol groups at accessible sites, e.g., for use in conjugation of agents and linker-agents, to produce immunoconjugates. Cysteine engineered antibodies are described, e.g., in U.S. Pat. Nos. 7,855,275 and 7,521,541.
In some embodiments, the antibodies are modified to contain additional nonproteinaceous moieties, including water soluble polymers. Exemplary polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
2 TCR-Like CARs
In some embodiments, the antibody or antigen-binding portion thereof is expressed on cells as part of a recombinant receptor, such as an antigen receptor. Among the antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Generally, a CAR containing an antibody or antigen-binding fragment that exhibits TCR-like specificity directed against a peptide in the context of an MHC molecule also may be referred to as a TCR-like CAR.
Thus, among the provided binding molecules, e.g., HPV 16 E6 or E7 binding molecules, are antigen receptors, such as those that include one of the provided antibodies, e.g., TCR-like antibodies. In some embodiments, the antigen receptors and other chimeric receptors specifically bind to a region or epitope of HPV16 E6 or E7, such as antigen receptors containing the provided anti-HPV 16 E6 or E7 antibodies or antibody fragments, e.g. TCR-like antibodies. Among the antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Also provided are cells expressing the CARs and uses thereof in adoptive cell therapy, such as treatment of diseases and disorders associated with HPV 16 E6 or E7 expression.
Thus, provided herein are TCR-like CARs that contain a non-TCR molecule that exhibits T cell receptor specificity, such as for a T cell epitope or peptide epitope when displayed or presented in the context of an MHC molecule. In some embodiments, a TCR-like CAR can contain an antibody or antigen-binding portion thereof, e.g., TCR-like antibody, such as described herein. In some embodiments, the antibody or antibody-binding portion thereof is reactive against specific peptide epitope in the context of an MHC molecule, wherein the antibody or antibody fragment can differentiate the specific peptide in the context of the MHC molecule from the MHC molecule alone, the specific peptide alone, and, in some cases, an irrelevant peptide in the context of an MHC molecule. In some embodiments, an antibody or antigen-binding portion thereof can exhibit a higher binding affinity than a T cell receptor.
Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in international patent application publication numbers WO2000/14257, WO2013/126726, WO2012/129514, WO2014/031687, WO2013/166321, WO2013/071154, WO2013/123061 U.S. patent application publication numbers US2002/131960, US2013/287748, US2013/0149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 Mar. 18(2): 160-75. In some aspects, the antigen receptors include a CAR as described in U.S. Pat. No. 7,446,190, and those described in International Patent Application Publication No.: WO2014/055668 A1. Exemplary of the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014/031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190, 8,389,282, e.g., and in which the antigen-binding portion, e.g., scFv, is replaced by an antibody, e.g., as provided herein.
In some embodiments, the CARs generally include an extracellular antigen (or ligand) binding domain, including as an antibody or antigen-binding fragment thereof specific for a peptide in the context of an MHC molecule, linked to one or more intracellular signaling components, in some aspects via linkers and/or transmembrane domain(s). In some embodiments, such molecules can typically mimic or approximate a signal through a natural antigen receptor, such as a TCR, and, optionally, a signal through such a receptor in combination with a costimulatory receptor.
In some embodiments, the CAR typically includes in its extracellular portion one or more antigen binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable domains, and/or antibody molecules. In some embodiments, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb). In some embodiments, the CAR contains a TCR-like antibody, such as an antibody or an antigen-binding fragment (e.g., scFv) that specifically recognizes a peptide epitope presented on the cell surface in the context of an MHC molecule.
In some aspects, the antigen-specific binding, or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR includes a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
In some embodiments, the CAR, e.g., TCR-like CAR, such as the antibody portion thereof, further includes a spacer, which may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region, e.g., an IgG4 hinge region, and/or a CH1/CL and/or Fc region. In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgG1. In some aspects, the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. In some examples, the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153 or international patent application publication number WO2014/031687.
In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgG1. In some embodiments, the spacer has the sequence ESKYGPPCPPCP (set forth in SEQ ID NO: 268), and is encoded by the sequence set forth in SEQ ID NO: 269. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 270. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 271. In some embodiments, the constant region or portion is of IgD. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 272. In some embodiments, the spacer has a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 268, 270, 271, or 272.
The antigen recognition domain generally is linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and/or signal via another cell surface receptor. Thus, in some embodiments, the antibody or antigen-binding fragment thereof is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and/or transmembrane domain(s).
Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
The CAR generally includes at least one intracellular signaling component or components. In some embodiments, the CAR includes an intracellular component of the TCR complex, such as a TCR CD3+ chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the antigen binding molecule is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the CAR further includes a portion of one or more additional molecules such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR includes a chimeric molecule between CD3-zeta (CD3-0 or Fc receptor γ and CD8, CD4, CD25 or CD16.
In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling domain of the CAR activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functional capability.
In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal. In some aspects, the cell comprises a first CAR which contains signaling domains to induce the primary signal and a second CAR which binds to a second antigen and contains the component for generating a costimulatory signal. For example, a first CAR can be an activating CAR and the second CAR can be a costimulatory CAR. In some aspects, both CARs must be ligated in order to induce a particular effector function in the cell, which can provide specificity and selectivity for the cell type being targeted.
T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.
In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from TCR or CD3 zeta, FcR gamma, CD3 gamma, CD3 delta or CD3 epsilon. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.
In some embodiments, the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR includes both the activating and costimulatory components; in other aspects, the activating domain is provided by one CAR whereas the costimulatory component is provided by another CAR recognizing another antigen.
In some embodiments, the activating domain is included within one CAR, whereas the costimulatory component is provided by another chimeric receptor recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, and costimulatory receptors, both expressed on the same cell (see WO2014/055668). In some aspects, the HPV 16 E6 or E7 antibody-containing receptor is the stimulatory or activating CAR; in other aspects, it is the costimulatory receptor. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as an inhibitory receptor recognizing a peptide epitope other than HPV 16 E6 or HPV16 E7, whereby an activating signal delivered through the HPV 16-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.
In some embodiments, the cell expressing the provided TCR or other binding molecule further expresses an additional receptor, such as a receptor capable of delivering a costimulatory or survival-promoting signal, such as a costimulatory receptor (see WO2014/055668) and/or to block or change the outcome of an inhibitory signal, such as one typically delivered via an immune checkpoint or other immunoinhibitory molecule, such as one expressed in the tumor microenvironment, e.g., in order to promote increased efficacy of such engineered cells. See, e.g., Tang et al., Am J Transl Res. 2015; 7(3): 460-473. In some embodiments, the cell may further include one or more other exogenous or recombinant or engineered components, such as one or more exogenous factors and/or costimulatory ligands, which are expressed on or in or secreted by the cells and can promote function, e.g., in the microenviroment. Exemplary of such ligands and components include, e.g., TNFR and/or Ig family receptors or ligands, e.g., 41BBL, CD40, CD40L, CD80, CD86, cytokines, chemokines, and/or antibodies or other molecules, such as scFvs. See, e.g., patent application publication Nos WO2008121420 A1, WO2014134165 A1, US20140219975 A1. In some embodiments, the cells comprise one or more inhibitory receptor ((iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013)), such as one that binds to a ligand or antigen not associated with the disease or condition or not expressed therein or thereon.
In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.
In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.
In some embodiments, the cell expressing the CAR or other antigen receptor further includes a marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor, such as a truncated version of a cell surface receptor, such as truncated EGFR (tEGFR). In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. See WO2014031687. In some embodiments, introduction of a construct encoding the CAR and EGFRt separated by a T2A ribosome switch can express two proteins from the same construct, such that the EGFRt can be used as a marker to detect cells expressing such construct. In some embodiments, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence. An exemplary polypeptide for a truncated EGFR (e.g. tEGFR) comprises the sequence of amino acids set forth in SEQ ID NO: 273 or 343 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 273 or 343. An exemplary T2A linker sequence comprises the sequence of amino acids set forth in SEQ ID NO: 211 or 274 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 211 or 274.
In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.
In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self” by the immune system of the host into which the cells will be adoptively transferred.
In some embodiments, the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive transfer and encounter with ligand.
In some cases, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR in some aspects is one that includes multiple costimulatory domains of different costimulatory receptors.
In some embodiments, the chimeric antigen receptor includes an extracellular portion containing a TCR-like antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment includes a scFv and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain includes a signaling domain of a zeta chain of a CD3-zeta (CD3) chain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some aspects, the transmembrane domain contains a transmembrane portion of CD28. The extracellular domain and transmembrane can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule, such as between the transmembrane domain and intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.
For example, in some embodiments, the CAR contains a TCR-like antibody, e.g., an antibody fragment, as provided herein, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some embodiments, the CAR contains a TCR-like antibody, e.g., antibody fragment, as provided herein, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4-1BB or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some such embodiments, the CAR further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4 hinge, such as a hinge-only spacer.
In some embodiments, the transmembrane domain of the receptor, e.g., the TCR-like CAR, is a transmembrane domain of human CD28 (e.g., Accession No. P01747.1) or variant thereof, such as a transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 275 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 275. In some embodiments, the transmembrane-domain containing portion of the CAR comprises the sequence of amino acids set forth in SEQ ID NO: 276 or a sequence of amino acids having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 276.
In some embodiments, the intracellular signaling component(s) of the CAR, e.g., the TCR-like CAR, contains an intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as a domain with an LL to GG substitution at positions 186-187 of a native CD28 protein. For example, the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 277 or 278 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 277 or 278. In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of 4-1BB (e.g. (Accession No. Q07011.1) or functional variant or portion thereof, such as the sequence of amino acids set forth in SEQ ID NO: 279 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 279.
In some embodiments, the intracellular signaling domain of the CAR, e.g. the TCR-like CAR, comprises a human CD3 zeta stimulatory signaling domain or functional variant thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human CD3 (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. No. 7,446,190 or U.S. Pat. No. 8,911,993. For example, in some embodiments, the intracellular signaling domain comprises the sequence of amino acids of SEQ ID NO: 280, 281, or 282, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 280, 281, or 282.
In some aspects, the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgG1, such as the hinge only spacer set forth in SEQ ID NO: 268. In other embodiments, the spacer is or contains an Ig hinge, e.g., an IgG4-derived hinge, optionally linked to a CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains, such as set forth in SEQ ID NO: 271. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a CH3 domain only, such as set forth in SEQ ID NO: 270. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers.
For example, in some embodiments, the TCR-like CAR includes a TCR-like antibody or fragment, such as any provided herein, including scFvs, a spacer such as any of the Ig-hinge containing spacers, a CD28 transmembrane domain, a CD28 intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the TCR-like CAR includes the a TCR-like antibody or fragment, such as any provided herein, including scFvs, a spacer such as any of the Ig-hinge containing spacers, a CD28 transmembrane domain, a CD28 intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, such TCR-like CAR constructs further includes a T2A ribosomal skip element and/or a tEGFR sequence, e.g., downstream of the CAR.
In some embodiments, such CAR constructs further includes a T2A ribosomal skip element and/or a tEGFR sequence, e.g., downstream of the CAR, such as set forth in SEQ ID NO: 211 or 274 and a tEGFR sequence set forth in SEQ ID NO: 273 or 343, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 211, 273, 343, or 274.
In some embodiments, the CAR includes an HPV 16 E6 or E7 antibody or fragment, such as any of the HPV16 E6 or E7 antibodies, including sdAbs (e.g. containing only the VH region) and scFvs, described herein, a spacer such as any of the Ig-hinge containing spacers, a CD28 transmembrane domain, a CD28 intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR includes the HPV 16 antibody or fragment, such as any of the HPV 16 E6 or E7 antibodies, including sdAbs and scFvs described herein, a spacer such as any of the Ig-hinge containing spacers, a CD28 transmembrane domain, a CD28 intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, such CAR constructs further includes a T2A ribosomal skip element and/or a tEGFR sequence, e.g., downstream of the CAR.
3. Exemplary Features of Binding Molecules and Engineered Cells
In some aspects, the provided binding molecules, e.g. TCRs or TCR-like CAR have one or more specified functional features, such as binding properties, including binding to particular epitopes, lack of off-target binding or activity and/or particular binding affinities. In some embodiments, any one or more of the features of a provided TCR can be assessed by expressing the TCR, e.g., by introducing one or more nucleic acid encoding the TCR, into a T cell, such a primary T cell or a T cell line. In some embodiments, the T cell line is a Jurkat cell or a Jurkat-derived cell line. Exemplary of a Jurkat-derived cell line is the J.RT3-T3.5 (ATCC® TIB-153™) cell line, produced by treatment of the Jurkat leukemia cell line with irradiation mutagenesis and negative selection with OKT3 monoclonal antibody (see Weiss & Stobo, J. Ex. Med. 160(5):1284-1299 (1984)).
In some embodiments, the provided binding molecules are capable of binding to a peptide epitope of HPV16, e.g. an epitope of HPV 16 E6 or E7 such as described above, with at least a certain affinity, as measured by any of a number of known methods. In some embodiments, the peptide epitope is a peptide in the context of an MHC molecule or ligand. In some embodiments, the affinity is represented by an equilibrium dissociation constant (KD) or an association constant (ka). In some embodiments, the affinity is represented by EC50.
In some embodiments, the binding molecule, e.g., TCR, binds, such as specifically binds, to a peptide epitope, e.g., in complex with an MHC molecule, with an affinity or KA (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M; equal to the ratio of the on-rate [kon or ka] to the off-rate [koff or kd] for this association reaction, assuming bimolecular interaction) equal to or greater than 105 M−1. In some embodiments, the TCR or fragment thereof exhibits a binding affinity for the peptide epitope with a KD (i.e., an equilibrium dissociation constant of a particular binding interaction with units of M; equal to the ratio of the off-rate [koff or kd] to the on-rate [kon or ka] for this association reaction, assuming bimolecular interaction) of equal to or less than 10−5 M. For example, the equilibrium dissociation constant KD ranges from or from about 10−5 M to or to about 10−12 M, such as from from about 10−6 M to or to about 1010 M, from or from about 10−7 M to or to about 10−11 M, from or from about 10−6 M to or to about 10−8 M, or from or from about 10−7 M to or to about 10−8 M. The on-rate (association rate constant; kon or ka; units of 1/Ms) and the off-rate (dissociation rate constant; koff or kd; units of 1/s) can be determined using any of the assay methods known in the art, for example, surface plasmon resonance (SPR).
In some embodiments, binding affinity may be classified as high affinity or as low affinity. In some cases, the binding molecule (e.g. TCR) that exhibits low to moderate affinity binding exhibits a KA of up to 107 M−1, up to 106 M−1, up to 105 M. In some cases, a binding molecule (e.g. TCR) that exhibits high affinity binding to a particular epitope interacts with such epitope with a KA of at least 107 M−1, at least 108 M−1, at least 109 M−1, at least 1010 M−1, at least 1011 M−1, at least 1012 M−1, or at least 1013 M. In some embodiments, the binding affinity (EC50) and/or the dissociation constant of the binding molecule to a peptide epitope of HPV 16 E6 or E7 is from or from about 0.1 nM to 1 μM, 1 nM to 1 μM, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 10 nM, 10 nM to 500 nM, 10 nM to 100 nM, 10 nM to 50 nM, 50 nM to 500 nM, 50 nM to 100 nM or 100 nM to 500 nM. In certain embodiments, the binding affinity (EC50) and/or the dissociation constant of the binding molecule to a peptide epitope of HPV 16 E6 or E7 is at or about or less than at or about 1 μM, 500 nm, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM.
A variety of assays are known for assessing binding affinity and/or determining whether a binding molecule specifically binds to a particular ligand (e.g. peptide in the context of an MHC molecule). It is within the level of a skilled artisan to determine the binding affinity of a binding molecule, e.g., TCR, for a T cell epitope of a target polypeptide, such as by using any of a number of binding assays that are well known in the art. For example, in some embodiments, a BIAcore machine can be used to determine the binding constant of a complex between two proteins. The dissociation constant for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), or determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blot, ELISA, analytical ultracentrifugation, spectroscopy and surface plasmon resonance (Biacore®) analysis (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Pat. Nos. 5,283,173, 5,468,614, or the equivalent), flow cytometry, sequencing and other methods for detection of expressed nucleic acids. In one example, apparent affinity for a TCR is measured by assessing binding to various concentrations of tetramers, for example, by flow cytometry using labeled tetramers. In one example, apparent KD of a TCR is measured using 2-fold dilutions of labeled tetramers at a range of concentrations, followed by determination of binding curves by non-linear regression, apparent KD being determined as the concentration of ligand that yielded half-maximal binding.
In some embodiments, the binding molecules display a binding preference for antigen recognition of HPV 16 E6- or E7-expressing cells as compared to HPV 16 E6- or E7-negative cells, such as particular cells known and/or described herein to express HPV 16 E6 or E7 and known not to express HPV 16 E6 or E7. In some embodiments, the binding preference is observed where a significantly greater degree of binding is measured to the HPV 16 E6- or E7-expressing, as compared to the non-HPV 16 E6- or E7-expressing cells. In some embodiments, the fold change in degree of binding detected, for example, as measured by mean fluorescence intensity in a flow cytometry-based assay and/or dissociation constant or EC50, to the HPV 16 E6- or E7-expressing cells as compared to the non-HPV 16 E6- or E7-expressing cells, is at least at or about 1.5, 2, 3, 4, 5, 6, or more.
In some embodiments, the binding molecule, e.g. TCR, does not exhibit cross-reactive or off-target binding, such as undesirable off-target binding, e.g. off-target binding to antigens present in healthy or normal tissues or cells. In some embodiments, the binding molecule, e.g. TCR, recognizes, such as specifically binds, only one peptide epitope or antigen complex, such as recognizes only a particular HPV 16 E6 or E7 epitope set forth in any of SEQ ID NOs: 232-239 or an antigen complex thereof. Thus, in some embodiments, the provided binding molecules, e.g. TCRs, have a reduced risk of causing unwanted side effects due to, for example, recognition of a non-target peptide epitope.
In some embodiments, the binding molecule, e.g., TCR, does not recognize, such as does not specifically bind, a sequence-related peptide epitope of the HPV 16 E6 or E7 epitope set forth in any of SEQ ID NOS: 232-239, i.e., does not recognize an epitope sharing some amino acids in common with an HPV 16 E6 or E7 epitope set forth in any of SEQ ID NOS: 232-239, such as does not recognize an epitope that differs in 1, 2, 3, 4, 5 or 6 amino acid residues from such epitope when the epitopes are aligned. In some embodiments, the binding molecule, e.g., TCR, does not recognize a sequence-unrelated epitope of the HPV 16 E6 or E7 epitope set forth in any of SEQ ID NOS: 232-239, i.e., does not recognize an epitope that is substantially different in sequence compared to an HPC 16 E6 or E7 epitope set forth in any of SEQ ID NOS: 232-239, such as differing in more than 6, 7, 8, 9, 10 or more amino acid residues from such epitope when the epitopes are aligned. In some embodiments, the binding molecule, e.g., TCR, does not recognize the HPV 16 E6 or E7 epitope set forth in any of SEQ ID NOS: 232-239 in the context of a different MHC allele, such as in the context of an MHC allele other than HLA-A2.
Typically, specific binding of binding molecule, e.g. TCR, to a peptide epitope, e.g. in complex with an MHC, is governed by the presence of an antigen-binding site containing one or more complementarity determining regions (CDRs). In general, it is understood that specifically binds does not mean that the particular peptide epitope, e.g. in complex with an MHC, is the only thing to which the MHC-peptide molecule may bind, since non-specific binding interactions with other molecules may also occur. In some embodiments, binding of binding molecule to a peptide in the context of an MHC molecule is with a higher affinity than binding to such other molecules, e.g. another peptide in the context of an MHC molecule or an irrelevant (control) peptide in the context of an MHC molecule, such as at least about 2-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or at least about 100-fold higher than binding affinity to such other molecules.
In some embodiments, the binding molecule, e.g., TCR, can be assessed for safety or off-target binding activity using any of a number of screening assays known in the art. In some embodiments, generation of an immune response to a particular binding molecule, e.g., TCR, can be measured in the presence of cells that are known not to express the target peptide epitope, such as cells derived from normal tissue(s), allogenic cell lines that express one or more different MHC types or other tissue or cell sources. In some embodiments, the cells or tissues include normal cells or tissues. For example, in some cases, cells or tissues can include brain, muscle, liver, colon, kidney, lung, ovary, placenta, heart, pancreas, prostate, epithelium or skin, testis, adrenal, intestine, bone marrow or spleen. In some embodiments, the binding to cells can be tested in 2 dimensional cultures. In some embodiments, the binding to cells can be tested in 3 dimensional cultures. In some embodiments, as a control, the tissues or cells can be ones that are known to express the target epitope. The immune response can be assessed directly or indirectly, such as by assessing activation of immune cells such as T cells (e.g. cytotoxic activity), production of cytokine (e.g. interferon gamma), or activation of a signaling cascade.
In some embodiments, potential off-targets can be identified by performing a homology scan of the human genome using the particular target epitope, e.g., to identify potential sequence-related epitopes. In some cases, a protein sequence database can be analyzed to identify peptides with similarity to the target peptide epitope. In some embodiments, to facilitate identification of potential sequence-related epitopes of interest, a binding motif can first be identified. In some embodiments, the binding motif can be identified by peptide scanning, such as an alanine mutagenesis scan, of the target epitope (e.g., HPV 16 E6 or E7 epitope set forth in any of SEQ ID NOS: 232-239) to identify the binding motif recognized by the binding molecule, see e.g. WO2014/096803. In some embodiments, the binding motif can be identified by mutagenesis of the target peptide so that a series of mutants are generated in which each amino acid or a subset thereof is changed to another amino acid residue, tested for its activity relative to the original target epitope, and those residues that are involved in or required for binding are identified. In some embodiments, a series of mutants may be made in which the amino acid residue at each position of the target epitope is mutated to all alternative amino acids. In some cases, once the binding motif is identified (i.e. amino acid residues that are non-tolerated and are involved in or are required for binding), protein databases may be searched for proteins that contain the binding motif.
In some embodiments, suitable protein databases include but are not limited to UniProtKB/Swiss-Prot (http://www.uniprot.org/), Protein Information Resource (PI R) (http://pir.georgetown.edu/pirwww/index.shtml), and/or Reference Sequence (RefSeq) (www.ncbi.nlm.nih.gov/RefSeq). Searching for a peptide motif may be carried out using any one of a number of tools, which may be found on bioinformatics resource sites such as ExPASY (http://www.expasy.org/). For example, the search tool ScanProsite identifies user-defined motifs in all protein sequences in the UniProtKB/Swiss-Prot Protein Knowledgebase (De Castro et al. Nucleic Acids Res. 2006 Jul. 1; 34 (Web Server issue):W362-5). In some cases, the search may be carried out for peptides that are of human origin or of organisms which are commonly present in humans, such as viral or bacterial pathogens, or commensal bacteria.
In some embodiments, if a potential off-target epitope is identified, the binding molecule, e.g., TCR, can be redesigned so that there is no longer any cross reactivity to the off target peptide(s), while maintaining binding, preferably with high affinity, to the target peptide epitope. For example, T cell receptors can be redesigned by mutagenesis using the methods described in WO 03/020763.
In some embodiments, the binding molecules, e.g., engineered cells comprising the binding molecules, e.g., TCRs, elicit an immune response to HPV 16. In some embodiments, cytotoxic T lymphocytes (CTL) may be activated when cells containing the binding molecules, e.g., TCRs, are contacted with target cells, such as those that express HPV 16, such as HPV 16 E6 or HPV 16 E7. For example, cells containing the TCRs may induce lysis of target cells, such as HPV 16-expressing, e.g., HPV 16 E6- or E7-expressing cells. In some aspects, the ability of the binding molecules, such as cells expressing the binding molecules, e.g., TCRs or CARs, to elicit an immune response can be determined by measuring cytokine release. In some embodiments, in response to coculture with or exposure to cells expressing the binding molecules, e.g., TCRs or CARs, a variety of cytokines are released when the cells are stimulated by an appropriate target cell known to express HPV 16, such as HPV 16 E6 or HPV 16 E7. Non-limiting examples of such cytokines include IFN-γ, TNF-α, and GM-CSF. Exemplary cells known to express HPV 16 include, but are not limited to, CaSki cells (ATCC No. CRL-1550, which contain about 600 copies of integrated HPV16) or other tumor cell expressing the relevant MHC molecule and the corresponding peptide epitope, e.g., HPV 16 E6 or E7 epitope, such as any of those set forth in SEQ ID NOs: 232-239.
In some embodiments, CTL activation can be determined. A variety of techniques exist for assaying the activity of CTL. In some embodiments, CTL activity can be assessed by assaying the culture for the presence of CTLs that lyse radio-labeled target cells, such as specific peptide-pulsed targets. These techniques include the labeling of target cells with radionuclides such as Na2, 51CrO4 or 3H-thymidine, and measuring the release or retention of the radionuclides from the target cells as an index of cell death. In some embodiments, CTL are known to release a variety of cytokines when they are stimulated by an appropriate target cell, such as a tumor cell expressing the relevant MHC molecule and the corresponding peptide epitope, and the presence of such epitope-specific CTLs can be determined by measuring cytokine release. Non-limiting examples of such cytokines include IFN-γ, TNF-α, and GM-CSF. Assays for these cytokines are well known in the art, and their selection is left to the skilled artisan. Methodology for measuring both target cell death and cytokine release as a measure of CTL reactivity are given in Coligan, J. E. et al. (Current Protocols in Immunology, 1999, John Wiley & Sons, Inc., New York).
In some embodiments, cytokine production can be measured as an indicator of an immune response. In some cases, such measured cytokines can include, without limitation, interlekukin-2 (IL-2), interferon-gamma (IFNγ), interleukin-4 (IL-4), TNF-alpha, interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12) or TGF-beta. Assays to measure cytokines are well known in the art, and include, without limitation, ELISA, intracellular cytokine staining, cytometric bead array, RT-PCR, ELISPOT, flow cytometry and bio-assays in which cells responsive to the relevant cytokine are tested for responsiveness (e.g. proliferation) in the presence of a test sample.
In some embodiments, cells exposed to the binding molecules, e.g. cells containing the binding molecules, such as TCRs or CARs, are assessed for an immunological readout, such as using a T cell assay. In some embodiments, the binding molecule-containing cells can activate a CD8+ T cell response. In one embodiment, CD8+ T cell responses can be assessed by monitoring CTL reactivity using assays that include, but are not limited to, target cell lysis via 51Cr release or detection of interferon gamma release, such as by enzyme-linked immunosorbent spot assay (ELISA), intracellular cytokine staining or ELISPOT. In some embodiments, the binding molecules, e.g., cells containing the binding molecules, such as TCRs or CARs, can activate a CD4+ T cell response. In some aspects, CD4+ T cell responses can be assessed by assays that measure proliferation, such as by incorporation of [3H]-thymidine into cellular DNA and/or by the production of cytokines, such as by ELISA, intracellular cytokine staining or ELISPOT. In some cases, the cytokine can include, for example, interleukin-2 (IL-2), interferon-gamma (IFN-gamma), interleukin-4 (IL-4), TNF-alpha, interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12) or TGF beta. In some embodiments, recognition or binding of the peptide epitope, such as a MHC class II epitope, by the binding molecule can elicit or activate a CD4+ T cell response and/or a CD8+ T cell response.
In some embodiments, the binding specificity and/or function (e.g., ability to elicit an immune response to HPV 16) of the binding molecule, e.g., TCR or antigen-binding fragment thereof, is at least partially CD8-independent. In some cases, TCR recognition of a peptide in the context of an MHC molecule and subsequent T cell activation is facilitated in the presence of a CD8 co-receptor. For example, CD8 coreceptor engagement can facilitate low- to moderate-TCR affinity interactions and/or T cell activation (See, for example, Kerry et al. J. Immunology (2003) 171(9): 4493-4503 and Robbins et al. J Immunology (2008) 180(9): 6116-6131). Among the provided binding molecules are molecules, e.g. TCRs, that exhibit CD8-independent binding for an HPV E6 or E7 peptide epitope. In some embodiments, such binding molecules, e.g. TCR, may have higher functional avidity or affinity than TCRs or antigen binding fragments thereof that require the presence of CD8 co-expression. In some aspects, the provided CD8-independent binding molecules, such as TCRs, can be expressed or engineered in cells, e.g. T cells, that do not express CD8, such as can be expressed or engineered in CD4+ cells. In some embodiments, among the provided engineered non-CD8-expressing cells, e.g. CD4+ cells, are cells expressing a recombinant binding molecule, e.g., TCR or antigen-binding fragment, that exhibit at least or at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the binding specificity, affinity and/or avidity for a peptide in the context of an MHC molecule as the same binding molecule (e.g., TCR or antigen-binding fragment thereof) that is expressed on a CD8+ T cell.
II. Nucleic Acids, Vectors and Methods of Expression
Also provided are nucleic acids encoding any of the provided binding molecules, e.g., TCRs or antigen-binding fragments thereof or antibodies or antigen-binding fragments thereof or CARs containing such antibodies, such as those described herein. The nucleic acids may include those encompassing natural and/or non-naturally occurring nucleotides and bases, e.g., including those with backbone modifications. The terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide.
In some embodiments, the binding molecule, e.g. TCR, or antigen binding portion thereof may be a recombinantly produced natural protein or mutated form thereof in which one or more property, such as binding characteristic, has been altered. In some aspects, the nucleic acid is synthetic. In some cases, the nucleic acid is or contains cDNA. In some aspects, the nucleic acid molecule can be modified for use in the constructs described herein, such as for codon optimization. In some cases, the sequences can be designed to contain terminal restriction site sequences for purposes of cloning into vectors.
In some embodiments, nucleic acid molecule encoding the binding molecule, e.g. TCR, can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of encoding nucleic acids within or isolated from a given cell or cells. In some embodiments, the TCR is obtained from a biological source, such as from cells such as from a T cell (e.g. cytotoxic T cell), T cell hybridomas or other publicly available source. In some embodiments, a TCR may be derived from one of various animal species, such as human, mouse, rat, or other mammal, such as generally from a human. In some embodiments, the T cells can be obtained from in vivo isolated cells, such as from normal (or healthy) subjects or diseased subjects, including T cells present in peripheral blood mononuclear cells (PBMCs) or tumor-infiltrating lymphocytes (TILs). In some embodiments, the T cells can be a cultured T cell hybridoma or clone. For example, in some embodiments, to generate a vector encoding a TCR, the α and β chains can be PCR amplified from total cDNA isolated from a T cell clone expressing the TCR of interest and cloned into an expression vector. In some embodiments, the α and β chains can be synthetically generated. In some embodiments, the α and β chains are cloned into the same vector.
In some embodiments, the TCR or antigen-binding portion thereof can be synthetically generated from knowledge of the sequence of the TCR.
In some embodiments, the nucleic acid molecule contains a nucleic acid sequence encoding an alpha chain and/or a nucleotide sequence encoding a beta chain.
In some embodiments, the nucleic acid sequence encoding the alpha chain comprises one of the following: residues 61-816 of SEQ ID NO: 20, residues 58-804 of SEQ ID NO: 30, residues 61-825 of SEQ ID NO: 40, residues 64-813 of SEQ ID NO: 50, residues 64-816 of SEQ ID NO: 60, residues 58-807 of SEQ ID NO: 70, residues 61-825 of SEQ ID NO: 80, residues 67-831 of SEQ ID NO: 90, residues 58-801 of SEQ ID NO: 100, residues 64-810 of SEQ ID NO: 183, residues 58-801 of SEQ ID NO: 202, residues 67-813 of SEQ ID NO: 219, a degenerate sequence thereof or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some aspects, the nucleotide sequence encoding the beta chain comprises one of the following: residues 58-936 of SEQ ID NO: 17, residues 58-930 of SEQ ID NO: 16, residues 58-939 of SEQ ID NO: 24, residues 64-930 of SEQ ID NO: 34 or 44, residues 58-933 of SEQ ID NO: 55, residues 58-927 of SEQ ID NO: 64, residues 64-936 of SEQ ID NO: 74, residues 58-933 of SEQ ID NO: 84, residues 63-930 of SEQ ID NO: 94, residues 46-936 of SEQ ID NO: 104, residues 58-933 of SEQ ID NO: 108, a degenerate sequence thereof or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
In some embodiments, the nucleotide sequence encoding the alpha chain and/or the nucleotide sequence encoding the beta chain is codon-optimized. Typically, codon optimization involves balancing the percentages of codons selected with the published abundance of human transfer RNAs so that none is overloaded or limiting. This may be necessary in some cases because most amino acids are encoded by more than one codon, and codon usage varies from organism to organism. Differences in codon usage between transfected genes and host cells can have effects on protein expression and immunogenicity of a nucleic acid construct. In general, for codon optimization, codons are chosen to select for those codons that are in balance with human usage frequency. Typically, the redundancy of the codons for amino acids is such that different codons code for one amino acid. In some embodiments, in selecting a codon for replacement, it may be desired that the resulting mutation is a silent mutation such that the codon change does not affect the amino acid sequence. Generally, the last nucleotide of the codon can remain unchanged without affecting the amino acid sequence.
In some cases, the nucleic acid sequence encoding the alpha chain contains one of the following: residues 67-825 of SEQ ID NO: 10, residues 58-813 of SEQ ID NO: 11, residues 64-822 of SEQ ID NO: 12 residues 61-825 of SEQ ID NO: 21, residues 58-813 of SEQ ID NO: 31, residues 61-834 of SEQ ID NO: 41, residues 63-822 of SEQ ID NO: 51, residues 64-825 of SEQ ID NO: 61, residues 58-816 of SEQ ID NO: 71, residues 61-834 of SEQ ID NO: 81, residues 67-840 of SEQ ID NO: 91, residues 58-810 of SEQ ID NO: 101, a degenerate sequence thereof or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some examples, the nucleotide sequence encoding the beta chain contains one of the following: residues 58-930 of SEQ ID NO: 7, residues 58-936 of SEQ ID NO: 8, residues 58-933 of SEQ ID NO: 9 residues 58-939 of SEQ ID NO: 25, residues 64-930 of SEQ ID NO: 35, 45, or 95, residues 58-933 of SEQ ID NO: 54 or 85, residues 58-927 of SEQ ID NO: 65, residues 64-936 of SEQ ID NO: 75, residues 46-936 of SEQ ID NO: 105, a degenerate sequence thereof or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
In some embodiments, the nucleic acid molecule encoding an alpha chain and/or beta chain of a TCR comprises a nucleic acid sequence corresponding to a SEQ ID NO. set forth in Table 11. Also among the provided nucleic acid molecules encoding a TCR are those containing sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences. Exemplary TCRs encoded by such sequences, or their modified versions, also are set forth in the Table 11.
TABLE 11
HPV16 E6 & E7 TCR Nucleotide SEQ ID NOs.
Exemplary TCR
Alpha
Beta
or modified
Codon-
Codon-
version thereof
Native
Optimized
Native
Optimized
TCR 3
20
21
24
25
TCR 4
30
31
34
35
TCR 5
40
41
44
45
TCR 8
70
71
74
75
TCR 9
80
81
84
85
TCR 10
90
91
94
95
TCR 6
50
51
54
55
TCR 7
60
61
64
65
TCR 11
100
101
104
105
TCR 12
183
12
108
9
TCR 13
202
11
17
8
TCR 14
219
10
16
7
TCR 15
389
1097
390
1098
TCR 16
430
1099
431
1100
TCR 17
1019
1101
1020
1102
TCR 18
1021
1103
1022
1104
TCR 19
1023
1105
1024
1106
TCR 20
1025
1107
1026
1108
TCR 21
1027
1109
1028
1110
TCR 22
1029
1111
1030
1112
TCR 23
1031
1113
1032
1114
TCR 24
1033
1115
1034
1116
TCR 25
1035
1117
1036
1118
TCR 26
1037
1119
1038
1120
TCR 27
1039
1121
1040
1122
TCR 28
1041
1123
1042
1124
TCR 29
1043
1125
1044
1126
TCR 30
1045
1127
1046
1128
TCR 31
1225
1129
1224
1130
TCR 32
1049
1131
1050
1132
TCR 33
1051
1133
1052
1134
TCR 34
1226
1135
1227
1136
TCR 35
1055
1137
1056
1138
TCR 36
1057
1139
1058
1140
TCR 37
1059
1141
1060
1142
TCR 38
1061
1143
1062
1144
TCR 39
1063
1145
1064
1146
TCR 40
1065
1147
1066
1148
TCR 41
1067
1149
1068
1150
TCR 42
1069
1151
1070
1152
TCR 43
1071
1153
1072
1154
TCR 44
1073
1155
1074
1156
TCR 45
1075
1157
1076
1158
TCR 46
1077
1159
1078
1160
TCR 47
1079
1161
1080
1162
TCR 48
1081
1163
1082
1164
TCR 49
1083
1165
1084
1166
TCR 50
1085
1167
1086
1168
TCR 51
1087
1169
1088
1170
TCR 52
1089
1171
1090
1172
TCR 53
1091
1173
1092
1174
TCR 54
1093
1175
1094
1176
TCR 55
1095
1177
1228
1178
Also provided are vectors or constructs containing such nucleic acid molecules. In some embodiments, the vectors or constructs contain one or more promoters operatively linked to the nucleotide encoding the alpha chain and/or beta chain. In some embodiments, the promoter is operatively linked to one or more than one nucleic acid molecule.
In some embodiments, the vector or construct can contain a single promoter that drives the expression of one or more nucleic acid molecules. In some embodiments, such promoters can be multicistronic (bicistronic or tricistronic, see e.g., U.S. Pat. No. 6,060,273). For example, in some embodiments, transcription units can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows coexpression of gene products (e.g. encoding an alpha chain and/or beta chain of a TCR) by a message from a single promoter. Alternatively, in some cases, a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF), two or three genes (e.g. encoding an alpha chain and/or beta chain of a TCR) separated from one another by sequences encoding a self-cleavage peptide (e.g., T2A) or a protease recognition site (e.g., furin). The ORF thus encodes a single polyprotein, which, either during (in the case of 2A e.g., T2A) or after translation, is cleaved into the individual proteins. In some cases, the peptide, such as T2A, can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream (see, for example, de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and deFelipe et al. Traffic 5:616-626 (2004)). Examples of 2A cleavage peptides, including those that can induce ribosome skipping, are Thosea asigna virus (T2A, e.g., SEQ ID NO: 211 or 274), porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 204 or 345), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 346) and 2A sequences from the foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 344) as described in U.S. Patent Publication No. 2007/0116690.
In some cases, the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a nucleotide sequence encoding an internal ribosome entry site (IRES) or a peptide sequence that causes ribosome skipping. In some instances, the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a peptide sequence that causes ribosome skipping. In some such instances, the peptide that causes ribosome skipping is a P2A or T2A peptide and/or contains the sequence of amino acids set forth in SEQ ID NO: 204, 211, 274 or 345. In some aspects, the nucleotide sequence encoding the peptide that causes ribosome skipping contains the sequence set forth in SEQ ID NO: 4, 5, 6, 207, 208, 209, or 210, 347, 1096, 1179, 1180, or 1181.
In some embodiments, the nucleic acid sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are present in any order, separated by the nucleotide sequence encoding an internal ribosome entry site (IRES) or a peptide sequence that causes ribosome skipping. For example, in some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a beta chain, a nucleic acid sequence encoding an IRES or peptide sequence that causes ribosome skipping, e.g., a P2A or T2A sequence as described herein, and a nucleic acid sequence that encodes an alpha chain, in that order. In other embodiments, the nucleic acid molecule contains a nucleic acid sequence that encodes an alpha chain, a nucleic acid sequence that encodes an IRES or peptide sequence that causes ribosome skipping, and a nucleic acid sequence that encodes a beta chain, in that order.
Thus, in some aspects, the nucleic acid molecule encodes a polypeptide comprising a beta chain, an IRES or peptide that causes ribosome skipping, and an alpha chain, in that order. In other aspects, the nucleic acid molecule encodes a polypeptide comprising an alpha chain, an IRES or peptide that causes ribosome skipping, and a beta chain, in that order.
In some embodiments, the nucleic acid molecule encodes a polypeptide containing an amino acid sequence set forth in Table 12, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule encodes a polypeptide set forth in any of SEQ ID NOS: 1, 2, 3, 27, 37, 47, 57, 67, 77, 87, 97, 107, 223, 224, 225, 226, 227, 228, 229, 230, 231, 340-342, 350-388, or 391-429, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule comprises the nucleic acid sequence set forth in any of SEQ ID NOs: 13, 14, 15, 26, 36, 46, 56, 66, 76, 86, 96, 106, 432-472, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
Also provided are polypeptides containing a sequence encoded by any of the provided nucleic acids. In some aspects, the polypeptide comprises an amino acid sequence corresponding to a SEQ ID NO. shown in Table 12, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the polypeptide comprises the sequence set forth in any of SEQ ID NOS 1, 2, 3, 27, 37, 47, 57, 67, 77, 87, 97, 107, 223, 224, 225, 226, 227, 228, 229, 230, 231, 340-342, 350-388, or 391-429, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Exemplary of such TCRs, or their modified versions, also are set forth in the Table 12.
TABLE 12
HPV16 E6 & E7 TCR SEQ ID NOs.
Full
Full Encoded
Nucleotide
Exemplary TCR or
Amino Acid
Codon-
modified version
Native
Modified
Optimized
TCR 3
223
27
26
TCR 4
224
37
36
TCR 5
225
47
46
TCR 8
228
77
76
TCR 9
229
87
86
TCR 10
230
97
96
TCR 6
226
57
56
TCR 7
227
67
66
TCR 11
231
107
106
TCR 12
340
3
15
TCR 13
341
2
14
TCR 14
342
1
13
TCR 15
391
350
432
TCR 16
392
351
433
TCR 17
393
352
434
TCR 18
394
353
435
TCR 19
395
354
436
TCR 20
396
355
437
TCR 21
397
356
438
TCR 22
398
357
439
TCR 23
399
358
440
TCR 24
400
359
441
TCR 25
401
360
442
TCR 26
402
361
443
TCR 27
403
362
444
TCR 28
404
363
445
TCR 29
405
364
446
TCR 30
406
365
447
TCR 31
407
366
448
TCR 32
408
367
449
TCR 33
409
368
450
TCR 34
410
369
451
TCR 35
411
370
452
TCR 36
412
371
453
TCR 37
413
372
454
TCR 38
414
373
455
TCR 39
415
374
456
TCR 40
416
375
457
TCR 41
417
376
458
TCR 42
418
377
459
TCR 43
419
378
460
TCR 44
420
379
461
TCR 45
421
380
462
TCR 46
422
381
463
TCR 47
423
382
464
TCR 48
424
383
465
TCR 49
425
384
466
TCR 50
426
385
467
TCR 51
427
386
468
TCR 52
428
387
469
TCR 53
429
388
470
TCR 54
227
67
471
TCR 55
340
3
472
In some embodiments, the nucleic acid molecule may further encode a marker (e.g. EGFRt or other marker as described) that is separated from the CAR or separated from the TCR chains by a linker, such as a cleavable linker sequence or a peptide sequence that causes ribosome skipping, e.g., T2A or P2A.
In some embodiments, the construct can be arranged in any order so that the encoding marker sequence is either 3′ to the alpha and/or beta sequence, 5′ to the alpha and/or beta sequence and/or between the alpha and beta sequence, where, in some cases, each separate component is separated by a cleavable linker sequence or a peptide that causes ribosome skipping (e.g. T2A or P2A) or an IRES. In some embodiments, the nucleic acid molecule contains a nucleic acid sequence that encodes a marker (e.g., EGFRt), cleavable linker or ribosome skip sequence (e.g. T2A or P2A), beta chain, cleavable linker or ribosome skip sequence (e.g. T2A or P2A), and alpha chain, in that order. In some embodiments, the nucleic acid molecule contains a nucleic acid sequence that encodes a marker (e.g., EGFRt), cleavable linker or ribosome skip sequence (e.g., T2A or P2A), alpha chain, cleavable linker or ribosome skip sequence (e.g., T2A or P2A), and beta chain, in that order. In some embodiments, the nucleic acid molecule contains a nucleic acid sequence that encodes a beta chain, cleavable linker or ribosome skip sequence (e.g., T2A or P2A), an alpha chain, a cleavable linker or ribosome skip sequence (e.g., T2A or P2A) and a marker (e.g. EGFRt), in that order. In some embodiments, the nucleic acid molecule contains a nucleic acid sequence that encodes a alpha chain, cleavable linker or ribosome skip sequence (e.g. T2A or P2A), a beta chain, a cleavable linker or ribosome skip sequence (e.g., T2A or P2A) and a marker (e.g., EGFRt), in that order. In some embodiments, the nucleic acid molecule contains a nucleic acid sequence that encodes a alpha chain, cleavable linker or ribosome skip sequence (e.g., T2A or P2A), a marker (e.g., EGFRt), a cleavable linker or ribosome skip sequence (e.g., T2A or P2A) and a beta chain, in that order. In some embodiments, the nucleic acid molecule contains a nucleic acid sequence that encodes a beta chain, cleavable linker or ribosome skip sequence (e.g., T2A or P2A), a marker (e.g. EGFRt), a cleavable linker or ribosome skip sequence (e.g., T2A or P2A) and a alpha chain, in that order.
In some embodiments, introduction of a construct encoding the CAR and EGFRt separated by a T2A ribosome switch can express two proteins from the same construct, such that the EGFRt can be used as a marker to detect cells expressing such construct.
The nucleic acid may encode an amino acid sequence comprising the variable alpha (Vα) region or variable light (VL) region of the TCR or antibody, respectively. In some cases, the nucleic acid encodes an amino acid sequence comprising the variable beta (Vβ) region or variable heavy (VH) region of the TCR or antibody, respectively. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided.
Also provided are vectors, such as those containing any of the nucleic acids described herein. In some embodiments, nucleic acid or nucleic acids encoding one or both chains of a binding molecule, e.g., TCR, are cloned into a suitable expression vector or vectors. The expression vector can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. In some embodiments, the vector is an expression vector.
In some embodiments, the vector can a vector of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), or the pEX series (Clontech, Palo Alto, Calif.). In some cases, bacteriophage vectors, such as λG10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used. In some embodiments, plant expression vectors can be used and include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). In some cases, the vector is a viral vector. In some such aspects, the viral vector is a retroviral vector, such as a lentiviral vector. In some instances, the lentiviral vector is derived from HIV-1.
In some embodiments, the recombinant expression vectors can be prepared using standard recombinant DNA techniques. In some embodiments, vectors can contain regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA-based. In some embodiments, the vector can contain a nonnative promoter operably linked to the nucleotide sequence encoding the binding molecule, such as TCR, antibody or antigen-binding fragment thereof. In some embodiments, the promoter can be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus. Other promoters known to a skilled artisan also are contemplated.
Also provided are methods of making the binding molecules (including antigen-binding fragments). In some embodiments, a host cell comprising such nucleic acid is provided. For recombinant production of the binding molecules, nucleic acid encoding the binding molecule, e.g., as described above, may be isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the alpha and beta chains of the TCR or the heavy and light chains of the antibody). In some embodiments, a method of making the binding molecule is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the binding molecule, as provided above, under conditions suitable for expression of the binding molecule, and optionally recovering the binding molecule from the host cell (or host cell culture medium).
In one such embodiment, a host cell comprises (e.g., has been transformed with): a vector comprising a nucleic acid that encodes an amino acid sequence comprising the Vβ region of the TCR or antigen-binding fragment thereof and a nucleic acid that encodes an amino acid sequence comprising the Vα region of the TCR or antigen-binding fragment thereof. In another such embodiment, a host cell comprises (e.g. has been transformed with): a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody or antigen-binding fragment thereof and the VL of the antibody or antigen-binding fragment thereof. In some aspects, a host cell comprises (e.g., has been transformed with): a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the Vα region of the TCR or antigen-binding fragment thereof and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the Vβ region of the TCR or antigen-binding fragment thereof. In other aspects, a host cell comprises (e.g. has been transformed with): a first vector comprising a nucleic acid that encodes an amino acid sequence or comprising the VL of the antibody or antigen-binding fragment thereof and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody or antigen-binding fragment thereof.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for binding molecule-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been modified to mimic or approximate those in human cells. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lec13 CHO cells, and FUT8 CHO cells; PER.C6® cells; and NSO cells. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the binding molecule. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells. In some embodiments, the binding molecule is produced in a cell-free system. Exemplary cell-free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713 (2003).
III. Methods for Identifying and Generating T Cell Receptors
In some embodiments, provided are methods for identifying and generating T cell receptors directed towards a target antigen. In some aspects, the methods involve subjecting biological samples containing T cells, such as primary T cells, including those derived from normal donors or patients having a disease or condition of interest, to multiple rounds of antigen exposure and assessment. In some aspects, the rounds involve the use of artificial or engineered antigen presenting cells, such as autologous dendritic cells or other APCs pulsed with a desired peptide antigen, to promote presentation on an MHC, such as a class I or II MHC. In some aspects, multiple rounds of antigen exposure are carried out and in some aspects T cells are sorted following one or more of the rounds, e.g., based on ability to bind to the desired antigen (such as peptide-MHC tetramers). In some aspects sorting is carried out by flow cytometry. In some aspects, cells from cells deemed to bind to the desired antigen (positive fraction) and cells deemed not to bind to the antigen, are assessed, e.g., by single-cell sequencing methods. In some aspects, the methods sequence and identify, at a single-cell level, TCR pairs present in each sample. In some aspects, the methods can quantify the number of copies of a given TCR pair present in a sample, and as such can assess the abundance of a given TCR in a given sample, and/or enrichment thereof over another sample, such as enrichment or abundance in the positive (antigen-binding) fraction, e.g., over one or more rounds, for example, as compared to the negative fraction. In some aspects, such assays are performed to generate antigen-specific T cell receptors (TCRs) that specifically bind to human papillomavirus 16 or 18 peptide antigens such as peptides derived from E6 or E7, such as E6(29-38) or E7(11-19) peptide, e.g., presented on MHC-I molecules and survived and/or were enriched over time, following multiple rounds of antigen-stimulation. In some aspects, clonal T cell lines are generated and the sequences of individual paired TCR alpha and beta chains and abundance thereof in various populations were determined on a single-cell basis, using high-throughput paired TCR sequencing.
In some aspects, peptide-pulsed HLA:A02:01APCs were generated with HPV 16 E6(29-38) peptide (TIHDIILECV; SEQ ID NO:233) or E7(11-19) peptide (YMLDLQPET; SEQ ID NO:236). Autologous CD8+ T cells from normal human donors are incubated over multiple rounds with the peptide-pulsed cells, and selections were carried out based on binding to peptide-loaded autologous MHC tetramers.
In some aspects, cells were subjected to multiple, such as a total of two or three or more, rounds of stimulation, in the presence of peptide-pulsed cells (such as with a particular peptide concentration of 1000 ng/mL maintained over the three rounds). Following one or more of, such as following the first and/or following the second and third rounds of stimulation, cells were sorted by flow cytometry into populations positive and negative, respectively, for binding to peptide-MHC tetramers containing the appropriate tetramer. Cells of the tetramer-positive and negative populations following each or one or more of of the one or more, such as the second and third, rounds in some aspects are subjected to single-cell TCR sequencing, to assess the presence and frequency of individual TCRs in the different populations, and the persistence of TCR clones over multiple rounds of antigen stimulation.
In some aspects, cell populations from the positive and negative fractions (i.e., sorted by flow cytometry based on positive and negative staining, respectively, for binding to the relevant antigen such as peptide-MHC such as loaded tetramers, e.g., as determined by flow cytometry), following the one or more rounds, are subject to high-throughput single-cell sequencing for TCR alpha and beta chain pairs. High throughput single cell TCR sequencing in some aspects is performed as generally described in published PCT patent applications, publication numbers WO2012/048340, WO2012/048341 and WO2016/044227. The sequencing methods thus in some aspects employ single-cell droplets and sample and molecular barcodes, to identify individual pairs of TCR alpha and beta chain sequences at a single-cell level, for each of a large number (e.g., millions) of single cells present in a single starting composition, and to assess abundance of each TCR pair in various populations assessed. The ability to identify and quantify TCR pairs at a single-cell level in some embodiments permits the assessment of the frequency of each of various TCR pairs in each of the individual positive and negative fractions, and to assess enrichment and persistence of TCRs over multiple rounds of antigen stimulation.
In some aspects, the methods generate, identify, isolate and/or select TCR pairs that are enriched in antigen-binding, e.g., peptide-binding, fractions following at least one and in some aspects a plurality of, multiple rounds of stimulation. In some aspects, the TCRs are present in and/or present at a desired abundance in and/or preferentially enriched following, rounds 1, 2 and/or and 3 and in some aspects at least multiple rounds, of antigen exposure. In some aspects, the TCRs are enriched in the population over time following multiple rounds of exposure to antigen. Also provided are TCRs generated or identified using such methods, such as TCRs having such properties, such as the ability to survive and/or expand over multiple rounds of antigen exposure, such as in a peptide-pulsed APC assay.
IV. Engineered Cells
Also provided are cells such as cells that have been engineered to contain the binding molecule described herein. Also provided are populations of such cells, compositions containing such cells and/or enriched for such cells, such as in which cells expressing the binding molecule make up at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more percent of the total cells in the composition or cells of a certain type such as T cells or CD8+ or CD4+ cells. In some embodiments, the cells are primary T cells. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.
Thus also provided are genetically engineered cells expressing the binding molecules. The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, as described herein, and re-introducing them into the same patient, before or after cryopreservation.
Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naïve T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MALT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
In some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.
In some embodiments, genes and/or gene products (and/or expression thereof) in the provided cells, and/or compositions containing such cells, are reduced, deleted, eliminated, knocked-out or disrupted. Such genes and/or gene products in some aspects include one or more of the gene encoding (or product thereof) TCR alpha constant region (TRAC) and/or TCR beta constant region (TRBC; encoded in humans by TRBC1 or TRBC2), e.g., to reduce or prevent expression of the endogenous TCR in the cell, e.g. T cell, and/or α chain thereof. In some embodiments, the genes and/or gene products, such as TRAC and/or TRBC, is reduced, deleted, eliminated, knocked-out or disrupted in any of the engineered cells provided herein and/or in any of the methods for producing engineered cells provided herein. In some embodiments, engineered cells and/or engineered cells produced by the methods are cells that have been engineered to express the binding molecule described herein, populations of such cells, compositions containing such cells and/or enriched for such cells. In some embodiments, genes and/or gene products, such as the TRAC and/or TRBC, is reduced, deleted, eliminated, knocked-out or disrupted in primary T cells, to reduce, delete, eliminate, knock-out or disrupt the expression of the endogenous TCR in primary T cells, e.g., that are engineered to express any of the binding molecules, e.g., TCRs, described herein.
In some embodiments, the genes and/or gene products targeted for reduction, deletion, elimination, knock-out or disruption are endogenous genes encoding the TCR or α chain, a domain and/or a region thereof. In some embodiments, a target site for disruption is in a T cell receptor alpha constant (TRAC) gene. In some embodiments, a target site for disruption is in a T cell receptor beta constant 1 (TRBC1) or T cell receptor beta constant 2 (TRBC2) gene. In some embodiments, the one or more target site(s) is in a TRAC gene and one or both of a TRBC1 and a TRBC2 gene.
In some embodiments, the endogenous TCR Cα is encoded by the TRAC gene (IMGT nomenclature). An exemplary nucleotide sequence of the human T cell receptor alpha constant chain (TRAC) gene locus is set forth in SEQ ID NO: 348 (NCBI Reference Sequence: NG 001332.3, TRAC). In some embodiments, the endogenous TCR Cβ is encoded by TRBC1 or TRBC2 genes (IMGT nomenclature). An exemplary nucleotide sequence of the human T cell receptor beta constant chain 1 (TRBC1) gene locus is set forth in SEQ ID NO:349 (NCBI Reference Sequence: NG 001333.2, TRBC1); and an exemplary nucleotide sequence of the human T cell receptor beta constant chain 2 (TRBC2) gene locus is set forth in SEQ ID NO:1047 (NCBI Reference Sequence: NG 001333.2, TRBC2).
In some embodiments, gene(s) targeted for disruption or knock-out is at or near one or more of the TRAC, TRBC1 and/or TRBC2 loci. In some embodiments, the TRAC gene is knocked out. In some embodiments, the TRBC1 gene is knocked out. In some embodiments, the TRBC2 gene is knocked out. In some embodiments, the TRAC gene and the TRBC1 gene are knocked out. In some embodiments, the TRAC gene and the TRBC2 gene are knocked out. In some embodiments, the TRAC gene and both the TRBC1 and TRBC2 genes are knocked out, e.g., targeting a sequence that is conserved between TRBC1 and TRBC2.
In some embodiments, reducing or preventing endogenous TCR expression can lead to a reduced risk or chance of mispairing between chains of the engineered TCR and the endogenous TCR, thereby creating a new TCR that could potentially result in a higher risk of undesired or unintended antigen recognition and/or side effects, and/or could reduce expression levels of the desired exogenous TCR. In some aspects, reducing or preventing endogenous TCR expression can increase expression of the engineered TCR in the cells as compared to cells in which expression of the TCR is not reduced or prevented, such as increased by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or more. For example, in some cases, suboptimal expression of an engineered or recombinant TCR can occur due to competition with an endogenous TCR and/or with TCRs having mispaired chains, for the invariant CD3 signaling molecules that are involved in permitting expression of the complex on the cell surface.
In some embodiments, the reduction, deletion, elimination, knockout or disruption involve the use of one or more agent(s) capable of introducing a genetic disruption, a cleavage, a double strand break (DSB) and/or a nick at a target site in the genomic DNA, resulting in a the reduction, deletion, elimination, knockout or disruption after repair by various cellular DNA repair mechanisms.
In some embodiments, the one or more agent(s) capable of introducing a cleavage comprises a DNA binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to a target site in the genome, e.g., in TRAC and/or TRBC genes. In some aspects, the targeted cleavage, e.g., DNA break, of the endogenous genes encoding TCR is achieved using a protein or a nucleic acid is coupled to or complexed with a gene editing nuclease, such as in a chimeric or fusion protein. In some embodiments, the one or more agent(s) capable of introducing a cleavage comprises a fusion protein comprising a DNA-targeting protein and a nuclease or an RNA-guided nuclease.
In some embodiments, reduction, deletion, elimination, knockout or disruption is carried out by gene editing methods, such as using a zinc finger nuclease (ZFN), TALEN or a CRISPR/Cas system with an engineered single guide RNA that cleaves a TCR gene. In some embodiments, reducing expression of an endogenous TCR is carried out using an inhibitory nucleic acid molecule against a target nucleic acids encoding specific TCRs (e.g., TCR-α and TCR-β). In some embodiments, the inhibitory nucleic acid is or contains or encodes a small interfering RNA (siRNA), a microRNA-adapted shRNA, a short hairpin RNA (shRNA), a hairpin siRNA, a microRNA (miRNA-precursor) or a microRNA (miRNA). Exemplary methods for reducing or preventing endogenous TCR expression are known in the art, see e.g. U.S. Pat. No. 9,273,283; U.S. publication no. US2014/0301990; and PCT publication No. WO2015/161276.
In some embodiments, the agent capable of introducing a targeted cleavage comprises various components, such as a fusion protein comprising a DNA-targeting protein and a nuclease or an RNA-guided nuclease. In some embodiments, the targeted cleavage is carried out using a DNA-targeting molecule that includes a DNA-binding protein such as one or more zinc finger protein (ZFP) or transcription activator-like effectors (TALEs), fused to a nuclease, such as an endonuclease. In some embodiments, the targeted cleavage is carried out using RNA-guided nucleases such as a clustered regularly interspaced short palindromic nucleic acid (CRISPR)-associated nuclease (Cas) system (including Cas and/or Cfp1). In some embodiments, the targeted cleavage is carried using agents capable of introducing a cleavage, such as sequence-specific or targeted nucleases, including DNA-binding targeted nucleases and gene editing nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases such as a CRISPR-associated nuclease (Cas) system, specifically engineered and/or designed to be targeted to the at least one target site(s), sequence of a gene or a portion thereof.
In some embodiments, the one or more agent(s) specifically targets the at least one target site(s), e.g., at or near TRAC and/or TRBC genes. In some embodiments, the agent comprises a ZFN, TALEN or a CRISPR/Cas9 combination that specifically binds to, recognizes, or hybridizes to the target site(s). In some embodiments, the CRISPR/Cas9 system includes an engineered crRNA/tracr RNA (“single guide RNA”) to guide specific cleavage. In some embodiments, the agent comprises nucleases based on the Argonaute system (e.g., from T. thermophilus, known as ‘TtAgo’, (Swarts et at (2014) Nature 507(7491): 258-261).
Zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs), and CRISPR system binding domains can be “engineered” to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring ZFP or TALE protein. Engineered DNA binding proteins (ZFPs or TALEs) are proteins that are non-naturally occurring. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP and/or TALE designs and binding data. See, e.g., U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Publication No. 20110301073. Exemplary ZFNs, TALEs, and TALENs are described in, e.g., Lloyd et al., Frontiers in Immunology, 4(221): 1-7 (2013).
In some embodiments, the TRAC and/or TRBC genes can be targeted for cleavage by engineered ZFNs. Exemplary ZFN that target endogenous T cell receptor (TCR) genes include those described in, e.g., US 2015/0164954, US 2011/0158957, U.S. Pat. No. 8,956,828 and Torikawa et al. (2012) Blood 119:5697-5705, the disclosures of which are incorporated by reference in their entireties.
In some embodiments, the TRAC and/or TRBC genes can be targeted for cleavage by engineered TALENs. Exemplary TALEN that target endogenous T cell receptor (TCR) genes include those described in, e.g., WO 2017/070429, WO 2015/136001, US20170016025 and US20150203817, the disclosures of which are incorporated by reference in their entireties.
In some embodiments, the TRAC and/or TRBC genes can be targeted for cleavage using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. See Sander and Joung, Nature Biotechnology, 32(4): 347-355. In some embodiments, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus.
In some aspects, the CRISPR/Cas nuclease or CRISPR/Cas nuclease system includes a non-coding guide RNA (gRNA), which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality.
In some embodiments, the CRISPR/Cas nuclease system comprises at least one of: a guide RNA (gRNA) having a targeting domain that is complementary with a target site of a TRAC gene; a gRNA having a targeting domain that is complementary with a target site of one or both of a TRBC1 and a TRBC2 gene; or at least one nucleic acid encoding the gRNA.
In general, a guide sequence, e.g., guide RNA, is any polynucleotide sequences comprising at least a sequence portion, e.g., targeting domain, that has sufficient complementarity with a target site sequence, such as a target site in the TRAC, TRBC1 and/or TRBC2 genes in humans, to hybridize with the target sequence at the target site and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, in the context of formation of a CRISPR complex, “target site” (also known as “target position,” “target DNA sequence” or “target location”) generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a domain, e.g., targeting domain, of the guide RNA promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. Generally, a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.
In some aspects, a CRISPR enzyme (e.g. Cas9 nuclease) in combination with (and optionally complexed with) a guide sequence is delivered to the cell. For example, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. For example, one or more elements of a CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes, Staphylococcus aureus or Neisseria meningitides.
In some embodiments, a guide RNA (gRNA) specific to the target site (e.g. TRAC, TRBC1 and/or TRBC2 in humans) is used to RNA-guided nucleases, e.g., Cas, to introduce a DNA break at the target site or target position. Methods for designing gRNAs and exemplary targeting domains can include those described in, e.g., in International PCT Publication No. WO2015/161276. Targeting domains of can be incorporated into the gRNA that is used to target Cas9 nucleases to the target site or target position. Methods for selection and validation of target sequences as well as off-target analyses are described, e.g., in Mali et al., 2013 Science 339(6121): 823-826; Hsu et al. Nat Biotechnol, 31(9): 827-32; Fu et al., 2014 Nat Biotechnol; Heigwer et al., 2014 Nat Methods 11(2):122-3; Bae et al., 2014 Bioinformatics; Xiao A et al., 2014 Bioinformatics. A genome-wide gRNA database for CRISPR genome editing is publicly available, which contains exemplary single guide RNA (sgRNA) sequences targeting constitutive exons of genes in the human genome or mouse genome (see e.g., genescript.com/gRNA-database.html; see also, Sanjana et al. (2014) Nat. Methods, 11:783-4). In some aspects, the gRNA sequence is or comprises a sequence with minimal off-target binding to a non-target site or position.
In some embodiments, the gRNA for targeting TRAC, TRBC1 and/or TRBC2 can be any that are described herein, or are described elsewhere. In some embodiments, the sequence targeted by the CRISPR/Cas9 gRNA in the TRAC gene locus is ATTCACCGATTTTGATTCTC (SEQ ID NO:1182). In some embodiments, the sequence targeted by the CRISPR/Cas9 gRNA in the TRBC1 and/or TRBC2 gene loci is GATCGTCAGCGCCGAGGCC (SEQ ID NO:1054). In some embodiments, the gRNA targeting domain sequence for targeting a target site in the TRAC gene locus is GAGAAUCAAAAUCGGUGAAU (SEQ ID NO: 1048). In some embodiments, the gRNA targeting domain sequence for targeting a target site in the TRBC1 and/or TRBC2 gene loci is GGCCUCGGCGCUGACGAUCU (SEQ ID NO: 1053). Other exemplary gRNA sequences, or targeting domains contained in the gRNA and/or other methods of gene editing and/or knock-out targeting endogenous TCR genes, e.g., TRAC and/or TRBC genes, include any described in, e.g., in International PCT Publication Nos. WO2015/161276, WO2014/191128, WO2015/136001, WO2016/069283, WO2016/016341; U.S. Publication Nos. US2011/0158957, US2014/0301990, US2015/0098954 and US2016/0208243; and Osborn et al. (2016) Mol. Ther. 24(3):570-581. Any of the known methods can be used to generate a cleavage of the endogenous genes encoding TCR domains or regions can be used in the embodiments provided herein, e.g., for engineering in cell lines and/or in primary T cells.
In some embodiments, t reduction, deletion, elimination, knockout or disruption of the endogenous genes encoding TCR, such as TRAC and TRBC1 or TRBC2, is carried out by delivering or introducing one or more agent(s) capable of introducing a cleavage, e.g., Cas9 and/or gRNA components, to a cell, using any of a number of known delivery method or vehicle for introduction or transfer to cells, for example, using lentiviral delivery vectors, or any of the known methods or vehicles for delivering Cas9 molecules and gRNAs. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505. In some embodiments, nucleic acid sequences encoding one or more components of one or more agent(s) capable of introducing a cleavage, e.g., DNA break, is introduced into the cells, e.g., by any methods for introducing nucleic acids into a cell described herein or known. In some embodiments, a vector encoding components of one or more agent(s) capable of introducing a cleavage such as a CRISPR guide RNA and/or a Cas9 enzyme can be delivered into the cell.
In some embodiments, the one or more agent(s) capable of introducing a cleavage, e.g., a Cas9/gRNA system, is introduced into the cell as a ribonucleoprotein (RNP) complex. RNP complexes include a sequence of ribonucleotides, such as an RNA or a gRNA molecule, and a protein, such as a Cas9 protein or variant thereof. For example, the Cas9 protein is delivered as RNP complex that comprises a Cas9 protein and a gRNA molecule targeting the target sequence, e.g., using electroporation or other physical delivery method. In some embodiments, the RNP is delivered into the cell via electroporation or other physical means, e.g., particle gun, calcium phosphate transfection, cell compression or squeezing. In some embodiments, the RNP can cross the plasma membrane of a cell without the need for additional delivery agents (e.g., small molecule agents, lipids, etc.).
A. Preparation of Cells for Genetic Engineering
In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for introduction of the binding molecule, e.g., TCR or CAR, may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, or pig.
In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.
In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++/Mg++ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.
In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques.
For example, CD3+, CD28+ T cells can be positively selected using anti-CD3/anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (markerhigh) on the positively or negatively selected cells, respectively.
In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood.1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.
In embodiments, memory T cells are present in both CD62L+ and CD62L− subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L−CD8+ and/or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.
In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for Tcm cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TQM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.
CD4+ T helper cells are sorted into naïve, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L− and CD45RO−.
In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher© Humana Press Inc., Totowa, N.J.).
In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.
In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.
The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.
In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.
In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.
In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.
In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Number WO2009/072003, or US 2011/0003380 A1.
In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.
In some aspects, the separation and/or other steps is carried out using CliniMACS system (Miltenyi Biotec), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.
The CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.
In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood may be automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakuraet al. (2012) Blood.1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.
In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.
In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to −80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.
In some embodiments, the provided methods include cultivation, incubation, culture, and/or genetic engineering steps. For example, in some embodiments, provided are methods for incubating and/or engineering the depleted cell populations and culture-initiating compositions.
Thus, in some embodiments, the cell populations are incubated in a culture-initiating composition. The incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.
In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of an antigen receptor.
The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3. In some embodiments, the stimulating conditions include one or more agent, e.g. ligand, which is capable of stimulating a costimulatory receptor, e.g., anti-CD28. In some embodiments, such agents and/or ligands may be, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agents include IL-2, IL-15 and/or IL-7. In some aspects, the IL-2 concentration is at least about 10 units/mL.
In some aspects, incubation is carried out in accordance with techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakuraet al. (2012) Blood.1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, the T cells are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.
In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.
In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigen-specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.
B. Vectors and Methods for Genetic Engineering
Also provided are methods, nucleic acids, compositions, and kits, for expressing the binding molecules, and for producing the genetically engineered cells expressing such binding molecules. The genetic engineering generally involves introduction of a nucleic acid encoding the binding molecule, e.g. TCR or CAR, e.g. TCR-like CAR, into the cell, such as by retroviral transduction, transfection, or transformation.
In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.
In some contexts, overexpression of a stimulatory factor (for example, a lymphokine or a cytokine) may be toxic to a subject. Thus, in some contexts, the engineered cells include gene segments that cause the cells to be susceptible to negative selection in vivo, such as upon administration in adoptive immunotherapy. For example in some aspects, the cells are engineered so that they can be eliminated as a result of a change in the in vivo condition of the patient to which they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell 2:223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).
In some aspects, the cells further are engineered to promote expression of cytokines or other factors. Various methods for the introduction of genetically engineered components are well known and may be used with the provided methods and compositions. Exemplary methods include those for transfer of nucleic acids encoding the binding molecules, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.
In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 Nov. 29(11): 550-557.
In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
Methods of lentiviral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.
In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).
Other approaches and vectors for transfer of the nucleic acids encoding the binding molecules or recombinant products are those described, e.g., in international patent application, Publication No.: WO2014/055668, and U.S. Pat. No. 7,446,190.
Among additional nucleic acids, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.
Thus, provided in some embodiments are engineered cells, such as those containing a binding molecule (such as TCR or antigen-binding fragment thereof or antibody or antigen-binding fragment thereof), nucleic acid, or vector as described herein. In some aspects, the cell is produced by transducing the cell in vitro or ex vivo with a vector described herein. In some aspects, the cell is a T cell, such as a CD8+ or CD4+ T cell. In some embodiments, the binding molecule is heterologous to the cell.
In some cases, the engineered cell contains a heterologous TCR or antigen-binding fragment thereof that recognizes or binds a peptide epitope derived from HPV16 E6. In some cases, the TCR or antigen-binding fragment thereof does not recognize or bind the epitope E6(29-38) comprising the amino acid sequence TIHDIILECV (SEQ ID NO. 233). In some instances, the TCR or antigen-binding fragment thereof that recognizes or binds a peptide epitope derived from HPV16 E6 is or comprises the sequence set forth in SEQ ID NO: 232 or SEQ ID NO: 234.
In some embodiments, the engineered cell contains a heterologous TCR or antigen-binding fragment thereof that recognizes or binds a peptide epitope derived from HPV16 E7. In some embodiments, the TCR or antigen-binding fragment thereof does not recognize or bind the epitope E7 (11-19) comprising the amino acid sequence YMLDLQPET (SEQ ID NO. 236). In some instances, the TCR or antigen-binding fragment thereof that recognizes or binds a peptide epitope derived from HPV16 E7 is or contains the sequence set forth in any of SEQ ID NOs: 235-239. In some cases, the peptide derived from HPV16 E7 is or contains the sequence set forth in SEQ ID NO: 235.
V. Compositions, Methods, and Uses
Also provided are compositions including the binding molecules, e.g. TCRs, and engineered cells, including pharmaceutical compositions and formulations, and methods of using and uses of the molecules and compositions, such as in the treatment of diseases, conditions, and disorders in which HPV16 E6 or E7 is expressed, and/or detection, diagnostic, and prognostic methods.
A. Pharmaceutical Compositions and Formulations
Provided are pharmaceutical formulations including the binding molecules, e.g., TCR or antigen binding fragment thereof or antibody or antigen-binding fragment thereof, and/or the engineered cells expressing the binding molecules. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.
The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
In some aspects, the choice of carrier is determined in part by the particular cell or binding molecule, and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).
Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
Formulations of the binding molecules can include lyophilized formulations and aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the binding molecules or cells, preferably those with activities complementary to the binding molecule or cell, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the cells or binding molecules are administered in the form of a salt, e.g., a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p-toluenesulphonic acid.
Active ingredients may be entrapped in microcapsules, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. In certain embodiments, the pharmaceutical composition is formulated as an inclusion complex, such as cyclodextrin inclusion complex, or as a liposome. Liposomes can serve to target the host cells (e.g., T-cells or NK cells) to a particular tissue. Many methods are available for preparing liposomes, such as those described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
The pharmaceutical composition in some aspects can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Many types of release delivery systems are available and known. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.
The pharmaceutical composition in some embodiments contains the binding molecules and/or cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
In certain embodiments, in the context of genetically engineered cells containing the binding molecules, a subject is administered the range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges, and/or such a number of cells per kilogram of body weight of the subject.
The cells or binding molecules may be administered using standard administration techniques, formulations, and/or devices. Provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. Administration of the cells can be autologous or heterologous. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, intracranial, intrathoracic, and intraperitoneal administration. In some embodiments, the cell populations are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
Sterile injectable solutions can be prepared by incorporating the binding molecule in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.
Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
B. Therapeutic and Prophylactic Methods and Uses
Also provided are methods of administering and uses, such as therapeutic and prophylactic uses, of the binding molecules, including TCRs and antigen-binding fragments thereof and antibodies or antigen-binding fragments thereof, and/or engineered cells expressing the binding molecules. Such methods and uses include therapeutic methods and uses, for example, involving administration of the molecules, cells, or compositions containing the same, to a subject having a disease, condition, or disorder expressing or associated with HPV, e.g., HPV16, and/or in which cells or tissues express, e.g., specifically express, HPV16, e.g., HPV16 E6 or E7. In some embodiments, the molecule, cell, and/or composition is administered in an effective amount to effect treatment of the disease or disorder. Uses include uses of the binding molecules and cells in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods are carried out by administering the binding molecules or cells, or compositions comprising the same, to the subject having, having had, or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject.
As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.
As used herein, “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.
“Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided molecules and compositions are used to delay development of a disease or to slow the progression of a disease.
As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, a binding molecule or composition or cell which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the binding molecule or composition or cell.
An “effective amount” of an agent, e.g., a pharmaceutical formulation, binding molecule, or cells, or composition, in the context of administration, refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.
A “therapeutically effective amount” of an agent, e.g., a pharmaceutical formulation, binding molecule, or cells, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered. In some embodiments, the provided methods involve administering the binding molecules, cells, and/or compositions at effective amounts, e.g., therapeutically effective amounts.
A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human.
Among the diseases to be treated are cancers, typically HPV-associated cancers, and any HPV-associated, e.g., HPV 16-associated, diseases or conditions or diseases or conditions in which an HPV oncoprotein, e.g., E6 or E7, such as an HPV 16 oncoprotein, e.g., HPV 16 E6 or E7 is expressed. In certain diseases and conditions, the viral protein such as the oncoprotein such as the HPV 16 E6 or E7 is expressed in or by malignant cells and cancers, and/or a peptide epitope thereof is expressed on such malignant cancers or tissues, such as by way of MHC presentation. In some embodiments, the disease or condition is an HPV16-expressing cancer. In some embodiments, the cancer is a carcinoma, melanoma or other precancerous or cancerous state caused by or otherwise associated with HPV, such as HPV-16. In some embodiments, the carcinoma can be a squamous cell or adenocarionma. In some embodiments, the disease or condition can be characterized by an epithelial cell abnormality associated with oncogenic HPV infection, such as koilocytosis; hyperkeratosis; precancerous conditions encompasssing intraepithelial neoplasias or intraepithelial lesion; high-grade dysplasias; and invasive or malignant cancers. Among the HPV 16-associated diseases or conditions that can be treated include, but are not limited to, cervical cancer, uterine cancer, anal cancer, colorectal cancer, vaginal cancer, vulvar cancer, penile cancer, oropharyngeal cancers, tonsil cancer, pharyngeal cancers (pharynx cancer), laryngeal cancer (larynx cancer), oral cancer, skin cancer, esophageal cancer, head and neck cancer such as a squamous cell carcinoma (SCC) head and neck cancer, or small cell lung cancer. In some embodiments, the disease or condition is a cervical carcinoma.
In some embodiments, the methods may include steps or features to identify a subject who has, is suspected to have, or is at risk for developing an HPV 16-associated disease or disorder (see e.g. U.S. Pat. Nos. 6,355,424 and 8,968,995) and/or the subject to be treated may be a subject identified to have or to be so at risk for having or developing such HPV-associated disease or condition or cancer. Hence, provided in some aspects are methods for identifying subjects with diseases or disorders associated with HPV 16 E6 or E7 expression and selecting them for treatment and/or treating such subjects, e.g., selectively treating such subjects, with a provided HPV 16 binding molecule, including in some aspects with cells engineered to express such binding molecules, including in some aspects any of the HPV 16 E6 or E7 TCRs or antigen binding fragments thereof or anti-HPV 16 E6 or E7 antibodies, e.g., antibody fragments and proteins containing the same, such as the chimeric receptors, e.g., TCR-like CARs, and/or engineered cells expressing the TCRs or CARs.
For example, a subject may be screened for the presence of a disease or disorder associated with HPV 16 E6 or E7 expression, such as an HPV 16 E6- or E7-expressing cancer.
In some embodiments, the methods include screening for or detecting the presence of an HPV 16 E6- or E7-associated disease, e.g. a tumor. Thus, in some aspects, a sample may be obtained from a patient suspected of having a disease or disorder associated with HPV 16 E6 or E7 expression and assayed for the expression level of HPV 16 E6 or E7. In some aspects, a subject who tests positive for an HPV 16 E6- or E7-associated disease or disorder may be selected for treatment by the present methods, and may be administered a therapeutically effective amount of a binding molecule described herein, a CAR expressing such a binding molecule, cells containing the binding molecule, or a pharmaceutical composition thereof as described herein. In some embodiments, the methods can be used to monitor the size or density of an HPV 16 E6- or E7-expressing tissue, e.g. tumor, over time, e.g., before, during, or after treatment by the methods. In some aspects, subjects treated by methods provided herein have been selected or tested positive for HPV expression according to such methods, e.g., prior to initiation of or during treatment.
In some embodiments, administration of a provided HPV 16 binding molecule, including any of the HPV 16 E6 or E7 TCRs or antigen binding fragments thereof or anti-HPV 16 E6 or E7 antibodies, e.g., antibody fragments and proteins containing the same, such as the chimeric receptors, e.g., TCR-like CARs, and/or engineered cells expressing the TCRs or CARs, can be combined with another therapeutic for the treatment of an HPV disease. For example, the additional therapeutic treatment can include treatment with another anti-cancer agent for the treatment of cervical cancer. Suitable dosages for such a co-administered agent may be lowered due to the combined action (synergy) of the agent and the provide HPV 16 binding molecule.
In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another HPV 16-specific binding molecule and/or cells expressing an HPV 16-targeting binding molecule and/or other therapy, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another HPV 16-targetetd therapy. In some embodiments, the subject has not relapsed but is determined to be at risk for relapse, such as at a high risk of relapse, and thus the compound or composition is administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.
In some embodiments, the treatment does not induce an immune response by the subject to the therapy, and/or does not induce such a response to a degree that prevents effective treatment of the disease or condition. In some aspects, the degree of immunogenicity and/or graft versus host response is less than that observed with a different but comparable treatment. For example, in the case of adoptive cell therapy using cells expressing TCRs or CARs including the provided binding molecules, the degree of immunogenicity in some embodiments is reduced compared to TCRs or CARs including a different binding molecule.
In some embodiments, the methods include adoptive cell therapy, whereby genetically engineered cells expressing the provided binding molecules are administered to subjects. Such administration can promote activation of the cells (e.g., T cell activation) in an HPV 16-targeted manner, such that the cells of the disease or disorder are targeted for destruction.
Thus, the provided methods and uses include methods and uses for adoptive cell therapy. In some embodiments, the methods include administration of the cells or a composition containing the cells to a subject, tissue, or cell, such as one having, at risk for, or suspected of having the disease, condition or disorder. In some embodiments, the cells, populations, and compositions are administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells or compositions are administered to the subject, such as a subject having or at risk for the disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptom of the disease or condition, such as by lessening tumor burden in an HPV 16 E6- or E7-expressing cancer.
Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.
In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.
In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.
In some embodiments, the subject, to whom the cells, cell populations, or compositions are administered, is a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent. In some examples, the patient or subject is a validated animal model for disease, adoptive cell therapy, and/or for assessing toxic outcomes such as cytokine release syndrome (CRS).
The provided binding molecules, such as TCRs and antigen-binding fragments thereof and antibodies and antigen-binding fragments thereof, and cells expressing the same, can be administered by any suitable means, for example, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intracranial, intrathoracic, or subcutaneous administration. Dosing and administration may depend in part on whether the administration is brief or chronic. Various dosing schedules include but are not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion.
For the prevention or treatment of disease, the appropriate dosage of the binding molecule or cell may depend on the type of disease to be treated, the type of binding molecule, the severity and course of the disease, whether the binding molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the binding molecule, and the discretion of the attending physician. The compositions and molecules and cells are in some embodiments suitably administered to the patient at one time or over a series of treatments.
In certain embodiments, in the context of genetically engineered cells containing the binding molecules, a subject is administered the range of about one million to about 100 billion cells and/or that amount of cells per kilogram of body weight, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges and/or per kilogram of body weight. Again, dosages may vary depending on attributes particular to the disease or disorder and/or patient and/or other treatments.
In some embodiments, the binding molecules or cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as another TCR, antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.
The cells or antibodies in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells or antibodies are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells or antibodies are administered after to the one or more additional therapeutic agents.
Once the cells are administered to a mammal (e.g., a human), the biological activity of the engineered cell populations and/or binding molecules in some aspects is measured by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells also can be measured by assaying expression and/or secretion of certain cytokines, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
In certain embodiments, engineered cells are modified in any number of ways, such that their therapeutic or prophylactic efficacy is increased. For example, the engineered TCRs or antibody-expressing CARs expressed by the engineered cells in some embodiments are conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating compounds, e.g., the TCR or CAR, to targeting moieties is known in the art. See, for instance, Wadwa et al., J. Drug Targeting 3: 1 1 1 (1995), and U.S. Pat. No. 5,087,616.
C. Diagnostic and Detection Methods
Also provided are methods involving use of the provided binding molecules, e.g., TCRs or antigen-binding fragments thereof and antibodies and antigen-binding fragments thereof, in detection of HPV 16, e.g., HPV 16 E6 or HPV 16 E7, for example, in diagnostic and/or prognostic methods in association with a HPV 16-expressing disease or condition. The methods in some embodiments include incubating a biological sample with the binding molecule and/or administering the binding molecule to a subject. In certain embodiments, a biological sample includes a cell or tissue, such as tumor or cancer tissue. In certain binding molecule to a region or peptide epitope of HPV 16, e.g., HPV 16 E6 or E7, and detecting whether a complex is formed between the binding molecule and peptide epitope. Such a method may be an in vitro or in vivo method. In one embodiment, an anti-HPV 16 binding molecule is used to select subjects eligible for therapy with an anti-HPV 16 binding molecules or engineered cells comprising such molecules, e.g. where HPV 16, e.g., HPV 16 E6 or E7 is a biomarker for selection of patients.
In some embodiments, a sample, such as a cell, tissue sample, lysate, composition, or other sample derived therefrom is contacted with the binding molecule and binding or formation of a complex between the binding molecule and the sample (e.g., region or epitope of HPV16 in the sample) is determined or detected. When binding in the test sample is demonstrated or detected as compared to a reference cell of the same tissue type, it may indicate the presence of an associated disease or condition. In some embodiments, the sample is from human tissues.
Various methods known in the art for detecting specific binding molecule-antigen binding can be used. Exemplary immunoassays include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). An indicator moiety, or label group, can be attached to the subject binding molecules and may be selected so as to meet the needs of various uses of the method which are often dictated by the availability of assay equipment and compatible immunoassay procedures. Exemplary labels include radionuclides (e.g. 125I, 131I, 35S, 3H, or 32P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or β-glactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot™ nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, Calif.). General techniques to be used in performing the various immunoassays noted above are known to those of ordinary skill in the art.
For purposes of diagnosis, the binding molecules can be labeled with a detectable moiety including but not limited to radioisotopes, fluorescent labels, and various enzyme-substrate labels know in the art. Methods of conjugating labels to binding molecules, e.g., TCRs or antibodies, are known in the art. In some embodiments, the binding molecules need not be labeled, and the presence thereof can be detected using a labeled antibody which binds to the binding molecules.
The provided binding molecules in some embodiments can be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. The binding molecules can also be used for in vivo diagnostic assays, such as in vivo imaging. Generally, the binding molecule is labeled with a radionuclide (such as 111In, 99Tc, 14C, 131I, 125I, or 3H) so that the cells or tissue of interest can be localized in vivo following administration to a subject. The binding molecule may also be used as staining reagent in pathology, e.g., using known techniques.
VI. Articles of Manufacture
Also provided are articles of manufacture containing the provided binding molecules, e.g., TCRs, antibodies, and CARs and/or engineered cells, and/or compositions. The articles of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container in some embodiments holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition. In some embodiments, the container has a sterile access port. Exemplary containers include an intravenous solution bags, vials, including those with stoppers pierceable by a needle for injection. The label or package insert may indicate that the composition is used for treating the HPV 16 E6- or E7-expressing or -associated disease or condition. The article of manufacture may include (a) a first container with a composition contained therein, wherein the composition includes the antibody or engineered antigen receptor; and (b) a second container with a composition contained therein, wherein the composition includes a further agent, such as a cytotoxic or otherwise therapeutic agent. The article of manufacture may further include a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further include another or the same container comprising a pharmaceutically-acceptable buffer. It may further include other materials such as other buffers, diluents, filters, needles, and/or syringes.
VII. Definitions
Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains and other peptides, e.g., linkers, may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
“Isolated nucleic acid encoding a TCR or an antibody” refers to one or more nucleic acid molecules encoding TCR alpha or beta chains (or fragments thereof) or antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
As used herein, “percent (%) amino acid sequence identity” and “percent identity” when used with respect to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence (e.g., the subject antibody or fragment) that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
An amino acid substitution may include replacement of one amino acid in a polypeptide with another amino acid. Amino acid substitutions may be introduced into a binding molecule, e.g., TCR or antibody, of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved cytolytic activity.
Amino acids generally can be grouped according to the following common side-chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro;
(6) aromatic: Trp, Tyr, Phe.
In some embodiments, conservative substitutions can involve the exchange of a member of one of these classes for another member of the same class. In some embodiments, non-conservative amino acid substitutions can involve exchanging a member of one of these classes for another class.
The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include “consisting” and/or “consisting essentially of” aspects and variations.
Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.
The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.
As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
As used herein, a statement that a cell or population of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.
As used herein, a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.
All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
VIII. Exemplary Embodiments
Among the provided embodiments are:
1. A binding molecule, comprising:
a first variable region comprising a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 138, 144, 147, 153, 159, 163, 167, 173, 175, 301, 304, 308, 478, 493, 505, 511, 523, 539, 555, 572, 588, 600, 612, 624, 638, 650, 662, 679, 694, 712, 729, 744, 762, 776, 788, 802, 818, 832, 846, 858, 870, 882, 896, 911, 926, 940, 952, 964, 976, 988, or 1002, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 117, 119, 121, 123, 125, 127, 295, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, 676, 691, 709, 726, 741, 759, 775, 787, 799, 815, 830, 845, 857, 869, 881, 895, 908, 925, 937, 951, 963, 975, 987, or 999; and/or a second variable region comprising a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 141, 146, 150, 156, 160, 164, 170, 174, 178, 305, 309, 486, 499, 517, 531, 548, 563, 581, 594, 606, 618, 630, 644, 656, 670, 686, 703, 721, 736, 753, 769, 782, 794, 809, 825, 840, 852, 864, 876, 888, 902, 919, 932, 946, 958, 970, 982, 994, or 1010, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 118, 120, 122, 124, 126, 128, 296, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, 685, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008.
2. The binding molecule of embodiment 1, wherein the first variable region further comprises:
a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 136, 142, 151, 157, 161, 165, 171, 302, 306, 537, 570, 677, 692, 710, 727, 742, 760, 800, 816, 909, 938, or 1000, or a CDR-1 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 117, 119, 121, 123, 125, 127, 295, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, 676, 691, 709, 726, 741, 759, 775, 787, 799, 815, 830, 845, 857, 869, 881, 895, 908, 925, 937, 951, 963, 975, 987, or 999; and/or a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 137, 143, 152, 158, 162, 166, 172, 303, 307, 538, 571, 678, 693, 711, 728, 743, 761, 801, 817, 831, 833, 910, 939, or 1001, or a CDR-2 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 117, 119, 121, 123, 125, 127, 295, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, 676, 691, 709, 726, 741, 759, 775, 787, 799, 815, 830, 845, 857, 869, 881, 895, 908, 925, 937, 951, 963, 975, 987, or 999.
3. The binding molecule of embodiment 1 or embodiment 2, wherein the second variable region comprises:
a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 139, 145, 148, 154, 168, 176, 484, 546, 561, 579, 668, 701, 719, or 751 or a CDR-1 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 118, 120, 122, 124, 126, 128, 296, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, 685, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008; and/or a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 140, 149, 155, 169, 177, 485, 547, 562, 580, 669, 702, 720, 752, 918, or 1009, or a CDR-2 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 118, 120, 122, 124, 126, 128, 296, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, 685, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008.
4. The binding molecule of any of embodiments 1-3, wherein the binding molecule is an antibody or antigen-binding fragment thereof.
5. The binding molecule of any of embodiments 1-3, wherein the binding molecule is a T cell receptor (TCR) or antigen-binding fragment thereof.
6. A T cell receptor (TCR) or antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein:
said Vα region comprises the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 117, 119, 121, 123, 125, 127, 295, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, 676, 691, 709, 726, 741, 759, 775, 787, 799, 815, 830, 845, 857, 869, 881, 895, 908, 925, 937, 951, 963, 975, 987, or 999, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or said Vβ region comprises the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 118, 120, 122, 124, 126, 128, 296, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, 685, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
7. The T cell receptor (TCR) or antigen-binding fragment thereof of embodiment 6, wherein:
said Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18(SEQ ID NO: 251), wherein X1 is A, I, or V; X2 is M, L, V, E or A; X3 is R, L, N, or S; X4 is E, V, P, T, F, I, R or A; X5 is G, I, L, A, P, R, D, or H; X6 is R, T, G, S, N or H; X7 is G, R, A, N, or null; X8 is T, G, or null; X9 is null, A or G; X10 is null or G; X11 is null or G; X12 is null or T; X13 is F, Y, A, S or null; X14 is G, Y, or N; X15 is F, G, T, N, Q, or Y; X16 is K, P, V, N or A; X17 is T, L, or F; and X18 is I, V, T, H, or N; and/or said Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15 (SEQ ID NO: 261), wherein X1 is A or S; X2 is S, I, or V; X3 is S, T, or V; X4 is H, P, L, Y, T, D, or Q; X5 is L, G, W, F, S, or R; X6 is A, G, L, S, or T; X7 is G, E, A, T, R, or null; X8 is null or G; X9 is null or G; X10 is null, F, G, T, S, or A; X11 is T, N, H, A, S, or F; X12 is G, T, Q, D, Y, or L; X13 is E, P, T, G or W; X14 is L, A, Q, Y, or K; and X15 is F, H, Y, or T.
8. The T cell receptor (TCR) or antigen-binding fragment thereof of embodiment 7, wherein:
said Vα region comprises:
a complementarity determining region 1 (CDR-1) comprising the amino acid sequence X1X2X3X4X5X6X7(SEQ ID NO: 243), wherein X1 is T, D, N, or V; X2 is I or S; X3 is S, D, A, P, or M; X4 is G, Q, P, or null; X5 is T, S, I, or F; X6 is D, Y, Q, T, or S; and X7 is Y, G, N, or Q; or
a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6X7X8(SEQ ID NO: 247), wherein X1 is G, Q, I, V, or M; X2 is L, S, Q, Y, F, T, or G; X3 is T, G, S, or F; X4 is Y, S, N, I, or null; X5 is null or D; X6 is null, E, Q, S, M, or K; X7 is S, Q, R, G, D, or N; and X8 is N, E, M, T, or K; and/or
said Vβ region comprises:
a complementarity determining region 1 (CDR-1) comprising the amino acid sequence X1X2X3X4X5 (SEQ ID NO: 254), wherein X1 is S, M, or L; X2 is G, E, D, N, or Q; X3 is H or V; X4 is V, N, E, L, or T; and X5 is S, R, N, Y, A, or M; or
a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6X7(SEQ ID NO: 257), wherein X1 is F, Y, S, or A; X2 is Q, Y, V, or N; X3 is N, D, G, F, or Q; X4 is null or G; X5 is E, V, N, K, or S; X6 is A, K, G, or E; and X7 is Q, M, T, I, or A.
9. The binding molecule of any of embodiments 1-5 or TCR or antigen-binding fragment thereof of any of embodiments 6-8, wherein the binding molecule or TCR or antigen-binding fragment thereof binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E6 or E7 in the context of an MHC molecule.
10. The binding molecule or TCR or antigen-binding fragment thereof of embodiment 9,
wherein the binding molecule or TCR or antigen-binding fragment thereof binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E6 in the context of an MHC molecule.
11. The binding molecule or TCR or antigen-binding fragment thereof of embodiment 10, wherein the peptide epitope derived from HPV16 E6 is or comprises the amino acid sequence set forth in any of SEQ ID NOs: 232-234.
12. The binding molecule or TCR or antigen-binding fragment thereof of embodiment 10 or embodiment 11, wherein the peptide epitope derived from HPV16 E6 is or comprises E6(29-38) TIHDIILECV (SEQ ID NO:233).
13. The binding molecule or TCR or antigen-binding fragment of any of embodiments 1-12, wherein:
said Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18 (SEQ ID NO: 248), wherein X1 is A, I, or V; X2 is M, L, or V; X3 is R, L, or N; X4 is E, V, T, P, or F; X5 is G, I, L, A, or P; X6 is R, T, G, or S; X7 is G, R, or null; X8 is T, G, or null; X9 is null or A; X10 is null or G; X11 is null or G; X12 is null or T; X13 is null or S; X14 is G, Y, or N; X15 is F, G, or T; X16 is K or P; X17 is T or L; and X18 is I, V or T; and/or
said Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4X5X6X7X8X9X10X11X12X13 (SEQ ID NO: 258), wherein X4 is H, P, L, or Y; X5 is L, G, W, F, or S; X6 is A, G, or L; X7 is G, E, A, T, or null; X8 is F, G, T, or S; X9 is T, N, H, or A; X10 is G, T, Q, D, or Y; X11 is E, P, T, or G; X12 is L, A, Q, or Y; and X13 is F, H, Y, or T.
14. The TCR or antigen-binding fragment thereof of embodiment 13, wherein:
said Vα region comprises: a complementarity determining region 1 (CDR-1) comprising the amino acid sequence X1X2X3X4X5X6X7 (SEQ ID NO: 240), wherein X1 is T, D, or N; X2 is I, or S; X3 is S, D, or A; X4 is G, Q, P, or null; X5 is T, S, or I; X6 is D, Y, or Q; and X7 is Y, G, N, or Q; or a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6X7X8(SEQ ID NO: 244), wherein X1 is G, Q, I, or V; X2 is L, S, Q, or Y; X3 is T, G, or S; X4 is Y, S, or null; X5 is null or D; X6 is null, E, Q, or S; X7 is S, Q, R, or G; and X8 is N or E; and/or
said Vβ region comprises: a complementarity determining region 1 (CDR-1) comprising the amino acid sequence X1X2HX4X5 (SEQ ID NO: 252), wherein X1 is S or M; X2 is G, E, D, or N; X4 is V, N, or E; and X5 is S, R, N, or Y; or a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6(SEQ ID NO: 255), wherein X1 is F or S; X2 is Q, Y, or V; X3 is N, D, or G; X4 is E or V; X5 is A, K, or G; and X6 is Q, M, or T.
15. The TCR or antigen-binding fragment of any of embodiments 6-14, wherein:
said Vα region comprises a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 138, 144, 147, 163, 167 173, 304, 308, 478, 493, 505, 511, 523, 539, 555, 572, 588, 600, 612, 624, 638, 650, 662, or 679, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123 125, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, or 676; and/or
a Vβ region comprising a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 141, 146, 150, 164, 170, 174, 305, 309, 486, 499, 517, 531, 548, 563, 581, 594, 606, 618, 630, 644, 656, 670, or 686, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 122, 124 126, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, or 685.
16. The TCR or antigen-binding fragment of embodiment 15, wherein the Vα region further comprises:
a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 136, 142, 161, 165, 171, 302, 306, 537, 570, or 677, or a CDR-1 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123, 125, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, or 676; and/or
a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 137, 143, 162, 166, 172, 303, 307, 538, 571, or 678, or a CDR-2 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123, 125, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, or 676.
17. The TCR or antigen-binding fragment of embodiment 15 or embodiment 16, wherein the Vβ region comprises:
a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 139, 145, 148, 168, 484, 546, 561, 579, or 668, or a CDR-1 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 122, 124, 126, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, or 685; and/or
a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 140, 149, 169, 177, 485, 547, 562, 580, or 669, or a CDR-2 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 122, 124, 126, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, or 685.
18. The TCR or antigen-binding fragment thereof of any of embodiments 6-17, wherein:
said Vα region comprises: a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 136, 142, 161, 165, 171, 302, 306, 537, 570, or 677; a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 137, 143, 162, 166, 172, 303, 307, 538, 571, or 678; and/or a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 138, 144, 147, 163, 167, 173, 304, 308, 478, 493, 505, 511, 523, 539, 555, 572, 588, 600, 612, 624, 638, 650, 662, 679; and/or
said Vβ region comprises: a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 139, 145, 148, 168, 484, 546, 561, 579, or 668; a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 140, 149 or 169; and/or a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 141, 146, 150, 164, 170, 174, 305, 309, 486, 499, 517, 531, 548, 563, 581, 594, 606, 618, 630, 644, 656, 670, or 686.
19. The TCR or antigen-binding fragment thereof of any of embodiments 6-18, wherein:
said Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137, and 138, respectively, and said Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 139, 140, and 141, respectively;
said Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 142, 143, and 144, respectively, and said Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 145, 140, and 146, respectively;
said Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137, and 147, respectively, and said Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 150, respectively;
said Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 161, 162, and 163, respectively, and said Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 164, respectively;
said Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 165, 166, and 167, respectively, and said Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 168, 169, and 170, respectively;
said Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 171, 172, and 173, respectively, and said Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 174, respectively;
said Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively, and said Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 139, 140, and 305, respectively; or said Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 306, 307, and 308, respectively, and said Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 309, respectively.
20. The TCR or antigen-binding fragment thereof of any of embodiments 6-19, wherein:
said Vα region comprises a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vα region amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123, 125, 297, 299, 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661, or 676; and/or
said Vβ region comprises a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vβ region amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 122, 124, 126, 298, 300, 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667, or 685.
21. The TCR or antigen-binding fragment thereof of any of embodiments 6-20, wherein:
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 111 and 112, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 113 and 114, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 115 and 116, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 121 and 122, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 123 and 124, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 125 and 126, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 297 and 298, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 299 and 300, respectively.
22. The binding molecule or TCR or antigen-binding fragment thereof of embodiment 9,
wherein the binding molecule or TCR or antigen-binding fragment thereof binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule.
23. A T cell receptor (TCR) or antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein the TCR or antigen-binding fragment thereof binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule.
24. The binding molecule or TCR or antigen-binding fragment thereof of embodiment 22 or embodiment 23, wherein the peptide epitope derived from HPV16 E7 is or comprises the amino acid sequence set forth in any of SEQ ID NOs: 235-239.
25. The binding molecule or TCR or antigen-binding fragment thereof of embodiment 26, wherein the peptide epitope derived from HPV16 E7 is or comprises E7(11-19) YMLDLQPET (SEQ ID NO:236).
26. The TCR or antigen-binding fragment thereof of any of embodiments 5-8 and 22-25, wherein:
said Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2SX4X5X6X7X8X9X10X11 (SEQ ID NO: 249), wherein X1 is A or V; X2 is E or V; X4 is I or R; X5 is R or D; X6 is G or N; X7 is F or Y; X8 is N or Q; X9 is V or N; X10 is L or F; and X11 is H or V; and/or
said Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2TX4RX6X7YX9X10X11 (SEQ ID NO: 259), wherein X2 is S or I; X4 is T or D; X6 is S or T; X7 is S or N; X9 is E or G; X10 is Q or Y; and X11 is Y or T.
27. The TCR or antigen-binding fragment thereof of embodiment 26, wherein:
said Vα region comprises: a complementarity determining region 1 (CDR-1) comprising the amino acid sequence X1SX3X4X5X6(SEQ ID NO: 241), wherein X1 is D or V; X3 is S, or P; X4 is S or F; X5 is T or S; and X6 is Y or N; or a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6X7 (SEQ ID NO: 245), wherein X1 is I or M; X2 is F or T; X3 is S or F; X4 is N or S; X5 is M or E; X6 is D or N; and X7 is M or T; and/or
said Vβ region comprises: a complementarity determining region 1 (CDR-1) comprising the amino acid sequence set forth in SEQ ID NO: 154; or a complementarity determining region 2 (CDR-2) comprising the amino acid sequence set forth in SEQ ID NO: 155.
28. The TCR or antigen-binding fragment of any of embodiments 5-8 and 22-27, wherein:
said Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence set forth in any of SEQ ID NOs: 153, 159, 301, 694, 712, 729, 744, 762, 776, 788, 802, 818, 832, 846, 858, 870, 882, 896, 911, 926, 940, 952, 964, 976, 988, or 1002, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 117, 119, or 295; and/or
said Vβ region comprises a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 156, 160, 703, 721, 736, 753, 769, 782, 794, 809, 825, 840, 852, 864, 876, 888, 902, 919, 932, 946, 958, 970, 982, 994, or 1010, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 118, 120, 296, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008.
29. The TCR or antigen-binding fragment thereof of embodiment 28, wherein the Vα region further comprises:
a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 151, 157, 692, 710, 727, 742, 760, 800, 816, 909, 938, or 1000; and/or
a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 152, 158, 693, 711, 728, 743, 761, 801, 817, 831, 833, 910, 939, or 1001.
30. The TCR or antigen-binding fragment thereof of embodiment 28 or embodiment 29, wherein the Vβ region comprises:
a complementarity determining region 1 (CDR-1) comprising the amino acid sequence set forth in SEQ ID NO: 154; and/or
a complementarity determining region 2 (CDR-2) comprising the amino acid sequence set forth in SEQ ID NO: 155.
31. The TCR or antigen-binding fragment thereof of any of embodiments 5-8 and 22-30, wherein:
said Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 151, 152, and 153, respectively, and said Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 156, respectively;
said Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 157, 158, and 159, respectively, and said Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 160, respectively; or
said Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 151, 152, and 301, respectively, and said Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 156, respectively.
32. The TCR or antigen-binding fragment thereof of any of embodiments 5-8 and 22-31, wherein:
said Vα region comprises a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vα region amino acid sequence set forth in any of SEQ ID NOs: 117, 119, or 295; and/or
said Vβ region comprises a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vβ region amino acid sequence set forth in any of SEQ ID NOs: 118, 120, 296, 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993, or 1008.
33. The TCR or antigen-binding fragment thereof of any of embodiments 5-8 and 22-32, wherein:
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 117 and either 118 or 296, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 119 and 120, respectively; or
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 295 and either 118 or 296, respectively.
34. The binding molecule or TCR or antigen-binding fragment thereof of embodiment 22, wherein the peptide epitope derived from HPV16 E7 is or comprises E7(86-93) TLGIVCPI (SEQ ID NO:235).
35. The TCR or antigen-binding fragment thereof of any of embodiments 5-8, 22-24 and 34, wherein:
said Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence set forth in SEQ ID NO: 175; and/or
said Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence set forth in SEQ ID NO: 178.
36. The TCR or antigen-binding fragment thereof of embodiment 35, wherein the Vα region comprises:
a complementarity determining region 1 (CDR-1) comprising the amino acid sequence set forth in any of SEQ ID NOs: 136 or 142; and/or
a complementarity determining region 2 (CDR-2) comprising the amino acid sequence set forth in any of SEQ ID NOs: 137 or 143.
37. The TCR or antigen-binding fragment thereof of embodiment 35 or embodiment 36, wherein said Vβ region comprises:
a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in SEQ ID NO: 176; and/or
a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in SEQ ID NO: or 177.
38. The TCR or antigen-binding fragment thereof of any of embodiments 5-8, 22-24 and 34-37, wherein:
said Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 142, 143, and 175, respectively, and said Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 176, 177, and 178, respectively.
39. The TCR or antigen-binding fragment thereof of any of embodiments 5-8, 22-24 and 34-38, wherein:
said Vα region comprises a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vα region amino acid sequence set forth in SEQ ID NO: 127; and/or
said Vβ region comprises a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vβ region amino acid sequence set forth in SEQ ID NO: 128.
40. The TCR or antigen-binding fragment thereof of any of embodiments 5-8, 22-24 and 34-39, wherein:
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 127 and 128, respectively.
41. The TCR or antigen-binding fragment thereof of any of embodiments 5-40, wherein the alpha chain further comprises an alpha constant (Cα) region and/or the beta chain further comprises a beta constant (Cβ) region.
42. The TCR or antigen-binding fragment thereof of embodiment 41, wherein the Cα and Cβ regions are mouse constant regions.
43. The TCR or antigen-binding fragment thereof of embodiment 41 or embodiment 42, wherein:
said Cα region comprises the amino acid sequence set forth in SEQ ID NO: 262, or a sequence of amino acids that has at least 90% sequence identity thereto; and/or
said Cβ region comprises the amino acid sequence set forth in SEQ ID NO: 263, or a sequence of amino acids that has at least 90% sequence identity thereto.
44. The TCR or antigen-binding fragment thereof of embodiment 41, wherein the Cα and Cβ regions are human constant regions.
45. The TCR or antigen-binding fragment thereof of embodiment 41 or embodiment 44, wherein:
said Cα region comprises the amino acid sequence set forth in any of SEQ ID NOs: 212, 213, 215, 217, 218, 220, or 524, or a sequence of amino acids that has at least 90% sequence identity thereto; and/or
said Cβ region comprises the amino acid sequence set forth in any of SEQ ID NOs: 214, 216, 631, or 889, or a sequence of amino acids that has at least 90% sequence identity thereto.
46. The TCR or antigen-binding fragment thereof of any of embodiments 5-45, comprising one or more modifications in the α chain and/or β chain such that when the TCR or antigen-binding fragment thereof is expressed in a cell, the frequency of mispairing between the TCR α chain and β chain and an endogenous TCR α chain and β chain is reduced, the expression of the TCR α chain and β chain is increased and/or the stability of the TCR α chain and β chain is increased.
47. The TCR or antigen-binding fragment thereof of embodiment 46, wherein the one or more modifications is a replacement, deletion, or insertion of one or more amino acids in the Cα region and/or the Cβ region.
48. The TCR or antigen-binding fragment thereof of embodiment 46 or embodiment 47, wherein the one or more modifications comprise replacement(s) to introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and beta chain.
49. The TCR or antigen-binding fragment thereof of any of embodiments 5-41 and 44-48, comprising a Cα region comprising a cysteine at a position corresponding to position 48 with numbering as set forth in SEQ ID NO: 212, 213, 215, 217, 218, 220, or 524, and/or a Cβ region comprising a cysteine at a position corresponding to position 57 with numbering as set forth in SEQ ID NO: 214, 216, 631, or 889.
50. The TCR or antigen-binding fragment thereof of any of embodiments 41, 44, and 46-49, wherein:
said Cα region comprises the amino acid sequence set forth in any of SEQ ID NOs: 196, 198, 200, 201, 203, or 525, or a sequence of amino acids that has at least 90% sequence identity thereto comprising one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; and/or
said Cβ region comprises the amino acid sequence set forth in any of SEQ ID NOs: 197, 199, 632, or 890, or a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain.
51. The TCR or antigen-binding fragment thereof of any of embodiments 5-50, wherein the TCR or antigen-binding fragment thereof is encoded by a nucleotide sequence that has been codon-optimized.
52. The TCR or antigen-binding fragment thereof of any of embodiments 5-21 and 41-45, wherein:
a) said alpha chain comprises:
the amino acid sequence set forth in any of SEQ ID NOs: 18, 28, 38, 68, 78, 88, 287, 291, 473, 488, 500, 506, 518, 532, 550, 565, 583, 595, 607, 619, 633, 645, 657, or 672, a sequence of amino acids that has at least 90% sequence identity thereto; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 20, 30, 40, 70, 80, 90, 100, 202, 219, 389, 430, 1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, or a nucleotide sequence that has at least 90% sequence identity thereto; and/or
said beta chain comprises an amino acid sequence set forth in any of SEQ ID NOs: 22, 32, 42, 72, 82, 92, 289, 293, 479, 494, 512, 526, 541, 556, 574, 589, 601, 613, 625, 639, 651, 663, or 681, a sequence of amino acids that has at least 90% sequence identity thereto; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOS: 16, 17, 24, 34, 44, 74, 84, 94, 104, 390, 431, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, or a nucleotide sequence that has at least 90% sequence identity thereto; or
b) the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 18 and 22, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 28 and 32, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 38 and 42, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 68 and 72, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 78 and 82, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 88 and 92, respectively, the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 287 and 289, respectively, or the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 291 and 293, respectively.
53. The TCR or antigen-binding fragment thereof of any of embodiments 5-21 and 41-51, wherein:
a) said alpha chain comprises:
the amino acid sequence set forth in any of SEQ ID NOs: 19, 29, 39, 69, 89, 288, 292, 474, 489, 501, 507, 519, 533, 551, 566, 584, 596, 608, 620, 634, 646, 658, or 673, a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 10, 11, 21, 31, 41, 71, 81, 91, 101, 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117, 1119, 1121, 1123, 1125, 1127, or a nucleotide sequence that has at least 90% sequence identity thereto and encodes an alpha chain that contains one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; and/or
said beta chain comprises an amino acid sequence set forth in any of SEQ ID NOs: 23, 33, 43, 73, 83, 93, 290, 294, 480, 495, 513, 527, 542, 557, 575, 590, 602, 614, 626, 640, 652, 664, or 682, a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 7, 8, 25, 35, 45, 75, 85, 95, 105, 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126, 1128, or a nucleotide sequence that has at least 90% sequence identity thereto and encodes a beta chain that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain; or
b) the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 19 and 23, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 29 and 33, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 39 and 43, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 69 and 73, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 79 and 83, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 89 and 93, respectively, the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 288 and 290, or the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 292 and 294.
54. The TCR or antigen-binding fragment thereof of any of embodiments 5-8, 22-33 and 41-4, wherein:
a) said alpha chain comprises:
the amino acid sequence set forth in SEQ ID NOs: 48, 58, 283, 687, 705, 722, 737, 755, 771, 783, 795, 811, 826, 841, 853, 865, 877, 891, 904, 921, 933, 947, 959, 971, 983, or 995, a sequence of amino acids that has at least 90% sequence identity thereto; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 50, 60, 183, 1049, 1051, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1093, 1095, 1225, 1226, or a nucleotide sequence that has at least 90% sequence identity thereto; and/or
said beta chain comprises an amino acid sequence set forth in SEQ ID NOs: 52, 62, 285, 696, 714, 731, 746, 764, 777, 789, 804, 820, 835, 847, 859, 871, 883, 897, 913, 927, 941, 953, 965, 977, 989, or 1004, a sequence of amino acids that has at least 90% sequence identity thereto; or an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NOS: 54, 64, 108, 1050, 1052, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, 1094, 1224, 1227, 1228 or a nucleotide sequence that has at least 90% sequence identity thereto; or
b) the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 48 and either 52 or 285, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 58 and 62, respectively; or the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 283 and either 52 or 285, respectively.
55. The TCR or antigen-binding fragment thereof of any of embodiments 5-8, 22-33 and 41-51, wherein:
a) said alpha chain comprises:
the amino acid sequence set forth in SEQ ID NOs: 49, 59, 284, 688, 706, 723, 738, 756, 772, 784, 796, 812, 827, 842, 854, 866, 878, 892, 905, 922, 934, 948, 960, 972, 984, or 996, a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 51, 61, 12, 1129, 1131, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171, 1173, 1175, 1177, or a nucleotide sequence that has at least 90% sequence identity thereto and encodes an alpha chain that contains one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; and/or
said beta chain comprises an amino acid sequence set forth in SEQ ID NOs: 53, 63, 286, 697, 715, 732, 747, 765, 778, 790, 805, 821, 836, 848, 860, 872, 884, 898, 914, 928, 942, 954, 966, 978, 990, or 1005, a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain; or an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NOS: 54, 65, 9, 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172, 1174, 1176, 1178, or a nucleotide sequence that has at least 90% sequence identity thereto and encodes a beta chain that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain; or
b) the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 49 and 53, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 59 and 63, respectively; or the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 284 and 286, respectively.
56. The TCR or antigen-binding fragment thereof of any of embodiments 5-8 and 34-45, wherein:
a) said alpha chain comprises:
the amino acid sequence set forth in SEQ ID NO: 98, a sequence of amino acids that has at least 90% sequence identity thereto; or an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 100, or a nucleotide sequence that has at least 90% sequence identity thereto; and/or
said beta chain comprises an amino acid sequence set forth in any of SEQ ID NOs: 9 or 102, a sequence of amino acids that has at least 90% sequence identity thereto; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOS: 11 or 104, or a nucleotide sequence that has at least 90% sequence identity thereto; or
b) the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 98 and 102, respectively.
57. The TCR or antigen-binding fragment thereof of any of embodiments 5-8 and 34-51, wherein:
a) said alpha chain comprises:
the amino acid sequence set forth in SEQ ID NO: 99, a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; or an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 101, or a nucleotide sequence that has at least 90% sequence identity thereto and encodes an alpha chain that contains one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; and/or
said beta chain comprises an amino acid sequence set forth in any of SEQ ID NOs: 10 or 103, a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain; or an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 105, or a nucleotide sequence that has at least 90% sequence identity thereto and encodes a beta chain that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain; or
b) the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 99 and 103, respectively.
58. The TCR or antigen-binding fragment thereof of any of embodiments 5-57, further comprising a signal peptide.
59. The TCR or antigen-binding fragment thereof of embodiment 58, wherein the signal peptide comprises the amino acid sequence set forth in any of SEQ ID NOs: 181, 184, 187, 189, 190, 192, 193, 310, 311, 182, 185, 186, 188, 191, 194, 487, 540, 549, 564, 573, 582, 671, 680, 695, 704, 713, 730, 745, 754, 763, 770, 803, 810, 819, 834, 903, 912, 920, 1003, or 1011.
60. The binding molecule or TCR or antigen-binding fragment thereof of any of embodiments 1-59, that is isolated or purified or is recombinant.
61. The binding molecule or TCR or antigen-binding fragment thereof of any of embodiments 1-60, that is human.
62. The binding molecule or TCR or antigen-binding fragment thereof of any of embodiments 1-61, that is monoclonal.
63. The binding molecule or TCR or antigen-binding fragment thereof of any of embodiments 1-62, wherein the binding molecule or TCR or antigen-binding fragment thereof is single chain.
64. The binding molecule of or TCR or antigen-binding fragment thereof of any of embodiments 1-62, wherein the binding molecule or TCR or antigen-binding fragment thereof comprises two chains.
65. The binding molecule or TCR or antigen-binding fragment thereof of any of embodiments 1-64, wherein the antigen-specificity is at least partially CD8-independent.
66. The binding molecule or TCR or antigen-binding fragment of any of embodiments 9-65 wherein the MHC molecule is an HLA-A2 molecule.
67. A nucleic acid molecule encoding the binding molecule or the TCR or antigen-binding fragment thereof of any of embodiments 1-66.
68. The nucleic acid molecule of embodiment 67, comprising a nucleotide sequence encoding an alpha chain and/or a nucleotide sequence encoding a beta chain, wherein:
said nucleotide sequence encoding an alpha chain comprises the sequence selected from the group consisting of: residues 61-816 of SEQ ID NO: 20, residues 58-804 of SEQ ID NO: 30, residues 61-825 of SEQ ID NO: 40, residues 64-813 of SEQ ID NO: 50, residues 64-816 of SEQ ID NO: 60, residues 58-807 of SEQ ID NO: 70, residues 61-825 of SEQ ID NO: 80, residues 67-831 of SEQ ID NO: 90, residues 58-801 of SEQ ID NO: 100, residues 64-810 of SEQ ID NO: 183, residues 58-801 of SEQ ID NO: 202, residues 67-813 of SEQ ID NO: 219, or a sequence having at least 90% sequence identity thereto; and/or
said nucleotide sequence encoding a beta chain comprises the sequence selected from the group consisting of: residues 58-930 of SEQ ID NO: 16, residues 58-936 of SEQ ID NO: 17, residues 58-939 of SEQ ID NO: 24, residues 64-930 of SEQ ID NO: 34 or 44, residues 58-933 of SEQ ID NO: 54, residues 58-927 of SEQ ID NO: 64, residues 64-936 of SEQ ID NO: 74, residues 58-933 of SEQ ID NO: 84, residues 63-930 of SEQ ID NO: 94, residues 46-936 of SEQ ID NO: 104, residues 58-933 of SEQ ID NO: 108, or a sequence having at least 90% sequence identity thereto.
69. The nucleic acid molecule of embodiment 67, wherein the nucleotide sequence is codon-optimized.
70. The nucleic acid molecule of embodiment 67 or embodiment 69, comprising a nucleotide sequence encoding an alpha chain and/or a nucleotide sequence encoding a beta chain, wherein:
said nucleotide sequence encoding an alpha chain comprises the sequence selected from the group consisting of: residues 67-825 of SEQ ID NO: 10, residues 58-813 of SEQ ID NO: 11, residues 64-822 of SEQ ID NO: 12 residues 61-825 of SEQ ID NO: 21, residues 58-813 of SEQ ID NO: 31, residues 61-834 of SEQ ID NO: 41, residues 63-822 of SEQ ID NO: 51, residues 64-825 of SEQ ID NO: 61, residues 58-816 of SEQ ID NO: 71, residues 61-834 of SEQ ID NO: 81, residues 67-840 of SEQ ID NO: 91, residues 58-810 of SEQ ID NO: 101, or a sequence having at least 90% sequence identity thereto; and/or
said nucleotide sequence encoding a beta chain comprises the sequence selected from the group consisting of: residues 58-930 of SEQ ID NO: 7, residues 58-936 of SEQ ID NO: 8, residues 58-933 of SEQ ID NO: 9 residues 58-939 of SEQ ID NO: 25, residues 64-930 of SEQ ID NO: 35, 45, or 95, residues 58-933 of SEQ ID NO: 54 or 85, residues 58-927 of SEQ ID NO: 65, residues 64-936 of SEQ ID NO: 75, residues 46-936 of SEQ ID NO: 105, or a sequence having at least 90% sequence identity thereto.
71. The nucleic acid molecule of any of embodiments 67-71, wherein the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a nucleotide sequence encoding an internal ribosome entry site (IRES) or a peptide sequence that causes ribosome skipping.
72. The nucleic acid molecule of embodiment 71, wherein the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a peptide sequence that causes ribosome skipping.
73. The nucleic acid molecule of embodiment 71 or embodiment 742 wherein the peptide that causes ribosome skipping is a P2A or T2A peptide and/or comprises the sequence of amino acids set forth in SEQ ID NO: 204 or 211.
74. The nucleic acid of any of embodiments 67-73, comprising the nucleotide sequence set forth in any of SEQ ID NOs: 13, 14, 15, 26, 36, 46, 56, 66, 76, 86, 96, 106, 432-472, or a nucleotide sequence having at least 90% sequence identity thereto.
75. The nucleic acid of any of embodiments 67-74, wherein the nucleic acid is synthetic.
76. The nucleic acid of any of embodiments 67-75, wherein the nucleic acid is cDNA.
77. A vector comprising the nucleic acid of any of embodiments 67-76.
78. The vector of embodiment 77, wherein the vector is an expression vector.
79. The vector of embodiment 77 or embodiment 78, wherein the vector is a viral vector.
80. The vector of embodiment 79, wherein the viral vector is a retroviral vector.
81. The vector of embodiment 79 or embodiment 80, wherein the viral vector is a lentiviral vector.
82. The vector of embodiment 81, wherein the lentiviral vector is derived from HIV-1.
83. An engineered cell comprising the vector of any of embodiments 77-82.
84. An engineered cell, comprising the binding molecule or the TCR or antigen-binding fragment thereof of any of embodiments 1-66.
85. The engineered cell of embodiment 83 or embodiment 84, wherein the binding molecule or TCR or antigen-binding fragment thereof is heterologous to the cell.
86. An engineered cell, comprising a heterologous TCR or antigen-binding fragment thereof that binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E6 in the context of an MHC molecule, wherein the TCR or antigen-binding fragment thereof does not bind to or recognize the epitope E6(29-38) comprising the amino acid sequence TIHDIILECV (SEQ ID NO. 233).
87. The engineered cell of embodiment 86, wherein the TCR or antigen-binding fragment thereof that binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E6 in the context of an MHC molecule is or comprises the sequence set forth in SEQ ID NO: 232 or SEQ ID NO: 234.
88. An engineered cell, comprising a heterologous TCR or antigen-binding fragment thereof that binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule.
89. The engineered cell of embodiment 88, wherein the peptide derived from HPV16 E7 is or comprises the sequence set forth in any of SEQ ID NOs: 235-239.
90. The engineered cell of embodiment 88 or embodiment 89, wherein the peptide derived from HPV16 E7 is or comprises the sequence set forth in SEQ ID NO: 236.
91. The engineered cell of any of embodiments 88-90, wherein the TCR or antigen-binding fragment thereof is a TCR or antigen-binding fragment thereof of any of embodiments 25-33, 55 or 56.
92. The engineered cell of embodiment 88 or embodiment 89, wherein the peptide derived from HPV16 E7 is or comprises the sequence set forth in SEQ ID NO: 235.
93. The engineered cell of embodiment 88, 89 or 92, wherein the TCR or antigen-binding fragment thereof is a TCR or antigen-binding fragment thereof of any of embodiments 34-42, 58 or 59.
94. The engineered cell of any of embodiments 83-93, wherein the engineered cell is a T cell.
95. The engineered cell of embodiment 94, wherein the T cell is CD8+.
96. The engineered cell of embodiment 94, wherein the T cell is CD4+.
97. A method for producing a cell of any of embodiments 83-96, comprising transducing a cell in vitro or ex vivo with a vector according to any of embodiments 77-82.
98. A composition, comprising the binding molecule or the TCR or antigen-binding fragment thereof of any of embodiments 1-66, or the engineered cell of any of embodiments 83-96.
99. A composition, comprising an engineered CD8+ cell of embodiment 95 and an engineered CD4+ cell of embodiment 96.
100. The composition of embodiment 99, wherein the TCR or antigen-binding fragment thereof binds to or recognizes a peptide epitope of HPV 16 in the context of an MHC molecule that is at least partially CD8-independent.
101. The composition of embodiment 99 or embodiment 100, wherein the CD8+ cell and CD4+ cell are engineered with the same TCR or antigen-binding fragment thereof and/or are each engineered with a TCR or antigen-binding fragment thereof that binds to or recognizes the same peptide epitope of HPV 16 in the context of an MHC molecule.
102. The composition of any of embodiments 99-101, further comprising a pharmaceutically acceptable excipient.
103. A method of treatment, comprising administering the engineered cell of any of embodiments 83-96 to a subject having a disease or disorder associated with HPV.
104. A method of treatment, comprising administering the composition of any of embodiments 98-102 to a subject having a disease or disorder associated with HPV.
105. The method of embodiment 103 or embodiment 104, wherein the disease or disorder is associated with HPV16.
106. The method of any of embodiments 103-105, wherein the disease or disorder is cancer.
107. A T cell receptor (TCR) or antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein:
the Vα region comprises the amino acid sequence set forth in any of SEQ ID NOs: 117, 119 or 295 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or
the Vβ region comprises the amino acid sequence set forth in any of SEQ ID NOs: 118, 120, or 296, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
108. The TCR or antigen-binding fragment thereof of any of embodiment 107, wherein:
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 1183), wherein X1 is A or V; X2 is V, A, G, Q, M, or E; X3 is S, G, A, N, Y, R, T, or P; X4 is E, A, S, G, R. F, N, D, V, P, L, I, or M; X5 is R, N, H, T, D, G, S, A, P, L, Q, or F; X6 is G, H, N, A, S, L, or T; X7 is T, S, G, or null; X8 is G, or null; X9 is G, Y, N, S, or null; X10 is T, G, S, D, F, Y, A, N, or null; X11 is Y, F, Y, Q, N, or R; X12 is N, K, Q, or D; X13 is Y, L, T, F, M, or V; and X14 is I, T, S, V, R, or Y; and/or
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 1193), wherein X2 is 5, M, I, K, or V; X3 is S, T, N, or A; X4 is R, V, P, S, T, G, L, A, I, or D; X5 is F, G, R, Y, S, L, V, or T; X6 is L, G, D, A, S, T, V, R, or null; X7 is G, D, R, S, T, or null; X8 is S, or null; X9 is S, H, G, R, V, T, D, L, or null; X10 is T, S, A, Y, N, G, or P; X11 is D, Y, N, E, K, or G; X12 is T, E, G, or K; X13 is Q, Y, A, or L; and X14 is Y, F, T, or I.
109. A T cell receptor (TCR) or antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein:
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 1183), wherein X1 is A or V; X2 is V, A, G, Q, M, or E; X3 is S, G, A, N, Y, R, T, or P; X4 is E, A, S, G, R. F, N, D, V, P, L, I, or M; X5 is R, N, H, T, D, G, S, A, P, L, Q, or F; X6 is G, H, N, A, S, L, or T; X7 is T, S, G, or null; X8 is G, or null; X9 is G, Y, N, S, or null; X10 is T, G, S, D, F, Y, A, N, or null; X11 is Y, F, Y, Q, N, or R; X12 is N, K, Q, or D; X13 is Y, L, T, F, M, or V; and X14 is I, T, S, V, R, or Y; and/or
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 1193), wherein X2 is 5, M, I, K, or V; X3 is S, T, N, or A; X4 is R, V, P, S, T, G, L, A, I, or D; X5 is F, G, R, Y, S, L, V, or T; X6 is L, G, D, A, S, T, V, R, or null; X7 is G, D, R, S, T, or null; X8 is S, null; X9 is S, H, G, R, V, T, D, L, or null; X10 is T, S, A, Y, N, G, or P; X11 is D, Y, N, E, K, or G; X12 is T, E, G, or K; X13 is Q, Y, A, or L; and X14 is Y, F, T, or I.
110. The TCR or antigen-binding fragment thereof of embodiment 108 or embodiment 109, wherein the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence VVX3X4X5X6X7X8GX10X11X12X13 (SEQ ID NO:1184), wherein X3 is S, N, or T; X4 is R, or F; X5 is D, or A; X6 is N, or L; X7 is T, or null; X8 is Y, or G; X10 is Q, or F; X11 is N, or K; X12 is F, or T; and X13 is V, or I.
111. The TCR or antigen-binding fragment thereof of any of embodiments 108-110, wherein:
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2TX4X5X6X7X8X9X10X11X12 (SEQ ID NO:1194), wherein X2 is 5, M, I, or K; X4 is P, T, G, A, S, or D; X5 is R, or S; X6 is D, G, S, T, or V; X7 is R, S, or null; X8 is T, Y, G, N, or S; X9 is Y, N, or K; X10 is E, or G; X11 is Q, A, or Y; and X12 is Y, F, or T;
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 1195), wherein X2 is 5, M, I, or K; X3 is S, T, A, or N; X4 is R, V, S, P, T, G, L, or A; X5 is F, G, R, Y, S, V, or T; X6 is L, G, D, A, S, T, V, or null; X7 is G, D, R, T, or null; X8 is S, or null; X9 is S, H, G, R, V, T, L, or null; X10 is T, S, Y, A, N, G, or P; X11 is D, Y, N, K, E, or G; X12 is T, or E; X13 is Q, A, or L; and X14 is Y, or F;
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11 Q Y (SEQ ID NO: 1196), wherein X2 is 5, M, I, or K; X3 is S, T, A, or N; X4 is R, P, S, G, L, A, or T; X5 is F, R, Y, V, or T; X6 is L, D, A, S, T, V, or null; X7 is G, R, or null; X8 is S, G, V, or null; X9 is T, A, G, N, S, or P; X10 is D, Y, or E; and X11 is T, or E;
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9YEQY (SEQ ID NO: 1197), wherein X2 is 5, M, I, or K; X3 is S, T, A, or N; X4 is P, S, G, T, or A; X5 is R, or Y; X6 is D, A, S, T, or V; X7 is R, or null; X8 is G, V, or null; and X9 is S, T, A, or N;
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASTX4X5X6X7X8X9X10X11EX13X14 (SEQ ID NO:1198), wherein X4 is T, P, or G; X5 is R, or S; X6 is S, D, G, or V; X7 is D, or null; X8 is S, or null; X9 is S, R, or null; X10 is S, T, Y, or G; X11 is Y, N, or K; X13 is Q, or A; and X14 is Y, or F;
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8YGYT (SEQ ID NO: 1199), wherein X2 is S, or I; X3 is S, or T; X4 is L, A, or D; X5 is L, T, or R; X6 is L, T, or R; X7 is G, D, or null; and X8 is A, or N; or
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2TX4RX6X7YX9X10X11 (SEQ ID NO: 259), wherein X2 is S or I; X4 is T or D; X6 is S or T; X7 is S or N; X9 is E or G; X10 is Q or Y; and X11 is Y or T.
112. The TCR or antigen-binding fragment thereof of any of embodiments 107-111 wherein the Vα region comprises:
a complementarity determining region 1 (CDR-1) comprising the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO: 1191), wherein X1 is N, S, D, T, or V; X2 is 5, V, R, T, or I; X3 is M, F, G, S, N, A, L, V, or P; X4 is F, S, N, A, or null; X5 is D, S, Q, Y, N, V, T, or P; and X6 is Y, S, R, N, G, or T; and/or
a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6X7X8 (SEQ ID NO: 1192), wherein X1 is I, V, L, G, N, T, Y, or M; X2 is 5, V, Y, L, P, F, I, or T; X3 is S, Y, K, L, T, or F; X4 is I, G, N, A, S, or null; X5 is S, D, or null; X6 is K, G, N, S, D, T, or E; X7 is D, E, G, A, K, L, or N; and X8 is K, V, D, P, N, T, L, or M.
113. The TCR or antigen-binding fragment thereof of any of embodiments 107-112, wherein the Vβ region comprises:
a complementarity determining region 1 (CDR-1) comprising the amino acid sequence SX2X3X4X5 (SEQ ID NO:1203), wherein X2 is G, or N; X3 is H, or D; X4 is T, L, N, or V; X5 is A, S, Y, or T; and/or
a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6, wherein X1 is F, or Y; X2 is Q, Y, or N; X3 is G, N, R, or Y; X4 is N, G, E, or T; X5 is S, E, A, or G; and X6 is A, E, I, or Q.
114. The TCR or antigen-binding fragment thereof of any of embodiments 107-113, wherein:
the Vα region comprises: a complementarity determining region 1 (CDR-1) comprising the amino acid sequence X1SX3X4X5X6 (SEQ ID NO: 241), wherein X1 is D or V; X3 is S, or P; X4 is S or F; X5 is T or S; and X6 is Y or N; or a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6X7 (SEQ ID NO: 245), wherein X1 is I or M; X2 is F or T; X3 is S or F; X4 is N or S; X5 is M or E; X6 is D or N; and X7 is M or T; and/or
the Vβ region comprises: a complementarity determining region 1 (CDR-1) comprising the amino acid sequence set forth in SEQ ID NO: 154; or a complementarity determining region 2 (CDR-2) comprising the amino acid sequence set forth in SEQ ID NO: 155.
115. The TCR or antigen-binding fragment thereof of any of embodiments 107-114, wherein the TCR or antigen-binding fragment thereof binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule, the peptide epitope is or comprises E7(11-19) YMLDLQPET (SEQ ID NO:236).
116. The TCR or antigen-binding fragment of any of embodiments 107-115, wherein:
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence set forth in any of SEQ ID NOs: 153, 159, or 301, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 117, 119, or 295; and/or
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 156 or 160 or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 118, 120, or 296.
117. The TCR or antigen-binding fragment thereof of any of embodiments 107-116, wherein the Vα region further comprises:
a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 151 or 157; and/or
a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 152 or 158.
118. The TCR or antigen-binding fragment thereof of any of embodiments 107-117, wherein the Vβ region comprises:
a complementarity determining region 1 (CDR-1) comprising the amino acid sequence set forth in SEQ ID NO: 154; and/or
a complementarity determining region 2 (CDR-2) comprising the amino acid sequence set forth in SEQ ID NO: 155.
119. The TCR or antigen-binding fragment thereof of any of embodiments 107-118, wherein:
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 151, 152, and 153, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 156, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 157, 158, and 159, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 160, respectively; or
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 151, 152, and 301, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155, and 156, respectively.
120. The TCR or antigen-binding fragment thereof of any of embodiments 107-119, wherein:
the Vα region comprises a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vα region amino acid sequence set forth in any of SEQ ID NOs: 117, 119, or 295; and/or
the Vβ region comprises a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vβ region amino acid sequence set forth in any of SEQ ID NOs: 118, 120, or 296.
121. The TCR or antigen-binding fragment thereof of any of embodiments 107-120, wherein:
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 117 and either 118 or 296, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 119 and 120, respectively; or
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 295 and either 118 or 296, respectively.
122. The TCR or antigen-binding fragment thereof of any of embodiments 107-121, wherein the alpha chain further comprises an alpha constant (Cα) region and/or the beta chain further comprises a beta constant (Cβ) region.
123. The TCR or antigen-binding fragment thereof of embodiment 122, wherein the Cα and Cβ regions are mouse constant regions.
124. The TCR or antigen-binding fragment thereof of embodiment 122 or embodiment 123, wherein:
the Cα region comprises the amino acid sequence set forth in SEQ ID NO: 262, 833, 1012, 1014, 1015, 1017, 1018, or a sequence of amino acids that has at least 90% sequence identity thereto; and/or
the Cβ region comprises the amino acid sequence set forth in SEQ ID NO: 263, 1013 or 1016 or a sequence of amino acids that has at least 90% sequence identity thereto.
125. The TCR or antigen-binding fragment thereof of embodiment 122, wherein the Cα and Cβ regions are human constant regions.
126. The TCR or antigen-binding fragment thereof of embodiment 122 or embodiment 19, wherein:
the Cα region comprises the amino acid sequence set forth in any of SEQ ID NOs: 212, 213, 215, 217, 218, 220 or 524, or a sequence of amino acids that has at least 90% sequence identity thereto; and/or
the Cβ region comprises the amino acid sequence set forth in any of SEQ ID NOs: 214, 216, 631 or 889, or a sequence of amino acids that has at least 90% sequence identity thereto.
127. The TCR or antigen-binding fragment thereof of any of embodiments 107-126, wherein:
a) the alpha chain comprises:
the amino acid sequence set forth in SEQ ID NOs: 48, 58, or 283, a sequence of amino acids that has at least 90% sequence identity thereto; or the amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 50. 60, 183, 1093 or 1095 or a nucleotide sequence that has at least 90% sequence identity thereto; and/or
the beta chain comprises:
the amino acid sequence set forth in SEQ ID NOs: 52, 62, or 285, a sequence of amino acids that has at least 90% sequence identity thereto; or the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NOS: 55, 64, 108, or 1094, or a nucleotide sequence that has at least 90% sequence identity thereto; or
b) the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 48 and either 52 or 285, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 58 and 62, respectively; or the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 283 and either 52 or 285, respectively.
128. The TCR or antigen-binding fragment thereof of any of embodiments 107-125, wherein the TCR or antigen-binding fragment comprises one or more modifications in the α chain and/or β chain such that when the TCR or antigen-binding fragment thereof is expressed in a cell, the frequency of mispairing between the TCR α chain and β chain and an endogenous TCR α chain and β chain is reduced, the expression of the TCR α chain and β chain is increased and/or the stability of the TCR α chain and β chain is increased, each compared to expression in a cell of the TCR or antigen-binding fragment thereof not containing the one or more modifications.
129. The TCR or antigen-binding fragment thereof of embodiment 128, wherein the one or more modifications is a replacement, deletion, or insertion of one or more amino acids in the Cα region and/or the Cβ region.
130. The TCR or antigen-binding fragment thereof of embodiment 128 or embodiment 129, wherein the one or more modifications comprise replacement(s) to introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and beta chain.
131. The TCR or antigen-binding fragment thereof of any of embodiments 107-122, 125 and 128-130, comprising a Cα region comprising a cysteine at a position corresponding to position 48 with numbering as set forth in SEQ ID NO: 212, 213, 217, 218, or 524 or at a position corresponding to position 49 with numbering as set forth in SEQ ID NO: 215 or 220; and/or a Cβ region comprising a cysteine at a position corresponding to position 57 with numbering as set forth in SEQ ID NO: 214 or 216 or at a position corresponding to position 58 with numbering as set forth in SEQ ID NO: 631 or 889.
132. The TCR or antigen-binding fragment thereof of any of embodiments 122, 125, and 128-130, wherein:
the Cα region comprises the amino acid sequence set forth in any of SEQ ID NOs: 196, 198, 200, 201, 203, or 525, or a sequence of amino acids that has at least 90% sequence identity thereto comprising one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; and/or
the Cβ region comprises the amino acid sequence set forth in any of SEQ ID NOs: 197, 199, 632, or 890 or a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain.
133. The TCR or antigen-binding fragment thereof of any of embodiments 107-132, wherein the TCR or antigen-binding fragment thereof is encoded by a nucleotide sequence that has been codon-optimized.
134. The TCR or antigen-binding fragment thereof of any of embodiments 107-132, wherein:
a) the alpha chain comprises:
the amino acid sequence set forth in SEQ ID NOs: 49, 59, or 284, a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 51, 61, 12, 1175 or 1177 or a nucleotide sequence that has at least 90% sequence identity thereto and encodes an alpha chain that contains one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; and/or
the beta chain comprises:
the amino acid sequence set forth in SEQ ID NOs: 53, 63, or 286, a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain; or an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NOS: 54, 65, 9, 1176 or 1178 or a nucleotide sequence that has at least 90% sequence identity thereto and encodes a beta chain that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain; or
b) the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 49 and 53, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 59 and 63, respectively; or the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 284 and 286, respectively.
135. The TCR or antigen-binding fragment thereof of any of embodiments 107-134, wherein the alpha and/or beta chain further comprises a signal peptide.
136. The TCR or antigen-binding fragment thereof of embodiment 135, wherein:
the alpha chain comprises the signal peptide comprising the amino acid sequence set forth in any of SEQ ID NOs: 181, 184, 187, 189, 190, 192, 193, 310, 311; and/or
the beta chain comprises the signal peptide comprising the amino acid sequence set forth in any of SEQ ID NOs: 182, 185, 186, 188, 191, or 194.
137. The TCR or antigen-binding fragment thereof of any of embodiments 107-136, that is isolated or purified or is recombinant.
138. The TCR or antigen-binding fragment thereof of any of embodiments 107-137, that is human.
139. The TCR or antigen-binding fragment thereof of any of embodiments 107-138, that is monoclonal.
140. The TCR or antigen-binding fragment thereof of any of embodiments 107-139, wherein the TCR or antigen-binding fragment thereof is single chain.
141. The TCR or antigen-binding fragment thereof of any of embodiments 107-139, wherein the TCR or antigen-binding fragment thereof comprises two chains.
142. The TCR or antigen-binding fragment thereof of any of embodiments 107-141, wherein the antigen-specificity is at least partially CD8-independent.
143. The TCR or antigen-binding fragment of any of embodiments 115-142 wherein the MHC molecule is an HLA-A2 molecule.
144. A nucleic acid molecule encoding the TCR or antigen-binding fragment thereof of any of embodiments 107-143, or an alpha or beta chain thereof.
145. The nucleic acid molecule of embodiment 144, comprising a nucleotide sequence encoding an alpha chain and/or a nucleotide sequence encoding a beta chain, wherein:
the nucleotide sequence encoding an alpha chain comprises residues 64-813 of SEQ ID NO: 50, residues 64-816 of SEQ ID NO: 60, or residues 64-810 of SEQ ID NO: 183, or a sequence having at least 90% sequence identity thereto; or comprises the sequence set forth in any of SEQ ID NOS: 50, 60, 183, 1093 or 1095, or a sequence having at least 90% sequence identity thereto; and/or
the nucleotide sequence encoding a beta chain comprises residues 58-933 of SEQ ID NO: 55, residues 58-927 of SEQ ID NO: 64, residues 58-933 of SEQ ID NO: 108, or a sequence having at least 90% sequence identity thereto, or comprises the sequence set forth in any of SEQ ID NOS: 55, 64, 108 or 1094 or a sequence having at least 90% sequence identity thereto.
146. The nucleic acid molecule of embodiment 144, wherein the nucleotide sequence is codon-optimized.
147. The nucleic acid molecule of embodiment 144 or embodiment 146, comprising a nucleotide sequence encoding an alpha chain and/or a nucleotide sequence encoding a beta chain, wherein:
the nucleotide sequence encoding an alpha chain comprises residues 64-822 of SEQ ID NO: 12, residues 63-822 of SEQ ID NO: 51, residues 64-825 of SEQ ID NO: 61, or a sequence having at least 90% sequence identity thereto, or comprises the sequence set forth in any of SEQ ID NOS: 12, 51, 61, 1175, or 1177, or a sequence having at least 90% sequence identity thereto; and/or
the nucleotide sequence encoding a beta chain comprises residues 58-933 of SEQ ID NO: 9; residues 58-933 of SEQ ID NO: 54, residues 58-927 of SEQ ID NO: 65, or a sequence having at least 90% sequence identity thereto, or comprises the sequence set forth in any of SEQ ID NOS: 9, 54, 65, 1176 or 1178, or a sequence having at least 90% sequence identity thereto.
148. The nucleic acid molecule of any of embodiments 144-147, wherein the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a peptide sequence that causes ribosome skipping.
149. The nucleic acid molecule of embodiment 148, wherein the peptide that causes ribosome skipping is a P2A or T2A peptide and/or comprises the sequence of amino acids set forth in SEQ ID NO: 204 or 211.
150. The nucleic acid of any of embodiments 144-149, comprising the nucleotide sequence set forth in any of SEQ ID NOs: 15, 56, 66, 471 or 472 or a nucleotide sequence having at least 90% sequence identity thereto.
151. A T cell receptor (TCR) or antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein:
the Vα region comprises the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123, 125, 297, or 299 or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or
the Vβ region comprises the amino acid sequence set forth in any of SEQ ID NOs: SEQ ID NOs: 112, 114, 116, 122, 124, 126, 298, or 300, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
152. The TCR or antigen-binding fragment thereof of any of embodiment 107, wherein:
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18 (SEQ ID NO: 1220), wherein X1 is A, I, or V; X2 is M, L, A, V, S, or E; X3 is R, L, N, S, Q, K, G, or W; X4 is E, V, P, T, F, A, G, N, D, or L; X5 is G, I, D, L, A, P, N, R, T, or null; X6 is G, N, R, T, M, S, P, or null; X7 is G, V, D, L, Q, T, R, or null; X8 is T, D, S, L, G, or null; X9 is A, G, Q, or null; X10 is G, or null; X11 is G, or null; X12 is T, or null; X13 is S, A, T, G, or null; X14 is G, Y, T, N, A, W, or null; X15 is F, G, N, T, Y, D, S, R, Q, or E; X16 is K, P, N, D, or Q; X17 is L, M, I, V, or T; and X18 is I, T, V, F, R, or Q; and/or
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 (SEQ ID NO: 1222), wherein X1 is A, S, or V; X2 is S, A, or V; X3 is S, R, or Q; X4 is H, P, Q, L, Y, G, T, F, S, R, or E; X5 is L, G, R, W, F, S, V, T, Y, Q, or null; X6 is A, G, L, E, P, or null; X7 is G, T, A, R, Q, N, S, or null; X8 is G, S, or null; X9 is G, or null; X10 is F, G, A, S, T, R, Q, L, or null; X11 is T, N, F, A, R, S, G, or null; X12 is G, T, L D, Y, N, Q, S, or E; X13 is E, W, T, G, K, N, or P; X14 is L, A, K, Q, Y, or I; and X15 is F, H, Y, T, or I.
153. A T cell receptor (TCR) or antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein:
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18 (SEQ ID NO: 1220), wherein X1 is A, I, or V; X2 is M, L, A, V, S, or E; X3 is R, L, N, S, Q, K, G, or W; X4 is E, V, P, T, F, A, G, N, D, or L; X5 is G, I, D, L, A, P, N, R, T, or null; X6 is G, N, R, T, M, S, P, or null; X7 is G, V, D, L, Q, T, R, or null; X8 is T, D, S, L, G, or null; X9 is A, G, Q, or null; X10 is G, or null; X11 is G, or null; X12 is T, or null; X13 is S, A, T, G, or null; X14 is G, Y, T, N, A, W, or null; X15 is F, G, N, T, Y, D, S, R, Q, or E; X16 is K, P, N, D, or Q; X17 is L, M, I, V, or T; and X18 is I, T, V, F, R, or Q; and/or
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 (SEQ ID NO: 1222), wherein X1 is A, S, or V; X2 is S, A, or V; X3 is S, R, or Q; X4 is H, P, Q, L, Y, G, T, F, S, R, or E; X5 is L, G, R, W, F, S, V, T, Y, Q, or null; X6 is A, G, L, E, P, or null; X7 is G, T, A, R, Q, N, S, or null; X8 is G, S, or null; X9 is G, or null; X10 is F, G, A, S, T, R, Q, L, or null; X11 is T, N, F, A, R, S, G, or null; X12 is G, T, L D, Y, N, Q, S, or E; X13 is E, W, T, G, K, N, or P; X14 is L, A, K, Q, Y, or I; and X15 is F, H, Y, T, or I.
154. The TCR or antigen-binding fragment thereof of embodiment 46 or embodiment 153, wherein the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16LT (SEQ ID NO:1206), wherein X1 is A, I, or V; X2 is L, M, V, or E; X3 is L, R, N, G, or S; X4 is V, T, F, N, E, P, G, or L; X5 is I, A, P, N, G, or T; X6 is R, G, S, or T; X7 is G, R, L, V, or T; X8 is T, G, L, or null; X9 is A, G, Q, or null; X10 is G, or null; X11 is G, or null; X12 is T, or null; X13 is S, T, or G; X14 is Y, A, G, or N; X15 is G, S, N, R, or E; and X16 is K, or Q;
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AMRX4X5X6X7X8X9X10X11X12X13X14X15 (SEQ ID NO:1207), wherein X4 is E, T, A, D, or L; X5 is G, A, N, or R; X6 is R, G, R, T, M, or S; X7 is G, V, D, L, or null; X8 is T, D, or null; X9 is G, or null; X10 is S, T, G, or null; X11 is G, Y, N, A, or W; X12 is F, G, N, D, S, or Y; X13 is K, D, or Q; X14 is T, L, M, or I; X15 is I, T, R, or Q;
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15KX17X18(SEQ ID NO:1208), X1 is I, or V; X2 is L, or V; X3 is L, N, or R; X4 is V, F, or G; X5 is I, P, G, or T; X6 is R, S, P, or G; X7 is G, R, Q, T, or V; X8 is T, G, S, or L; X9 is A, G, Q, or null; X10 is G, or null; X11 is G, or null; X12 is T, or null; X13 is G, or S; X14 is Y, or N; X15 is G, Q, or E; X17 is V, or L; and X18 is I, or T; or
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18 (SEQ ID NO: 248), wherein X1 is A, I, or V; X2 is M, L, or V; X3 is R, L, or N; X4 is E, V, T, P, or F; X5 is G, I, L, A, or P; X6 is R, T, G, or S; X7 is G, R, or null; X8 is T, G, or null; X9 is null or A; X10 is null or G; X11 is null or G; X12 is null or T; X13 is null or S; X14 is G, Y, or N; X15 is F, G, or T; X16 is K or P; X17 is T or L; and X18 is I, V or T.
155. The TCR or antigen-binding fragment thereof of any of embodiments 151-154, wherein:
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO:1212), wherein X4 is H, P, Q, L, Y, F, R, or E; X5 is L, G, R, W, F, S, V, T, Y, or Q; X6 is A, G, L, E or P; X7 is G, T, A, R, Q, S, or null; X8 is G, S, or null; X9 is F, G, A, S, T, R, L, or null; X10 is T, N, A, F, R, S, or G; X11 is G, T, L, D, Y, Q, S, E, or N; X12 is E, W, T, G, P, or K; X13 is L, A, K, Q, Y, or I; and X14 is F, H, Y, or T;
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2SX4X5X6X7X8X9X10X11X12X13QY (SEQ ID NO:1223), X1 is A, or S; X2 is 5, V, or A; X4 is L, Y, P, or S; X5 is W, F, V, L, or Y; X6 is G, or A; X7 is A, R, Q, S, or null; X8 is G, or null; X9 is G, or null; X10 is S, T, R, or G; X11 is T, A, R, S, or N; X12 is D, Y, T, or G; and X13 is T, or E;
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASX3 X4 X5 X6 X7 X8 X9 X10 X11 X12 F (SEQ ID NO:1214), wherein X3 is S, Q, or R; X4 is H, P, T, or E; X5 is L, G, W, or F; X6 is A, G, or null; X7 is G, N, S, R, or null; X8 is F, G, Q, L, A, or null; X9 is T, N, or A; X10 is G, T, N, or E; X11 is E, N, or K; and X12 is L, A, or Q;
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4 X5 X6 X7 X8 NYX11 YT (SEQ ID NO: 1215), X4 is L, or R; X5 is S, or T; X6 is G, T, or A; X7 is T, or null; X8 is G, or null; and X11 is G, or null; or the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4X5X6X7X8X9X10X11X12X13 (SEQ ID NO: 258), wherein X4 is H, P, L, or Y; X5 is L, G, W, F, or S; X6 is A, G, or L; X7 is G, E, A, T, or null; X8 is F, G, T, or S; X9 is T, N, H, or A; X10 is G, T, Q, D, or Y; X11 is E, P, T, or G; X12 is L, A, Q, or Y; and X13 is F, H, Y, or T.
156. The TCR or antigen-binding fragment thereof of any of embodiments 151-155, wherein the Vα region comprises a complementarity determining region 1 (CDR-1) comprising:
the amino acid sequence X1 X2 X3 X4 X5 X6 X7 (SEQ ID NO:1209), wherein X1 is T, N, D, or S; X2 is 5, I, or R; X3 is D, S, M, A, Y, N, or G; X4 is Q, G, P, or null; X5 is S, T, F, I, or N; X6 is Y, D, Q, P, N, or E; and X7 is G, Y, N, S, or A; or
the amino acid sequence X1X2X3X4X5X6X7 (SEQ ID NO: 240), wherein X1 is T, D, or N; X2 is I, or S; X3 is S, D, or A; X4 is G, Q, P, or null; X5 is T, S, or I; X6 is D, Y, or Q; and X7 is Y, G, N, or Q.
157. The TCR or antigen-binding fragment thereof of any of embodiments 151-156, wherein the Vα region comprises a complementarity determining region 2 (CDR-2) comprising:
the amino acid sequence X1 X2 X3 X4 X5 X6 X7 X8 (SEQ ID NO:1210), wherein X1 is Q, G, I, V, Y, M, R, or N; X2 is G, L, S, Q, Y, T, N, or V; X3 is S, T, L, or K; X4 is Y, I, S, A, N, F, or null; X5 is D, A, or null; X6 is E, K, Q, S, T, G, D, or null; X7 is Q, S, N, R, G, L, or D; and X8 is N, K, E, V, or L; or
the amino acid sequence X1X2X3X4X5X6X7X8 (SEQ ID NO: 244), wherein X1 is G, Q, I, or V; X2 is L, S, Q, or Y; X3 is T, G, or S; X4 is Y, S, or null; X5 is null or D; X6 is null, E, Q, or S; X7 is S, Q, R, or G; and X8 is N or E.
158. The TCR or antigen-binding fragment thereof of any of embodiments 151-157, wherein the Vβ region comprises a complementarity determining region 1 (CDR-1) comprising:
the amino acid sequence X1 X2 X3 X4 X5 X6 (SEQ ID NO:1218), wherein X1 is S, M, D, or L; X2 is G, E, D, N, Q, S, or F; X3 is H, V, Y, N, or Q; X4 is A, S, F, or null; X5 is W V, N, E, T, P, Y, K, D, or L; and X6 is S, R, A, N, Y, M, or T; or
the amino acid sequence X1X2HX4X5 (SEQ ID NO: 252), wherein X1 is S or M; X2 is G, E, D, or N; X4 is V, N, or E; and X5 is S, R, N, or Y.
159. The TCR or antigen-binding fragment thereof of any of embodiments 151-158, wherein the Vβ region comprises a complementarity determining region 2 (CDR-2) comprising:
the amino acid sequence X1 X2 X3 X4 X5 X6 X7 (SEQ ID NO:1219), wherein X1 is F, Y, S, A or M; X2 is N, Q, V, T, Y, or A; X3 is N, D, E, S, G, I, F, Q, or L; X4 is G, A, N, or null; X5 is E, K, V, E, S, T, G, or N; X6 is A, E, K, G, L, D, V, or N; X7 is Q, M, T, A, V, E, P, D, or I; or
the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO: 255), wherein X1 is F or S; X2 is Q, Y, or V; X3 is N, D, or G; X4 is E or V; X5 is A, K, or G; and X6 is Q, M, or T.
160. The TCR or antigen-binding fragment thereof of any of embodiments 151-159, wherein the TCR or antigen-binding fragment thereof binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E6 in the context of an MHC molecule, the peptide epitope is or comprises E6(29-38) TIHDIILECV (SEQ ID NO:233).
161. The TCR or antigen-binding fragment of any of embodiments 151-160, wherein:
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 138, 144, 147, 163, 167, 173, 304, or 308, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123, 125, 297, or 299; and/or
a Vβ region comprising a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 141, 146, 150, 164, 170, 174, 305, or 309, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 122, 124, 126, 298, or 300.
162. The TCR or antigen-binding fragment of any of embodiments 151-161, wherein the Vα region further comprises:
a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 136, 142, 161, 165, 171, 302, or 306, or a CDR-1 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123, 125, 297, or 299; and/or
a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 137, 143, 162, 166, 172, 303, or 307, or a CDR-2 contained within the amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123, 125, 297, or 299.
163. The TCR or antigen-binding fragment of any of embodiments 151-152, wherein the Vβ region comprises:
a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 139, 145, 148, 168, or a CDR-1 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 122, 124, 126, 298, or 300; and/or
a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 140, 149, or 169 or a CDR-2 contained within the amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 122, 124, 126, 298, or 300.
164. The TCR or antigen-binding fragment thereof of any of embodiments 151-163, wherein:
the Vα region comprises: a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 136, 142, 161, 165, 171, 302, or 306; a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 137, 143, 162, 166, 172, 303, or 307; and/or a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 138, 144, 147, 163, 167, 173, 304, 308; and/or
the Vβ region comprises: a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 139, 145, 148, or 168; a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 140, 149, or 169; and/or a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 141, 146, 150, 164, 170, 174, 305, or 309.
165. The TCR or antigen-binding fragment thereof of any of embodiments 151-164, wherein:
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137, and 138, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 139, 140, and 141, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 142, 143, and 144, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 145, 140, and 146, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137, and 147, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 150, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 161, 162, and 163, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 164, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 165, 166, and 167, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 168, 169, and 170, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 171, 172, and 173, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 174, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 302, 303, and 304, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 139, 140, and 305, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 306, 307, and 308, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149, and 309, respectively.
166. The TCR or antigen-binding fragment thereof of any of embodiments 151-165, wherein:
the Vα region comprises a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vα region amino acid sequence set forth in any of SEQ ID NOs: 111, 113, 115, 121, 123, 125, 297, or 299; and/or
the Vβ region comprises a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vβ region amino acid sequence set forth in any of SEQ ID NOs: 112, 114, 116, 122, 124, 126, 298, or 300.
167. The TCR or antigen-binding fragment thereof of any of embodiments 151-166, wherein:
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 111 and 112, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 113 and 114, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 115 and 116, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 121 and 122, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 123 and 124, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 125 and 126, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 297 and 298, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 299 and 300, respectively.
168. The TCR or antigen-binding fragment thereof of any of embodiments 151-167, wherein the alpha chain further comprises an alpha constant (Cα) region and/or the beta chain further comprises a beta constant (Cβ) region.
169. The TCR or antigen-binding fragment thereof of embodiment 168, wherein the Cα and Cβ regions are mouse constant regions.
170. The TCR or antigen-binding fragment thereof of embodiment 168 or embodiment 63, wherein:
the Cα region comprises the amino acid sequence set forth in SEQ ID NO: 262, 833, 1012, 1014, 1015, 1017, 1018, or a sequence of amino acids that has at least 90% sequence identity thereto; and/or
the Cβ region comprises the amino acid sequence set forth in SEQ ID NO: 263, 1013 or 1016 or a sequence of amino acids that has at least 90% sequence identity thereto.
171. The TCR or antigen-binding fragment thereof of embodiment 168, wherein the Cα and Cβ regions are human constant regions.
172. The TCR or antigen-binding fragment thereof of embodiment 168 or embodiment 65, wherein:
the Cα region comprises the amino acid sequence set forth in any of SEQ ID NOs: 212, 213, 215, 217, 218, 220 or 524, or a sequence of amino acids that has at least 90% sequence identity thereto; and/or
the Cβ region comprises the amino acid sequence set forth in any of SEQ ID NOs: 214, 216, 631 or 889, or a sequence of amino acids that has at least 90% sequence identity thereto.
173. The TCR or antigen-binding fragment thereof of any of embodiments 151-172, comprising one or more modifications in the α chain and/or β chain such that when the TCR or antigen-binding fragment thereof is expressed in a cell, the frequency of mispairing between the TCR α chain and β chain and an endogenous TCR α chain and β chain is reduced, the expression of the TCR α chain and β chain is increased and/or the stability of the TCR α chain and β chain is increased, each compared to expression in a cell of the TCR or antigen-binding fragment thereof not containing the one or more modifications.
174. The TCR or antigen-binding fragment thereof of embodiment 173, wherein the one or more modifications is a replacement, deletion, or insertion of one or more amino acids in the Cα region and/or the Cβ region.
175. The TCR or antigen-binding fragment thereof of embodiment 173 or embodiment 68, wherein the one or more modifications comprise replacement(s) to introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and beta chain.
176. The TCR or antigen-binding fragment thereof of any of embodiments 151-168 and 171-175, comprising a Cα region comprising a cysteine at a position corresponding to position 48 with numbering as set forth in SEQ ID NO: 212, 213, 217, 218, or 524 or at a position corresponding to position 49 with numbering as set forth in SEQ ID NO: 215 or 220; and/or a Cβ region comprising a cysteine at a position corresponding to position 57 with numbering as set forth in SEQ ID NO: 214 or 216 or at a position corresponding to position 58 with numbering as set forth in SEQ ID NO: 631 or 889.
177. The TCR or antigen-binding fragment thereof of any of embodiments 168, 171, and 173-176, wherein:
the Cα region comprises the amino acid sequence set forth in any of SEQ ID NOs: 196, 198, 200, 201, 203, or 525, or a sequence of amino acids that has at least 90% sequence identity thereto comprising one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; and/or
the Cβ region comprises the amino acid sequence set forth in any of SEQ ID NOs: 197, 199, 632, or 890 or a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain.
178. The TCR or antigen-binding fragment thereof of any of embodiments 151-177, wherein the TCR or antigen-binding fragment thereof is encoded by a nucleotide sequence that has been codon-optimized.
179. The TCR or antigen-binding fragment thereof of any of embodiments 151-178, wherein:
a) the alpha chain comprises:
the amino acid sequence set forth in any of SEQ ID NOs: 18, 28, 38, 68, 78, 88, 287, or 291, a sequence of amino acids that has at least 90% sequence identity thereto; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 20, 30, 40, 70, 80, 90, 202 or 219 or a nucleotide sequence that has at least 90% sequence identity thereto; and/or
the beta chain comprises:
the amino acid sequence set forth in any of SEQ ID NOs: 22, 32, 42, 72, 82, 92, 289, or 293, a sequence of amino acids that has at least 90% sequence identity thereto; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOS: 16, 17, 24, 34, 44, 74, 84, 94, or a nucleotide sequence that has at least 90% sequence identity thereto; or
b) the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 18 and 22, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 28 and 32, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 38 and 42, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 68 and 72, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 78 and 82, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 88 and 92, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 287 and 289, respectively; or the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 291 and 293, respectively.
180. The TCR or antigen-binding fragment thereof of any of embodiments 151-178, wherein:
a) the alpha chain comprises:
the amino acid sequence set forth in any of SEQ ID NOs: 19, 29, 39, 69, 79, 89, 288 or 292, a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 10, 11, 21, 31, 41, 71, 81, 91, or a nucleotide sequence that has at least 90% sequence identity thereto and encodes an alpha chain that contains one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; and/or
the beta chain comprises
the amino acid sequence set forth in any of SEQ ID NOs: 23, 33, 43, 73, 83, 93, 290, or 294, a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain; or an amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 7, 8, 25, 35, 45, 75, 85, 95, or a nucleotide sequence that has at least 90% sequence identity thereto and encodes a beta chain that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain; or
b) the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 19 and 23, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 29 and 33, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 39 and 43, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 69 and 73, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 79 and 83, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 89 and 93, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 288 and 290, respectively; the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 292 and 294, respectively.
181. The TCR or antigen-binding fragment thereof of any of embodiments 151-180, wherein the alpha and/or beta chain further comprises a signal peptide.
182. The TCR or antigen-binding fragment thereof of embodiment 181, wherein:
the alpha chain comprises the signal peptide comprising the amino acid sequence set forth in any of SEQ ID NOs: 181, 184, 187, 189, 190, 192, 193, 310, 311; and/or
the beta chain comprises the signal peptide comprising the amino acid sequence set forth in any of SEQ ID NOs: 182, 185, 186, 188, 191, or 194.
183. The TCR or antigen-binding fragment thereof of any of embodiments 151-182, that is isolated or purified or is recombinant.
184. The TCR or antigen-binding fragment thereof of any of embodiments 151-183, that is human.
185. The TCR or antigen-binding fragment thereof of any of embodiments 151-184, that is monoclonal.
186. The TCR or antigen-binding fragment thereof of any of embodiments 151-185, wherein the TCR or antigen-binding fragment thereof is single chain.
187. The TCR or antigen-binding fragment thereof of any of embodiments 151-185, wherein the TCR or antigen-binding fragment thereof comprises two chains.
188. The TCR or antigen-binding fragment thereof of any of embodiments 151-187, wherein the antigen-specificity is at least partially CD8-independent.
189. The TCR or antigen-binding fragment of any of embodiments 151-188 wherein the MHC molecule is an HLA-A2 molecule.
190. A nucleic acid molecule encoding the TCR or antigen-binding fragment thereof of any of embodiments 151-189, or an alpha or beta chain thereof.
191. The nucleic acid molecule of embodiment 190, comprising a nucleotide sequence encoding an alpha chain and/or a nucleotide sequence encoding a beta chain, wherein:
the nucleotide sequence encoding an alpha chain comprises the sequence selected from the group consisting of: residues 61-816 of SEQ ID NO: 20, residues 58-804 of SEQ ID NO: 30, residues 61-825 of SEQ ID NO: 40, residues 58-807 of SEQ ID NO: 70, residues 61-825 of SEQ ID NO: 80, residues 67-831 of SEQ ID NO: 90, residues 58-801 of SEQ ID NO: 202, residues 67-813 of SEQ ID NO: 219, or a sequence having at least 90% sequence identity thereto; and/or
the nucleotide sequence encoding a beta chain comprises the sequence selected from the group consisting of: residues 58-930 of SEQ ID NO: 16, residues 58-936 of SEQ ID NO: 17, residues 58-939 of SEQ ID NO: 24, residues 64-930 of SEQ ID NO: 34 or 44, residues 64-936 of SEQ ID NO: 74, residues 58-933 of SEQ ID NO: 84, residues 63-930 of SEQ ID NO: 94, or a sequence having at least 90% sequence identity thereto.
192. The nucleic acid molecule of embodiment 190, wherein the nucleotide sequence is codon-optimized.
193. The nucleic acid molecule of embodiment 190 or embodiment 192, comprising a nucleotide sequence encoding an alpha chain and/or a nucleotide sequence encoding a beta chain, wherein:
the nucleotide sequence encoding an alpha chain comprises the sequence selected from the group consisting of: residues 67-825 of SEQ ID NO: 10, residues 58-813 of SEQ ID NO: 11, residues 61-825 of SEQ ID NO: 21, residues 58-813 of SEQ ID NO: 31, residues 61-834 of SEQ ID NO: 41, residues 58-816 of SEQ ID NO: 71, residues 61-834 of SEQ ID NO: 81, residues 67-840 of SEQ ID NO: 91, or a sequence having at least 90% sequence identity thereto; and/or
the nucleotide sequence encoding a beta chain comprises the sequence selected from the group consisting of: residues 58-930 of SEQ ID NO: 7, residues 58-936 of SEQ ID NO: 8, residues 58-939 of SEQ ID NO: 25, residues 64-930 of SEQ ID NO: 35, 45, or 95, residues 58-933 of SEQ ID NO: 85, residues 64-936 of SEQ ID NO: 75, or a sequence having at least 90% sequence identity thereto.
194. The nucleic acid molecule of any of embodiments 190-193, wherein the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a peptide sequence that causes ribosome skipping.
195. The nucleic acid molecule of embodiment 194, wherein the peptide that causes ribosome skipping is a P2A or T2A peptide and/or comprises the sequence of amino acids set forth in SEQ ID NO: 204 or 211.
196. The nucleic acid of any of embodiments 190-195, comprising the nucleotide sequence set forth in any of SEQ ID NOs: 13, 14, 26, 36, 46, 76, 86, 96, or a nucleotide sequence having at least 90% sequence identity thereto.
197. The nucleic acid of any of embodiments 144-151 and 190-196, wherein the nucleic acid is synthetic.
198. The nucleic acid of any of embodiments 144-151 and 190-197, wherein the nucleic acid is cDNA.
199. A vector comprising the nucleic acid of any of embodiments 144-150 and 190-198.
200. The vector of embodiment 199, wherein the vector is an expression vector.
201. The vector of embodiment 199 or embodiment 200, wherein the vector is a viral vector.
202. The vector of embodiment 201, wherein the viral vector is a retroviral vector.
203. The vector of embodiment 201 or embodiment 202, wherein the viral vector is a lentiviral vector.
204. The vector of embodiment 203, wherein the lentiviral vector is derived from HIV-1.
205. An engineered cell comprising the nucleic acid molecule of any of embodiments 144-150 and 190-198 or vector of any of embodiments 199-204.
206. An engineered cell, comprising the TCR or antigen-binding fragment thereof of any of embodiments 107-143 and 151-189.
207. The engineered cell of embodiment 205 or embodiment 206, wherein the TCR or antigen-binding fragment thereof is heterologous to the cell.
208. The engineered cell of any of embodiments 205-207, wherein the engineered cell is a cell line.
209. The engineered cell of any of embodiments 205-207, wherein the engineered cell is a primary cell obtained from a subject.
210. The engineered cell of embodiment 209, wherein the subject is a mammalian subject.
211. The engineered cell of embodiment 209 or embodiment 210, wherein the subject is a human.
212. The engineered cell of any of embodiments 205-211, wherein the engineered cell is a T cell.
213. The engineered cell of embodiment 212, wherein the T cell is CD8+.
214. The engineered cell of embodiment 212, wherein the T cell is CD4+.
215. The engineered cell of any of embodiments 205-214, comprising a genetic disruption of a T cell receptor alpha constant (TRAC) gene and/or a T cell receptor beta constant (TRBC) gene.
216. The engineered cell of embodiment 215, wherein the TRBC gene is one or both of a T cell receptor beta constant 1 (TRBC1) or T cell receptor beta constant 2 (TRBC2) gene.
217. A method for producing a cell of any of embodiments 205-216, comprising introducing a vector of any of embodiments 199-204 into a cell in vitro or ex vivo.
218. The method of embodiment 217, wherein the vector is a viral vector and the introducing is carried out by transduction.
219. The method of embodiment 217 or embodiment 218, further comprising introducing into the cell one or more agent, wherein each of the one or more agent is independently capable of inducing a genetic disruption of a T cell receptor alpha constant (TRAC) gene and/or a T cell receptor beta constant (TRBC) gene.
220. The method of any of embodiment 219, wherein the one or more agent capable of inducing a genetic disruption comprises a DNA binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to the target site.
221. The method of embodiment 220, wherein the one or more agent capable of inducing a genetic disruption comprises (a) a fusion protein comprising a DNA-targeting protein and a nuclease or (b) an RNA-guided nuclease.
222. The method of embodiment 221, wherein the DNA-targeting protein or RNA-guided nuclease comprises a zinc finger protein (ZFP), a TAL protein, or a clustered regularly interspaced short palindromic nucleic acid (CRISPR)-associated nuclease (Cas) specific for a target site within the TRAC and/or TRBC gene.
223. The method of embodiment 222, wherein the one or more agent comprises a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or and a CRISPR-Cas9 combination that specifically binds to, recognizes, or hybridizes to the target site.
224. The method of embodiment 222 or embodiment 223, wherein the each of the one or more agent comprises a guide RNA (gRNA) having a targeting domain that is complementary to the at least one target site.
225. The method of embodiment 224, wherein the one or more agent is introduced as a ribonucleoprotein (RNP) complex comprising the gRNA and a Cas9 protein.
226. The method of embodiment 225, wherein the RNP is introduced via electroporation, particle gun, calcium phosphate transfection, cell compression or squeezing.
227. The method of embodiment 225 or embodiment 226, wherein the RNP is introduced via electroporation.
228. The method of any of embodiments 224-227, wherein the one or more agent is introduced as one or more polynucleotide encoding the gRNA and/or a Cas9 protein.
229. A composition comprising engineered cells of any of embodiments 205-216.
230. The composition of embodiment 229, wherein the engineered cells comprise CD4+ and/or CD8+ T cells.
231. The composition of embodiment 229 or embodiment 230, wherein the engineered cells comprise CD4+ and CD8+ T cells.
232. A composition, comprising an engineered CD8+ cell of embodiment 107 and an engineered CD4+ cell of embodiment 214.
233. The composition of any of embodiments 229-232, wherein the TCR or antigen-binding fragment thereof binds to or recognizes a peptide epitope of HPV 16 in the context of an MHC molecule that is at least partially CD8-independent.
234. The composition of any of embodiments 230-233, wherein the CD8+ cell and CD4+ cell are engineered with the same TCR or antigen-binding fragment thereof and/or are each engineered with a TCR or antigen-binding fragment thereof that binds to or recognizes the same peptide epitope of HPV 16 in the context of an MHC molecule.
235. The composition of any of embodiments 229-234, further comprising a pharmaceutically acceptable excipient.
236. A method of treatment, comprising administering the engineered cell of any of embodiments 205-216 to a subject having a disease or disorder associated with HPV.
237. A method of treatment, comprising administering the composition of any of embodiments 229-235 to a subject having a disease or disorder associated with HPV.
238. The method of embodiment 236 or embodiment 237, wherein the disease or disorder is associated with HPV16.
239. The method of any of embodiments 236-237, wherein the disease or disorder is cancer.
240. The method of any of embodiments 236-239, wherein the subject is a human.
241. A composition of any of embodiments 229-235 for use in treating a disease or disorder associated with HPV.
242. Use of a composition of any of embodiments 229-235 for the manufacture of a medicament for treating a disease or disorder associated with HPV.
243. The composition of embodiment 241 or use of embodiment 136, wherein the disease or disorder is associated with HPV16.
244. The composition or use of any of embodiments 241-243, wherein the disease or disorder is cancer.
245. The composition or use of any of embodiments 241-244, wherein the subject is a human.
246. A T cell receptor (TCR) or antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein:
the Vα region comprises the amino acid sequence set forth in any of SEQ ID NOs: 691, 709, 726, 741, 759, 775, 787, 799, 815, 830, 845, 857, 869, 881, 895, 908, 925, 937, 951, 963, 975, 987 or 999, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or
the Vβ region comprises the amino acid sequence set forth in any of SEQ ID NOs: 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993 or 1008, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
247. The TCR or antigen-binding fragment thereof of embodiment 246, wherein:
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO:1185), wherein X2 is A, G, V, Q, M, or E; X3 is S, G, N, A, Y, R, or P; X4 is E, S, A, G, F, N, D, V, P, L, I, M, or R; X5 is R, N, H, T, D, G, S, P, L, Q, or F; X6 is G, H, A, S, T, or null; X7 is T, S, G, or null; X8 is G, or null; X9 is G, N, S, or null; X10 is T, G, S, D, F, Y, A, or N; X11 is Y, F, Q, R, or N; X12 is K, Q, or D; X13 is Y, L, T, M, F, or V; X14 is I, T, S, R, Y, or V;
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10KX12I (SEQ ID NO:1186), wherein X1 is A, or V; X2 is A, V, or E; X3 is S, N, T, R, or P; X4 is E, A, G, F, V, P, I, D, or S; X5 is R, H, T, A P, S, G, or F; X6 is G, H, L, T, S, or A, null; X7 is S, T, or null; X8 is G, or null; X9 is G, T, or null; X10 is F, Y, or N; X12 is Y, T, or L;
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9YKYI (SEQ ID NO:1187), wherein X2 is A, V, or E; X3 is S, N, or R; X4 is E, G, V, P, I, or D; X5 is R, T, P, S, G, or F; X6 is G, T, S, or null; X7 is S, or null; X8 is G, or null; X9 is T, or null;
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO:1188), wherein X2 is G, V, Q, or M; X3 is G, A, Y, S, N, or R; X4 is S, G, L, I, M, or R; X5 is N, D, G, S, L, Q, or R; X6 is A, S, G, or null; X7 is G, or null; X8 is G, or null; X9 is G, N, S, or null; X10 is S, D, Y, A, N, or null; X11 is Y, Q, or R; X12 is K, or Q; X13 is L, or V; X14 is S, T, or V;
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11X12X13T (SEQ ID NO: 1189), wherein X2 is G, V, or Q; X3 is G, Y, S, or N; X4 is S, L, or M; X5 is N, G, L, or R; X6 is A, S, G, or null; X7 is G, or null; X8 is G, or null; X9 is G, S, or null; X10 is S, Y, A, N, or null; X11 is Y, Q, or R; X12 is K, or Q; X13 is L, or V;
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7YKLS (SEQ ID NO:1190), wherein X2 is G, or V; X3 is A, or Y; X4 is G, S, or R; X5 is D, or S; X6 is N, or null; X7 is D, or null.
248. The TCR or antigen-binding fragment thereof of embodiment 246 or embodiment 247, wherein:
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO:1200), X2 is 5, V, or I; X3 is S, N, or A; X4 is R, V, S, L, P, G, I, or A; X5 is F, G, Y, L, V, R, T, or S; X6 is L, G, A, D, R, V, or null; X7 is G, D, R, S, T, or null; X8 is S, or null; X9 is S, H, G, V, T, D, L, or null; X10 is T, S, A, G, P, N, or Y; X11 is D, Y, E, G, or N; X12 is T, E, G, or K; X13 is Q, Y, or L; X14 is Y, F, T, or I;
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO:1201), wherein X4 is R, V, S, L, G, or A; X5 is F, G, Y, L, V, T, or S; X6 is A, L, R, D, G, or null; X7 is G, D, T, or null; X8 is S, or null; X9 is S, H, G, T, D, L, or null; X10 is T, S, A, G, P, N, or Y; X11 is D, Y, E, G, or N; X12 is T, E, or G; X13 is Q, Y, or L; X14 is Y, F, or T;
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4X5X6X7X8X9X10TQY (SEQ ID NO: 1202), wherein X4 is R, L, or G; X5 is F, V, T, or Y; X6 is L, or A, null; X7 is G, or null; X8 is S, G, or null; X9 is T, G, P, or S; X10 is D, or E.
249. A T cell receptor (TCR) or antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein:
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO:1185), wherein X2 is A, G, V, Q, M, or E; X3 is S, G, N, A, Y, R, or P; X4 is E, S, A, G, F, N, D, V, P, L, I, M, or R; X5 is R, N, H, T, D, G, S, P, L, Q, or F; X6 is G, H, A, S, T, or null; X7 is T, S, G, or null; X8 is G, or null; X9 is G, N, S, or null; X10 is T, G, S, D, F, Y, A, or N; X11 is Y, F, Q, R, or N; X12 is K, Q, or D; X13 is Y, L, T, M, F, or V; X14 is I, T, S, R, Y, or V;
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10KX12I (SEQ ID NO:1186), wherein X1 is A, or V; X2 is A, V, or E; X3 is S, N, T, R, or P; X4 is E, A, G, F, V, P, I, D, or S; X5 is R, H, T, A P, S, G, or F; X6 is G, H, L, T, S, or A, null; X7 is S, T, or null; X8 is G, or null; X9 is G, T, or null; X10 is F, Y, or N; X12 is Y, T, or L;
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9YKYI (SEQ ID NO:1187), wherein X2 is A, V, or E; X3 is S, N, or R; X4 is E, G, V, P, I, or D; X5 is R, T, P, S, G, or F; X6 is G, T, S, or null; X7 is S, or null; X8 is G, or null; X9 is T, or null;
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO:1188), wherein X2 is G, V, Q, or M; X3 is G, A, Y, S, N, or R; X4 is S, G, L, I, M, or R; X5 is N, D, G, S, L, Q, or R; X6 is A, S, G, or null; X7 is G, or null; X8 is G, or null; X9 is G, N, S, or null; X10 is S, D, Y, A, N, or null; X11 is Y, Q, or R; X12 is K, or Q; X13 is L, or V; X14 is S, T, or V;
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11X12X13T (SEQ ID NO: 1189), wherein X2 is G, V, or Q; X3 is G, Y, S, or N; X4 is S, L, or M; X5 is N, G, L, or R; X6 is A, S, G, or null; X7 is G, or null; X8 is G, or null; X9 is G, S, or null; X10 is S, Y, A, N, or null; X11 is Y, Q, or R; X12 is K, or Q; X13 is L, or V;
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7YKLS (SEQ ID NO:1190), wherein X2 is G, or V; X3 is A, or Y; X4 is G, S, or R; X5 is D, or S; X6 is N, or null; X7 is D, or null.
250. A T cell receptor (TCR) or antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein:
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO:1200), X2 is 5, V, or I; X3 is S, N, or A; X4 is R, V, S, L, P, G, I, or A; X5 is F, G, Y, L, V, R, T, or S; X6 is L, G, A, D, R, V, or null; X7 is G, D, R, S, T, or null; X8 is S, or null; X9 is S, H, G, V, T, D, L, or null; X10 is T, S, A, G, P, N, or Y; X11 is D, Y, E, G, or N; X12 is T, E, G, or K; X13 is Q, Y, or L; X14 is Y, F, T, or I;
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO:1201), wherein X4 is R, V, S, L, G, or A; X5 is F, G, Y, L, V, T, or S; X6 is A, L, R, D, G, or null; X7 is G, D, T, or null; X8 is S, or null; X9 is S, H, G, T, D, L, or null; X10 is T, S, A, G, P, N, or Y; X11 is D, Y, E, G, or N; X12 is T, E, or G; X13 is Q, Y, or L; X14 is Y, F, or T;
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4X5X6X7X8X9X10TQY (SEQ ID NO: 1202), wherein X4 is R, L, or G; X5 is F, V, T, or Y; X6 is L, or A, null; X7 is G, or null; X8 is S, G, or null; X9 is T, G, P, or S; X10 is D, or E.
251. A T cell receptor (TCR) or antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein:
the Vα region comprises a complementarity determining region 3 (CDR-3) set forth in any of SEQ ID NOs: 694, 712, 729, 744, 762, 776, 788, 802, 818, 832, 846, 858, 870, 882, 896, 911, 926, 940, 952, 964, 976, 988, 1002 or a sequence that exhibits at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto;
the Vβ region comprises a complementarity determining region 3 (CDR-3) set forth in any of SEQ ID NOs: 703, 721, 736, 753, 769, 782, 794, 809, 825, 840, 852, 864, 876, 888, 902, 919, 932, 946, 958, 970, 982, 994, or 1010 or a sequence that exhibits at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto.
252. The TCR or antigen-binding fragment thereof of any of embodiments 246-251, wherein the Vα region comprises:
a complementarity determining region 1 (CDR-1) comprising the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO: 1191), wherein X1 is N, S, D, T, or V; X2 is 5, V, R, T, or I; X3 is M, F, G, S, N, A, L, V, or P; X4 is F, S, N, A, or null; X5 is D, S, Q, Y, N, V, T, or P; and X6 is Y, S, R, N, G, or T; and/or
a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6X7X8 (SEQ ID NO: 1192), wherein X1 is I, V, L, G, N, T, Y, or M; X2 is 5, V, Y, L, P, F, I, or T; X3 is S, Y, K, L, T, or F; X4 is I, G, N, A, S, or null; X5 is S, D, or null; X6 is K, G, N, S, D, T, or E; X7 is D, E, G, A, K, L, or N; and X8 is K, V, D, P, N, T, L, or M.
253. The TCR or antigen-binding fragment thereof of any of embodiments 246-252, wherein the Vβ region comprises:
a complementarity determining region 1 (CDR-1) comprising the amino acid sequence SX2X3X4X5 (SEQ ID NO:1203), wherein X2 is G, or N; X3 is H, or D; X4 is T, L, N, or V; and X5 is A, S, Y, or T; and/or
a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO:1204), wherein X1 is F, or Y; X2 is Q, Y, or N; X3 is G, N, R, or Y; X4 is N, G, E, or T; X5 is S, E, A, or G; and X6 is A, E, I, or Q.
254. The TCR or antigen-binding fragment thereof of any of embodiments 246-8, wherein the TCR or antigen-binding fragment thereof binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule, the peptide epitope is or comprises E7(11-19) YMLDLQPET (SEQ ID NO:236).
255. The TCR or antigen-binding fragment of any of embodiments 246-254, wherein:
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence set forth in any of SEQ ID NOs: 694, 712, 729, 744, 762, 776, 788, 802, 818, 832, 846, 858, 870, 882, 896, 911, 926, 940, 952, 964, 976, 988 or 1002, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 691, 709, 726, 741, 759, 775, 787, 799, 815, 830, 845, 857, 869, 881, 895, 908, 925, 937, 951, 963, 975, 987 or 999; and/or
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 703, 721, 736, 753, 769, 782, 794, 809, 825, 840, 852, 864, 876, 888, 902, 919, 932, 946, 958, 970, 982, 994, or 1010 or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993 or 1008.
256. The TCR or antigen-binding fragment thereof of any of embodiments 246-255, wherein the Vα region further comprises:
a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 692, 710, 727, 742, 760, 171, 800, 816, 570, 909, 938, 151, or 1000; and/or
a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 693, 711, 728, 743, 761, 172, 801, 817, 831, 833, 571, 910, 939, 152, or 1001.
257. The TCR or antigen-binding fragment thereof of any of embodiments 246-256, wherein the Vβ region comprises:
a complementarity determining region 1 (CDR-1) comprising the amino acid sequence set forth in any of SEQ ID NOs: 701, 719, 154, 751 or 139; and/or
a complementarity determining region 2 (CDR-2) comprising the amino acid sequence set forth in any of SEQ ID NOs: 702, 720, 155, 752, 140 or 918.
258. The TCR or antigen-binding fragment thereof of any of embodiments 246-257, wherein:
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 692, 693, and 694, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 701, 702 and 703, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 710, 711, and 712, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 719, 720 and 721, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 727, 728 and 729, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155 and 736, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 742, 743 and 744, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 751, 752 and 753, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 760, 761 and 762, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 719, 720 and 769, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 171, 172 and 776, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155 and 782, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 742, 743 and 788, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 139, 140 and 794, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 800, 801 and 802, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 751, 752 and 809, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 816, 817 and 818, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155 and 825, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 816, 831 and 832, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155 and 840, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 171, 172 and 846, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155 and 852, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 816, 833 and 858, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155 and 864, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 727, 728 and 870, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155 and 876, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 570, 571 and 882, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 719, 720 and 888, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 816, 817 and 896, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 701, 702 and 902, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 909, 910 and 911, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 701, 702 and 919, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 727, 728 and 926, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155 and 932, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 938, 939 and 940, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155 and 946, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 727, 728 and 952, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155 and 958, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 151, 152 and 964, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 719, 720 and 970, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 727, 728 and 976, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155 and 982, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 710, 711 and 988, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 719, 729 and 994, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 1000, 1001 and 1002, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 139, 1009 and 1010, respectively;
259. The TCR or antigen-binding fragment thereof of any of embodiments 246-258, wherein:
the Vα region comprises a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vα region amino acid sequence set forth in any of SEQ ID NOs: 691, 709, 726, 741, 759, 775, 787, 799, 815, 830, 845, 857, 869, 881, 895, 908, 925, 937, 951, 963, 975, 987 or 999; and/or
the Vβ region comprises a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vβ region amino acid sequence set forth in any of SEQ ID NOs: 700, 718, 735, 750, 768, 781, 793, 808, 824, 839, 851, 863, 875, 887, 901, 917, 931, 945, 957, 969, 981, 993 or 1008.
260. The TCR or antigen-binding fragment thereof of any of embodiments 246-259, wherein:
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 691 and 700, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 709 and 718, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:726 and 735, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:741 and 750, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:759 and 768, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:775 and 781, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:787 and 793, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:799 and 808, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:815 and 824, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:830 and 839, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:845 and 851, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:857 and 863, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:869 and 875, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:881 and 887, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:895 and 901, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:908 and 917, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:925 and 931, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:937 and 945, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:951 and 957, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:963 and 969, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:975 and 981, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:987 and 993, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:999 and 1008, respectively.
261. The TCR or antigen-binding fragment thereof of any of embodiments 246-260, wherein the alpha chain further comprises an alpha constant (Cα) region and/or the beta chain further comprises a beta constant (Cβ) region.
262. The TCR or antigen-binding fragment thereof of embodiment 261, wherein the Cα and Cβ regions are mouse constant regions.
263. The TCR or antigen-binding fragment thereof of embodiment 261 or embodiment 262, wherein:
the Cα region comprises the amino acid sequence set forth in SEQ ID NO: 262, 833, 1012, 1014, 1015, 1017, 1018, or a sequence of amino acids that has at least 90% sequence identity thereto; and/or
the Cβ region comprises the amino acid sequence set forth in SEQ ID NO: 263, 1013 or 1016 or a sequence of amino acids that has at least 90% sequence identity thereto.
264. The TCR or antigen-binding fragment thereof of embodiment 261, wherein the Cα and Cβ regions are human constant regions.
265. The TCR or antigen-binding fragment thereof of embodiment 261 or embodiment 19, wherein:
the Cα region comprises the amino acid sequence set forth in any of SEQ ID NOs: 212, 213, 215, 217, 218, 220 or 524, or a sequence of amino acids that has at least 90% sequence identity thereto; and/or
the Cβ region comprises the amino acid sequence set forth in any of SEQ ID NOs: 214, 216, 631 or 889, or a sequence of amino acids that has at least 90% sequence identity thereto.
266. The TCR or antigen-binding fragment thereof of any of embodiments 246-265, wherein:
a) the alpha chain comprises:
the amino acid sequence set forth in any of SEQ ID NOs: 687, 705, 722, 737, 755, 771, 783, 795, 811, 826, 841, 853, 865, 877, 891, 904, 921, 933, 947, 959, 971, 983, 995, a sequence of amino acids that has at least 90% sequence identity thereto; or the amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 1049, 1051, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, or a nucleotide sequence that has at least 90% sequence identity thereto; and/or
the beta chain comprises:
the amino acid sequence set forth in any of SEQ ID NOs: 696, 714, 731, 746, 764, 777, 789, 804, 820, 835, 847, 859, 871, 883, 897, 913, 927, 941, 953, 965, 977, 989, or 1004, a sequence of amino acids that has at least 90% sequence identity thereto; or the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NOS: 1050, 1052, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090 or 1092, or a nucleotide sequence that has at least 90% sequence identity thereto.
267. The TCR or antigen-binding fragment thereof of any of embodiments 246-265, wherein:
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 687 and 696, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 705 and 714, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 722 and 731, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 737 and 746, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 755 and 764, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 771 and 777, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 783 and 789, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 795 and 804, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 811 and 820, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 826 and 835, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 841 and 847, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 853 and 859, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 865 and 871, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 877 and 883, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 891 and 897, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 904 and 913, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 921 and 927, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 933 and 941, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 947 and 953, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 959 and 965, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 971 and 977, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 983 and 989, respectively; or the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 995 and 1004, respectively.
268. The TCR or antigen-binding fragment thereof of any of embodiments 246-264, wherein the TCR or antigen-binding fragment comprises one or more modifications in the α chain and/or β chain such that when the TCR or antigen-binding fragment thereof is expressed in a cell, the frequency of mispairing between the TCR α chain and β chain and an endogenous TCR α chain and β chain is reduced, the expression of the TCR α chain and β chain is increased and/or the stability of the TCR α chain and β chain is increased, each compared to expression in a cell of the TCR or antigen-binding fragment thereof not containing the one or more modifications.
269. The TCR or antigen-binding fragment thereof of embodiment 268, wherein the one or more modifications is a replacement, deletion, or insertion of one or more amino acids in the Cα region and/or the Cβ region.
270. The TCR or antigen-binding fragment thereof of embodiment 268 or embodiment 269, wherein the one or more modifications comprise replacement(s) to introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and beta chain.
271. The TCR or antigen-binding fragment thereof of any of embodiments 246-16, 19 and 23-25, comprising a Cα region comprising a cysteine at a position corresponding to position 48 with numbering as set forth in SEQ ID NO: 212, 213, 217, 218, or 524 or at a position corresponding to position 49 with numbering as set forth in SEQ ID NO: 215 or 220; and/or a Cβ region comprising a cysteine at a position corresponding to position 57 with numbering as set forth in SEQ ID NO: 214 or 216 or at a position corresponding to position 58 with numbering as set forth in SEQ ID NO: 631 or 889.
272. The TCR or antigen-binding fragment thereof of any of embodiments 261, 264, and 268-271, wherein:
the Cα region comprises the amino acid sequence set forth in any of SEQ ID NOs: 196, 198, 200, 201, 203, or 525, or a sequence of amino acids that has at least 90% sequence identity thereto comprising one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; and/or
the Cβ region comprises the amino acid sequence set forth in any of SEQ ID NOs: 197, 199, 632, or 890 or a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain.
273. The TCR or antigen-binding fragment thereof of any of embodiments 246-272, wherein the TCR or antigen-binding fragment thereof is encoded by a nucleotide sequence that has been codon-optimized.
274. The TCR or antigen-binding fragment thereof of any of embodiments 246-264 and 268-273, wherein:
a) the alpha chain comprises:
the amino acid sequence set forth in any of SEQ ID NOs: 688, 706, 723, 738, 756, 772, 784, 796, 812, 827, 842, 854, 866, 878, 892, 905, 922, 934, 948, 960, 972, 984 or 996, a sequence of amino acids that has at least 90% sequence identity thereto; or the amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 1129, 1131, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171 or 1173, or a nucleotide sequence that has at least 90% sequence identity thereto; and/or
the beta chain comprises:
the amino acid sequence set forth in any of SEQ ID NOs: 697, 715, 732, 747, 765, 778, 790, 805, 821, 836, 848, 860, 872, 884, 898, 914, 928, 942, 954, 966, 978, 990 or 1005, a sequence of amino acids that has at least 90% sequence identity thereto; or the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NOS: 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172 or 1174, or a nucleotide sequence that has at least 90% sequence identity thereto.
275. The TCR or antigen-binding fragment thereof of any of embodiments 246-264 and 268-274, wherein:
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 688 and 697, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 706 and 715, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 723 and 732, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 738 and 747, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 756 and 765, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 772 and 778, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 784 and 790, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 796 and 805, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 812 and 821, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 827 and 836, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 842 and 848, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 854 and 860, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 866 and 872, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 878 and 884, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 892 and 898, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 905 and 914, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 922 and 928, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 934 and 942, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 948 and 954, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 960 and 966, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 972 and 978, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 984 and 990, respectively; or
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 996 and 1005, respectively.
276. The TCR or antigen-binding fragment thereof of any of embodiments 1-30, wherein the alpha and/or beta chain further comprises a signal peptide.
277. The TCR or antigen-binding fragment thereof of embodiment 31, wherein:
the alpha chain comprises the signal peptide comprising the amino acid sequence set forth in any of SEQ ID NOs: 181, 184, 187, 189, 190, 192, 193, 310, 311; and/or
the beta chain comprises the signal peptide comprising the amino acid sequence set forth in any of SEQ ID NOs: 182, 185, 186, 188, 191, or 194.
278. The TCR or antigen-binding fragment thereof of any of embodiments 246-277, that is isolated or purified or is recombinant.
279. The TCR or antigen-binding fragment thereof of any of embodiments 246-279, that is human.
280. The TCR or antigen-binding fragment thereof of any of embodiments 246-279, that is monoclonal.
281. The TCR or antigen-binding fragment thereof of any of embodiments 246-280, wherein the TCR or antigen-binding fragment thereof is single chain.
282. The TCR or antigen-binding fragment thereof of any of embodiments 246-281, wherein the TCR or antigen-binding fragment thereof comprises two chains.
283. The TCR or antigen-binding fragment thereof of any of embodiments 246-282, wherein the antigen-specificity is at least partially CD8-independent.
284. The TCR or antigen-binding fragment of any of embodiments 254-283 wherein the MHC molecule is an HLA-A2 molecule.
285. A nucleic acid molecule encoding the TCR or antigen-binding fragment thereof of any of embodiments 246-284, or an alpha or beta chain thereof.
286. The nucleic acid molecule of embodiment 285, comprising a nucleotide sequence encoding an alpha chain and/or a nucleotide sequence encoding a beta chain, wherein:
the nucleotide sequence encoding an alpha chain comprises the sequence set forth in any of SEQ ID NOS: 1049, 1051, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1073, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, or a nucleotide sequence that has at least 90% sequence identity thereto;
the nucleotide sequence encoding a beta chain comprises the sequence set forth in SEQ ID NOS: 1050, 1052, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1072, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090 or 1092, or a nucleotide sequence that has at least 90% sequence identity thereto.
287. The nucleic acid molecule of embodiment 285, wherein the nucleotide sequence is codon-optimized.
288. The nucleic acid molecule of embodiment 285 or embodiment 287, comprising a nucleotide sequence encoding an alpha chain and/or a nucleotide sequence encoding a beta chain, wherein:
the nucleotide sequence encoding an alpha chain comprises the sequence to set forth in any of SEQ ID NOS: 1129, 1131, 1133, 1135, 1137, 1139, 1141, 1143, 1145, 1147, 1149, 1151, 1153, 1155, 1157, 1159, 1161, 1163, 1165, 1167, 1169, 1171 or 1173, or a nucleotide sequence that has at least 90% sequence identity thereto;
the nucleotide sequence encoding a beta chain comprises the sequence set forth in SEQ ID NOS: 1130, 1132, 1134, 1136, 1138, 1140, 1142, 1144, 1146, 1148, 1150, 1152, 1154, 1156, 1158, 1160, 1162, 1164, 1166, 1168, 1170, 1172 or 1174, or a nucleotide sequence that has at least 90% sequence identity thereto.
289. The nucleic acid molecule of any of embodiments 285-288, wherein the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a peptide sequence that causes ribosome skipping.
290. The nucleic acid molecule of embodiment 289, wherein the peptide that causes ribosome skipping is a P2A or T2A peptide and/or comprises the sequence of amino acids set forth in SEQ ID NO: 204 or 211.
291. The nucleic acid of any of embodiments 285-290, comprising the nucleotide sequence set forth in any of SEQ ID NOs: 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469 or 470, or a nucleotide sequence having at least 90% sequence identity thereto.
292. A T cell receptor (TCR) or antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein:
the Vα region comprises the amino acid sequence set forth in any of SEQ ID NOs: 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661 or 676, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or
the Vβ region comprises the amino acid sequence set forth in any of SEQ ID NOs: 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667 or 685, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
293. The TCR or antigen-binding fragment thereof of embodiment 292, wherein the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2RX4AX6NNDMR, wherein X2 is V, or M; X4 is P, or D; and X6 is N, or R.
294. The TCR or antigen-binding fragment thereof of embodiment 292 or embodiment 293, wherein:
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4WGX7SNQPX12H, wherein X4 is L, F, or P; X7 is R, or Q; and X12 is Q, or L; or
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4X5X6X7X8SGNTIY, wherein X4 is L, or R; X5 is W, or Q; X6 is G, or P; X7 is R, or S; and X8 is S, or null.
295. A T cell receptor (TCR) or antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence AX2RX4AX6NNDMR, wherein X2 is V, or M; X4 is P, or D; and X6 is N, or R.
296. A T cell receptor (TCR) or antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein:
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4WGX7SNQPX12H, wherein X4 is L, F, or P; X7 is R, or Q; and X12 is Q, or L; or
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence ASSX4X5X6X7X8SGNTIY, wherein X4 is L, or R; X5 is W, or Q; X6 is G, or P; X7 is R, or S; and X8 is S, or null.
297. A T cell receptor (TCR) or antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein:
the Vα region comprises a complementarity determining region 3 (CDR-3) set forth in any of SEQ ID NOs: 478, 493, 505, 511, 523, 539, 555, 572, 588, 600, 612, 624, 638, 650, 662 or 679, or a sequence that exhibits at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto;
the Vβ region comprises a complementarity determining region 3 (CDR-3) set forth in any of SEQ ID NOs: 486, 499, 517, 531, 548, 563, 581, 594, 606, 618, 630, 644, 656, 670 or 686, or a sequence that exhibits at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto.
298. The TCR or antigen-binding fragment thereof of any of embodiments 292-297, wherein the Vα region comprises:
a complementarity determining region 1 (CDR-1) comprising the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO: 1191), wherein X1 is N, S, D, T, or V; X2 is 5, V, R, T, or I; X3 is M, F, G, S, N, A, L, V, or P; X4 is F, S, N, A, or null; X5 is D, S, Q, Y, N, V, T, or P; and X6 is Y, S, R, N, G, or T; and/or
a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6X7X8 (SEQ ID NO:1192), wherein X1 is I, V, L, G, N, T, Y, or M; X2 is 5, V, Y, L, P, F, I, or T; X3 is S, Y, K, L, T, or F; X4 is I, G, N, A, S, or null; X5 is S, D, or null; X6 is K, G, N, S, D, T, or E; X7 is D, E, G, A, K, L, or N; and X8 is K, V, D, P, N, T, L, or M.
299. The TCR or antigen-binding fragment thereof of any of embodiments 292-298, wherein the Vβ region comprises:
a complementarity determining region 1 (CDR-1) comprising the amino acid sequence SX2X3X4X5 (SEQ ID NO:1203), wherein X2 is G, or N; X3 is H, or D; X4 is T, L, N, or V; and X5 is A, S, Y, or T; and/or
a complementarity determining region 2 (CDR-2) comprising the amino acid sequence X1X2X3X4X5X6 (SEQ ID NO:1204), wherein X1 is F, or Y; X2 is Q, Y, or N; X3 is G, N, R, or Y; X4 is N, G, E, or T; X5 is S, E, A, or G; and X6 is A, E, I, or Q.
300. The TCR or antigen-binding fragment thereof of any of embodiments 292-299, wherein the TCR or antigen-binding fragment thereof binds to or recognizes a peptide epitope of human papillomavirus (HPV) 16 E6 in the context of an MHC molecule, the peptide epitope is or comprises E6(29-38) TIHDIILECV (SEQ ID NO:233).
301. The TCR or antigen-binding fragment of any of embodiments 292-300, wherein:
the Vα region comprises a complementarity determining region 3 (CDR-3) comprising the amino acid sequence set forth in any of SEQ ID NOs: 478, 493, 505, 511, 523, 539, 555, 572, 588, 600, 612, 624, 638, 650, 662 or 679, or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661 or 676; and/or
the Vβ region comprises a complementarity determining region 3 (CDR-3) comprising an amino acid sequence set forth in any of SEQ ID NOs: 486, 499, 517, 531, 548, 563, 581, 594, 606, 618, 630, 644, 656, 670 or 686 or a CDR3 contained within the amino acid sequence set forth in any of SEQ ID NOs: 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667 or 685.
302. The TCR or antigen-binding fragment thereof of any of embodiments 292-301, wherein the Vα region further comprises:
a complementarity determining region 1 (CDR-1) comprising an amino acid sequence set forth in any of SEQ ID NOs: 136, 161, 165, 537, 570, 142, 171 or 677; and/or
a complementarity determining region 2 (CDR-2) comprising an amino acid sequence set forth in any of SEQ ID NOs: 137, 162, 166, 538, 571, 143, 172 or 678.
303. The TCR or antigen-binding fragment thereof of any of embodiments 292-301, wherein the Vβ region comprises:
a complementarity determining region 1 (CDR-1) comprising the amino acid sequence set forth in any of SEQ ID NOs: 484, 148, 546, 561, 579, 168, 668 or 154; and/or
a complementarity determining region 2 (CDR-2) comprising the amino acid sequence set forth in any of SEQ ID NOs: 485, 149, 547, 562, 580, 169, 669 or 155.
304. The TCR or antigen-binding fragment thereof of any of embodiments 292-303, wherein:
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137 and 478, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 484, 485 and 486, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 161, 162 and 493, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149 and 499, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 165, 166 and 505, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149 and 499, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 161, 162 and 511, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149 and 517, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137 and 523, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149 and 531, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 537, 538, and 539, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 546, 547 and 548, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137 and 555, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 561, 562 and 563, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 570, 571 and 572, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 579, 580 and 581, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137 and 600, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149 and 594, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137 and 600, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149 and 606, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137 and 612, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149 and 618, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137 and 624, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 168, 169 and 630, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 142, 143 and 638, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 561, 562 and 644, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 171, 172 and 650, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 148, 149 and 656, respectively;
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 136, 137 and 662, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 668, 669 and 670, respectively; or
the Vα region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 677, 678 and 679, respectively, and the Vβ region comprises a CDR-1, CDR-2, and CDR-3, comprising the amino acid sequences of SEQ ID NOs: 154, 155 and 686, respectively.
305. The TCR or antigen-binding fragment thereof of any of embodiments 292-304, wherein:
the Vα region comprises a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vα region amino acid sequence set forth in any of SEQ ID NOs: 477, 492, 504, 510, 522, 536, 554, 569, 587, 599, 611, 623, 637, 649, 661 or 676; and/or
the Vβ region comprises a complementarity determining region 1 (CDR-1), a CDR-2, and a CDR-3, respectively comprising the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within a Vβ region amino acid sequence set forth in any of SEQ ID NOs: 483, 498, 498, 516, 530, 545, 560, 578, 593, 605, 617, 629, 643, 655, 667 or 685.
306. The TCR or antigen-binding fragment thereof of any of embodiments 292-305, wherein:
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 477 and 403, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 492 and 498, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 504 and 498, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 510 and 516, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 522 and 530, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 536 and 545, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 554 and 560, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 569 and 578, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 587 and 593, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 599 and 605, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 611 and 617, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 623 and 629, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 637 and 643, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 649 and 655, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs: 661 and 667, respectively;
the Vα and Vβ regions comprise the amino acid sequences of SEQ ID NOs:676 and 685, respectively.
307. The TCR or antigen-binding fragment thereof of any of embodiments 292-306, wherein the alpha chain further comprises an alpha constant (Cα) region and/or the beta chain further comprises a beta constant (Cβ) region.
308. The TCR or antigen-binding fragment thereof of embodiment 307, wherein the Cα and Cβ regions are mouse constant regions.
309. The TCR or antigen-binding fragment thereof of embodiment 307 or embodiment 308, wherein:
the Cα region comprises the amino acid sequence set forth in SEQ ID NO: 262, 833, 1012, 1014, 1015, 1017, 1018, or a sequence of amino acids that has at least 90% sequence identity thereto; and/or
the Cβ region comprises the amino acid sequence set forth in SEQ ID NO: 263, 1013 or 1016 or a sequence of amino acids that has at least 90% sequence identity thereto.
310. The TCR or antigen-binding fragment thereof of embodiment 307, wherein the Cα and Cβ regions are human constant regions.
311. The TCR or antigen-binding fragment thereof of embodiment 307 or embodiment 310, wherein:
the Cα region comprises the amino acid sequence set forth in any of SEQ ID NOs: 212, 213, 215, 217, 218, 220 or 524, or a sequence of amino acids that has at least 90% sequence identity thereto; and/or
the Cβ region comprises the amino acid sequence set forth in any of SEQ ID NOs: 214, 216, 631 or 889, or a sequence of amino acids that has at least 90% sequence identity thereto.
312. The TCR or antigen-binding fragment thereof of any of embodiments 292-311, wherein:
a) the alpha chain comprises:
the amino acid sequence set forth in any of SEQ ID NOs: 473, 488, 500, 506, 518, 532, 550, 565, 583, 595, 607, 619, 633, 645, 657 or 672, a sequence of amino acids that has at least 90% sequence identity thereto; or the amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 389, 430, 1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043 or 1045, or a nucleotide sequence that has at least 90% sequence identity thereto; and/or
the beta chain comprises:
the amino acid sequence set forth in any of SEQ ID NOs: 479, 494, 494, 512, 526, 541, 556, 574, 589, 601, 613, 625, 639, 651, 663 or 681, a sequence of amino acids that has at least 90% sequence identity thereto; or the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NOS: 390, 431, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034 1036, 1038, 1040, 1042, 1044 or 1046, or a nucleotide sequence that has at least 90% sequence identity thereto.
313. The TCR or antigen-binding fragment thereof of any of embodiments 292-312, wherein:
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 473 and 479, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 488 and 494, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 500 and 494, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 506 and 512, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 518 and 526, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 532 and 541, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 550 and 556, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 565 and 574, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 583 and 589, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 595 and 601, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 607 and 613, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 619 and 625, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 633 and 639, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 645 and 651, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 657 and 663, respectively; or
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 672 and 681, respectively.
314. The TCR or antigen-binding fragment thereof of any of embodiments 292-313, wherein the TCR or antigen-binding fragment comprises one or more modifications in the α chain and/or β chain such that when the TCR or antigen-binding fragment thereof is expressed in a cell, the frequency of mispairing between the TCR α chain and β chain and an endogenous TCR α chain and β chain is reduced, the expression of the TCR α chain and β chain is increased and/or the stability of the TCR α chain and β chain is increased, each compared to expression in a cell of the TCR or antigen-binding fragment thereof not containing the one or more modifications.
315. The TCR or antigen-binding fragment thereof of embodiment 314, wherein the one or more modifications is a replacement, deletion, or insertion of one or more amino acids in the Cα region and/or the Cβ region.
316. The TCR or antigen-binding fragment thereof of embodiment 314 or embodiment 315, wherein the one or more modifications comprise replacement(s) to introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and beta chain.
317. The TCR or antigen-binding fragment thereof of any of embodiments 292-307, 310 and 314-316, comprising a Cα region comprising a cysteine at a position corresponding to position 48 with numbering as set forth in SEQ ID NO: 212, 213, 217, 218, or 524 or at a position corresponding to position 49 with numbering as set forth in SEQ ID NO: 215 or 220; and/or a Cβ region comprising a cysteine at a position corresponding to position 57 with numbering as set forth in SEQ ID NO: 214 or 216 or at a position corresponding to position 58 with numbering as set forth in SEQ ID NO: 631 or 889.
318. The TCR or antigen-binding fragment thereof of any of embodiments 307, 310, and 314-317, wherein:
the Cα region comprises the amino acid sequence set forth in any of SEQ ID NOs: 196, 198, 200, 201, 203, or 525, or a sequence of amino acids that has at least 90% sequence identity thereto comprising one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain; and/or
the Cβ region comprises the amino acid sequence set forth in any of SEQ ID NOs: 197, 199, 632, or 890 or a sequence of amino acids that has at least 90% sequence identity thereto that contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain.
319. The TCR or antigen-binding fragment thereof of any of embodiments 292-318, wherein the TCR or antigen-binding fragment thereof is encoded by a nucleotide sequence that has been codon-optimized.
320. The TCR or antigen-binding fragment thereof of any of embodiments 292-307, 310, and 314-319, wherein:
a) the alpha chain comprises:
the amino acid sequence set forth in any of SEQ ID NOs: 474, 489, 501, 507, 519, 533, 551, 566, 584, 596, 608, 620, 634, 646, 658 or 673, a sequence of amino acids that has at least 90% sequence identity thereto; or the amino acid sequence encoded by the nucleotide sequence set forth in any of SEQ ID NOs: 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117, 1119, 1121, 1123, 1125 or 1127, or a nucleotide sequence that has at least 90% sequence identity thereto; and/or
the beta chain comprises:
the amino acid sequence set forth in any of SEQ ID NOs: 480, 495, 495, 513, 527, 542, 557, 575, 590, 602, 614, 626, 640, 652, 664 or 682, a sequence of amino acids that has at least 90% sequence identity thereto; or the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NOS: 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126 or 1128, or a nucleotide sequence that has at least 90% sequence identity thereto.
321. The TCR or antigen-binding fragment thereof of any of embodiments 292-307, 310, and 314-320, wherein:
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 474 and 482, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 489 and 497, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 501 and 497, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 507 and 515, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 519 and 529, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 533 and 544, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 551 and 559, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 566 and 577, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 584 and 592, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 596 and 604, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 608 and 616, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 620 and 628, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 634 and 642, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 646 and 654, respectively;
the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 658 and 666, respectively; or the alpha and beta chains comprise the amino acid sequences of SEQ ID NOs: 673 and 684, respectively.
322. The TCR or antigen-binding fragment thereof of any of embodiments 292-321, wherein the alpha and/or beta chain further comprises a signal peptide.
323. The TCR or antigen-binding fragment thereof of embodiment 322, wherein:
the alpha chain comprises the signal peptide comprising the amino acid sequence set forth in any of SEQ ID NOs: 181, 184, 187, 189, 190, 192, 193, 310, 311; and/or
the beta chain comprises the signal peptide comprising the amino acid sequence set forth in any of SEQ ID NOs: 182, 185, 186, 188, 191, or 194.
324. The TCR or antigen-binding fragment thereof of any of embodiments 292-323, that is isolated or purified or is recombinant.
325. The TCR or antigen-binding fragment thereof of any of embodiments 292-324, that is human.
326. The TCR or antigen-binding fragment thereof of any of embodiments 292-325, that is monoclonal.
327. The TCR or antigen-binding fragment thereof of any of embodiments 292-326, wherein the TCR or antigen-binding fragment thereof is single chain.
328. The TCR or antigen-binding fragment thereof of any of embodiments 292-327, wherein the TCR or antigen-binding fragment thereof comprises two chains.
329. The TCR or antigen-binding fragment thereof of any of embodiments 292-328, wherein the antigen-specificity is at least partially CD8-independent.
330. The TCR or antigen-binding fragment of any of embodiments 292-329 wherein the MHC molecule is an HLA-A2 molecule.
331. A nucleic acid molecule encoding the TCR or antigen-binding fragment thereof of any of embodiments 292-330, or an alpha or beta chain thereof.
332. The nucleic acid molecule of embodiment 331, comprising a nucleotide sequence encoding an alpha chain and/or a nucleotide sequence encoding a beta chain, wherein:
the nucleotide sequence encoding an alpha chain comprises the sequence set forth in any of SEQ ID NOS: 389, 430, 1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043 or 1045, or a nucleotide sequence that has at least 90% sequence identity thereto;
the nucleotide sequence encoding a beta chain comprises the sequence set forth in SEQ ID NOS: 390, 431, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034 1036, 1038, 1040, 1042, 1044 or 1046, or a nucleotide sequence that has at least 90% sequence identity thereto.
333. The nucleic acid molecule of embodiment 331, wherein the nucleotide sequence is codon-optimized.
334. The nucleic acid molecule of embodiment 331 or embodiment 332, comprising a nucleotide sequence encoding an alpha chain and/or a nucleotide sequence encoding a beta chain, wherein:
the nucleotide sequence encoding an alpha chain comprises the sequence to set forth in any of SEQ ID NOS: 1097, 1099, 1101, 1103, 1105, 1107, 1109, 1111, 1113, 1115, 1117, 1119, 1121, 1123, 1125 or 1127, or a nucleotide sequence that has at least 90% sequence identity thereto;
the nucleotide sequence encoding a beta chain comprises the sequence set forth in SEQ ID NOS: 1098, 1100, 1102, 1104, 1106, 1108, 1110, 1112, 1114, 1116, 1118, 1120, 1122, 1124, 1126 or 1128, or a nucleotide sequence that has at least 90% sequence identity thereto.
335. The nucleic acid molecule of any of embodiments 331-334, wherein the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a peptide sequence that causes ribosome skipping.
336. The nucleic acid molecule of embodiment 335, wherein the peptide that causes ribosome skipping is a P2A or T2A peptide and/or comprises the sequence of amino acids set forth in SEQ ID NO: 204 or 211.
337. The nucleic acid of any of embodiments 331-336, comprising the nucleotide sequence set forth in any of SEQ ID NOs: 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446 or 447, or a nucleotide sequence having at least 90% sequence identity thereto.
338. The nucleic acid of any of embodiments 285-291 and 331-337, wherein the nucleic acid is synthetic.
339. The nucleic acid of any of embodiments 285-291 and 331-338, wherein the nucleic acid is cDNA.
340. A vector comprising the nucleic acid of any of embodiments 285-291 and 331-339.
341. The vector of embodiment 340, wherein the vector is an expression vector.
342. The vector of embodiment 340 or embodiment 341, wherein the vector is a viral vector.
343. The vector of embodiment 342, wherein the viral vector is a retroviral vector.
344. The vector of embodiment 342 or embodiment 343, wherein the viral vector is a lentiviral vector.
345. The vector of embodiment 344, wherein the lentiviral vector is derived from HIV-1.
346. An engineered cell comprising the nucleic acid molecule of any of embodiments 40-46 and 86-94 or vector of any of embodiments 340-345.
347. An engineered cell, comprising the TCR or antigen-binding fragment thereof of any of embodiments 246-384 and 292-330.
348. The engineered cell of embodiment 346 or embodiment 347, wherein the TCR or antigen-binding fragment thereof is heterologous to the cell.
349. The engineered cell of any of embodiments 346-348, wherein the engineered cell is a cell line.
350. The engineered cell of any of embodiments 346-349, wherein the engineered cell is a primary cell obtained from a subject.
351. The engineered cell of embodiment 350, wherein the subject is a mammalian subject.
352. The engineered cell of embodiment 350 or embodiment 351, wherein the subject is a human.
353. The engineered cell of any of embodiments 346-352, wherein the engineered cell is a T cell.
354. The engineered cell of embodiment 353, wherein the T cell is CD8+.
355. The engineered cell of embodiment 353, wherein the T cell is CD4+.
356. The engineered cell of any of embodiments 346-355, comprising a genetic disruption of a T cell receptor alpha constant (TRAC) gene and/or a T cell receptor beta constant (TRBC) gene.
357. The engineered cell of embodiment 356, wherein the TRBC gene is one or both of a T cell receptor beta constant 1 (TRBC1) or T cell receptor beta constant 2 (TRBC2) gene.
358. A method for producing a cell of any of embodiments 346-357, comprising introducing a vector of any of embodiments 93-98 into a cell in vitro or ex vivo.
359. The method of embodiment 358, wherein the vector is a viral vector and the introducing is carried out by transduction.
360. The method of embodiment 358 or embodiment 359, further comprising introducing into the cell one or more agent, wherein each of the one or more agent is independently capable of inducing a genetic disruption of a T cell receptor alpha constant (TRAC) gene and/or a T cell receptor beta constant (TRBC) gene.
361. The method of any of embodiment 360, wherein the one or more agent capable of inducing a genetic disruption comprises a DNA binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to the target site.
362. The method of embodiment 361, wherein the one or more agent capable of inducing a genetic disruption comprises (a) a fusion protein comprising a DNA-targeting protein and a nuclease or (b) an RNA-guided nuclease.
363. The method of embodiment 362, wherein the DNA-targeting protein or RNA-guided nuclease comprises a zinc finger protein (ZFP), a TAL protein, or a clustered regularly interspaced short palindromic nucleic acid (CRISPR)-associated nuclease (Cas) specific for a target site within the TRAC and/or TRBC gene.
364. The method of embodiment 363, wherein the one or more agent comprises a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or and a CRISPR-Cas9 combination that specifically binds to, recognizes, or hybridizes to the target site.
365. The method of embodiment 363 or embodiment 364, wherein the each of the one or more agent comprises a guide RNA (gRNA) having a targeting domain that is complementary to the at least one target site.
366. The method of embodiment 365, wherein the one or more agent is introduced as a ribonucleoprotein (RNP) complex comprising the gRNA and a Cas9 protein.
367. The method of embodiment 366, wherein the RNP is introduced via electroporation, particle gun, calcium phosphate transfection, cell compression or squeezing.
368. The method of embodiment 366 or embodiment 367, wherein the RNP is introduced via electroporation.
369. The method of any of embodiments 365-368, wherein the one or more agent is introduced as one or more polynucleotide encoding the gRNA and/or a Cas9 protein.
370. A composition comprising engineered cells of any of embodiments 346-357.
371. The composition of embodiment 370, wherein the engineered cells comprise CD4+ and/or CD8+ T cells.
372. The composition of embodiment 370 or embodiment 371, wherein the engineered cells comprise CD4+ and CD8+ T cells.
373. A composition, comprising an engineered CD8+ cell of embodiment 354 and an engineered CD4+ cell of embodiment 355.
374. The composition of any of embodiments 370-373, wherein the TCR or antigen-binding fragment thereof binds to or recognizes a peptide epitope of HPV 16 in the context of an MHC molecule that is at least partially CD8-independent.
375. The composition of any of embodiments 371-374, wherein the CD8+ cell and CD4+ cell are engineered with the same TCR or antigen-binding fragment thereof and/or are each engineered with a TCR or antigen-binding fragment thereof that binds to or recognizes the same peptide epitope of HPV 16 in the context of an MHC molecule.
376. The composition of any of embodiments 370-375, further comprising a pharmaceutically acceptable excipient.
377. A method of treatment, comprising administering the engineered cell of any of embodiments 346-357 to a subject having a disease or disorder associated with HPV.
378. A method of treatment, comprising administering the composition of any of embodiments 370-376 to a subject having a disease or disorder associated with HPV.
379. The method of embodiment 377 or embodiment 378, wherein the disease or disorder is associated with HPV16.
380. The method of any of embodiments 377-379, wherein the disease or disorder is cancer.
381. The method of any of embodiments 377-380, wherein the subject is a human.
382. A composition of any of embodiments 370-376 for use in treating a disease or disorder associated with HPV.
383. Use of a composition of any of embodiments 370-376 for the manufacture of a medicament for treating a disease or disorder associated with HPV.
384. The composition of embodiment 382 or use of embodiment 383, wherein the disease or disorder is associated with HPV16.
385. The composition or use of any of embodiments 382-384, wherein the disease or disorder is cancer.
386. The composition or use of any of embodiments 382-385, wherein the subject is a human.
IX. Examples
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1: Screening and Selection of HPV-16 E6 and E7 Epitope-Specific T Cell Receptors from Normal Donors
An exemplary autologous screening process using autologous dendritic and T cells, generally as described by Ho et al., J. Immunol. Methods, 310:1-2, 40-52, with indicated modifications, was performed to generate antigen-specific T cells that specifically bound to peptide epitopes of human papillomavirus 16 (HPV16) E6 and E7 proteins presented on MHC-I molecules. Clonal T cell lines were generated and their TCR sequences cloned by this method were cloned.
1A. Generation and Cloning of Human HPV-Specific T Cells and TCRs
Briefly, dendritic cells were derived from adherent fractions of peripheral blood mononuclear cell (PBMC) samples obtained from normal human HLA-A02:01 donors, by culturing over two days in the presence of GM-CSF and IL-4, followed by incubation beginning at day 3 in the presence of pro-inflammatory cytokines to produce mature dendritic cells. On Day 4, the resulting mature dendritic cells were harvested, washed and pulsed with HPV-16 E6- or E7-derived peptides, such as some of those shown in Table 13, including peptide epitopes E6 (29-38), E7 (11-19), and E7 (86-93).
TABLE 13
HPV-16 Epitopes
Epitope
Epitope
SEQ ID
Description
Name
NO.
KLPQLCTEL
E6(18-26)
232
TIHDIILECV
E6(29-38)
233
FAFRDLCIV
E6(52-60)
234
TLGIVCPI
E7(86-93)
235
YMLDLQPET
E7(11-19)
236
GTLGIVCPI
E7(85-93)
237
LLMGTLGIV
E7(82-90)
238
TLHEYMLDL
E7(7-15)
239
On Day 5, autologous CD8+ T cells from normal human donors were incubated with the peptide-pulsed dendritic cells.
On Day 8, IFNγ in the cultures was measured as an indicator for cultures containing antigen-specific T cells. Cells from reactive co-cultures were selected and re-stimulated two or three times with peptide-pulsed dendritic cells to enrich for specific T cells. Following the repeated stimulations, populations of cells staining positive for peptide-loaded autologous MHC tetramers were identified by flow cytometry. Clonal lines were generated by cell sorting and/or limiting dilution cloning essentially as described by Ho et al. 2006.
Clones were cultured with peptide-pulsed T2 cells (cells deficient in transporter associated with antigen transport (TAP) but expressing MHC-I and thus able to present peptides loaded onto the cells), pulsed with the relevant peptide, e.g. E6 (29-38), E7 (11-19) or E7 (86-93). Level of IFNγ in the cultures, as compared to those resulting from co-culture with cells loaded with a non-HPV-derived (negative control) peptide, was measured as an indicator of T cell specificity for the peptide-MHC and functional activity. Flow cytometry-based staining was used to assess the ability of the clonal cell lines to bind, in a peptide-specific manner, to labeled peptide-MHC (HLA-A02:01) tetramers (either HLA-A2/E6 (29-38), HLA-A2/E7 (11-19) or HLA-A2/E7 (86-93)); tetramers containing an irrelevant peptide served as a negative control).
Table 14 lists sequence identifiers corresponding to TCR alpha and beta chains expressed by clonal T cell lines generated via this process.
The ability of clonal lines to lyse target cells in an antigen-specific manner was assessed using peptide-pulsed T2 cells and/or cells of an antigen-expressing cancer cell line.
In an exemplary assay, monoclonal cell lines expressing the TCRs were incubated with the CaSki target cells (ATCC No. CRL-1550, containing approximately 600 copies of integrated HPV16) at various effector:target (E:T) ratios. Lytic activity was assessed by measuring caspase in the target cells and assessing the percentage of such cells that were positive to caspase at various time-points following initiation of incubation with the T cells, over 50 hours. Negative controls included incubation of T cells with SiHa cells (ATCC No. HTB-35, essentially negative for the endogenous target antigen, having no more than approximately one or two copies of integrated HPV16 genome) and Caski cells not incubated with T cell clones. The results for two exemplary clonal T cell lines are shown in FIG. 1. As shown, the monoclonal T cell lines were observed to exhibit lytic activity against cells presenting the subject HPV16-derived peptide in the context of HLA-A02:01. A number of CD8+ clones were generated and confirmed to exhibit antigen-specific binding and functionality by this process.
The ability of T cells of clonal lines to specifically bind to peptide epitopes independently of the CD8 co-receptor was assessed using a mutant MHC class I tetramer containing a D227K mutation in its CD8 binding site, rendering it unable to engage the CD8 co-receptor on T cells. See Kerry et al. J Immunol (2003) 171:4493-4503; Kerry et al. Immunology (2005) 114: 44-52. Table 14 lists exemplary TCRs expressed by exemplary clonal cell lines generated by this method. Each of these cell lines was observed in this study to bind the indicated peptide-MHC complex in an antigen-specific manner, as indicated by tetramer staining in comparison to control. Additionally, the indicated clonal lines were observed to specifically bind the relevant peptide in the context of the mutant (non-CD8 interacting) tetramers, indicating the ability of the TCRs expressed by these clonal lines to specifically bind to cognate antigen independently of CD8.
1B. Cloning of TCRs Expressed by Clonal Cell Lines
Polynucleotides having sequences encoding the polypeptide chains of TCRs from clonal lines generated as described above were amplified from T cell lines and sequenced using 5′ rapid amplification of cDNA ends (RACE). Table 14 provides the sequence identifier (SEQ ID NO) for the alpha and beta chain nucleotide and amino acid sequences, respectively, for a plurality of TCRs generated by this process. Table 14 also lists the SEQ ID NO corresponding to an exemplary full-length encoded amino acid sequence containing the beta and alpha chain sequences of each respective TCR, separated by a ribosome-skip P2A sequence (P2A linker set forth in SEQ ID NO: 204, which may be encoded by a sequence of nucleotides set forth in any of SEQ ID NOs: 4, 5, 6, 207-210) (designated “beta-P2A-alpha”). A nucleotide sequence encoding such a full-length sequence for each of a number of TCRs was inserted into a vector for transfer into a host cell, such as a primary human cell, e.g., a T cell, as described below. Following translation of the nucleotide sequence and self-cleavage of the P2A sequence separating the TCR chains, the recombinant alpha and beta chain of the TCR were exogenously expressed in host cells, such as a primary T cell. The Table 14 also lists the specific Valpha and Vbeta usage for each cloned TCR.
TABLE 14
Amino Acid and Nucleotide Sequences of HPV-Specific TCRs
Binding to
Peptide in
SEQ ID NO.
Complex with
Full-
Mutant (non-
length
CD8-binding)
beta-
MHC
P2A-
tetramers by
alpha
alpha
beta
TCR
Epitope
Clonal Line
Valpha Usage
Vbeta Usage
aa
nt
aa
nt
aa
TCR 3
E6(29-38)
Yes
TRAV14/DV4*02
TRBV7-8*01
223
20
18
24
22
TCR 4
E6(29-38)
Yes
TRAV26-2*01
TRBV7-9*03
224
30
28
34
32
TCR 5
E6(29-38)
No
TRAV14/DV4*02
TRBV28*01
225
40
38
44
42
TCR 7
E7(11-19)
No
TRAV10*01
TRBV2*01
227
60
58
64
62
TCR 8
E6(29-38)
No
TRAV21*02
TRBV28*01
228
70
68
74
72
TCR 9
E6(29-38)
Yes
TRAV14/DV4*01
TRBV6-2*01
229
80
78
84
82
TCR 10
E6(29-38)
Yes
TRAV12-1*01
TRBV28*01
230
90
88
94
92
TCR 11
E7(86-93)
No
TRAV26-2*01
TRBV29-1*01
231
100
98
104
102
TCR 12
E7(11-19)
Yes
TRBV2*01
340
183
283
108
52, 285
TCR 13
E6(29-38)
Yes
TRAV8-2
TRBV10-3
341
202
287
17
289
TCR 14
E6(29-38)
TRAV24
TRBV28
342
219
291
16
293
1C. Codon Optimization, Modification and Lentiviral Expression
Nucleotide sequences encoding TCRs generated as described above were modified by codon optimization and/or by mutation(s) to promote the formation of a non-native disulfide bond in the interface between the TCR constant domains to increase pairing and stability of the TCR. The non-native disulfide bond was promoted by modifying the TCR chains at residue 48 in the Cα region from Thr to Cys and residue 57 of the Cβ region from Ser to Cys (see Kuball et al. (2007) Blood, 109:2331-2338). The corresponding SEQ ID NO for the resulting modified nucleotide sequences and corresponding encoded amino acid sequences for the modified version of each TCR are shown in Table 15.
For individual TCRs modified as described above, constructs were generated that contained the modified nucleotide sequences encoding the beta chain and alpha chain, respectively, of the cloned TCRs, separated by a sequence encoding a P2A polypeptide were generated and inserted into a lentiviral vector, which were used to transduce T cell lines and primary T cells using standard methods, to express the encoded TCR chains.
TABLE 15
Codon Optimized, Cysteine Modified Version of the TCRs
SEQ ID NO. of Modified Version of TCR
Full-length
alpha
beta
TCR
Epitope
nt
nt
aa
nt
aa
TCR 3
E6(29-38)
26
21
19
25
23
TCR 4
E6(29-38)
36
31
29
35
33
TCR 5
E6(29-38)
46
41
39
45
43
TCR 6
E7(11-19)
56
51
49
54
53, 286
TCR 7
E7(11-19)
66
61
59
65
63
TCR 8
E6(29-38)
76
71
69
75
73
TCR 9
E6(29-38)
86
81
79
85
83
TCR 10
E6(29-38)
96
91
89
95
93
TCR 11
E7(86-93)
106
101
99
105
103
TCR 12
E7(11-19)
15
12
284
9
53, 286
TCR 13
E6(29-38)
14
11
288
8
290
TCR 14
E6(29-38)
13
10
292
7
294
Example 2: Expression and Antigen-Binding of Exemplary TCRs in Jurkat Cells
Exemplary E6-specific and E7-specific T cell receptors (TCRs), generated as described above, were assessed for surface expression on T cells and antigen-specific binding with or without CD8 interaction. Specifically, cells derived from the Jurkat human T cell line that did not express the endogenous TCR on their surfaces (CD4+ Jurkat-derived cells), with or without exogenously expressed CD8, referred to in FIG. 2A, FIG. 2B, FIG. 3 and FIG. 4, as CD8+ and CD4+, respectively, were engineered to express the modified version of the TCRs. For each TCR assessed in this process, the Jurkat-derived cells were transduced with a lentiviral vector particle generated as described above encoding the particular modified version of the TCR. Cells (those containing or not containing exogenous CD8) not transduced with a TCR were used as controls. At day 6 post-transduction with the sequence encoding each TCR, TCR expression and functional activity were assessed by flow cytometry, following staining with labeled tetramers complexed with the respective E6- or E7-peptide (either HLA-A2/E6 (29-38), HLA-A2/E7 (11-19) or HLA-A2/E7 (86-93) tetramer). A reference TCR capable of binding to HLA-A2/E6 (29-38) also was assessed in this study.
Exemplary results are shown in FIG. 2A and FIG. 2B (E6(29-38)-loaded tetramer binding), FIG. 3 (E7 (11-19)-loaded tetramer binding) and FIG. 4 (E7(86-93)-loaded tetramer binding). The percentage of cells in the indicated quadrants in flow cytometry plots shown in FIGS. 2A, 2B, 3 and 4 are also summarized below in Table 16 (FIG. 2A), Table 17 (FIG. 2B), Table 18 (FIG. 3) and Table 19 (FIG. 4).
TABLE 16
Percentage of cells present in each indicated quadrant
in Flow Cytometry Plots Shown in FIG. 2A
E6
E6
E6
E6
tet+/CD8−
tet+/CD8+
tet−/CD8+
tet−/CD8−
TCR/Cells
quadrant
quadrant
quadrant
quadrant
Reference/Neg Ctrl (CD4+)
0.1
4.24E−03
0.17
99.7
Reference/CD4+ TCR − E6(29)
7.53
8.63E−03
0.056
92.4
TCR 5/Neg Ctrl (CD4+)
0.14
0
0.1
99.8
TCR 5/CD4+ TCR − E6(29)
0.094
0
0.026
99.9
TCR 4/Neg Ctrl (CD4+)
0.1
0
0.12
99.8
TCR 4/CD4+ TCR − E6(29)
2.52
4.42E−03
0.04
97.4
Reference/CD8
8.73E−03
0.27
98
1.69
Reference/CD8+ TCR − E6(29)
0.041
15.8
82.5
1.65
TCR 5/CD8
8.90E−03
0.18
97.5
2.33
TCR 5/CD8+ TCR − E6(29)
0.018
3.28
94.5
2.22
TCR 4/CD8
0
0.26
98.1
1.6
TCR 4/CD8+ TCR − E6(29)
0.023
24.4
73.5
2.04
TABLE 17
Percentage of cells present in each indicated quadrant
in Flow Cytometry Plots Shown in FIG. 2B
E6
E6
E6
E6
tet+/CD8−
tet+/CD8+
tet−/CD8+
tet−/CD8−
TCR/Cells
quadrant
quadrant
quadrant
quadrant
Reference/Neg Ctrl (CD4+)
0.1
4.24E−03
0.17
99.7
Reference/CD4+ TCR − E6(29)
7.53
8.63E−03
0.056
92.4
TCR 3/Neg Ctrl (CD4+)
0.15
4.29E−03
0.1
99.7
TCR 3/CD4+ TCR − E6(29)
8.05
0
0.022
91.9
TCR 8/Neg Ctrl (CD4+)
0.15
0
0.11
99.7
TCR 8/CD4+ TCR − E6(29)
0.12
0
0.044
99.8
Reference/CD8
8.73E−03
0.27
98
1.69
Reference/CD8+ TCR − E6(29)
0.041
15.8
82.5
1.65
TCR 3/CD8
4.58E−03
0.31
97.8
1.9
TCR 3/CD8+ TCR − E6(29)
0.083
18
80
1.84
TCR 8/CD8
0
0.22
97.2
2.57
TCR 8/CD8+ TCR − E6(29)
0
4.09
93.6
2.34
TABLE 18
Percentage of cells present in each indicated quadrant
in Flow Cytometry Plots Shown in FIG. 3
E7
E7
E7
E7
tet+/CD8−
tet+/CD8+
tet−/CD8+
tet−/CD8−
TCR/Cells
quadrant
quadrant
quadrant
quadrant
TCR 7/Neg Ctrl (CD4+)
0.098
0
0.29
99.6
TCR 7/CD4+ TCR − E7(11)
0.095
4.11E−03
0.3
99.6
TCR 12/Neg Ctrl (CD4+)
0.32
0
0
99.7
TCR 12/CD4+ TCR − E7(11)
0.3
0.015
0.049
99.6
TCR 7/CD8
0
0.15
97.9
1.95
TCR 7/CD8+ TCR − E7(11)
4.28E−03
2.05
96
1.93
TCR 12/CD8
0
0.21
99.8
0
TCR 12/CD8+ TCR − E7(11)
0
9.66
90.3
0
TABLE 19
Percentage of cells present in each indicated quadrant
in Flow Cytometry Plots Shown in FIG. 4
E7
E7
E7
E7
tet+/CD8−
tet+/CD8+
tet−/CD8+
tet−/CD8−
TCR/Cells
quadrant
quadrant
quadrant
quadrant
TCR 11/Neg Ctrl (CD4+)
0.1
4.54E−03
0.027
99.9
TCR 11/CD4+ TCR − E7(86)
0.11
0
0.045
99.8
TCR 11/CD8
9.41E−03
2.09
95.3
2.62
TCR 11/CD8+ TCR − E7(86)
0.015
8.04
89
2.96
As shown, TCRs generated by these methods were cloned and observed to be expressed on the surface of T cells and to bind HPV peptide in the context of MHC tetramers, in some cases independently of CD8 co-receptor.
Example 3: Functional Assessment of Cells Transduced with HPV-16 E6 and E7 Epitope-Specific T Cell Receptors
Primary CD8+ T cells were transduced with a lentiviral vector particle generated as described above encoding chains of modified versions of TCRs specific for E6(29-38) in the context of HLA:A2:01, including exemplary modified versions of TCRs TCR 5, TCR 4, TCR 3, TCR 8, TCR 9, TCR 10 and TCR15. Such transduced T cells were assessed for functional activity, including the ability to generate cytokines and exhibit lytic activity in response to cells expressing the peptide:MHC. An exemplary E7(11-19)-specific TCR was used as a negative control in these studies.
A. Cytokine Production
To assess the production of cytokines in response to antigen, the cells were incubated for 4 hours at a 10:1 E:T ratio with T2 cells that had been pulsed overnight with 10 μM of E6(29-38) peptide or, as a control, 10 μM of E7(11-19) peptide. As a positive control, cytokine activity also was assessed in cultures of transduced T cells stimulated with either phorbol myristate acetate (PMA) and Brefeldin A (BFA) or with BFA alone. Intracellular IFNγ was measured in the cultured cells by flow cytometry. The percent of CD8 and intracellular IFNγ positive (% CD8+/IC IFNγ+) cells was determined by flow cytometry.
The results are shown in Table 20. These results confirmed the ability of primary human T cells expressing E6(29-38)-specific TCRs generated by these methods to produce cytokine in response to target cells in an antigen-specific manner.
TABLE 20
Cytokine activity
Peptide/Treatment
TCR
% CD8+/IC IFNγ+
E6(29-38)
TCR 5
43.7
TCR 7
70.5
TCR 4
94.2
TCR 3
95.1
TCR 8
95.0
TCR 9
91.1
TCR 10
98.9
E7(11-19)
TCR 5
7.22
TCR 7
62.4
TCR 4
2.5
TCR 3
2.51
TCR 8
11.4
TCR 9
19.5
TCR 10
1.17
T cells + PMA + BFA
TCR 5
22.4
TCR 7
89.4
TCR 4
27.9
TCR 3
94.4
TCR 8
98.4
TCR 9
22.3
TCR 10
27.5
T cells + BFA
TCR 5
4.83
TCR 7
57.9
TCR 4
1.87
TCR 3
1.82
TCR 8
8.18
TCR 9
11.1
TCR 10
0.63
B. Lytic Activity
Lytic activity of the transduced primary T cells against cells expressing HPV16 was assessed by incubating CaSki cells (in the presence or absence of IFNγ) at a 10:1 E:T ratio. Samples in which SiHa cells were used as the target cells at the same E:T ratio served as a negative control. Lytic activity also was assessed against T2 cells pulsed with peptide E6(29-38). The ability of the T cells to antigen-specifically cause lytic activity was assessed by measuring active-caspase in the target cells 4 hours post co-culture.
Example 4: Screening and Selection of HPV-16 E6 and E7 Epitope-Specific T Cell Receptors from Normal Donors
A screening process using autologous dendritic and T cells was performed to generate antigen-specific T cell receptors (TCRs) that specifically bound to human papillomavirus 16 (HPV16) E6(29-38) or E7(11-19) peptide presented on MHC-I molecules and survived and/or were enriched over time, following multiple rounds of antigen-stimulation. Clonal T cell lines were generated and the sequences of individual paired TCR alpha and beta chains and abundance thereof in various populations were determined on a single-cell basis, using high-throughput paired TCR sequencing.
A. Generation and Cloning of Human HPV-Specific T Cells and TCRs
Briefly, peptide-pulsed antigen-presenting cells were generated from PBMCs substantially as described in Example 1. Specifically, peptide-pulsed HLA:A02:01APCs were generated with HPV 16 E6(29-38) peptide (TIHDIILECV; SEQ ID NO:233) or E7(11-19) peptide (YMLDLQPET; SEQ ID NO:236). Autologous CD8+ T cells from normal human donors were incubated over multiple rounds with the peptide-pulsed cells, and selections were carried out based on binding to peptide-loaded autologous MHC tetramers. Generally, cells were subjected to a total of three rounds of stimulation, in the presence of peptide-pulsed cells (with a peptide concentration of 1000 ng/mL maintained over the three rounds). Following the second and third rounds of stimulation, cells were sorted by flow cytometry into populations positive and negative, respectively, for binding to peptide-MHC tetramers containing the appropriate tetramer. Cells of the tetramer-positive and negative populations following each of the second and third rounds were subjected to single-cell TCR sequencing, to assess the presence and frequency of individual TCRs in the different populations, and the persistence of TCR clones over multiple rounds of antigen stimulation.
B. Determination of TCR Sequences and Assessment of TCRs
Cell populations from the positive and negative fractions (i.e., sorted by flow cytometry based on positive and negative staining, respectively, for binding to the E6(29-38) peptide-loaded, or E7(11-19) peptide-loaded, MHC tetramers, as determined by flow cytometry) following rounds 2 and 3 of stimulation were subject to high-throughput single-cell sequencing for TCR alpha and beta chain pairs. High throughput single cell TCR sequencing was performed as generally described in published PCT patent applications, publication numbers WO2012/048340, WO2012/048341 and WO2016/044227. The sequencing methods employed single-cell droplets and sample and molecular barcodes, to identify individual pairs of TCR alpha and beta chain sequences at a single-cell level, for each of a large number (e.g., millions) of single cells present in a single starting composition, and to assess abundance of each TCR pair in various populations assessed. The ability to identify and quantify TCR pairs at a single-cell level permitted the assessment of the frequency of each of various TCR pairs in each of the individual positive and negative fractions, and to assess enrichment and persistence of TCRs over multiple rounds of antigen stimulation. TCR pairs identified in this assay were selected based on their presence in the peptide-binding fractions following rounds 2 and 3, higher abundance in positive versus negative fractions in each of these rounds, and enrichment over time following multiple rounds of exposure to antigen.
Tables 21 and 22 list exemplary E6(29-38)- and E7(11-19)-specific TCRs isolated according to this method, respectively, and the sequence identifiers (SEQ ID NO:) for the alpha and beta chain nucleotide and amino acid sequences for each TCR. Tables 21 and 22 also list the sequence identifier (SEQ ID NO) corresponding to an exemplary full-length encoded amino acid sequence containing the beta and alpha chain sequences of each respective TCR, separated by a sequence encoding a ribosome-skip P2A sequence (P2A linker set forth in SEQ ID NO: 204) (designated “beta-P2A-alpha”). A nucleotide sequence encoding such a full-length sequence for each of a number of TCRs was inserted into a vector for transfer into a host cell, such as a primary human cell, e.g., a T cell, as described below. Following translation of the nucleotide sequence and self-cleavage of the P2A sequence separating the TCR chains, the recombinant alpha and beta chain of the TCR were exogenously expressed in host cells.
TABLE 21
Amino Acid and Nucleotide Sequences
of HPV 16 E6(29-38)-Specific TCRs
SEQ ID NO.
Full length beta-
P2A-alpha
sequence
alpha
beta
TCR
Epitope
aa
nt
aa
nt
aa
TCR 15
E6(29-38)
391
389
473
390
479
TCR 16
E6(29-38)
392
430
488
431
494
TCR 17
E6(29-38)
393
1019
500
1020
494
TCR 18
E6(29-38)
394
1021
506
1022
512
TCR 19
E6(29-38)
395
1023
518
1024
526
TCR 20
E6(29-38)
396
1025
532
1026
541
TCR 21
E6(29-38)
397
1027
550
1028
556
TCR 22
E6(29-38)
398
1029
565
1030
574
TCR 23
E6(29-38)
399
1031
583
1032
589
TCR 24
E6(29-38)
400
1033
595
1034
601
TCR 25
E6(29-38)
401
1035
607
1036
613
TCR 26
E6(29-38)
402
1037
619
1038
625
TCR 27
E6(29-38)
403
1039
633
1040
639
TCR 28
E6(29-38)
404
1041
645
1042
651
TCR 29
E6(29-38)
405
1043
657
1044
663
TCR 30
E6(29-38)
406
1045
672
1046
681
TABLE 22
Amino Acid and Nucleotide Sequences
of HPV 16 E7(11-19)-Specific TCRs
SEQ ID NO.
Full length beta-
P2A-alpha
sequence
alpha
beta
TCR
Epitope
aa
nt
aa
nt
aa
TCR 31
E7(11-19)
407
1225
687
1224
696
TCR 32
E7(11-19)
408
1049
705
1050
714
TCR 33
E7(11-19)
409
1051
722
1052
731
TCR 34
E7(11-19)
410
1226
737
1227
746
TCR 35
E7(11-19)
411
1055
755
1056
764
TCR 36
E7(11-19)
412
1057
771
1058
777
TCR 37
E7(11-19)
413
1059
783
1060
789
TCR 38
E7(11-19)
414
1061
795
1062
804
TCR 39
E7(11-19)
415
1063
811
1064
820
TCR 40
E7(11-19)
416
1065
826
1066
835
TCR 41
E7(11-19)
417
1067
841
1068
847
TCR 42
E7(11-19)
418
1069
853
1070
859
TCR 43
E7(11-19)
419
1071
865
1072
871
TCR 44
E7(11-19)
420
1073
877
1074
883
TCR 45
E7(11-19)
421
1075
891
1076
897
TCR 46
E7(11-19)
422
1077
904
1078
913
TCR 47
E7(11-19)
423
1079
921
1080
927
TCR 48
E7(11-19)
424
1081
933
1082
941
TCR 49
E7(11-19)
425
1083
947
1084
953
TCR 50
E7(11-19)
426
1085
959
1086
965
TCR 51
E7(11-19)
427
1087
971
1088
977
TCR 52
E7(11-19)
428
1089
983
1090
989
TCR 53
E7(11-19)
429
1091
995
1092
1004
TCR 54
E7(11-19)
227
1093
58
1094
62
TCR 55
E7(11-19)
340
1095
283
1228
285
C. Codon Optimization and Modification
Nucleotide sequences encoding TCRs generated as described above were modified by codon optimization and/or by mutation(s) to promote the formation of a non-native disulfide bond in the interface between the TCR constant domains to increase pairing and stability of the TCR. The non-native disulfide bond was promoted by modifying the TCR chains at residue 48 in the Cα region from Thr to Cys and residue 57 of the Cβ region from Ser to Cys (see Kuball et al. (2007) Blood, 109:2331-2338). The corresponding SEQ ID NO for the resulting modified nucleotide sequences and corresponding encoded amino acid sequences for the modified version of each TCR are shown in Table 23 (E6(29-38)-specific TCR) and Table 24 (E7(11-19)-specific TCRs).
For individual TCRs modified as described above, constructs were generated that contained the modified nucleotide sequences encoding the beta chain and alpha chain, respectively, of the cloned TCRs, separated by a sequence encoding a P2A polypeptide and inserted into a vector, e.g. lentiviral vector, which were used for expressing the TCR chain in T cell lines and primary T cells using standard methods.
TABLE 23
Codon Optimized, Cysteine Modified Version
of HPV 16 E6(29-38)-Specific TCRs
SEQ ID NO. of Modified Version of TCR
Full-length
alpha
beta
TCR
Epitope
nt
nt
aa
nt
aa
TCR 15
E6(29-38)
432
1097
474
1098
480
TCR 16
E6(29-38)
433
1099
489
1100
495
TCR 17
E6(29-38)
434
1101
501
1102
495
TCR 18
E6(29-38)
435
1103
507
1104
513
TCR 19
E6(29-38)
436
1105
519
1106
527
TCR 20
E6(29-38)
437
1107
533
1108
542
TCR 21
E6(29-38)
438
1109
551
1110
557
TCR 22
E6(29-38)
439
1111
566
1112
575
TCR 23
E6(29-38)
440
1113
584
1114
590
TCR 24
E6(29-38)
441
1115
596
1116
602
TCR 25
E6(29-38)
442
1117
608
1118
614
TCR 26
E6(29-38)
443
1119
620
1120
626
TCR 27
E6(29-38)
444
1121
634
1122
640
TCR 28
E6(29-38)
445
1123
646
1124
652
TCR 29
E6(29-38)
446
1125
658
1126
664
TCR 30
E6(29-38)
447
1127
673
1128
682
TABLE 24
Codon Optimized, Cysteine Modified Version
of HPV 16 E7(11-19)-Specific TCRs
SEQ ID NO. of Modified Version of TCR
Full-length
alpha
beta
TCR
Epitope
nt
nt
aa
nt
aa
TCR 31
E7(11-19)
448
1129
688
1130
697
TCR 32
E7(11-19)
449
1131
706
1132
715
TCR 33
E7(11-19)
450
1133
723
1134
732
TCR 34
E7(11-19)
451
1135
738
1136
747
TCR 35
E7(11-19)
452
1137
756
1138
765
TCR 36
E7(11-19)
453
1139
772
1140
778
TCR 37
E7(11-19)
454
1141
784
1142
790
TCR 38
E7(11-19)
455
1143
796
1144
805
TCR 39
E7(11-19)
456
1145
812
1146
821
TCR 40
E7(11-19)
457
1147
827
1148
836
TCR 41
E7(11-19)
458
1149
842
1150
848
TCR 42
E7(11-19)
459
1151
854
1152
860
TCR 43
E7(11-19)
460
1153
866
1154
872
TCR 44
E7(11-19)
461
1155
878
1156
884
TCR 45
E7(11-19)
462
1157
892
1158
898
TCR 46
E7(11-19)
463
1159
905
1160
914
TCR 47
E7(11-19)
464
1161
922
1162
928
TCR 48
E7(11-19)
465
1163
934
1164
942
TCR 49
E7(11-19)
466
1165
948
1166
954
TCR 50
E7(11-19)
467
1167
960
1168
966
TCR 51
E7(11-19)
468
1169
972
1170
978
TCR 52
E7(11-19)
469
1171
984
1172
990
TCR 53
E7(11-19)
470
1173
996
1174
1005
TCR 54
E7(11-19)
471
1175
59
1176
63
TCR 55
E7(11-19)
472
1177
284
1178
286
Example 5: Expression and Antigen-Binding of Exemplary E6- and E7-Specific TCRs
Exemplary E6- and E7-specific T cell receptors (TCRs), identified as described in Example 4 above, were expressed in T cells and assessed for surface expression and antigen-specific binding, with or without CD8 interaction substantially as described in Example 2 above. Specifically, CD4+ Jurkat-derived cells that did not express endogenous TCR on their surfaces, that either had or had not been modified by introduction of exogenous CD8 (modification resulting in CD4+/CD8+ cells), were mixed in a 1:1 mixture for transfection with plasmid DNA encoding the TCRs, to assess CD8-independent binding activity of the TCRs. For transfection, the CD4+ and CD4+/CD8+ cell mixtures were transiently transfected with TCR-encoding plasmids and 48 hours after transfection, cells were assessed by flow cytometry for (1) binding of the target peptide in the context of an MHC molecule (HLA:A02:01) by staining with an E6(29-38) peptide- or an E7(11-19) peptide-MHC tetramer reagent, and/or (2) CD8+ independent binding of the target by co-staining the tetramer-labeled cells with an anti-CD8 antibody. Cells that had been mock transfected (mock) and cells expressing a reference TCR capable of binding to HLA-A2/E6(29-38) also were assessed in this study.
Exemplary results are shown in FIGS. 5A-5F (E6(29-38)-loaded tetramer binding) and FIGS. 6A-6F (E7 (11-19)-loaded tetramer binding). The percentage of cells in the indicated quadrants in flow cytometry plots shown in FIGS. 5A-5H and 6A-6H are also summarized below in Table 25 (flow cytometry plots showing E6(29) tetramer and CD8+ staining results for CD8+ cells from TCR-transfected compositions; FIGS. 5A-5C), Table 26 (flow cytometry plots showing results for E6(29)-specific TCR-transfected cell compositions; FIGS. 5D-5F) and Table 27 (flow cytometry plots showing results for E7(11)-specific TCRs; FIG. 6A-6F). Specifically, FIGS. 5A-5C depict flow cytometry plots for tetramer and CD8 staining in CD8+ populations; FIGS. 5D-5F and 6A-6F depict plots reflecting staining of CD8+ and CD8− populations.
TABLE 25
Percentage of cells present in each indicated quadrant
in Flow Cytometry Plots Shown in FIGS. 5A-5C
E6
E6
E6
E6
tet+/CD8−
tet+/CD8+
tet−/CD8+
tet−/CD8−
E6 TCRs
quadrant
quadrant
quadrant
quadrant
Mock
0.046
12.5
83.7
3.75
Reference
0.07
32
65.9
1.95
TCR
TCR 9
0.051
42.5
55.6
1.89
TCR 13
0.064
38.6
59.5
1.82
TCR 14
0.04
38.4
59.7
1.8
Mock
5.85E−03
4.44
88.9
6.64
Reference
0.16
40
57.9
1.93
TCR
TCR 17
0.17
34.7
63.6
1.53
TCR 18
0.045
50.4
47.7
1.86
TCR 21
0.22
51.6
46
2.18
TCR 22
0.14
51.2
47.3
1.38
TCR 23
0.18
43.6
54.1
2.14
TCR 24
0.13
29.1
66.2
4.51
TCR 27
0.02
24.5
73.5
1.96
TABLE 26
Percentage of cells present in each indicated
quadrant in flow cytometry plots in FIGS. 5D-5F
E6
E6
E6
E6
tet+/CD8−
tet+/CD8+
tet−/CD8+
tet−/CD8−
E6 TCRs
quadrant
quadrant
quadrant
quadrant
TCR 15
40.2
21.4
13.6
24.8
TCR 16
28.2
35.6
9.51
26.7
TCR 17
21.3
36.2
7.72
34.8
TCR 18
3.61
23.3
12
61.1
TCR 19
20.8
35.5
7.71
36
TCR 20
34.1
38.2
5.17
22.6
TCR 21
32.7
28.8
7.16
31.3
TCR 23
22.5
52.5
5.19
19.7
TCR 24
23.5
55
5.56
16
TCR 25
14.7
34
10.2
41.1
TCR 26
47.4
42.3
1.58
8.73
TCR 27
3.5
15.8
20.1
60.6
TCR 28
0.15
13.1
31.4
55.4
TCR 29
44.5
35.6
2
17.9
TCR 30
0.74
31
13.9
54.3
TABLE 27
Percentage of cells identified in each indicated
quadrant in flow cytometry plots in FIGS. 6A-6F
E7
E7
E7
E7
tet+/CD8−
tet+/CD8+
tet−/CD8+
tet−/CD8−
E7 TCRs
quadrant
quadrant
quadrant
quadrant
Mock
0.01
0.1
96.1
3.77
TCR 12
8.48E−03
1.89
96.2
1.86
TCR 12
0.001
18.6
78.6
2.82
TCR 31
0.042
4.52
21.1
74.3
TCR 32
33.5
25.3
7.53
33.7
TCR 33
14
22.6
12.8
50.6
TCR 34
26
26.3
6.85
40.9
TCR 35
7.18
14.5
35.1
43.2
TCR 36
16.7
23.4
25.4
34.5
TCR 37
19.5
25.5
22.7
32.2
TCR 38
5.44
15.7
33.3
45.5
TCR 39
2.61
12.3
37
48
TCR 40
1.37
7.84
42.4
48.4
TCR 41
2.41
6.07
43.6
47.9
TCR 42
1.65
1.21
39.5
57.4
TCR 43
1.88
3.82
37.6
56.7
TCR 44
1.43
2.96
39.9
55.7
TCR 45
16.9
22.4
19.5
41.3
TCR 46
1.21
1.27
38.9
58.6
TCR 47
0.71
1.98
40.6
56.7
TCR 48
1.29
5.36
37
56.4
TCR 49
3.06
5.54
27.2
64.3
TCR 50
0.25
3.28
30.7
65.8
TCR 51
2.06
5.7
27.5
64.7
TCR 53
0.43
3.35
28.7
67.5
TCR 54
11.3
9.66
21.2
57.6
TCR 54
0.63
2.75
48.3
48.3
TCR 55
0.28
1.45
50.4
47.9
As shown, the exemplary assessed TCRs were expressed on the surface of T cells and recognized HPV peptide in the context of MHC tetramers. In some cases, the binding was independent of CD8 co-receptor, as indicated by tetramer+ cells in the CD8− population in FIGS. 5D-5F (percentages listed in Table 26) and FIGS. 6A-6F (percentages listed in Table 27).
Example 6: Expression and Assessment of Exemplary Recombinant T Cell Receptors (TCRs) in Primary T Cells
Expression and function of exemplary recombinant E7-specific TCRs in primary human T cells was assessed.
Primary human CD4+ and CD8+ T cells were transduced with lentiviral preparations encoding TCR 16, specific for HPV 16 E6(29-38); and TCR 49, TCR 53 and TCR 37, each specific for HPV 16 E7(11-19) (described above in Example 4 above). Approximately 5×106 primary human CD4+ and CD8+ T cells were isolated by immunoaffinity-based selection from human peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. The cells were stimulated for 24 hours by culturing with an anti-CD3/anti-CD28 reagent in media containing human serum and cytokines, at 37° C. prior to lentiviral transduction. Stimulated cells were transduced with a lentiviral preparation encoding TCR 16, TCR 49, TCR 53 or TCR 37, or a mock transduction control (cells treated under the same conditions used for lentiviral transduction but without addition of lentivirus). The lentiviral constructs also contained sequences encoding EGFRt as a surrogate marker for transduction and expression, separated from the recombinant TCR encoding sequences by a sequence encoding a T2A ribosome skip sequence. Following transduction, the cells were cultured in media containing human serum and cytokines. On day 13 after transduction, the cells were assessed by flow cytometry for staining with an anti-CD3 antibody, an anti-CD8 antibody, and a HPV 16 E6(29-38)- or HPV16 E7(11-19)-peptide-MHC tetramer complex. (interferon-gamma (IFNγ) production was assessed following incubation of recombinant TCR-expressing cells with a squamous cell carcinoma cell line UPCI:SCC152 (ATCC® CRL-3240™), an antigen-specific target cell line which is HPV+, at an E:T ratio of 7.5:1 or 3.25:1 for TCR 16-expressing cells, and E:T ratio of 2.5:1 for TCR 49-, TCR 53- or TCR 37-expressing cells.
The results showed binding of the respective peptide-MHC tetramer complex specific for each TCR. TCR 16-expressing cells produced IFNγ at levels above background at both E:T ratios tested. CD8+ cells expressing TCR 49, TCR 53 or TCR 37 produced IFNγ at levels above background, and CD4+ cells expressing TCR 53 and TCR 37 produced IFNγ at levels above background, consistent with CD8-independent function of these TCRs in primary T cells. The results are consistent with expression, cell surface expression and antigen-specific function of the recombinant TCRs in primary T cells.
The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.
SEQUENCE TABLE
SEQ
ID
NO.
SEQUENCE
DESCRIPTION
1
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 14
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Full sequence
YLCASTFWGQRRTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATL
Cysteine-modified
VCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
Homo sapiens
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQ
AGDVEENPGPMEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTC
SFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYI
KGSQPEDSATYLCASQTGANNLFFGTGTRLTVIPYIQNPDPAVYQLRDSKSSDKS
VCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACA
NAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFN
LLMTLRLWSS
2
MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHVSLF
TCR 13
WYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDS
Full sequence
AVYLCASSPTGTERELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATL
Cysteine-modified
VCLATGFYPDHVELSWWV
Homo sapiens
NGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQF
(aa)
YGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLG
KATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQAGDVEENPGPMLLLL
VPVLEVIFTLGGTRAQSVTQLDSHVSVSEGTPVLLRCNYSSSYSPSLFWYVQHPN
KGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCVVRG
GKLIFGQGTELSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDS
DVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSC
DVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
3
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 12/ TCR 55
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Full sequence
CASTTRSSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Cysteine-modified
TGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQ
AGDVEENPGPMKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCT
YTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRI
ADTQTGDSAIYFCAVPSGATNKLIFGTGTLLAVQPNIQNPDPAVYQLRDSKSSDK
SVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFAC
ANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGF
NLLMTLRLWSS
4
GGCTCCGGCGCCACAAACTTTTCTCTGCTGAAGCAGGCAGGCGATGTGGAGG
TCR 14
AGAACCCTGGACCA
P2A
Artificial
(nt)
5
GGAAGCGGAGCCACCAACTTTTCCCTGCTGAAGCAGGCCGGCGATGTGGAG
TCR 13
GAGAATCCTGGCCCA
P2A
Artificial
(nt)
6
GGATCTGGAGCCACCAACTTCTCCCTGCTGAAGCAGGCCGGCGATGTGGAGG
TCR 12
AGAATCCTGGCCCA
P2A
Artificial
(nt)
7
ATGGGCATCCGGCTGCTGTGCAGAGTGGCCTTCTGTTTTCTGGCCGTGGGCCT
TCR 14 - Beta
GGTGGACGTGAAGGTGACCCAGAGCTCCCGGTATCTGGTGAAGAGAACAGG
Codon-optimized/
CGAGAAGGTGTTTCTGGAGTGCGTGCAGGACATGGATCACGAGAACATGTTC
cysteine-modified
TGGTACAGGCAGGATCCAGGCCTGGGCCTGAGACTGATCTATTTCAGCTACG
Homo sapiens
ATGTGAAGATGAAGGAGAAGGGCGACATCCCTGAGGGCTATTCTGTGAGCA
(nt)
GGGAGAAGAAGGAGCGGTTCAGCCTGATCCTGGAGTCCGCCTCTACCAACC
AGACATCTATGTACCTGTGCGCAAGCACCTTCTGGGGACAGAGGAGAACAG
AGGCCTTCTTTGGCCAGGGCACCAGGCTGACAGTGGTGGAGGACCTGAATAA
GGTGTTCCCCCCTGAGGTGGCCGTGTTTGAGCCATCCGAGGCCGAGATCTCT
CACACCCAGAAGGCCACCCTGGTGTGCCTGGCAACCGGCTTCTTTCCCGATC
ACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAGGTGCACTCTGGCGTGTG
CACAGACCCACAGCCCCTGAAGGAGCAGCCTGCCCTGAATGATAGCCGCTAT
TGTCTGTCTAGCAGGCTGCGCGTGTCCGCCACCTTTTGGCAGAACCCAAGGA
ATCACTTCCGCTGCCAGGTGCAGTTTTACGGCCTGTCCGAGAATGACGAGTG
GACCCAGGATAGGGCCAAGCCAGTGACACAGATCGTGTCTGCCGAGGCATG
GGGCAGAGCCGACTGTGGCTTCACCAGCGTGTCCTACCAGCAGGGCGTGCTG
AGCGCCACCATCCTGTATGAGATCCTGCTGGGCAAGGCCACACTGTACGCCG
TGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTGAAGCGGAAGGACTTC
8
ATGGGAACCAGGCTGCTGTGCTGGGTGGTGCTGGGCTTTCTGGGAACCGACC
TCR 13 - Beta
ACACAGGAGCAGGCGTGTCCCAGTCTCCAAGGTACAAGGTGGCCAAGAGAG
Codon-optimized/
GCCAGGATGTGGCCCTGAGATGTGACCCCATCTCCGGCCACGTGTCTCTGTT
cysteine-modified
CTGGTACCAGCAGGCCCTGGGACAGGGACCAGAGTTCCTGACATATTTTCAG
Homo sapiens
AACGAGGCCCAGCTGGATAAGAGCGGCCTGCCTTCCGACAGGTTCTTTGCAG
(nt)
AGCGCCCAGAGGGAAGCGTGTCCACCCTGAAGATCCAGAGGACACAGCAGG
AGGACTCCGCCGTGTACCTGTGCGCAAGCTCCCCTACCGGAACAGAGAGGG
AGCTGTTCTTTGGAGAGGGCAGCCGCCTGACCGTGCTGGAGGATCTGAAGAA
CGTGTTCCCCCCTGAGGTGGCCGTGTTTGAGCCTAGCGAGGCCGAGATCTCC
CACACCCAGAAGGCCACCCTGGTGTGCCTGGCAACCGGCTTCTATCCAGACC
ACGTGGAGCTGAGCTGGTGGGTGAACGGCAAGGAGGTGCACTCCGGCGTGT
GCACAGACCCACAGCCCCTGAAGGAGCAGCCCGCCCTGAATGATAGCCGCT
ACTGTCTGTCTAGCCGGCTGAGAGTGTCCGCCACCTTTTGGCAGAACCCTAG
GAATCACTTCCGCTGCCAGGTGCAGTTTTATGGCCTGTCCGAGAACGACGAG
TGGACCCAGGATCGGGCCAAGCCCGTGACACAGATCGTGTCTGCCGAGGCAT
GGGGCAGAGCCGATTGTGGCTTCACATCTGAGAGCTACCAGCAGGGCGTGCT
GTCCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTATGCC
GTGCTGGTGAGCGCCCTGGTGCTGATGGCCATGGTGAAGAGGAAGGACTCTA
GAGGA
9
ATGGACACCTGGCTGGTGTGCTGGGCCATCTTCAGCCTGCTGAAGGCAGGCC
TCR 12 - Beta
TGACCGAGCCTGAGGTGACCCAGACACCATCCCACCAGGTGACACAGATGG
Codon-optimized/
GCCAGGAAGTGATCCTGCGGTGCGTGCCTATCTCCAACCACCTGTACTTTTAT
cysteine-modified
TGGTACAGACAGATCCTGGGCCAGAAGGTGGAGTTTCTGGTGAGCTTCTACA
Homo sapiens
ACAATGAGATCAGCGAGAAGTCCGAGATCTTTGACGATCAGTTCTCTGTGGA
(nt)
GAGGCCCGACGGCAGCAACTTCACCCTGAAGATCCGCTCCACAAAGCTGGA
GGATTCTGCCATGTATTTCTGCGCCAGCACCACACGGAGCTCCTACGAGCAG
TATTTTGGCCCTGGCACCAGACTGACCGTGACAGAGGACCTGAAGAACGTGT
TCCCCCCTGAGGTGGCCGTGTTCGAGCCATCTGAGGCCGAGATCAGCCACAC
CCAGAAGGCCACCCTGGTGTGCCTGGCAACCGGCTTCTACCCCGATCACGTG
GAGCTGAGCTGGTGGGTGAACGGCAAGGAGGTGCACTCCGGCGTGTGCACA
GACCCACAGCCCCTGAAGGAGCAGCCTGCCCTGAATGATAGCAGATACTGTC
TGTCTAGCCGGCTGAGAGTGTCCGCCACCTTCTGGCAGAACCCAAGGAATCA
CTTTCGCTGCCAGGTGCAGTTCTATGGCCTGTCTGAGAACGACGAGTGGACC
CAGGATAGGGCCAAGCCAGTGACACAGATCGTGAGCGCCGAGGCATGGGGC
AGAGCCGATTGTGGCTTTACAAGCGAGTCCTATCAGCAGGGCGTGCTGTCCG
CCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTATGCCGTGCT
GGTGTCTGCCCTGGTGCTGATGGCCATGGTGAAGAGGAAGGACTCCAGAGG
A
10
ATGGAGAAGAATCCTCTGGCCGCCCCACTGCTGATCCTGTGGTTCCACCTGG
TCR 14 - Alpha
ACTGCGTGTCCTCTATCCTGAATGTGGAGCAGAGCCCACAGTCCCTGCACGT
Codon-optimized/
GCAGGAGGGCGATAGCACCAACTTCACATGTTCCTTTCCTAGCTCCAACTTCT
cysteine-modified
ACGCCCTGCACTGGTACCGGTGGGAGACAGCCAAGAGCCCAGAGGCCCTGT
Homo sapiens
TCGTGATGACACTGAACGGCGACGAGAAGAAGAAGGGCAGAATCAGCGCCA
(nt)
CCCTGAATACAAAGGAGGGCTACTCCTATCTGTACATCAAGGGCAGCCAGCC
CGAGGATTCCGCCACCTACCTGTGCGCCTCCCAGACAGGCGCCAACAATCTG
TTCTTTGGCACCGGCACAAGGCTGACCGTGATCCCTTATATCCAGAACCCAG
ACCCTGCCGTGTACCAGCTGAGGGACTCTAAGTCTAGCGATAAGAGCGTGTG
CCTGTTCACCGACTTTGATTCTCAGACAAACGTGAGCCAGAGCAAGGACAGC
GACGTGTACATCACCGACAAGTGCGTGCTGGATATGAGAAGCATGGACTTTA
AGTCCAACTCTGCCGTGGCCTGGTCTAATAAGAGCGATTTCGCCTGCGCCAA
CGCCTTTAACAATTCCATCATCCCCGAGGATACATTCTTTCCATCTCCCGAGT
CCTCTTGTGACGTGAAGCTGGTGGAGAAGAGCTTCGAGACAGATACAAACCT
GAATTTTCAGAACCTGAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTG
GCCGGCTTCAATCTGCTGATGACCCTGAGACTGTGGAGCTCCTGA
11
ATGCTGCTGCTGCTGGTGCCAGTGCTGGAAGTGATCTTCACCCTGGGAGGAA
TCR 13 - Alpha
CAAGGGCACAGTCTGTGACCCAGCTGGACAGCCACGTGTCCGTGTCTGAGGG
Codon-optimized/
CACACCCGTGCTGCTGAGATGCAACTACTCCTCTAGCTATAGCCCCTCCCTGT
cysteine-modified
TTTGGTACGTGCAGCACCCTAATAAGGGCCTGCAGCTGCTGCTGAAGTATAC
Homo sapiens
CTCCGCCGCCACACTGGTGAAGGGCATCAATGGCTTCGAGGCCGAGTTTAAG
(nt)
AAGAGCGAGACAAGCTTCCACCTGACAAAGCCTTCCGCCCACATGTCTGACG
CCGCCGAGTACTTTTGCGTGGTGCGGGGAGGCAAGCTGATCTTCGGACAGGG
AACCGAGCTGAGCGTGAAGCCAAACATCCAGAATCCCGATCCTGCCGTGTAT
CAGCTGCGCGACTCCAAGTCCTCTGATAAGAGCGTGTGCCTGTTCACCGACT
TTGATTCTCAGACAAACGTGTCTCAGAGCAAGGACAGCGACGTGTACATCAC
CGACAAGTGCGTGCTGGATATGCGGAGCATGGACTTTAAGTCCAACTCTGCC
GTGGCCTGGTCTAATAAGAGCGATTTCGCCTGCGCCAATGCCTTTAACAATT
CCATCATCCCCGAGGATACATTCTTTCCATCTCCCGAGAGCTCCTGTGACGTG
AAGCTGGTGGAGAAGAGCTTCGAGACAGATACAAACCTGAATTTTCAGAAC
CTGAGCGTGATCGGCTTCAGGATCCTGCTGCTGAAGGTGGCCGGCTTCAATC
TGCTGATGACCCTGCGCCTGTGGTCTAGCTGA
12
ATGAAGACATTTGCCGGCTTCTCTTTTCTGTTCCTGTGGCTGCAGCTGGATTG
TCR 12 - Alpha
CATGAGCAGGGGCGAGGACGTGGAGCAGAGCCTGTTCCTGTCCGTGCGCGA
Codon-optimized/
GGGCGATTCCTCTGTGATCAACTGTACCTACACAGACAGCTCCTCTACCTATC
cysteine-modified
TGTACTGGTATAAGCAGGAGCCAGGAGCAGGCCTGCAGCTGCTGACCTATAT
Homo sapiens
CTTTTCCAACATGGACATGAAGCAGGATCAGCGGCTGACAGTGCTGCTGAAT
(nt)
AAGAAGGACAAGCACCTGAGCCTGAGAATCGCTGACACCCAGACAGGCGAT
TCCGCCATCTACTTCTGCGCCGTGCCCTCTGGCGCCACCAATAAGCTGATCTT
TGGAACCGGCACACTGCTGGCAGTGCAGCCTAACATCCAGAATCCCGATCCT
GCCGTGTACCAGCTGCGGGACAGCAAGAGCTCCGATAAGTCCGTGTGCCTGT
TTACCGACTTCGATTCTCAGACAAACGTGTCTCAGAGCAAGGACAGCGACGT
GTACATCACCGACAAGTGCGTGCTGGATATGCGGAGCATGGACTTCAAGTCC
AACTCTGCCGTGGCCTGGTCTAATAAGAGCGACTTTGCCTGCGCCAATGCCT
TCAACAATTCCATCATCCCCGAGGATACATTCTTTCCATCTCCCGAGTCTAGC
TGTGACGTGAAGCTGGTGGAGAAGAGCTTCGAGACAGATACAAACCTGAAT
TTCCAGAACCTGTCTGTGATCGGCTTTAGGATCCTGCTGCTGAAGGTGGCCG
GCTTTAATCTGCTGATGACCCTGCGCCTGTGGTCCTCTTGA
13
ATGGGCATCCGGCTGCTGTGCAGAGTGGCCTTCTGTTTTCTGGCCGTGGGCCT
TCR 14 Codon-
GGTGGACGTGAAGGTGACCCAGAGCTCCCGGTATCTGGTGAAGAGAACAGG
optimized/ cysteine-
CGAGAAGGTGTTTCTGGAGTGCGTGCAGGACATGGATCACGAGAACATGTTC
modified full sequence
TGGTACAGGCAGGATCCAGGCCTGGGCCTGAGACTGATCTATTTCAGCTACG
Homo sapiens
ATGTGAAGATGAAGGAGAAGGGCGACATCCCTGAGGGCTATTCTGTGAGCA
(nt)
GGGAGAAGAAGGAGCGGTTCAGCCTGATCCTGGAGTCCGCCTCTACCAACC
AGACATCTATGTACCTGTGCGCAAGCACCTTCTGGGGACAGAGGAGAACAG
AGGCCTTCTTTGGCCAGGGCACCAGGCTGACAGTGGTGGAGGACCTGAATAA
GGTGTTCCCCCCTGAGGTGGCCGTGTTTGAGCCATCCGAGGCCGAGATCTCT
CACACCCAGAAGGCCACCCTGGTGTGCCTGGCAACCGGCTTCTTTCCCGATC
ACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAGGTGCACTCTGGCGTGTG
CACAGACCCACAGCCCCTGAAGGAGCAGCCTGCCCTGAATGATAGCCGCTAT
TGTCTGTCTAGCAGGCTGCGCGTGTCCGCCACCTTTTGGCAGAACCCAAGGA
ATCACTTCCGCTGCCAGGTGCAGTTTTACGGCCTGTCCGAGAATGACGAGTG
GACCCAGGATAGGGCCAAGCCAGTGACACAGATCGTGTCTGCCGAGGCATG
GGGCAGAGCCGACTGTGGCTTCACCAGCGTGTCCTACCAGCAGGGCGTGCTG
AGCGCCACCATCCTGTATGAGATCCTGCTGGGCAAGGCCACACTGTACGCCG
TGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTGAAGCGGAAGGACTTCGG
CTCCGGCGCCACAAACTTTTCTCTGCTGAAGCAGGCAGGCGATGTGGAGGAG
AACCCTGGACCAATGGAGAAGAATCCTCTGGCCGCCCCACTGCTGATCCTGT
GGTTCCACCTGGACTGCGTGTCCTCTATCCTGAATGTGGAGCAGAGCCCACA
GTCCCTGCACGTGCAGGAGGGCGATAGCACCAACTTCACATGTTCCTTTCCT
AGCTCCAACTTCTACGCCCTGCACTGGTACCGGTGGGAGACAGCCAAGAGCC
CAGAGGCCCTGTTCGTGATGACACTGAACGGCGACGAGAAGAAGAAGGGCA
GAATCAGCGCCACCCTGAATACAAAGGAGGGCTACTCCTATCTGTACATCAA
GGGCAGCCAGCCCGAGGATTCCGCCACCTACCTGTGCGCCTCCCAGACAGGC
GCCAACAATCTGTTCTTTGGCACCGGCACAAGGCTGACCGTGATCCCTTATA
TCCAGAACCCAGACCCTGCCGTGTACCAGCTGAGGGACTCTAAGTCTAGCGA
TAAGAGCGTGTGCCTGTTCACCGACTTTGATTCTCAGACAAACGTGAGCCAG
AGCAAGGACAGCGACGTGTACATCACCGACAAGTGCGTGCTGGATATGAGA
AGCATGGACTTTAAGTCCAACTCTGCCGTGGCCTGGTCTAATAAGAGCGATT
TCGCCTGCGCCAACGCCTTTAACAATTCCATCATCCCCGAGGATACATTCTTT
CCATCTCCCGAGTCCTCTTGTGACGTGAAGCTGGTGGAGAAGAGCTTCGAGA
CAGATACAAACCTGAATTTTCAGAACCTGAGCGTGATCGGCTTCCGGATCCT
GCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGAGACTGTGGAGC
TCCTGA
14
ATGGGAACCAGGCTGCTGTGCTGGGTGGTGCTGGGCTTTCTGGGAACCGACC
TCR 13 Codon-
ACACAGGAGCAGGCGTGTCCCAGTCTCCAAGGTACAAGGTGGCCAAGAGAG
optimized/ cysteine-
GCCAGGATGTGGCCCTGAGATGTGACCCCATCTCCGGCCACGTGTCTCTGTT
modified full sequence
CTGGTACCAGCAGGCCCTGGGACAGGGACCAGAGTTCCTGACATATTTTCAG
Homo sapiens
AACGAGGCCCAGCTGGATAAGAGCGGCCTGCCTTCCGACAGGTTCTTTGCAG
(nt)
AGCGCCCAGAGGGAAGCGTGTCCACCCTGAAGATCCAGAGGACACAGCAGG
AGGACTCCGCCGTGTACCTGTGCGCAAGCTCCCCTACCGGAACAGAGAGGG
AGCTGTTCTTTGGAGAGGGCAGCCGCCTGACCGTGCTGGAGGATCTGAAGAA
CGTGTTCCCCCCTGAGGTGGCCGTGTTTGAGCCTAGCGAGGCCGAGATCTCC
CACACCCAGAAGGCCACCCTGGTGTGCCTGGCAACCGGCTTCTATCCAGACC
ACGTGGAGCTGAGCTGGTGGGTGAACGGCAAGGAGGTGCACTCCGGCGTGT
GCACAGACCCACAGCCCCTGAAGGAGCAGCCCGCCCTGAATGATAGCCGCT
ACTGTCTGTCTAGCCGGCTGAGAGTGTCCGCCACCTTTTGGCAGAACCCTAG
GAATCACTTCCGCTGCCAGGTGCAGTTTTATGGCCTGTCCGAGAACGACGAG
TGGACCCAGGATCGGGCCAAGCCCGTGACACAGATCGTGTCTGCCGAGGCAT
GGGGCAGAGCCGATTGTGGCTTCACATCTGAGAGCTACCAGCAGGGCGTGCT
GTCCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTATGCC
GTGCTGGTGAGCGCCCTGGTGCTGATGGCCATGGTGAAGAGGAAGGACTCTA
GAGGAGGAAGCGGAGCCACCAACTTTTCCCTGCTGAAGCAGGCCGGCGATG
TGGAGGAGAATCCTGGCCCAATGCTGCTGCTGCTGGTGCCAGTGCTGGAAGT
GATCTTCACCCTGGGAGGAACAAGGGCACAGTCTGTGACCCAGCTGGACAG
CCACGTGTCCGTGTCTGAGGGCACACCCGTGCTGCTGAGATGCAACTACTCC
TCTAGCTATAGCCCCTCCCTGTTTTGGTACGTGCAGCACCCTAATAAGGGCCT
GCAGCTGCTGCTGAAGTATACCTCCGCCGCCACACTGGTGAAGGGCATCAAT
GGCTTCGAGGCCGAGTTTAAGAAGAGCGAGACAAGCTTCCACCTGACAAAG
CCTTCCGCCCACATGTCTGACGCCGCCGAGTACTTTTGCGTGGTGCGGGGAG
GCAAGCTGATCTTCGGACAGGGAACCGAGCTGAGCGTGAAGCCAAACATCC
AGAATCCCGATCCTGCCGTGTATCAGCTGCGCGACTCCAAGTCCTCTGATAA
GAGCGTGTGCCTGTTCACCGACTTTGATTCTCAGACAAACGTGTCTCAGAGC
AAGGACAGCGACGTGTACATCACCGACAAGTGCGTGCTGGATATGCGGAGC
ATGGACTTTAAGTCCAACTCTGCCGTGGCCTGGTCTAATAAGAGCGATTTCG
CCTGCGCCAATGCCTTTAACAATTCCATCATCCCCGAGGATACATTCTTTCCA
TCTCCCGAGAGCTCCTGTGACGTGAAGCTGGTGGAGAAGAGCTTCGAGACAG
ATACAAACCTGAATTTTCAGAACCTGAGCGTGATCGGCTTCAGGATCCTGCT
GCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGCGCCTGTGGTCTAGC
TGA
15
ATGGACACCTGGCTGGTGTGCTGGGCCATCTTCAGCCTGCTGAAGGCAGGCC
TCR 12
TGACCGAGCCTGAGGTGACCCAGACACCATCCCACCAGGTGACACAGATGG
Codon-optimized/
GCCAGGAAGTGATCCTGCGGTGCGTGCCTATCTCCAACCACCTGTACTTTTAT
cysteine-modified full
TGGTACAGACAGATCCTGGGCCAGAAGGTGGAGTTTCTGGTGAGCTTCTACA
sequence
ACAATGAGATCAGCGAGAAGTCCGAGATCTTTGACGATCAGTTCTCTGTGGA
Homo sapiens
GAGGCCCGACGGCAGCAACTTCACCCTGAAGATCCGCTCCACAAAGCTGGA
(nt)
GGATTCTGCCATGTATTTCTGCGCCAGCACCACACGGAGCTCCTACGAGCAG
TATTTTGGCCCTGGCACCAGACTGACCGTGACAGAGGACCTGAAGAACGTGT
TCCCCCCTGAGGTGGCCGTGTTCGAGCCATCTGAGGCCGAGATCAGCCACAC
CCAGAAGGCCACCCTGGTGTGCCTGGCAACCGGCTTCTACCCCGATCACGTG
GAGCTGAGCTGGTGGGTGAACGGCAAGGAGGTGCACTCCGGCGTGTGCACA
GACCCACAGCCCCTGAAGGAGCAGCCTGCCCTGAATGATAGCAGATACTGTC
TGTCTAGCCGGCTGAGAGTGTCCGCCACCTTCTGGCAGAACCCAAGGAATC A
CTTTCGCTGCCAGGTGCAGTTCTATGGCCTGTCTGAGAACGACGAGTGGACC
CAGGATAGGGCCAAGCCAGTGACACAGATCGTGAGCGCCGAGGCATGGGGC
AGAGCCGATTGTGGCTTTACAAGCGAGTCCTATCAGCAGGGCGTGCTGTCCG
CCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACACTGTATGCCGTGCT
GGTGTCTGCCCTGGTGCTGATGGCCATGGTGAAGAGGAAGGACTCCAGAGG
AGGATCTGGAGCCACCAACTTCTCCCTGCTGAAGCAGGCCGGCGATGTGGAG
GAGAATCCTGGCCCAATGAAGACATTTGCCGGCTTCTCTTTTCTGTTCCTGTG
GCTGCAGCTGGATTGCATGAGCAGGGGCGAGGACGTGGAGCAGAGCCTGTT
CCTGTCCGTGCGCGAGGGCGATTCCTCTGTGATCAACTGTACCTACACAGAC
AGCTCCTCTACCTATCTGTACTGGTATAAGCAGGAGCCAGGAGCAGGCCTGC
AGCTGCTGACCTATATCTTTTCCAACATGGACATGAAGCAGGATCAGCGGCT
GACAGTGCTGCTGAATAAGAAGGACAAGCACCTGAGCCTGAGAATCGCTGA
CACCCAGACAGGCGATTCCGCCATCTACTTCTGCGCCGTGCCCTCTGGCGCC
ACCAATAAGCTGATCTTTGGAACCGGCACACTGCTGGCAGTGCAGCCTAACA
TCCAGAATCCCGATCCTGCCGTGTACCAGCTGCGGGACAGCAAGAGCTCCGA
TAAGTCCGTGTGCCTGTTTACCGACTTCGATTCTCAGACAAACGTGTCTCAGA
GCAAGGACAGCGACGTGTACATCACCGACAAGTGCGTGCTGGATATGCGGA
GCATGGACTTCAAGTCCAACTCTGCCGTGGCCTGGTCTAATAAGAGCGACTT
TGCCTGCGCCAATGCCTTCAACAATTCCATCATCCCCGAGGATACATTCTTTC
CATCTCCCGAGTCTAGCTGTGACGTGAAGCTGGTGGAGAAGAGCTTCGAGAC
AGATACAAACCTGAATTTCCAGAACCTGTCTGTGATCGGCTTTAGGATCCTG
CTGCTGAAGGTGGCCGGCTTTAATCTGCTGATGACCCTGCGCCTGTGGTCCTC
TTGA
16
ATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCT
TCR 14 - Beta
CGTAGATGTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGG
Native
AGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCATGAAAATATGTTC
Homo sapiens
TGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCTATTTCTCATATG
(nt)
ATGTTAAAATGAAAGAAAAAGGAGATATTCCTGAGGGGTACAGTGTCTCTA
GAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCCAGCACCAACCA
GACATCTATGTACCTCTGTGCCAGCACCTTCTGGGGACAGCGAAGGACTGAA
GCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAGGACCTGAACAAGG
TGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCA
CACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCTGACCAC
GTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGC
ACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACT
GCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAA
CCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGG
ACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGG
GGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGT
CTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGT
GCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTCTGA
17
ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGACAGATC
TCR 13 - Beta
ACACAGGTGCTGGAGTCTCCCAGTCCCCTAGGTACAAAGTCGCAAAGAGAG
Native
GACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCATGTATCCCTTTTT
Homo sapiens
TGGTACCAACAGGCCCTGGGGCAGGGGCCAGAGTTTCTGACTTATTTCCAGA
(nt)
ATGAAGCTCAACTAGACAAATCGGGGCTGCCCAGTGATCGCTTCTTTGCAGA
AAGGCCTGAGGGATCCGTCTCCACTCTGAAGATCCAGCGCACACAGCAGGA
GGACTCCGCCGTGTATCTCTGTGCCAGCAGCCCGACAGGGACTGAGAGGGA
GCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAGGACCTGAAAAAC
GTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCC
ACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCA
CGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAG
CACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATAC
TGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCA
ACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTG
GACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTG
GGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTG
TCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGT
GCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGA
GGCTAG
18
AQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSY
TCR 3 - Alpha
DEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMREGRGFKTIFGAGT
Native
RLFVKANIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKT
Homo sapiens
VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPADTFFPSPESSCDVKLVEKS
(aa)
FETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
19
AQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSY
TCR 3 - Alpha
DEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMREGRGFKTIFGAGT
Cysteine-modified
RLFVKANIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKC
Homo sapiens
VLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPADTFFPSPESSCDVKLVEKS
(aa)
FETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
20
ATGTCACTTTCTAGCCTGCTGAAGGTGGTCACAGCTTCACTGTGGCTAGGAC
TCR 3 - Alpha
CTGGCATTGCCCAGAAGATAACTCAAACCCAACCAGGAATGTTCGTGCAGGA
Native
AAAGGAGGCTGTGACTCTGGACTGCACATATGACACCAGTGATCAAAGTTAT
Homo sapiens
GGTCTCTTCTGGTACAAGCAGCCCAGCAGTGGGGAAATGATTTTTCTTATTTA
(nt)
TCAGGGGTCTTATGACGAGCAAAATGCAACAGAAGGTCGCTACTCATTGAAT
TTCCAGAAGGCAAGAAAATCCGCCAACCTTGTCATCTCCGCTTCACAACTGG
GGGACTCAGCAATGTATTTCTGTGCAATGAGAGAGGGGCGAGGCTTCAAAA
CTATCTTTGGAGCAGGAACAAGACTATTTGTTAAAGCAAATATCCAGAAGCC
TGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTC
TGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTC
TGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTC
AAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAA
ACGCCTTCAACAACAGCATTATTCCAGCAGACACCTTCTTCCCCAGCCCAGA
AAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAAC
CTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGT
GGCCGGGTTTAATCTGCTCATGACGCTGCGGCTG
21
ATGTCACTTTCTAGCCTGCTGAAGGTGGTCACAGCTTCACTGTGGCTAGGAC
TCR 3 - Alpha
CTGGCATTGCCCAGAAGATAACTCAAACCCAACCAGGAATGTTCGTGCAGGA
Codon-optimized/
AAAGGAGGCTGTGACTCTGGACTGCACATATGACACCAGTGATCAAAGTTAT
cysteine-modified
GGTCTCTTCTGGTACAAGCAGCCCAGCAGTGGGGAAATGATTTTTCTTATTTA
Homo sapiens
TCAGGGGTCTTATGACGAGCAAAATGCAACAGAAGGTCGCTACTCATTGAAT
(nt)
TTCCAGAAGGCAAGAAAATCCGCCAACCTTGTCATCTCCGCTTCACAACTGG
GGGACTCAGCAATGTATTTCTGTGCAATGAGAGAGGGGCGAGGCTTCAAAA
CTATCTTTGGAGCAGGAACAAGACTATTTGTTAAAGCAAATATCCAGAAGCC
TGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTC
TGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTC
TGATGTGTATATCACAGACAAATGTGTGCTAGACATGAGGTCTATGGACTTC
AAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAA
ACGCCTTCAACAACAGCATTATTCCAGCAGACACCTTCTTCCCCAGCCCAGA
AAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAAC
CTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGT
GGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCTTCC
22
GAGVSQSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQNEA
TCR 3 - Beta
QLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSHLAGFTGELFFGE
Native
GSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV
Homo sapiens
NGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY
(aa)
GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGK
ATLYAVLVSALVLMAMVKRKDSRG
23
GAGVSQSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQNEA
TCR 3 - Beta
QLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSHLAGFTGELFFGE
Cysteine-modified
GSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV
Homo sapiens
NGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQF
(aa)
YGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLG
KATLYAVLVSALVLMAMVKRKDSRG
24
ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGACAGATC
TCR 3 - Beta
ACACAGGTGCTGGAGTCTCCCAGTCCCCTAGGTACAAAGTCGCAAAGAGAG
Native
GACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCATGTATCCCTTTTT
Homo sapiens
TGGTACCAACAGGCCCTGGGGCAGGGGCCAGAGTTTCTGACTTATTTCCAGA
(nt)
ATGAAGCTCAACTAGACAAATCGGGGCTGCCCAGTGATCGCTTCTTTGCAGA
AAGGCCTGAGGGATCCGTCTCCACTCTGAAGATCCAGCGCACACAGCAGGA
GGACTCCGCCGTGTATCTCTGTGCCAGCAGCCACCTCGCCGGGTTCACCGGG
GAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAGGACCTGAAAA
ACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTC
CCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGAC
CACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTC
AGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGAT
ACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCG
CAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAG
TGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCT
GGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCT
GTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCC
GTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCA
GAGGC
25
ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGACAGATC
TCR 3 - Beta
ACACAGGTGCTGGAGTCTCCCAGTCCCCTAGGTACAAAGTCGCAAAGAGAG
Codon-optimized/
GACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCATGTATCCCTTTTT
cysteine-modified
TGGTACCAACAGGCCCTGGGGCAGGGGCCAGAGTTTCTGACTTATTTCCAGA
Homo sapiens
ATGAAGCTCAACTAGACAAATCGGGGCTGCCCAGTGATCGCTTCTTTGCAGA
(nt)
AAGGCCTGAGGGATCCGTCTCCACTCTGAAGATCCAGCGCACACAGCAGGA
GGACTCCGCCGTGTATCTCTGTGCCAGCAGCCACCTCGCCGGGTTCACCGGG
GAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAGGACCTGAAAA
ACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTC
CCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGAC
CACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTC
TGTACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGAT
ACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCG
CAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAG
TGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCT
GGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCT
GTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCC
GTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCA
GAGGC
26
GCGGCCGCCACCATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCC
TCR 3
TAGGGACAGATCACACAGGTGCTGGAGTCTCCCAGTCCCCTAGGTACAAAGT
Codon-optimized/
CGCAAAGAGAGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCAT
cysteine-modified full
GTATCCCTTTTTTGGTACCAACAGGCCCTGGGGCAGGGGCCAGAGTTTCTGA
sequence
CTTATTTCCAGAATGAAGCTCAACTAGACAAATCGGGGCTGCCCAGTGATCG
Homo sapiens
CTTCTTTGCAGAAAGGCCTGAGGGATCCGTCTCCACTCTGAAGATCCAGCGC
(nt)
ACACAGCAGGAGGACTCCGCCGTGTATCTCTGTGCCAGCAGCCACCTCGCCG
GGTTCACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGA
GGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAA
GCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCT
TCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGC
ACAGTGGGGTCTGTACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAA
TGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGG
CAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGG
AGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCA
GCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCA
GCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCC
ACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGA
GAAAGGATTCCAGAGGCGGATCCGGAGCTACCAACTTCTCTCTGCTGAAACA
GGCAGGCGATGTGGAGGAAAATCCTGGGCCAATGTCACTTTCTAGCCTGCTG
AAGGTGGTCACAGCTTCACTGTGGCTAGGACCTGGCATTGCCCAGAAGATAA
CTCAAACCCAACCAGGAATGTTCGTGCAGGAAAAGGAGGCTGTGACTCTGG
ACTGCACATATGACACCAGTGATCAAAGTTATGGTCTCTTCTGGTACAAGCA
GCCCAGCAGTGGGGAAATGATTTTTCTTATTTATCAGGGGTCTTATGACGAG
CAAAATGCAACAGAAGGTCGCTACTCATTGAATTTCCAGAAGGCAAGAAAA
TCCGCCAACCTTGTCATCTCCGCTTCACAACTGGGGGACTCAGCAATGTATTT
CTGTGCAATGAGAGAGGGGCGAGGCTTCAAAACTATCTTTGGAGCAGGAAC
AAGACTATTTGTTAAAGCAAATATCCAGAAGCCTGACCCTGCCGTGTACCAG
CTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGA
TTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGAC
AAATGTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGG
CCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCAT
TATTCCAGCAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAG
CTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGT
CAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTC
ATGACGCTGCGGCTGTGGTCTTCCTAAGGCGCGCC
27
MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHVSLF
TCR 3
WYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDS
Full sequence
AVYLCASSHLAGFTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKA
Cysteine-modified
TLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRL
Homo sapiens
RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCG
(aa)
FTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNF
SLLKQAGDVEENPGPMSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAV
TLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARK
SANLVISASQLGDSAMYFCAMREGRGFKTIFGAGTRLFVKANIQKPDPAVYQLR
DSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWS
NKSDFACANAFNNSIIPADTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRIL
LLKVAGFNLLMTLRLWSS
28
DAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNV
TCR 4 - (E6)29 alpha
NNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILLVIRGTSYGKLTFGQGTILT
Native
VHPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLD
Homo sapiens
MRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFET
(aa)
DTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
29
DAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNV
TCR 4 - (E6)29 alpha
NNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILLVIRGTSYGKLTFGQGTILT
Cysteine-modified
VHPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLD
Homo sapiens
MRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFET
(aa)
DTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
30
ATGAAGTTGGTGACAAGCATTACTGTACTCCTATCTTTGGGTATTATGGGTGA
TCR 4 - (E6)29 alpha
TGCTAAGACCACACAGCCAAATTCAATGGAGAGTAACGAAGAAGAGCCTGT
Native
TCACTTGCCTTGTAACCACTCCACAATCAGTGGAACTGATTACATACATTGGT
Homo sapiens
ATCGACAGCTTCCCTCCCAGGGTCCAGAGTACGTGATTCATGGTCTTACAAG
(nt)
CAATGTGAACAACAGAATGGCCTCTCTGGCAATCGCTGAAGACAGAAAGTC
CAGTACCTTGATCCTGCACCGTGCTACCTTGAGAGATGCTGCTGTGTACTACT
GCATCCTACTGGTAATCCGTGGTACTAGCTATGGAAAGCTGACATTTGGACA
AGGGACCATCTTGACTGTCCATCCAAATATCCAGAACCCTGACCCTGCCGTG
TACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCG
ATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATC
ACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGT
GCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACA
ACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGA
TGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAA
AACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTA
ATCTGCTCATGACGCTGCGGCTG
31
ATGAAACTGGTGACCAGCATCACAGTCCTGCTGTCCCTGGGAATTATGGGCG
TCR 4 - (E6)29 alpha
ACGCCAAGACCACACAGCCTAACTCTATGGAGAGTAATGAGGAAGAGCCTG
Codon-optimized/
TGCACCTGCCATGTAACCATTCAACTATCAGCGGCACCGATTACATTCACTG
cysteine-modified
GTATCGGCAGCTGCCCTCCCAGGGACCTGAATACGTGATCCATGGCCTGACC
Homo sapiens
TCAAATGTCAACAATCGCATGGCTAGCCTGGCTATCGCAGAGGACCGAAAGT
(nt)
CAAGCACCCTGATTCTGCACCGAGCCACACTGCGAGATGCAGCCGTGTACTA
TTGCATCCTGCTGGTCATTAGAGGGACCAGCTACGGAAAACTGACATTTGGC
CAGGGGACTATCCTGACCGTGCATCCTAACATTCAGAATCCCGACCCTGCCG
TGTATCAGCTGAGGGACTCTAAGTCCTCTGATAAAAGCGTGTGCCTGTTCAC
TGACTTTGATTCCCAGACCAACGTGTCCCAGTCTAAGGACTCTGACGTGTAC
ATCACAGACAAATGCGTCCTGGATATGCGCAGCATGGACTTCAAGAGTAACT
CAGCCGTGGCTTGGTCCAACAAGTCTGATTTCGCATGCGCCAACGCTTTTAA
CAACAGTATCATCCCAGAAGATACCTTCTTTCCATCACCCGAGAGTTCATGT
GACGTGAAGCTGGTCGAAAAATCTTTCGAGACTGATACCAACCTGAATTTTC
AGAACCTGAGTGTGATCGGGTTCAGGATTCTGCTGCTGAAGGTCGCCGGATT
CAATCTGCTGATGACACTGCGCCTGTGGAGCTCC
32
DTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQ
TCR 4 - (E6)29 Beta
LEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSPGGGNTEAFFGQGT
Native
RLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNG
Homo sapiens
KEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGL
(aa)
SENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKAT
LYAVLVSALVLMAMVKRKDF
33
DTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQ
TCR 4 - (E6)29 Beta
LEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSPGGGNTEAFFGQGT
Cysteine-modified
RLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNG
Homo sapiens
KEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGL
(aa)
SENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKAT
LYAVLVSALVLMAMVKRKDF
34
ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATC
TCR 4 - (E6)29 Beta
ACGCAGATACTGGAGTCTCCCAGGACCCCAGACACAAGATCACAAAGAGGG
Native
GACAGAATGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTA
Homo sapiens
TTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAG
(nt)
AATGAAGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAG
AGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGG
GGACTCGGCCATGTATCTCTGTGCCAGCAGCCCCGGCGGGGGGAACACTGAA
GCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAGGACCTGAACAAGG
TGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCA
CACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCTGACCAC
GTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGC
ACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACT
GCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAA
CCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGG
ACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGG
GGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGT
CTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGT
GCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC
35
ATGGGGACTAGCCTGCTGTGCTGGATGGCACTGTGCCTGCTGGGAGCAGACC
TCR 4 - (E6)29 Beta
ACGCAGATACCGGAGTGAGCCAGGACCCAAGACATAAGATCACAAAAAGGG
Codon-optimized/
GCCAGAACGTGACTTTTAGATGCGATCCCATTAGCGAACACAATAGACTGTA
cysteine-modified
CTGGTATAGGCAGACACTGGGACAGGGACCAGAGTTCCTGACTTACTTTCAG
Homo sapiens
AACGAAGCTCAGCTGGAGAAGAGTCGCCTGCTGTCAGACCGGTTCAGCGCC
(nt)
GAGCGACCAAAAGGCTCTTTCAGTACACTGGAAATCCAGCGAACTGAGCAG
GGGGATTCCGCCATGTATCTGTGCGCTAGCTCCCCAGGAGGAGGAAACACCG
AAGCCTTCTTTGGACAGGGCACACGGCTGACTGTGGTCGAGGACCTGAATAA
GGTGTTCCCCCCTGAAGTGGCCGTCTTTGAGCCTTCCGAAGCTGAGATTTCTC
ACACCCAGAAAGCCACCCTGGTGTGCCTGGCAACAGGCTTCTTTCCAGATCA
CGTGGAACTGAGCTGGTGGGTCAACGGAAAGGAGGTGCATAGCGGCGTCTG
CACTGACCCACAGCCCCTGAAAGAGCAGCCCGCACTGAATGATAGCAGGTA
CTGCCTGTCTAGTCGGCTGAGAGTGTCCGCCACCTTTTGGCAGAACCCTAGG
AATCATTTCCGCTGTCAGGTGCAGTTTTATGGCCTGTCCGAAAACGACGAGT
GGACTCAGGATCGGGCCAAGCCCGTGACCCAGATCGTCTCTGCAGAAGCCTG
GGGCAGAGCTGACTGCGGGTTCACCTCAGTGAGCTACCAGCAGGGAGTCCTG
TCCGCTACCATCCTGTACGAGATTCTGCTGGGCAAGGCTACACTGTATGCAG
TGCTGGTCTCTGCACTGGTGCTGATGGCCATGGTCAAGCGCAAAGACTTC
36
GCGGCCGCCACCATGGGGACTAGCCTGCTGTGCTGGATGGCACTGTGCCTGC
TCR 4 - (E6)29
TGGGAGCAGACCACGCAGATACCGGAGTGAGCCAGGACCCAAGACATAAGA
Codon-optimized/
TCACAAAAAGGGGCCAGAACGTGACTTTTAGATGCGATCCCATTAGCGAACA
cysteine-modified full
CAATAGACTGTACTGGTATAGGCAGACACTGGGACAGGGACCAGAGTTCCT
sequence
GACTTACTTTCAGAACGAAGCTCAGCTGGAGAAGAGTCGCCTGCTGTCAGAC
Homo sapiens
CGGTTCAGCGCCGAGCGACCAAAAGGCTCTTTCAGTACACTGGAAATCCAGC
(nt)
GAACTGAGCAGGGGGATTCCGCCATGTATCTGTGCGCTAGCTCCCCAGGAGG
AGGAAACACCGAAGCCTTCTTTGGACAGGGCACACGGCTGACTGTGGTCGA
GGACCTGAATAAGGTGTTCCCCCCTGAAGTGGCCGTCTTTGAGCCTTCCGAA
GCTGAGATTTCTCACACCCAGAAAGCCACCCTGGTGTGCCTGGCAACAGGCT
TCTTTCCAGATCACGTGGAACTGAGCTGGTGGGTCAACGGAAAGGAGGTGCA
TAGCGGCGTCTGCACTGACCCACAGCCCCTGAAAGAGCAGCCCGCACTGAAT
GATAGCAGGTACTGCCTGTCTAGTCGGCTGAGAGTGTCCGCCACCTTTTGGC
AGAACCCTAGGAATCATTTCCGCTGTCAGGTGCAGTTTTATGGCCTGTCCGA
AAACGACGAGTGGACTCAGGATCGGGCCAAGCCCGTGACCCAGATCGTCTCT
GCAGAAGCCTGGGGCAGAGCTGACTGCGGGTTCACCTCAGTGAGCTACCAG
CAGGGAGTCCTGTCCGCTACCATCCTGTACGAGATTCTGCTGGGCAAGGCTA
CACTGTATGCAGTGCTGGTCTCTGCACTGGTGCTGATGGCCATGGTCAAGCG
CAAAGACTTCGGGAGTGGAGCAACAAACTTTTCACTGCTGAAGCAGGCCGG
CGATGTGGAGGAAAATCCTGGGCCAATGAAACTGGTGACCAGCATCACAGT
CCTGCTGTCCCTGGGAATTATGGGCGACGCCAAGACCACACAGCCTAACTCT
ATGGAGAGTAATGAGGAAGAGCCTGTGCACCTGCCATGTAACCATTCAACTA
TCAGCGGCACCGATTACATTCACTGGTATCGGCAGCTGCCCTCCCAGGGACC
TGAATACGTGATCCATGGCCTGACCTCAAATGTCAACAATCGCATGGCTAGC
CTGGCTATCGCAGAGGACCGAAAGTCAAGCACCCTGATTCTGCACCGAGCCA
CACTGCGAGATGCAGCCGTGTACTATTGCATCCTGCTGGTCATTAGAGGGAC
CAGCTACGGAAAACTGACATTTGGCCAGGGGACTATCCTGACCGTGCATCCT
AACATTCAGAATCCCGACCCTGCCGTGTATCAGCTGAGGGACTCTAAGTCCT
CTGATAAAAGCGTGTGCCTGTTCACTGACTTTGATTCCCAGACCAACGTGTCC
CAGTCTAAGGACTCTGACGTGTACATCACAGACAAATGCGTCCTGGATATGC
GCAGCATGGACTTCAAGAGTAACTCAGCCGTGGCTTGGTCCAACAAGTCTGA
TTTCGCATGCGCCAACGCTTTTAACAACAGTATCATCCCAGAAGATACCTTCT
TTCCATCACCCGAGAGTTCATGTGACGTGAAGCTGGTCGAAAAATCTTTCGA
GACTGATACCAACCTGAATTTTCAGAACCTGAGTGTGATCGGGTTCAGGATT
CTGCTGCTGAAGGTCGCCGGATTCAATCTGCTGATGACACTGCGCCTGTGGA
GCTCCTGAGGCGCGCC
37
MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYW
TCR 4 - (E6)29
YRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAM
Full sequence
YLCASSPGGGNTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLV
Cysteine-modified
CLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVS
Homo sapiens
ATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQ
AGDVEENPGPMKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTIS
GTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDA
AVYYCILLVIRGTSYGKLTFGQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLFT
DFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNN
SIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTL
RLWSS
38
AQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSY
TCR 5 - (E6)29 - TCR
DEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMREGTGTSYGKLTF
alpha
GQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT
Native
DKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKL
Homo sapiens
VEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
(aa)
39
AQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSY
TCR 5 - (E6)29 - TCR
DEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMREGTGTSYGKLTF
alpha
GQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT
Cysteine-modified
DKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKL
Homo sapiens
VEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
(aa)
40
ATGTCACTTTCTAGCCTGCTGAAGGTGGTCACAGCTTCACTGTGGCTAGGAC
TCR 5 - (E6)29 - TCR
CTGGCATTGCCCAGAAGATAACTCAAACCCAACCAGGAATGTTCGTGCAGGA
alpha
AAAGGAGGCTGTGACTCTGGACTGCACATATGACACCAGTGATCAAAGTTAT
Native
GGTCTATTCTGGTACAAGCAGCCCAGCAGTGGGGAAATGATTTTTCTTATTTA
Homo sapiens
TCAGGGGTCTTATGACGAGCAAAATGCAACAGAAGGTCGCTACTCATTGAAT
(nt)
TTCCAGAAGGCAAGAAAATCCGCCAACCTTGTCATCTCCGCTTCACAACTGG
GGGACTCAGCAATGTATTTCTGTGCAATGAGAGAGGGCACAGGTACTAGCTA
TGGAAAGCTGACATTTGGACAAGGGACCATCTTGACTGTCCATCCAAATATC
CAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACA
AGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGT
AAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTA
TGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGC
ATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCC
AGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAG
ATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTC
CTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTG
41
ATGAGTCTGTCCTCTCTGCTGAAGGTGGTCACTGCATCACTGTGGCTGGGAC
TCR 5 - (E6)29 - TCR
CAGGAATCGCACAGAAAATTACCCAGACACAGCCTGGCATGTTTGTCCAGGA
alpha
GAAGGAAGCCGTGACCCTGGACTGTACTTACGACACCAGCGATCAGTCCTAC
Codon-optimized/
GGGCTGTTTTGGTATAAGCAGCCAAGTTCAGGAGAGATGATCTTCCTGATCT
cysteine-modified
ACCAGGGCAGCTATGACGAGCAGAACGCTACAGAAGGCAGGTATAGCCTGA
Homo sapiens
ATTTCCAGAAAGCCCGCAAGTCCGCTAACCTGGTCATCTCTGCCAGTCAGCT
(nt)
GGGGGATTCTGCCATGTACTTTTGCGCTATGAGGGAGGGAACTGGCACCAGC
TATGGAAAGCTGACCTTCGGGCAGGGAACAATCCTGACTGTCCATCCCAACA
TTCAGAATCCAGACCCTGCCGTGTACCAGCTGCGAGACAGTAAAAGCTCCGA
TAAGAGCGTGTGCCTGTTCACAGACTTTGATTCTCAGACTAACGTGAGCCAG
AGCAAAGACAGTGATGTCTATATTACCGACAAGTGCGTGCTGGATATGCGCA
GCATGGACTTTAAATCCAACTCTGCAGTGGCCTGGTCTAATAAGAGTGATTT
CGCTTGCGCAAACGCCTTTAACAATTCAATCATTCCCGAGGATACCTTCTTTC
CAAGCCCCGAATCTAGTTGTGACGTGAAACTGGTGGAGAAGTCTTTCGAAAC
AGATACTAACCTGAATTTTCAGAATCTGAGTGTCATCGGGTTCCGGATTCTGC
TGCTGAAGGTGGCCGGATTCAACCTGCTGATGACCCTGAGACTGTGGTCAAG
C
42
DVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDV
TCR 5 - (E6)29 - TCR
KMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSPWGETHQPQHFG
beta
DGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWV
Native
NGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY
Homo sapiens
GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGK
(aa)
ATLYAVLVSALVLMAMVKRKDF
43
DVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDV
TCR 5 - (E6)29 - TCR
KMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSPWGETHQPQHFG
beta
DGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWV
Cysteine-modified
NGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQF
Homo sapiens
YGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLG
(aa)
KATLYAVLVSALVLMAMVKRKDF
44
ATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCT
TCR 5 - (E6)29 - TCR
CGTAGATGTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGG
beta
AGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCATGAAAATATGTTC
Native
TGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCTATTTCTCATATG
Homo sapiens
ATGTTAAAATGAAAGAAAAAGGAGATATTCCTGAGGGGTACAGTGTCTCTA
(nt)
GAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCCAGCACCAACCA
GACATCTATGTACCTCTGTGCCAGCAGCCCATGGGGAGAAACTCATCAGCCC
CAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAGGACCTGAACAAGG
TGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCA
CACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCTGACCAC
GTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGC
ACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACT
GCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAA
CCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGG
ACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGG
GGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGT
CTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGT
GCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC
45
ATGGGAATCAGGCTGCTGTGCCGCGTCGCATTCTGTTTTCTGGCCGTGGGCCT
TCR 5 - (E6)29 - TCR
GGTGGACGTGAAAGTGACTCAGAGCTCCAGATACCTGGTGAAAAGGACCGG
beta
CGAGAAGGTCTTTCTGGAATGCGTGCAGGACATGGATCACGAGAATATGTTC
Codon-optimized/
TGGTATCGGCAGGATCCAGGCCTGGGGCTGAGACTGATCTACTTTTCCTATG
cysteine-modified
ATGTGAAGATGAAAGAGAAGGGCGACATTCCCGAAGGGTACTCCGTGTCTC
Homo sapiens
GCGAGAAGAAAGAACGATTCAGCCTGATCCTGGAGAGTGCTTCAACCAATC
(nt)
AGACATCCATGTATCTGTGCGCATCTAGTCCTTGGGGCGAGACACACCAGCC
ACAGCATTTCGGAGATGGCACTCGGCTGAGCATCCTGGAAGACCTGAACAA
AGTGTTCCCCCCTGAGGTCGCCGTGTTCGAACCTTCAGAGGCAGAAATTAGC
CACACTCAGAAGGCCACCCTGGTGTGCCTGGCCACTGGCTTCTTTCCAGACC
ACGTCGAGCTGTCCTGGTGGGTGAATGGGAAAGAAGTCCATAGTGGAGTGT
GCACCGACCCACAGCCCCTGAAGGAGCAGCCCGCACTGAACGATTCCAGAT
ACTGCCTGTCAAGCCGGCTGAGAGTGTCTGCCACTTTTTGGCAGAACCCTCG
AAATCATTTCCGGTGTCAGGTGCAGTTTTATGGCCTGAGCGAGAACGACGAA
TGGACCCAGGATCGAGCCAAACCTGTCACACAGATCGTGTCCGCCGAGGCTT
GGGGACGCGCTGATTGCGGCTTCACAAGCGTCTCCTACCAGCAGGGCGTGCT
GTCTGCCACCATCCTGTACGAAATTCTGCTGGGGAAGGCTACACTGTATGCC
GTGCTGGTGAGCGCCCTGGTGCTGATGGCAATGGTGAAAAGGAAGGACTTC
46
GCGGCCGCCACCATGGGAATCAGGCTGCTGTGCCGCGTCGCATTCTGTTTTCT
TCR 5 - (E6)29 - TCR
GGCCGTGGGCCTGGTGGACGTGAAAGTGACTCAGAGCTCCAGATACCTGGTG
Codon-optimized/
AAAAGGACCGGCGAGAAGGTCTTTCTGGAATGCGTGCAGGACATGGATCAC
cysteine-modified full
GAGAATATGTTCTGGTATCGGCAGGATCCAGGCCTGGGGCTGAGACTGATCT
sequence
ACTTTTCCTATGATGTGAAGATGAAAGAGAAGGGCGACATTCCCGAAGGGTA
Homo sapiens
CTCCGTGTCTCGCGAGAAGAAAGAACGATTCAGCCTGATCCTGGAGAGTGCT
(nt)
TCAACCAATCAGACATCCATGTATCTGTGCGCATCTAGTCCTTGGGGCGAGA
CACACCAGCCACAGCATTTCGGAGATGGCACTCGGCTGAGCATCCTGGAAGA
CCTGAACAAAGTGTTCCCCCCTGAGGTCGCCGTGTTCGAACCTTCAGAGGCA
GAAATTAGCCACACTCAGAAGGCCACCCTGGTGTGCCTGGCCACTGGCTTCT
TTCCAGACCACGTCGAGCTGTCCTGGTGGGTGAATGGGAAAGAAGTCCATAG
TGGAGTGTGCACCGACCCACAGCCCCTGAAGGAGCAGCCCGCACTGAACGA
TTCCAGATACTGCCTGTCAAGCCGGCTGAGAGTGTCTGCCACTTTTTGGCAG
AACCCTCGAAATCATTTCCGGTGTCAGGTGCAGTTTTATGGCCTGAGCGAGA
ACGACGAATGGACCCAGGATCGAGCCAAACCTGTCACACAGATCGTGTCCG
CCGAGGCTTGGGGACGCGCTGATTGCGGCTTCACAAGCGTCTCCTACCAGCA
GGGCGTGCTGTCTGCCACCATCCTGTACGAAATTCTGCTGGGGAAGGCTACA
CTGTATGCCGTGCTGGTGAGCGCCCTGGTGCTGATGGCAATGGTGAAAAGGA
AGGACTTCGGGTCCGGAGCCACAAATTTTTCTCTGCTGAAACAGGCTGGCGA
TGTGGAGGAAAACCCTGGGCCAATGAGTCTGTCCTCTCTGCTGAAGGTGGTC
ACTGCATCACTGTGGCTGGGACCAGGAATCGCACAGAAAATTACCCAGACA
CAGCCTGGCATGTTTGTCCAGGAGAAGGAAGCCGTGACCCTGGACTGTACTT
ACGACACCAGCGATCAGTCCTACGGGCTGTTTTGGTATAAGCAGCCAAGTTC
AGGAGAGATGATCTTCCTGATCTACCAGGGCAGCTATGACGAGCAGAACGCT
ACAGAAGGCAGGTATAGCCTGAATTTCCAGAAAGCCCGCAAGTCCGCTAAC
CTGGTCATCTCTGCCAGTCAGCTGGGGGATTCTGCCATGTACTTTTGCGCTAT
GAGGGAGGGAACTGGCACCAGCTATGGAAAGCTGACCTTCGGGCAGGGAAC
AATCCTGACTGTCCATCCCAACATTCAGAATCCAGACCCTGCCGTGTACCAG
CTGCGAGACAGTAAAAGCTCCGATAAGAGCGTGTGCCTGTTCACAGACTTTG
ATTCTCAGACTAACGTGAGCCAGAGCAAAGACAGTGATGTCTATATTACCGA
CAAGTGCGTGCTGGATATGCGCAGCATGGACTTTAAATCCAACTCTGCAGTG
GCCTGGTCTAATAAGAGTGATTTCGCTTGCGCAAACGCCTTTAACAATTCAA
TCATTCCCGAGGATACCTTCTTTCCAAGCCCCGAATCTAGTTGTGACGTGAAA
CTGGTGGAGAAGTCTTTCGAAACAGATACTAACCTGAATTTTCAGAATCTGA
GTGTCATCGGGTTCCGGATTCTGCTGCTGAAGGTGGCCGGATTCAACCTGCT
GATGACCCTGAGACTGTGGTCAAGCTGAGGCGCGCC
47
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 5 - (E6)29 - TCR
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Full sequence
YLCASSPWGETHQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATL
Cysteine-modified
VCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
Homo sapiens
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQ
AGDVEENPGPMSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCT
YDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLV
ISASQLGDSAMYFCAMREGTGTSYGKLTFGQGTILTVHPNIQNPDPAVYQLRDS
KSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNK
SDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLL
KVAGFNLLMTLRLWSS
48
GEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMD
TCR 6 - Alpha
MKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAESIRGFGNVLHCGSGTQV
Native
IVLPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLD
Homo sapiens
MRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFET
(aa)
DTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
49
GEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMD
TCR 6 - Alpha
MKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAESIRGFGNVLHCGSGTQV
Cysteine-modified
IVLPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLD
Homo sapiens
MRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFET
(aa)
DTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
50
ATGAAGACATTTGCTGGATTTTCGTTCCTGTTTTTGTGGCTGCAGCTGGACTG
TCR 6 - Alpha
TATGAGTAGAGGAGAGGATGTGGAGCAGAGTCTTTTCCTGAGTGTCCGAGAG
Native
GGAGACAGCTCCGTTATAAACTGCACTTACACAGACAGCTCCTCCACCTACT
Homo sapiens
TATACTGGTATAAGCAAGAACCTGGAGCAGGTCTCCAGTTGCTGACGTATAT
(nt)
TTTTTCAAATATGGACATGAAACAAGACCAAAGACTCACTGTTCTATTGAAT
AAAAAGGATAAACATCTGTCTCTGCGCATTGCAGACACCCAGACTGGGGACT
CAGCTATCTACTTCTGTGCAGAGAGTATAAGAGGCTTTGGGAATGTGCTGCA
TTGCGGGTCCGGCACTCAAGTGATTGTTTTACCACATATCCAGAACCCTGAC
CCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCC
TATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGAT
GTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGA
GCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGC
CTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGT
TCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAA
ACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCC
GGGTTTAATCTGCTCATGACGCTGCGGCTG
51
ATGAAGACATTTGCTGGATTTTCGTTCCTGTTTTTGTGGCTGCAGCTGGACTG
TCR 6 - Alpha
TATGAGTAGAGGAGAGGATGTGGAGCAGAGTCTTTTCCTGAGTGTCCGAGAG
Codon-optimized/
GGAGACAGCTCCGTTATAAACTGCACTTACACAGACAGCTCCTCCACCTACT
cysteine-modified
TATACTGGTATAAGCAAGAACCTGGAGCAGGTCTCCAGTTGCTGACGTATAT
Homo sapiens
TTTTTCAAATATGGACATGAAACAAGACCAAAGACTCACTGTTCTATTGAAT
(nt)
AAAAAGGATAAACATCTGTCTCTGCGCATTGCAGACACCCAGACTGGGGACT
CAGCTATCTACTTCTGTGCAGAGAGTATAAGAGGCTTTGGGAATGTGCTGCA
TTGCGGGTCCGGCACTCAAGTGATTGTTTTACCACATATCCAGAACCCTGAC
CCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCC
TATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGAT
GTGTATATCACAGACAAATGTGTGCTAGACATGAGGTCTATGGACTTCAAGA
GCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGC
CTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGT
TCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAA
ACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCC
GGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCTTCC
52
EPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEIS
TCR 6, TCR 12 - Beta
EKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASTTRSSYEQYFGPGTRLT
Native
VTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEV
Homo sapiens
HSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN
(aa)
DEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYA
VLVSALVLMAMVKRKDSRG
53
EPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEIS
TCR 6, TCR 12 - Beta
EKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASTTRSSYEQYFGPGTRLT
Cysteine-modified
VTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEV
Homo sapiens
HSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN
(aa)
DEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYA
VLVSALVLMAMVKRKDSRG
54
ATGGATACCTGGCTCGTATGCTGGGCAATTTTTAGTCTCTTGAAAGCAGGAC
TCR 6 - Beta
TCACAGAACCTGAAGTCACCCAGACTCCCAGCCATCAGGTCACACAGATGGG
Codon
ACAGGAAGTGATCTTGCGCTGTGTCCCCATCTCTAATCACTTATACTTCTATT
Optimized/Cysteine
GGTACAGACAAATCTTGGGGCAGAAAGTCGAGTTTCTGGTTTCCTTTTATAA
Modified
TAATGAAATCTCAGAGAAGTCTGAAATATTCGATGATCAATTCTCAGTTGAA
Homo sapiens
AGGCCTGATGGATCAAATTTCACTCTGAAGATCCGGTCCACAAAGCTGGAGG
(nt)
ACTCAGCCATGTACTTCTGTGCCAGCACAACGAGGAGCTCCTACGAGCAGTA
CTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTC
CCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCC
AAAAGGCCACACTGGTATGCCTGGCCACAGGCTTCTACCCCGACCACGTGGA
GCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCTGCACAGA
CCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTG
AGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACT
TCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCA
GGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAG
AGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCC
ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGG
TCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC
55
ATGGATACCTGGCTCGTATGCTGGGCAATTTTTAGTCTCTTGAAAGCAGGAC
TCR 6 - Beta
TCACAGAACCTGAAGTCACCCAGACTCCCAGCCATCAGGTCACACAGATGGG
Native
ACAGGAAGTGATCTTGCGCTGTGTCCCCATCTCTAATCACTTATACTTCTATT
Homo sapiens
GGTACAGACAAATCTTGGGGCAGAAAGTCGAGTTTCTGGTTTCCTTTTATAA
(nt)
TAATGAAATCTCAGAGAAGTCTGAAATATTCGATGATCAATTCTCAGTTGAA
AGGCCTGATGGATCAAATTTCACTCTGAAGATCCGGTCCACAAAGCTGGAGG
ACTCAGCCATGTACTTCTGTGCCAGCACAACGAGGAGCTCCTACGAGCAGTA
CTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTC
CCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCC
AAAAGGCCACACTGGTATGCCTGGCCACAGGCTTCTACCCCGACCACGTGGA
GCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGA
CCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTG
AGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACT
TCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCA
GGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAG
AGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCC
ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGG
TCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC
56
GCGGCCGCCACCATGGATACCTGGCTCGTATGCTGGGCAATTTTTAGTCTCTT
TCR 6
GAAAGCAGGACTCACAGAACCTGAAGTCACCCAGACTCCCAGCCATCAGGT
Codon-optimized/
CACACAGATGGGACAGGAAGTGATCTTGCGCTGTGTCCCCATCTCTAATCAC
cysteine-modified full
TTATACTTCTATTGGTACAGACAAATCTTGGGGCAGAAAGTCGAGTTTCTGG
sequence
TTTCCTTTTATAATAATGAAATCTCAGAGAAGTCTGAAATATTCGATGATCAA
Homo sapiens
TTCTCAGTTGAAAGGCCTGATGGATCAAATTTCACTCTGAAGATCCGGTCCA
(nt)
CAAAGCTGGAGGACTCAGCCATGTACTTCTGTGCCAGCACAACGAGGAGCTC
CTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCT
GAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAG
ATCTCCCACACCCAAAAGGCCACACTGGTATGCCTGGCCACAGGCTTCTACC
CCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTG
GGGTCTGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTC
CAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAAC
CCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATG
ACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCG
AGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGG
GGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGT
ATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGA
TTCCAGAGGCGGATCCGGAGCTACCAACTTCTCTCTGCTGAAACAGGCAGGC
GATGTGGAGGAAAATCCTGGGCCAATGAAGACATTTGCTGGATTTTCGTTCC
TGTTTTTGTGGCTGCAGCTGGACTGTATGAGTAGAGGAGAGGATGTGGAGCA
GAGTCTTTTCCTGAGTGTCCGAGAGGGAGACAGCTCCGTTATAAACTGCACT
TACACAGACAGCTCCTCCACCTACTTATACTGGTATAAGCAAGAACCTGGAG
CAGGTCTCCAGTTGCTGACGTATATTTTTTCAAATATGGACATGAAACAAGA
CCAAAGACTCACTGTTCTATTGAATAAAAAGGATAAACATCTGTCTCTGCGC
ATTGCAGACACCCAGACTGGGGACTCAGCTATCTACTTCTGTGCAGAGAGTA
TAAGAGGCTTTGGGAATGTGCTGCATTGCGGGTCCGGCACTCAAGTGATTGT
TTTACCACATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCT
AAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAA
TGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAATGTGTGCTA
GACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAAC
AAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAG
ACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAA
AAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGG
TTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCG
GCTGTGGTCTTCCTAAGGCGCGCC
57
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 6
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Full sequence
CASTTRSSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Cysteine-modified
TGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQ
AGDVEENPGPMKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCT
YTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRI
ADTQTGDSAIYFCAESIRGFGNVLHCGSGTQVIVLPHIQNPDPAVYQLRDSKSSD
KSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFA
CANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAG
FNLLMTLRLWSS
58
KNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIMTFSEN
TCR 7/ TCR 54 -
TKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSRDNYGQNFVFGPGTRLS
(E7)11 - alpha
VLPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLD
Native
MRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFET
Homo sapiens
DTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
(aa)
59
KNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIMTFSEN
TCR 7/ TCR 54 -
TKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSRDNYGQNFVFGPGTRLS
(E7)11 - alpha
VLPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLD
Cysteine-modified
MRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFET
Homo sapiens
DTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
(aa)
60
ATGAAAAAGCATCTGACGACCTTCTTGGTGATTTTGTGGCTTTATTTTTATAG
TCR 7 - (E7)11 - alpha
GGGGAATGGCAAAAACCAAGTGGAGCAGAGTCCTCAGTCCCTGATCATCCT
Native
GGAGGGAAAGAACTGCACTCTTCAATGCAATTATACAGTGAGCCCCTTCAGC
Homo sapiens
AACTTAAGGTGGTATAAGCAAGATACTGGGAGAGGTCCTGTTTCCCTGACAA
(nt)
TCATGACTTTCAGTGAGAACACAAAGTCGAACGGAAGATATACAGCAACTCT
GGATGCAGACACAAAGCAAAGCTCTCTGCACATCACAGCCTCCCAGCTCAGC
GATTCAGCCTCCTACATCTGTGTGGTGAGCCGGGATAACTATGGTCAGAATT
TTGTCTTTGGTCCCGGAACCAGATTGTCCGTGCTGCCCTATATCCAGAACCCT
GACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCT
GCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCT
GATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCA
AGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAA
CGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAA
AGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACC
TAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTG
GCCGGGTTTAATCTGCTCATGACGCTGCGGCTG
61
ATGAAGAAACACCTGACCACCTTCCTGGTCATCCTGTGGCTGTACTTCTACA
TCR 7 - (E7)11 - alpha
GAGGGAACGGAAAGAATCAGGTGGAACAGAGTCCACAGTCACTGATCATTC
Codon-optimized/
TGGAGGGCAAAAACTGCACTCTGCAGTGTAATTATACCGTGAGCCCATTTTC
cysteine-modified
CAATCTGCGATGGTACAAGCAGGACACTGGACGAGGACCCGTGAGCCTGAC
Homo sapiens
CATTATGACATTCTCCGAGAACACCAAGTCTAATGGCCGCTATACAGCCACT
(nt)
CTGGACGCTGATACTAAACAGTCTAGTCTGCATATCACCGCCTCTCAGCTGTC
TGATAGTGCTTCATATATTTGCGTGGTCAGTAGGGACAACTACGGGCAGAAT
TTCGTGTTTGGACCAGGAACCCGACTGTCCGTCCTGCCTTATATCCAGAACCC
CGACCCTGCCGTGTACCAGCTGAGGGACTCTAAGTCAAGCGATAAAAGCGTG
TGCCTGTTCACAGACTTTGATTCCCAGACTAATGTGAGCCAGTCCAAGGACT
CTGACGTGTACATTACTGACAAATGCGTCCTGGATATGCGCAGCATGGACTT
TAAGTCTAACAGTGCAGTGGCCTGGTCTAACAAGAGTGATTTCGCTTGCGCA
AACGCCTTTAACAATAGTATCATTCCCGAAGATACTTTCTTTCCATCACCCGA
GTCCTCTTGTGACGTGAAGCTGGTCGAAAAATCATTCGAGACCGATACAAAC
CTGAATTTTCAGAACCTGTCTGTGATCGGGTTCCGGATTCTGCTGCTGAAGGT
CGCCGGATTCAATCTGCTGATGACACTGAGACTGTGGAGTTCA
62
EPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEIS
TCR 7/ TCR 54 -
EKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCAITDRTNYGYTFGSGTRLT
(E7)11 -Beta
VVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEV
Native
HSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN
Homo sapiens
DEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYA
(aa)
VLVSALVLMAMVKRKDF
63
EPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEIS
TCR 7/ TCR 54 -
EKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCAITDRTNYGYTFGSGTRLT
(E7)11 -Beta
VVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEV
Cysteine-modified
HSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN
Homo sapiens
DEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYA
(aa)
VLVSALVLMAMVKRKDF
64
ATGGATACCTGGCTCGTATGCTGGGCAATTTTTAGTCTCTTGAAAGCAGGAC
TCR 7 - (E7)11 Beta
TCACAGAACCTGAAGTCACCCAGACTCCCAGCCATCAGGTCACACAGATGGG
Native
ACAGGAAGTGATCTTGCGCTGTGTCCCCATCTCTAATCACTTATACTTCTATT
Homo sapiens
GGTACAGACAAATCTTGGGGCAGAAAGTCGAGTTTCTGGTTTCCTTTTATAA
(nt)
TAATGAAATCTCAGAGAAGTCTGAAATATTCGATGATCAATTCTCAGTTGAA
AGGCCTGATGGATCAAATTTCACTCTGAAGATCCGGTCCACAAAGCTGGAGG
ACTCAGCCATGTACTTCTGTGCCATTACAGACCGCACTAACTATGGCTACAC
CTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGACCTGAACAAGGTGTTC
CCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCC
AAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCTGACCACGTGGA
GCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGA
CCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTG
AGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACT
TCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCA
GGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAG
AGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCC
ACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGG
TCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC
65
ATGGACACCTGGCTGGTGTGCTGGGCAATCTTTAGTCTGCTGAAGGCCGGAC
TCR 7 - (E7)11 -Beta
TGACCGAGCCTGAAGTGACTCAGACCCCATCCCACCAGGTCACACAGATGGG
Codon-optimized/
CCAGGAAGTGATCCTGCGGTGCGTGCCAATTTCCAACCATCTGTACTTCTATT
cysteine-modified
GGTACAGACAGATTCTGGGCCAGAAGGTGGAGTTCCTGGTCAGCTTTTATAA
Homo sapiens
CAACGAGATCTCAGAAAAGAGCGAGATTTTCGACGATCAGTTTTCAGTGGAA
(nt)
AGACCCGATGGGAGCAATTTCACCCTGAAGATCAGGAGTACAAAACTGGAG
GATTCAGCAATGTACTTTTGCGCCATTACTGACCGCACCAACTATGGATACA
CCTTCGGCTCCGGGACACGACTGACTGTGGTCGAGGACCTGAATAAGGTGTT
CCCCCCTGAAGTGGCTGTCTTTGAGCCTTCAGAGGCAGAAATCAGCCACACA
CAGAAAGCCACCCTGGTGTGCCTGGCTACAGGCTTCTTTCCAGATCACGTGG
AACTGAGCTGGTGGGTCAACGGCAAGGAGGTGCATTCCGGGGTCTGCACTG
ACCCACAGCCCCTGAAAGAGCAGCCCGCTCTGAATGATAGCAGGTATTGCCT
GAGCTCCCGGCTGAGAGTGTCCGCCACCTTTTGGCAGAACCCTAGGAATCAT
TTCCGCTGTCAGGTGCAGTTTTACGGCCTGTCTGAAAACGACGAGTGGACCC
AGGATCGAGCTAAGCCTGTGACACAGATCGTCAGCGCCGAAGCTTGGGGGC
GCGCAGACTGCGGATTCACCAGCGTGTCCTACCAGCAGGGCGTCCTGTCCGC
CACAATCCTGTATGAGATTCTGCTGGGGAAGGCTACTCTGTACGCAGTGCTG
GTCTCTGCTCTGGTGCTGATGGCAATGGTCAAGCGGAAAGACTTC
66
GCGGCCGCCACCATGGACACCTGGCTGGTGTGCTGGGCAATCTTTAGTCTGC
TCR 7 - (E7)11 -
TGAAGGCCGGACTGACCGAGCCTGAAGTGACTCAGACCCCATCCCACCAGGT
Codon-optimized/
CACACAGATGGGCCAGGAAGTGATCCTGCGGTGCGTGCCAATTTCCAACCAT
cysteine-modified full
CTGTACTTCTATTGGTACAGACAGATTCTGGGCCAGAAGGTGGAGTTCCTGG
sequence
TCAGCTTTTATAACAACGAGATCTCAGAAAAGAGCGAGATTTTCGACGATCA
Homo sapiens
GTTTTCAGTGGAAAGACCCGATGGGAGCAATTTCACCCTGAAGATCAGGAGT
(nt)
ACAAAACTGGAGGATTCAGCAATGTACTTTTGCGCCATTACTGACCGCACCA
ACTATGGATACACCTTCGGCTCCGGGACACGACTGACTGTGGTCGAGGACCT
GAATAAGGTGTTCCCCCCTGAAGTGGCTGTCTTTGAGCCTTCAGAGGCAGAA
ATCAGCCACACACAGAAAGCCACCCTGGTGTGCCTGGCTACAGGCTTCTTTC
CAGATCACGTGGAACTGAGCTGGTGGGTCAACGGCAAGGAGGTGCATTCCG
GGGTCTGCACTGACCCACAGCCCCTGAAAGAGCAGCCCGCTCTGAATGATAG
CAGGTATTGCCTGAGCTCCCGGCTGAGAGTGTCCGCCACCTTTTGGCAGAAC
CCTAGGAATCATTTCCGCTGTCAGGTGCAGTTTTACGGCCTGTCTGAAAACG
ACGAGTGGACCCAGGATCGAGCTAAGCCTGTGACACAGATCGTCAGCGCCG
AAGCTTGGGGGCGCGCAGACTGCGGATTCACCAGCGTGTCCTACCAGCAGG
GCGTCCTGTCCGCCACAATCCTGTATGAGATTCTGCTGGGGAAGGCTACTCT
GTACGCAGTGCTGGTCTCTGCTCTGGTGCTGATGGCAATGGTCAAGCGGAAA
GACTTCGGAAGCGGCGCAACAAACTTTTCCCTGCTGAAACAGGCCGGAGATG
TGGAGGAAAATCCTGGCCCAATGAAGAAACACCTGACCACCTTCCTGGTCAT
CCTGTGGCTGTACTTCTACAGAGGGAACGGAAAGAATCAGGTGGAACAGAG
TCCACAGTCACTGATCATTCTGGAGGGCAAAAACTGCACTCTGCAGTGTAAT
TATACCGTGAGCCCATTTTCCAATCTGCGATGGTACAAGCAGGACACTGGAC
GAGGACCCGTGAGCCTGACCATTATGACATTCTCCGAGAACACCAAGTCTAA
TGGCCGCTATACAGCCACTCTGGACGCTGATACTAAACAGTCTAGTCTGCAT
ATCACCGCCTCTCAGCTGTCTGATAGTGCTTCATATATTTGCGTGGTCAGTAG
GGACAACTACGGGCAGAATTTCGTGTTTGGACCAGGAACCCGACTGTCCGTC
CTGCCTTATATCCAGAACCCCGACCCTGCCGTGTACCAGCTGAGGGACTCTA
AGTCAAGCGATAAAAGCGTGTGCCTGTTCACAGACTTTGATTCCCAGACTAA
TGTGAGCCAGTCCAAGGACTCTGACGTGTACATTACTGACAAATGCGTCCTG
GATATGCGCAGCATGGACTTTAAGTCTAACAGTGCAGTGGCCTGGTCTAACA
AGAGTGATTTCGCTTGCGCAAACGCCTTTAACAATAGTATCATTCCCGAAGA
TACTTTCTTTCCATCACCCGAGTCCTCTTGTGACGTGAAGCTGGTCGAAAAAT
CATTCGAGACCGATACAAACCTGAATTTTCAGAACCTGTCTGTGATCGGGTT
CCGGATTCTGCTGCTGAAGGTCGCCGGATTCAATCTGCTGATGACACTGAGA
CTGTGGAGTTCATGAGGCGCGCC
67
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 7/ TCR 54-
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
(E7)11 -
CAITDRTNYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLA
Full sequence
TGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
Cysteine-modified
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSY
Homo sapiens
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQAG
(aa)
DVEENPGPMKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNY
TVSPFSNLRWYKQDTGRGPVSLTIMTFSENTKSNGRYTATLDADTKQSSLHITAS
QLSDSASYICVVSRDNYGQNFVFGPGTRLSVLPYIQNPDPAVYQLRDSKSSDKSV
CLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACAN
AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNL
LMTLRLWSS
68
KQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQR
TCR 8 - Alpha
EQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLGNTPLVFGKGTRLSV
Native
IANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMR
Homo sapiens
SMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT
(aa)
NLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
69
KQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQR
TCR 8 - Alpha
EQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLGNTPLVFGKGTRLSV
Cysteine-modified
IANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDM
Homo sapiens
RSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETD
(aa)
TNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
70
ATGGAGACCCTCTTGGGCCTGCTTATCCTTTGGCTGCAGCTGCAATGGGTGA
TCR 8 - Alpha
GCAGCAAACAGGAGGTGACACAGATTCCTGCAGCTCTGAGTGTCCCAGAAG
Native
GAGAAAACTTGGTTCTCAACTGCAGTTTCACTGATAGCGCTATTTACAACCTC
Homo sapiens
CAGTGGTTTAGGCAGGACCCTGGGAAAGGTCTCACATCTCTGTTGCTTATTC
(nt)
AGTCAAGTCAGAGAGAGCAAACAAGTGGAAGACTTAATGCCTCGCTGGATA
AATCATCAGGACGTAGTACTTTATACATTGCAGCTTCTCAGCCTGGTGACTCA
GCCACCTACCTCTGTGCTGTGAGGCCTCTCGGAAACACACCTCTTGTCTTTGG
AAAGGGCACAAGACTTTCTGTGATTGCAAATATCCAGAACCCTGACCCTGCC
GTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCA
CCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTA
TATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAAC
AGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCA
ACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTG
TGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTT
CAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGT
TTAATCTGCTCATGACGCTGCGGCTC
71
ATGGAGACCCTCTTGGGCCTGCTTATCCTTTGGCTGCAGCTGCAATGGGTGA
TCR 8 - Alpha
GCAGCAAACAGGAGGTGACACAGATTCCTGCAGCTCTGAGTGTCCCAGAAG
Codon-optimized/
GAGAAAACTTGGTTCTCAACTGCAGTTTCACTGATAGCGCTATTTACAACCTC
cysteine-modified
CAGTGGTTTAGGCAGGACCCTGGGAAAGGTCTCACATCTCTGTTGCTTATTC
Homo sapiens
AGTCAAGTCAGAGAGAGCAAACAAGTGGAAGACTTAATGCCTCGCTGGATA
(nt)
AATCATCAGGACGTAGTACTTTATACATTGCAGCTTCTCAGCCTGGTGACTCA
GCCACCTACCTCTGTGCTGTGAGGCCTCTCGGAAACACACCTCTTGTCTTTGG
AAAGGGCACAAGACTTTCTGTGATTGCAAATATCCAGAACCCTGACCCTGCC
GTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCA
CCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTA
TATCACAGACAAATGCGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAAC
AGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCA
ACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTG
TGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTT
CAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGT
TTAATCTGCTCATGACGCTGCGGCTCTGGTCTTCC
72
KVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKM
TCR 8 - Beta
KEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLWGASTDTQYFGPGT
Native
RLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNG
Homo sapiens
KEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGL
(aa)
SENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKAT
LYAVLVSALVLMAMVKRKDSRG
73
KVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKM
TCR 8 - Beta
KEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLWGASTDTQYFGPGT
Cysteine-modified
RLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNG
Homo sapiens
KEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGL
(aa)
SENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKAT
LYAVLVSALVLMAMVKRKDSRG
74
ATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCT
TCR 8 - Beta
CGTAGATGTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGG
Native
AGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCATGAAAATATGTTC
Homo sapiens
TGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCTATTTCTCATATG
(nt)
ATGTTAAAATGAAAGAAAAAGGAGATATTCCTGAGGGGTACAGTGTCTCTA
GAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCCAGCACCAACCA
GACATCTATGTACCTCTGTGCCAGCAGTTTATGGGGGGCTAGCACAGATACG
CAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACG
TGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCA
CACCCAAAAGGCCACACTGGTATGCCTGGCCACAGGCTTCTACCCCGACCAC
GTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGC
ACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACT
GCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAA
CCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGG
ACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGG
GGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGT
CTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGT
GCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGA
GGC
75
ATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCT
TCR 8 - Beta
CGTAGATGTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGG
Codon-optimized/
AGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCATGAAAATATGTTC
cysteine-modified
TGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCTATTTCTCATATG
Homo sapiens
ATGTTAAAATGAAAGAAAAAGGAGATATTCCTGAGGGGTACAGTGTCTCTA
(nt)
GAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCCAGCACCAACCA
GACATCTATGTACCTCTGTGCCAGCAGTTTATGGGGGGCTAGCACAGATACG
CAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACG
TGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCA
CACCCAAAAGGCCACACTGGTATGCCTGGCCACAGGCTTCTACCCCGACCAC
GTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCTGT
ACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACT
GCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAA
CCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGG
ACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGG
GGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGT
CTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGT
GCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGA
GGC
76
GCGGCCGCCACCATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCT
TCR 8
GGCTGTAGGCCTCGTAGATGTGAAAGTAACCCAGAGCTCGAGATATCTAGTC
Codon-optimized/
AAAAGGACGGGAGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCAT
cysteine-modified full
GAAAATATGTTCTGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCT
sequence
ATTTCTCATATGATGTTAAAATGAAAGAAAAAGGAGATATTCCTGAGGGGTA
Homo sapiens
CAGTGTCTCTAGAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCC
(nt)
AGCACCAACCAGACATCTATGTACCTCTGTGCCAGCAGTTTATGGGGGGCTA
GCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGA
CCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCA
GAGATCTCCCACACCCAAAAGGCCACACTGGTATGCCTGGCCACAGGCTTCT
ACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACA
GTGGGGTCTGTACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGA
CTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAG
AACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGA
ATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCG
CCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCA
AGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACC
TTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAA
AGGATTCCAGAGGCGGATCCGGAGCTACCAACTTCTCTCTGCTGAAACAGGC
AGGCGATGTGGAGGAAAATCCTGGGCCAATGGAGACCCTCTTGGGCCTGCTT
ATCCTTTGGCTGCAGCTGCAATGGGTGAGCAGCAAACAGGAGGTGACACAG
ATTCCTGCAGCTCTGAGTGTCCCAGAAGGAGAAAACTTGGTTCTCAACTGCA
GTTTCACTGATAGCGCTATTTACAACCTCCAGTGGTTTAGGCAGGACCCTGG
GAAAGGTCTCACATCTCTGTTGCTTATTCAGTCAAGTCAGAGAGAGCAAACA
AGTGGAAGACTTAATGCCTCGCTGGATAAATCATCAGGACGTAGTACTTTAT
ACATTGCAGCTTCTCAGCCTGGTGACTCAGCCACCTACCTCTGTGCTGTGAGG
CCTCTCGGAAACACACCTCTTGTCTTTGGAAAGGGCACAAGACTTTCTGTGA
TTGCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAA
ATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATG
TGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAATGCGTGCTAGA
CATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAA
ATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGAC
ACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAA
GCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTT
CCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGG
CTCTGGTCTTCCTAAGGCGCGCC
77
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 8
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Full sequence
YLCASSLWGASTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATL
Cysteine-modified
VCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
Homo sapiens
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
ESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLL
KQAGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNC
SFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAA
SQPGDSATYLCAVRPLGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSKSSDKSV
CLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACAN
AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNL
LMTLRLWSS
78
AQKITQTQPGMFVQEKEAVTLDCTYDTSDPSYGLFWYKQPSSGEMIFLIYQGSY
TCR 9 - Alpha
DQQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMRTAGGTSYGKLTF
Native
GQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT
Homo sapiens
DKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKL
(aa)
VEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
79
AQKITQTQPGMFVQEKEAVTLDCTYDTSDPSYGLFWYKQPSSGEMIFLIYQGSY
TCR 9 - Alpha
DQQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMRTAGGTSYGKLTF
Cysteine-modified
GQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYIT
Homo sapiens
DKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKL
(aa)
VEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
80
ATGTCACTTTCTAGCCTGCTGAAGGTGGTCACAGCTTCACTGTGGCTAGGAC
TCR 9 - Alpha
CTGGCATTGCCCAGAAGATAACTCAAACCCAACCAGGAATGTTCGTGCAGGA
Native
AAAGGAGGCTGTGACTCTGGACTGCACATATGACACCAGTGATCCAAGTTAT
Homo sapiens
GGTCTATTCTGGTACAAGCAGCCCAGCAGTGGGGAAATGATTTTTCTTATTTA
(nt)
TCAGGGGTCTTATGACCAGCAAAATGCAACAGAAGGTCGCTACTCATTGAAT
TTCCAGAAGGCAAGAAAATCCGCCAACCTTGTCATCTCCGCTTCACAACTGG
GGGACTCAGCAATGTACTTCTGTGCAATGAGAACTGCTGGTGGTACTAGCTA
TGGAAAGCTGACATTTGGACAAGGGACCATCTTGACTGTCCATCCAAATATC
CAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACA
AGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGT
AAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTA
TGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGC
ATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCC
AGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAG
ATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTC
CTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTG
81
ATGTCACTTTCTAGCCTGCTGAAGGTGGTCACAGCTTCACTGTGGCTAGGAC
TCR 9 - Alpha
CTGGCATTGCCCAGAAGATAACTCAAACCCAACCAGGAATGTTCGTGCAGGA
Codon-optimized/
AAAGGAGGCTGTGACTCTGGACTGCACATATGACACCAGTGATCCAAGTTAT
cysteine-modified
GGTCTATTCTGGTACAAGCAGCCCAGCAGTGGGGAAATGATTTTTCTTATTTA
Homo sapiens
TCAGGGGTCTTATGACCAGCAAAATGCAACAGAAGGTCGCTACTCATTGAAT
(nt)
TTCCAGAAGGCAAGAAAATCCGCCAACCTTGTCATCTCCGCTTCACAACTGG
GGGACTCAGCAATGTACTTCTGTGCAATGAGAACTGCTGGTGGTACTAGCTA
TGGAAAGCTGACATTTGGACAAGGGACCATCTTGACTGTCCATCCAAATATC
CAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACA
AGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGT
AAGGATTCTGATGTGTATATCACAGACAAATGTGTGCTAGACATGAGGTCTA
TGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGC
ATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCC
AGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAG
ATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTC
CTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCTTCC
82
NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSVGE
TCR 9 - Beta
GTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYFGTAYEQYFGP
Native
GTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV
Homo sapiens
NGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY
(aa)
GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGK
ATLYAVLVSALVLMAMVKRKDSRG
83
NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSVGE
TCR 9 - Beta
GTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYFGTAYEQYFGP
Cysteine-modified
GTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV
Homo sapiens
NGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQF
(aa)
YGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLG
KATLYAVLVSALVLMAMVKRKDSRG
84
ATGAGCCTCGGGCTCCTGTGCTGTGGGGCCTTTTCTCTCCTGTGGGCAGGTCC
TCR 9 - Beta
AGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCGGGTCCTGAAGACAGGA
Native
CAGAGCATGACACTGCTGTGTGCCCAGGATATGAACCATGAATACATGTACT
Homo sapiens
GGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGG
(nt)
TGAGGGTACAACTGCCAAAGGAGAGGTCCCTGATGGCTACAATGTCTCCAGA
TTAAAAAAACAGAATTTCCTGCTGGGGTTGGAGTCGGCTGCTCCCTCCCAAA
CATCTGTGTACTTCTGTGCCAGCAGTTACTTCGGGACAGCCTACGAGCAGTA
CTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTC
CCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCC
AAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGA
GCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGA
CCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTG
AGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACT
TCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCA
GGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAG
AGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCC
ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGG
TCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC
85
ATGAGCCTCGGGCTCCTGTGCTGTGGGGCCTTTTCTCTCCTGTGGGCAGGTCC
TCR 9 - Beta
AGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCGGGTCCTGAAGACAGGA
Codon-optimized/
CAGAGCATGACACTGCTGTGTGCCCAGGATATGAACCATGAATACATGTACT
cysteine-modified
GGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGG
Homo sapiens
TGAGGGTACAACTGCCAAAGGAGAGGTCCCTGATGGCTACAATGTCTCCAGA
(nt)
TTAAAAAAACAGAATTTCCTGCTGGGGTTGGAGTCGGCTGCTCCCTCCCAAA
CATCTGTGTACTTCTGTGCCAGCAGTTACTTCGGGACAGCCTACGAGCAGTA
CTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTC
CCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCC
AAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGA
GCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCTGTACAGA
CCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTG
AGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACT
TCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCA
GGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAG
AGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCC
ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGG
TCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC
86
GCGGCCGCCACCATGAGCCTCGGGCTCCTGTGCTGTGGGGCCTTTTCTCTCCT
TCR 9 -
GTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCGGGTC
Codon-optimized/
CTGAAGACAGGACAGAGCATGACACTGCTGTGTGCCCAGGATATGAACCAT
cysteine-modified full
GAATACATGTACTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTC
sequence
ATTACTCAGTTGGTGAGGGTACAACTGCCAAAGGAGAGGTCCCTGATGGCTA
Homo sapiens
CAATGTCTCCAGATTAAAAAAACAGAATTTCCTGCTGGGGTTGGAGTCGGCT
(nt)
GCTCCCTCCCAAACATCTGTGTACTTCTGTGCCAGCAGTTACTTCGGGACAGC
CTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCT
GAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAG
ATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACC
CCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTG
GGGTCTGTACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTC
CAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAAC
CCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATG
ACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCG
AGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGG
GGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGT
ATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGA
TTCCAGAGGCGGATCCGGAGCTACCAACTTCTCTCTGCTGAAACAGGCAGGC
GATGTGGAGGAAAATCCTGGGCCAATGTCACTTTCTAGCCTGCTGAAGGTGG
TCACAGCTTCACTGTGGCTAGGACCTGGCATTGCCCAGAAGATAACTCAAAC
CCAACCAGGAATGTTCGTGCAGGAAAAGGAGGCTGTGACTCTGGACTGCAC
ATATGACACCAGTGATCCAAGTTATGGTCTATTCTGGTACAAGCAGCCCAGC
AGTGGGGAAATGATTTTTCTTATTTATCAGGGGTCTTATGACCAGCAAAATG
CAACAGAAGGTCGCTACTCATTGAATTTCCAGAAGGCAAGAAAATCCGCCA
ACCTTGTCATCTCCGCTTCACAACTGGGGGACTCAGCAATGTACTTCTGTGCA
ATGAGAACTGCTGGTGGTACTAGCTATGGAAAGCTGACATTTGGACAAGGG
ACCATCTTGACTGTCCATCCAAATATCCAGAACCCTGACCCTGCCGTGTACC
AGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTT
GATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAG
ACAAATGTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGT
GGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGC
ATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCA
AGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCT
GTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGC
TCATGACGCTGCGGCTGTGGTCTTCCTAAGGCGCGCC
87
MSLGLLCCGAFSLLWAGPVNAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMY
TCR 9 -
WYRQDPGMGLRLIHYSVGEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQT
Full sequence
SVYFCASSYFGTAYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKAT
Cysteine-modified
LVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLR
Homo sapiens
VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF
(aa)
TSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFS
LLKQAGDVEENPGPMSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVT
LDCTYDTSDPSYGLFWYKQPSSGEMIFLIYQGSYDQQNATEGRYSLNFQKARKS
ANLVISASQLGDSAMYFCAMRTAGGTSYGKLTFGQGTILTVHPNIQNPDPAVYQ
LRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVA
WSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGF
RILLLKVAGFNLLMTLRLWSS
88
RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSG
TCR 10 - Alpha
NEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNFPSRGAGGTSYGKLTFGQ
Native
GTILTVHPNIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDK
Homo sapiens
TVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPADTFFPSPESSCDVKLVE
(aa)
KSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
89
RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSG
TCR 10 - Alpha
NEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNFPSRGAGGTSYGKLTFGQ
Cysteine-modified
GTILTVHPNIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDK
Homo sapiens
CVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPADTFFPSPESSCDVKLVE
(aa)
KSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
90
ATGATGATATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGCTG
TCR 10 - Alpha
GGTTTGGAGCCAACGGAAGGAGGTGGAGCAGGATCCTGGACCCTTCAATGTT
Native
CCAGAGGGAGCCACTGTCGCTTTCAACTGTACTTACAGCAACAGTGCTTCTC
Homo sapiens
AGTCTTTCTTCTGGTACAGACAGGATTGCAGGAAAGAACCTAAGTTGCTGAT
(nt)
GTCCGTATACTCCAGTGGTAATGAAGATGGAAGGTTTACAGCACAGCTCAAT
AGAGCCAGCCAGTATATTTCCCTGCTCATCAGAGACTCCAAGCTCAGTGATT
CAGCCACCTACCTCTGTGTGGTGAACTTCCCTTCTCGGGGTGCTGGTGGTACT
AGCTATGGAAAGCTGACATTTGGACAAGGGACCATCTTGACTGTCCATCCAA
ATATCCAGAAGCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAG
TGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCAC
AAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAG
GTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGAC
TTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGCAGACACCTTCTT
CCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAA
ACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCC
TCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTG
91
ATGATGATATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGCTG
TCR 10 - Alpha
GGTTTGGAGCCAACGGAAGGAGGTGGAGCAGGATCCTGGACCCTTCAATGTT
Codon-optimized/
CCAGAGGGAGCCACTGTCGCTTTCAACTGTACTTACAGCAACAGTGCTTCTC
cysteine-modified
AGTCTTTCTTCTGGTACAGACAGGATTGCAGGAAAGAACCTAAGTTGCTGAT
Homo sapiens
GTCCGTATACTCCAGTGGTAATGAAGATGGAAGGTTTACAGCACAGCTCAAT
(nt)
AGAGCCAGCCAGTATATTTCCCTGCTCATCAGAGACTCCAAGCTCAGTGATT
CAGCCACCTACCTCTGTGTGGTGAACTTCCCTTCTCGGGGTGCTGGTGGTACT
AGCTATGGAAAGCTGACATTTGGACAAGGGACCATCTTGACTGTCCATCCAA
ATATCCAGAAGCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAG
TGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCAC
AAAGTAAGGATTCTGATGTGTATATCACAGACAAATGTGTGCTAGACATGAG
GTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGAC
TTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGCAGACACCTTCTT
CCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAA
ACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCC
TCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCT
TCC
92
DVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDV
TCR 10 - Beta
KMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLSLTGNYGYTFGS
Native
GTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWV
Homo sapiens
NGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY
(aa)
GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGK
ATLYAVLVSALVLMAMVKRKDF
93
DVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDV
TCR 10 - Beta
KMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLSLTGNYGYTFGS
Cysteine-modified
GTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWV
Homo sapiens
NGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQF
(aa)
YGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLG
KATLYAVLVSALVLMAMVKRKDF
94
ATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCT
TCR 10 - Beta
CGTAGATGTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGG
Native
AGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCATGAAAATATGTTC
Homo sapiens
TGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCTATTTCTCATATG
(nt)
ATGTTAAAATGAAAGAAAAAGGAGATATTCCTGAGGGGTACAGTGTCTCTA
GAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCCAGCACCAACCA
GACATCTATGTACCTCTGTGCCAGCAGTTTATCCCTAACAGGGAACTATGGC
TACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGACCTGAACAAGG
TGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCA
CACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCTGACCAC
GTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGC
ACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACT
GCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAA
CCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGG
ACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGG
GGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGT
CTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGT
GCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC
95
ATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCTGGCTGTAGGCCT
TCR 10 - Beta
CGTAGATGTGAAAGTAACCCAGAGCTCGAGATATCTAGTCAAAAGGACGGG
Codon-optimized/
AGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCATGAAAATATGTTC
cysteine-modified
TGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCTATTTCTCATATG
Homo sapiens
ATGTTAAAATGAAAGAAAAAGGAGATATTCCTGAGGGGTACAGTGTCTCTA
(nt)
GAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCCAGCACCAACCA
GACATCTATGTACCTCTGTGCCAGCAGTTTATCCCTAACAGGGAACTATGGC
TACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGACCTGAACAAGG
TGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCA
CACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCTGACCAC
GTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCTGT
ACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACT
GCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAA
CCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGG
ACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGG
GGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGT
CTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGT
GCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC
96
GCGGCCGCCACCATGGGAATCAGGCTCCTCTGTCGTGTGGCCTTTTGTTTCCT
TCR 10
GGCTGTAGGCCTCGTAGATGTGAAAGTAACCCAGAGCTCGAGATATCTAGTC
Codon-optimized/
AAAAGGACGGGAGAGAAAGTTTTTCTGGAATGTGTCCAGGATATGGACCAT
cysteine-modified full
GAAAATATGTTCTGGTATCGACAAGACCCAGGTCTGGGGCTACGGCTGATCT
sequence
ATTTCTCATATGATGTTAAAATGAAAGAAAAAGGAGATATTCCTGAGGGGTA
Homo sapiens
CAGTGTCTCTAGAGAGAAGAAGGAGCGCTTCTCCCTGATTCTGGAGTCCGCC
(nt)
AGCACCAACCAGACATCTATGTACCTCTGTGCCAGCAGTTTATCCCTAACAG
GGAACTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGA
CCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCA
GAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCT
TCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACA
GTGGGGTCTGTACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGA
CTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAG
AACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGA
ATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCG
CCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCA
AGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACC
CTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAA
AGGATTTCGGATCCGGAGCTACCAACTTCTCTCTGCTGAAACAGGCAGGCGA
TGTGGAGGAAAATCCTGGGCCAATGATGATATCCTTGAGAGTTTTACTGGTG
ATCCTGTGGCTTCAGTTAAGCTGGGTTTGGAGCCAACGGAAGGAGGTGGAGC
AGGATCCTGGACCCTTCAATGTTCCAGAGGGAGCCACTGTCGCTTTCAACTG
TACTTACAGCAACAGTGCTTCTCAGTCTTTCTTCTGGTACAGACAGGATTGCA
GGAAAGAACCTAAGTTGCTGATGTCCGTATACTCCAGTGGTAATGAAGATGG
AAGGTTTACAGCACAGCTCAATAGAGCCAGCCAGTATATTTCCCTGCTCATC
AGAGACTCCAAGCTCAGTGATTCAGCCACCTACCTCTGTGTGGTGAACTTCC
CTTCTCGGGGTGCTGGTGGTACTAGCTATGGAAAGCTGACATTTGGACAAGG
GACCATCTTGACTGTCCATCCAAATATCCAGAAGCCTGACCCTGCCGTGTAC
CAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATT
TTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCAC
AGACAAATGTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCT
GTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACA
GCATTATTCCAGCAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTC
AAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAAC
CTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCT
GCTCATGACGCTGCGGCTGTGGTCTTCCTAAGGCGCGCC
97
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 10
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Full sequence
YLCASSLSLTGNYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATL
Cysteine-modified
VCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
Homo sapiens
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQ
AGDVEENPGPMMISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAF
NCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIR
DSKLSDSATYLCVVNFPSRGAGGTSYGKLTFGQGTILTVHPNIQKPDPAVYQLR
DSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWS
NKSDFACANAFNNSIIPADTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRIL
LLKVAGFNLLMTLRLWSS
98
DAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNV
TCR 11 - Alpha
NNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILSAHSNSGYALNFGKGTSLLV
Native
TPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDM
Homo sapiens
RSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETD
(aa)
TNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
99
DAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNV
TCR 11 - Alpha
NNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILSAHSNSGYALNFGKGTSLLV
Cysteine-modified
TPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDM
Homo sapiens
RSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETD
(aa)
TNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
100
ATGAAGTTGGTGACAAGCATTACTGTACTCCTATCTTTGGGTATTATGGGTGA
TCR 11 - Alpha
TGCTAAGACCACACAGCCAAATTCAATGGAGAGTAACGAAGAAGAGCCTGT
Native
TCACTTGCCTTGTAACCACTCCACAATCAGTGGAACTGATTACATACATTGGT
Homo sapiens
ATCGACAGCTTCCCTCCCAGGGTCCAGAGTACGTGATTCATGGTCTTACAAG
(nt)
CAATGTGAACAACAGAATGGCCTCTCTGGCAATCGCTGAAGACAGAAAGTC
CAGTACCTTGATCCTGCACCGTGCTACCTTGAGAGATGCTGCTGTGTACTACT
GCATCCTGAGCGCTCACTCAAATTCCGGGTATGCACTCAACTTCGGCAAAGG
CACCTCGCTGTTGGTCACACCCCATATCCAGAACCCTGACCCTGCCGTGTACC
AGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTT
GATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAG
ACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGT
GGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGC
ATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCA
AGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCT
GTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGC
TCATGACGCTGCGGCTG
101
ATGAAGTTGGTGACAAGCATTACTGTACTCCTATCTTTGGGTATTATGGGTGA
TCR 11 - Alpha
TGCTAAGACCACACAGCCAAATTCAATGGAGAGTAACGAAGAAGAGCCTGT
Codon-optimized/
TCACTTGCCTTGTAACCACTCCACAATCAGTGGAACTGATTACATACATTGGT
cysteine-modified
ATCGACAGCTTCCCTCCCAGGGTCCAGAGTACGTGATTCATGGTCTTACAAG
Homo sapiens
CAATGTGAACAACAGAATGGCCTCTCTGGCAATCGCTGAAGACAGAAAGTC
(nt)
CAGTACCTTGATCCTGCACCGTGCTACCTTGAGAGATGCTGCTGTGTACTACT
GCATCCTGAGCGCTCACTCAAATTCCGGGTATGCACTCAACTTCGGCAAAGG
CACCTCGCTGTTGGTCACACCCCATATCCAGAACCCTGACCCTGCCGTGTACC
AGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTT
GATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAG
ACAAATGTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGT
GGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGC
ATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCA
AGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCT
GTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGC
TCATGACGCTGCGGCTGTGGTCTTCC
102
SAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEA
TCR 11 - Beta
TYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVVPWTRGGSTDTQYFGP
Native
GTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV
Homo sapiens
NGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY
(aa)
GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGK
ATLYAVLVSALVLMAMVKRKDSRG
103
SAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEA
TCR 11 - Beta
TYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVVPWTRGGSTDTQYFGP
Cysteine-modified
GTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWV
Homo sapiens
NGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQF
(aa)
YGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLG
KATLYAVLVSALVLMAMVKRKDSRG
104
ATGCTGAGTCTTCTGCTCCTTCTCCTGGGACTAGGCTCTGTGTTCAGTGCTGT
TCR 11 - Beta
CATCTCTCAAAAGCCAAGCAGGGATATCTGTCAACGTGGAACCTCCCTGACG
Native
ATCCAGTGTCAAGTCGATAGCCAAGTCACCATGATGTTCTGGTACCGTCAGC
Homo sapiens
AACCTGGACAGAGCCTGACACTGATCGCAACTGCAAATCAGGGCTCTGAGG
(nt)
CCACATATGAGAGTGGATTTGTCATTGACAAGTTTCCCATCAGCCGCCCAAA
CCTAACATTCTCAACTCTGACTGTGAGCAACATGAGCCCTGAAGACAGCAGC
ATATATCTCTGCAGCGTTGTCCCTTGGACGCGCGGGGGGAGCACAGATACGC
AGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGT
GTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCAC
ACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACG
TGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCA
CAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTG
CCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAAC
CACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGA
CCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGG
GTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTC
TGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTG
CTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAG
GC
105
ATGCTGAGTCTTCTGCTCCTTCTCCTGGGACTAGGCTCTGTGTTCAGTGCTGT
TCR 11 - Beta
CATCTCTCAAAAGCCAAGCAGGGATATCTGTCAACGTGGAACCTCCCTGACG
Codon-optimized/
ATCCAGTGTCAAGTCGATAGCCAAGTCACCATGATGTTCTGGTACCGTCAGC
cysteine-modified
AACCTGGACAGAGCCTGACACTGATCGCAACTGCAAATCAGGGCTCTGAGG
Homo sapiens
CCACATATGAGAGTGGATTTGTCATTGACAAGTTTCCCATCAGCCGCCCAAA
(nt)
CCTAACATTCTCAACTCTGACTGTGAGCAACATGAGCCCTGAAGACAGCAGC
ATATATCTCTGCAGCGTTGTCCCTTGGACGCGCGGGGGGAGCACAGATACGC
AGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGT
GTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCAC
ACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACG
TGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCTGTA
CAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTG
CCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAAC
CACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGA
CCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGG
GTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTC
TGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTG
CTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAG
GC
106
GCGGCCGCCACCATGCTGAGTCTTCTGCTCCTTCTCCTGGGACTAGGCTCTGT
TCR 11
GTTCAGTGCTGTCATCTCTCAAAAGCCAAGCAGGGATATCTGTCAACGTGGA
Codon-optimized/
ACCTCCCTGACGATCCAGTGTCAAGTCGATAGCCAAGTCACCATGATGTTCT
cysteine-modified full
GGTACCGTCAGCAACCTGGACAGAGCCTGACACTGATCGCAACTGCAAATCA
sequence
GGGCTCTGAGGCCACATATGAGAGTGGATTTGTCATTGACAAGTTTCCCATC
Homo sapiens
AGCCGCCCAAACCTAACATTCTCAACTCTGACTGTGAGCAACATGAGCCCTG
(nt)
AAGACAGCAGCATATATCTCTGCAGCGTTGTCCCTTGGACGCGCGGGGGGAG
CACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGAC
CTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAG
AGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTA
CCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAG
TGGGGTCTGTACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGAC
TCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGA
ACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAA
TGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGC
CGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAA
GGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCT
TGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAA
GGATTCCAGAGGCGGATCCGGAGCTACCAACTTCTCTCTGCTGAAACAGGCA
GGCGATGTGGAGGAAAATCCTGGGCCAATGAAGTTGGTGACAAGCATTACT
GTACTCCTATCTTTGGGTATTATGGGTGATGCTAAGACCACACAGCCAAATT
CAATGGAGAGTAACGAAGAAGAGCCTGTTCACTTGCCTTGTAACCACTCCAC
AATCAGTGGAACTGATTACATACATTGGTATCGACAGCTTCCCTCCCAGGGT
CCAGAGTACGTGATTCATGGTCTTACAAGCAATGTGAACAACAGAATGGCCT
CTCTGGCAATCGCTGAAGACAGAAAGTCCAGTACCTTGATCCTGCACCGTGC
TACCTTGAGAGATGCTGCTGTGTACTACTGCATCCTGAGCGCTCACTCAAATT
CCGGGTATGCACTCAACTTCGGCAAAGGCACCTCGCTGTTGGTCACACCCCA
TATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGT
GACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACA
AAGTAAGGATTCTGATGTGTATATCACAGACAAATGTGTGCTAGACATGAGG
TCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACT
TTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTT
CCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAA
ACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCC
TCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCT
TCCTAAGGCGCGCC
107
MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQP
TCR 11
GQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVV
Full sequence
PWTRGGSTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Cysteine-modified
TGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQ
AGDVEENPGPMKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTIS
GTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDA
AVYYCILSAHSNSGYALNFGKGTSLLVTPHIQNPDPAVYQLRDSKSSDKSVCLFT
DFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNN
SIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTL
RLWSS
108
ATGGATACCTGGCTCGTATGCTGGGCAATTTTTAGTCTCTTGAAAGCAGGAC
TCR 12 - Beta
TCACAGAACCTGAAGTCACCCAGACTCCCAGCCATCAGGTCACACAGATGGG
Native
ACAGGAAGTGATCTTGCGCTGTGTCCCCATCTCTAATCACTTATACTTCTATT
Homo sapiens
GGTACAGACAAATCTTGGGGCAGAAAGTCGAGTTTCTGGTTTCCTTTTATAA
(nt)
TAATGAAATCTCAGAGAAGTCTGAAATATTCGATGATCAATTCTCAGTTGAA
AGGCCTGATGGATCAAATTTCACTCTGAAGATCCGGTCCACAAAGCTGGAGG
ACTCAGCCATGTACTTCTGTGCCAGCACAACGAGGAGCTCCTACGAGCAGTA
CTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTC
CCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCC
AAAAGGCCACACTGGTATGCCTGGCCACAGGCTTCTACCCCGACCACGTGGA
GCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGA
CCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTG
AGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACT
TCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCA
GGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAG
AGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCC
ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGG
TCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTA
G
109
DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHS
Mouse beta constant
GVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPE
sequence
GSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGL
Mus musculus
VLMAMVKRKNS
(aa)
110
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 8 - Beta
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Native
YLCASSLWGASTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATL
Homo sapiens
VCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRV
(aa)
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
ESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG
111
AQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSY
TCR 3
DEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMREGRGFKTIFGAGT
alpha variable region
RLFVKA
Homo sapiens
(aa)
112
GAGVSQSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQNEA
TCR 3
QLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSHLAGFTGELFFGE
beta variable region
GSRLTVL
Homo sapiens
(aa)
113
DAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNV
TCR 4 - (E6)29 alpha
NNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILLVIRGTSYGKLTFGQGTILT
variable region
VHP
Homo sapiens
(aa)
114
GVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLE
TCR 4 - (E6)29 Beta
KSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSPGGGNTEAFFGQGTRL
variable region
TVV
Homo sapiens
(aa)
115
AQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSY
TCR 5 - (E6)29 - TCR
DEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMREGTGTSYGKLTF
alpha variable region
GQGTILTVHP
Homo sapiens
(aa)
116
KVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKM
TCR 5 - (E6)29 - TCR
KEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSPWGETHQPQHFGDGT
beta variable region
RLSIL
Homo sapiens
(aa)
117
GEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMD
TCR 6
MKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAESIRGFGNVLHCGSGTQV
alpha variable region
IVLP
Homo sapiens
(aa)
118
EPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEIS
TCR 6, TCR 12
EKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASTTRSSYEQYFGPGTRLT
Beta variable region
VT
Homo sapiens
(aa)
119
KNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIMTFSEN
TCR 7/ TCR 54 -
TKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSRDNYGQNFVFGPGTRLS
(E7)11 - alpha variable
VLP
region
Homo sapiens
(aa)
120
EPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEIS
TCR 7/ TCR 54-
EKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCAITDRTNYGYTFGSGTRLT
(E7)11 -Beta variable
VV
region
Homo sapiens
(aa)
121
KQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQR
TCR 8
EQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLGNTPLVFGKGTRLSV
alpha variable region
IA
Homo sapiens
(aa)
122
KVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKM
TCR 8
KEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLWGASTDTQYFGPGT
Beta variable region
RLTVL
Homo sapiens
(aa)
123
AQKITQTQPGMFVQEKEAVTLDCTYDTSDPSYGLFWYKQPSSGEMIFLIYQGSY
TCR 9
DQQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMRTAGGTSYGKLTF
alpha variable region
GQGTILTVHP
Homo sapiens
(aa)
124
NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSVGE
TCR 9
GTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSYFGTAYEQYFGP
Beta variable region
GTRLTVT
Homo sapiens
(aa)
125
RKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSG
TCR 10
NEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVNFPSRGAGGTSYGKLTFGQ
alpha variable region
GTILTVHP
Homo sapiens
(aa)
126
KVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKM
TCR 10
KEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLSLTGNYGYTFGSGTR
Beta variable region
LTVV
Homo sapiens
(aa)
127
DAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNV
TCR 11
NNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILSAHSNSGYALNFGKGTSLLV
alpha variable region
TP
Homo sapiens
(aa)
128
SAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQPGQSLTLIATANQGSEA
TCR 11
TYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVVPWTRGGSTDTQYFGP
Beta variable region
GTRLTVL
Homo sapiens
(aa)
129
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 8 - Beta
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Cysteine-modified
YLCASSLWGASTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATL
Homo sapiens
VCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
(aa)
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
ESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG
130
MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSDPSYGL
TCR 9 - Alpha
FWYKQPSSGEMIFLIYQGSYDQQNATEGRYSLNFQKARKSANLVISASQLGDSA
Native
MYFCAMRTAGGTSYGKLTFGQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLF
Homo sapiens
TDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFN
(aa)
NSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMT
LRLWSS
131
MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSDPSYGL
TCR 9 - Alpha
FWYKQPSSGEMIFLIYQGSYDQQNATEGRYSLNFQKARKSANLVISASQLGDSA
Cysteine-modified
MYFCAMRTAGGTSYGKLTFGQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLF
Homo sapiens
TDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFN
(aa)
NSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMT
LRLWSS
132
MSLGLLCCGAFSLLWAGPVNAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMY
TCR 9 - Beta
WYRQDPGMGLRLIHYSVGEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQT
Native
SVYFCASSYFGTAYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKAT
Homo sapiens
LVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLR
(aa)
VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF
TSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG
133
MSLGLLCCGAFSLLWAGPVNAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMY
TCR 9 - Beta
WYRQDPGMGLRLIHYSVGEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQT
Cysteine-modified
SVYFCASSYFGTAYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKAT
Homo sapiens
LVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLR
(aa)
VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF
TSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG
134
MMISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQ
TCR 10 - Alpha
SFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATY
Native
LCVVNFPSRGAGGTSYGKLTFGQGTILTVHPNIQKPDPAVYQLRDSKSSDKSVCL
Homo sapiens
FTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAF
(aa)
NNSIIPADTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLM
TLRLWSS
135
MMISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQ
TCR 10 - Alpha
SFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATY
Cysteine-modified
LCVVNFPSRGAGGTSYGKLTFGQGTILTVHPNIQKPDPAVYQLRDSKSSDKSVCL
Homo sapiens
FTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAF
(aa)
NNSIIPADTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLM
TLRLWSS
136
TSDQSYG
TCR 3/ TCR 5/ TCR
15/ TCR 19/ TCR 21/
TCR 23/ TCR 24/ TCR
25/ TCR 26/ TCR 29 -
(E6)29 - TCR alpha
CDR1
Homo sapiens
(aa)
137
QGSYDEQN
TCR 3/
TCR 5/ TCR 15/ TCR
19/ TCR 21/ TCR 23/
TCR 25 / TCR 26/
TCR 29 - (E6)29/ -
TCR alpha CDR2
Homo sapiens
(aa)
138
AMREGRGFKTI
TCR 3 alpha CDR3
Homo sapiens
(aa)
139
SGHVS
TCR 3/ TCR 13/ TCR
37/ TCR 53 Beta
CDR1
140
FQNEAQ
Homo sapiens
(aa)
TCR 3/TCR 4/ TCR
13/ TCR 37-
(E6)29Beta CDR2
Homo sapiens
(aa)
141
ASSHLAGFTGELF
TCR 3 Beta CDR3
Homo sapiens
(aa)
142
TISGTDY
TCR 4/ TCR 27 -
(E6)29/
TCR 11
alpha CDR1
Homo sapiens
(aa)
143
GLTSN
TCR 4/ TCR 27 -
(E6)29/
TCR 11
alpha CDR2
Homo sapiens
(aa)
144
ILLVIRGTSYGKLT
TCR 4 - (E6)29 alpha
CDR3
Homo sapiens
(aa)
145
SEHNR
TCR 4 - (E6)29 Beta
CDR1
Homo sapiens
(aa)
146
ASSPGGGNTEAF
TCR 4 - (E6)29 Beta
CDR3
Homo sapiens
(aa)
147
AMREGTGTSYGKLT
TCR 5 - (E6)29 - TCR
alpha CDR3
Homo sapiens
(aa)
148
MDHEN
TCR 5/ TCR 16/ TCR
17 /TCR 18 /TCR 19/
TCR 23 /TCR 24 /TCR
25/TCR 28 - (E6)29/
TCR 8/
TCR 10/ TCR 14 -
TCR beta CDR1
Homo sapiens
(aa)
149
SYDVKM
TCR 5/ TCR 16/ TCR
17/ TCR 18/ TCR 19/
TCR 23/ TCR 24/ TCR
25/ TCR 28 - (E6)29 /
TCR 8/
TCR 10/ TCR 14 -
TCR beta CDR2
Homo sapiens
(aa)
150
ASSPWGETHQPQH
TCR 5 - (E6)29 - TCR
beta CDR3
Homo sapiens
(aa)
151
DSSSTY
TCR 6, TCR 12, TCR
50, TCR 55
alpha CDR1
Homo sapiens
(aa)
152
IFSNMDM
TCR 6, TCR 12, TCR
50, TCR 55
alpha CDR2
Homo sapiens
(aa)
153
AESIRGFGNVLH
TCR 6
alpha CDR3
Homo sapiens
(aa)
154
SNHLY
TCR 6/ TCR 7 -
(E7)11, E7(11-19)/
TCR 12 consensus,
TCR 30/ TCR 33/ TCR
36/ TCR 39 /TCR 40/
TCR 41/ TCR 42/ TCR
43/ TCR 47/ TCR 48/
TCR 49/ TCR 51/ TCR
54/ TCR 55
Beta CDR1
Homo sapiens
(aa)
155
FYNNEI
TCR 6/ TCR 7 -
(E7)11, E7(11-19)/
TCR 12 consensus,
TCR 30/ TCR 33/ TCR
36/ TCR 39/ TCR 42/
TCR 43 / TCR 47/
TCR 48/ TCR 49/ TCR
51/ TCR 54/ TCR 55
Beta CDR2
Homo sapiens
(aa)
156
ASTTRSSYEQY
TCR 6/ TCR 12/ TCR
55
Beta CDR3
Homo sapiens
(aa)
157
VSPFSN
TCR 7/ TCR 54
alpha CDR1
Homo sapiens
158
MTFSENT
TCR 7/ TCR 54-
(E7)11 - alpha CDR2
Homo sapiens
159
VVSRDNYGQNFV
TCR 7/ TCR 54 -
(E7)11 - alpha CDR3
Homo sapiens
(aa)
160
AITDRTNYGYT
TCR 7/ TCR 54-
(E7)11 -Beta CDR3
Homo sapiens
(aa)
161
DSAIYN
TCR 8/ TCR 16/ TCR
18
alpha CDR1
Homo sapiens
(aa)
162
IQSSQRE
TCR 8/ TCR 16/ TCR
18
alpha CDR2
Homo sapiens
163
AVRPLGNTPLV
TCR 8
alpha CDR3
Homo sapiens
164
ASSLWGASTDTQY
TCR 8
Beta CDR3
Homo sapiens
(aa)
165
TSDPSYG
TCR 9/ TCR 17
alpha CDR1
Homo sapiens
(aa)
166
QGSYDQQN
TCR 9/ TCR 17
alpha CDR2
Homo sapiens
(aa)
167
AMRTAGGTSYGKLT
TCR 9
alpha CDR3
Homo sapiens
(aa)
168
MNHEY
TCR 9/ TCR 26
Beta CDR1
Homo sapiens
(aa)
169
SVGEGT
TCR 9/ TCR 26
Beta CDR2
Homo sapiens
(aa)
170
ASSYFGTAYEQY
TCR 9
Beta CDR3
Homo sapiens
(aa)
171
NSASQS
TCR 10/ TCR 28/ TCR
36/ TCR 41
alpha CDR1
Homo sapiens
(aa)
172
VYSSGN
TCR 10/ TCR 28/ TCR
41
alpha CDR2
Homo sapiens
(aa)
173
VVNFPSRGAGGTSYGKLT
TCR 10
alpha CDR3
Homo sapiens
(aa)
174
ASSLSLTGNYGYT
TCR 10
Beta CDR3
Homo sapiens
(aa)
175
ILSAHSNSGYALN
TCR 11
alpha CDR3
Homo sapiens
(aa)
176
SQVTM
TCR 11
Beta CDR1
Homo sapiens
(aa)
177
ANQGSEA
TCR 11
Beta CDR2
Homo sapiens
(aa)
178
SVVPWTRGGSTDTQY
TCR 11
Beta CDR3
Homo sapiens
(aa)
179
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 10- Beta
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Native
YLCASSLSLTGNYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATL
Homo sapiens
VCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVS
(aa)
ATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF
180
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 10- Beta
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Cysteine-modified
YLCASSLSLTGNYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATL
Homo sapiens
VCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
(aa)
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF
181
MSLSSLLKVVTASLWLGPGI
TCR 3/ TCR 9/ TCR 5/
TCR 15/ TCR 17/ TCR
19/ TCR 21/ TCR 23/
TCR 24/ TCR 25/ TCR
26/ TCR 29 -(E6)29
TCR alpha signal
peptide
Homo sapiens
(aa)
182
MGTRLLCWVVLGFLGTDHT
TCR 3/ TCR 13/ TCR
37 - Beta signal peptide
Homo sapiens
(aa)
183
ATGAAGACATTTGCTGGATTTTCGTTCCTGTTTTTGTGGCTGCAGCTGGACTG
TCR 12 - Alpha
TATGAGTAGAGGAGAGGATGTGGAGCAGAGTCTTTTCCTGAGTGTCCGAGAG
Native
GGAGACAGCTCCGTTATAAACTGCACTTACACAGACAGCTCCTCCACCTACT
Homo sapiens
TATACTGGTATAAGCAAGAACCTGGAGCAGGTCTCCAGTTGCTGACGTATAT
(nt)
TTTTTCAAATATGGACATGAAACAAGACCAAAGACTCACTGTTCTATTGAAT
AAAAAGGATAAACATCTGTCTCTGCGCATTGCAGACACCCAGACTGGGGACT
CAGCTATCTACTTCTGTGCAGTCCCCTCGGGTGCTACAAACAAGCTCATCTTT
GGAACTGGCACTCTGCTTGCTGTCCAGCCAAATATCCAGAACCCTGACCCTG
CCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATT
CACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTG
TATATCACAGACAAATGCGTGCTAGACATGAGGTCTATGGACTTCAAGAGCA
ACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTT
CAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCC
TGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACT
TTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGG
GTTTAATCTGCTCATGACGCTGCGGCTG
184
MKLVTSITVLLSLGIMG
TCR 4/ TCR 27-
(E6)29 alpha signal
peptide
Homo sapiens
(aa)
185
MGTSLLCWMALCLLGADHADT
TCR 4 - (E6)29 Beta
signal peptide
Homo sapiens
(aa)
186
MGIRLLCRVAFCFLAVGLV
TCR 5/ TCR 16/ TCR
17/ TCR 18/ TCR 19/
TCR 23/ TCR 24/ TCR
25/ TCR 28 - (E6)29/
TCR 8/ TCR10/ TCR
14 -
TCR beta signal
peptide
Homo sapiens
(aa)
187
MKTFAGFSFLFLWLQLDCMSR
TCR 6/ TCR 12/ TCR
50/ TCR 55- alpha
signal peptide
Homo sapiens
(aa)
188
MDTWLVCWAIFSLLKAGLT
TCR 6/7/12/33/36/39/
43/47/49/51/54/55/30 -
Beta signal peptide
Homo sapiens
(aa)
189
MKKHLTTFLVILWLYFYRGNG
TCR 7/ TCR 54-
(E7)11 - alpha signal
peptide
Homo sapiens
(aa)
190
METLLGLLILWLQLQWVSS
TCR 8/ TCR 16/ TCR
18 - alpha signal
peptide
Homo sapiens
(aa)
191
MSLGLLCCGAFSLLWAGPV
TCR 9/ TCR 26- Beta
signal peptide
Homo sapiens
(aa)
192
MMISLRVLLVILWLQLSWVWSQ
TCR 10/ TCR 28/ TCR
36/ TCR 41 - alpha
signal peptide
Homo sapiens
(aa)
193
MKLVTSITVLLSLGIMG
TCR 11 - alpha signal
peptide
Homo sapiens
(aa)
194
MLSLLLLLLGLGSVF
TCR 11 - Beta signal
peptide
Homo sapiens
(aa)
195
MKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQ
TCR 11 - Alpha
LPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILSAH
Native
SNSGYALNFGKGTSLLVTPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQ
Homo sapiens
SKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSP
(aa)
ESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
196
NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRS
TCR 4 - (E6)29 /
MDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN
TCR 5 - (E6)29/ TCR
LNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
12/ TCR 55 - TCR
alpha constant region
Homo sapiens
(aa)
197
EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHS
TCR
GVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE
4/5/7/10/14/16/17/18/21/
WTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVL
22/23/25/27/28/30/37/
VSALVLMAMVKRKDF
39/50/54 - TCR beta
constant region
Homo sapiens
(aa)
198
NIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRS
TCR 3/
MDFKSNSAVAWSNKSDFACANAFNNSIIPADTFFPSPESSCDVKLVEKSFETDTN
TCR 10
LNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
TCR alpha constant
region
Homo sapiens
(aa)
199
EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHS
TCR 3/6/8/9/11/13/19/
GVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE
20/24/29/31/32/33/34/
WTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVL
35/36/38/ 40/41/42/43/
VSALVLMAMVKRKDSRG
45/46/47/48/49/51/52/55 -
TCR beta constant
region
Homo sapiens
(aa)
200
HIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRS
TCR 6/
MDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN
TCR 11
LNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
alpha constant region
Homo sapiens
(aa)
201
YIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRS
TCR 7/ TCR 14 /TCR
MDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN
15/ TCR 20/ TCR 36/
LNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
TCR 54 - alpha
constant region
Homo sapiens
(aa)
202
ATGCTCCTGCTGCTCGTCCCAGTGCTCGAGGTGATTTTTACTCTGGGAGGAAC
TCR 13 - Alpha
CAGAGCCCAGTCGGTGACCCAGCTTGACAGCCACGTCTCTGTCTCTGAAGGA
Native
ACCCCGGTGCTGCTGAGGTGCAACTACTCATCTTCTTATTCACCATCTCTCTT
Homo sapiens
CTGGTATGTGCAACACCCCAACAAAGGACTCCAGCTTCTCCTGAAGTACACA
(nt)
TCAGCGGCCACCCTGGTTAAAGGCATCAACGGTTTTGAGGCTGAATTTAAGA
AGAGTGAAACCTCCTTCCACCTGACGAAACCCTCAGCCCATATGAGCGACGC
GGCTGAGTACTTCTGTGTTGTGAGGGGAGGAAAGCTTATCTTCGGACAGGGA
ACGGAGTTATCTGTGAAACCCAATATCCAGAACCCTGACCCTGCCGTGTACC
AGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTT
GATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAG
ACAAATGCGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGT
GGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGC
ATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCA
AGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCT
GTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGC
TCATGACGCTGCGGCTG
203
NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRS
TCR
MDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN
8/9/13/16/17/18/21/26/
LNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
27/28/30/31/32/33/34/35/
37/38/39/40/41/42/43/
44/45/46/48/49/50/51/
52/53 - alpha constant
region
Homo sapiens
(aa)
204
GSGATNFSLLKQAGDVEENPGP
P2A
Artificial
(aa)
205
MKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQ
TCR 11 - Alpha
LPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILSAH
Cysteine-modified
SNSGYALNFGKGTSLLVTPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQ
Homo sapiens
SKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSP
(aa)
ESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
206
MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQP
TCR 11 - Beta
GQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVV
Native
PWTRGGSTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Homo sapiens
TGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATF
(aa)
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG
207
GGATCCGGAGCTACCAACTTCTCTCTGCTGAAACAGGCAGGCGATGTGGAGG
TCR 3/
AAAATCCTGGGCCA
TCR 6/
TCR 8/
TCR 9/
TCR 10
TCR 11
P2A
Artificial
(nt)
208
GGGAGTGGAGCAACAAACTTTTCACTGCTGAAGCAGGCCGGCGATGTGGAG
TCR 4
GAAAATCCTGGGCCA
P2A
Artificial
(nt)
209
GGGTCCGGAGCCACAAATTTTTCTCTGCTGAAACAGGCTGGCGATGTGGAGG
TCR 5
AAAACCCTGGGCCA
P2A
Artificial
(nt)
210
GGAAGCGGCGCAACAAACTTTTCCCTGCTGAAACAGGCCGGAGATGTGGAG
TCR 7
GAAAATCCTGGCCCA
P2A
Artificial
(nt)
211
EGRGSLLTCGDVEENPGP
T2A
Artificial
(aa)
212
NIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRS
TCR 3/
MDFKSNSAVAWSNKSDFACANAFNNSIIPADTFFPSPESSCDVKLVEKSFETDTN
TCR 10
LNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
Native TCR alpha
constant region
Homo sapiens
(aa)
213
NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRS
TCR
MDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN
4/5/12/8/9/13/16/17/18/
LNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
21/26/27/28/30/31/32/
33/34/35/37/38/39/40/
41/42/43/44/45/46/48/
49/50/51/52/53/55 -
Native TCR alpha
constant region
Homo sapiens
(aa)
214
EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHS
TCR
GVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE
4/5/16/17/18/21/22/23/
WTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVL
25/27/28/7/37/39/50/51/
VSALVLMAMVKRKDF
52/54/10/14 - Native
TCR beta constant
region
Homo sapiens
(aa)
215
PNIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMR
TCR 3/
SMDFKSNSAVAWSNKSDFACANAFNNSIIPADTFFPSPESSCDVKLVEKSFETDT
TCR 10
NLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
Native TCR alpha
constant region
Homo sapiens
(aa)
216
EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHS
TCR
GVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDE
3/6/12/8/9/11/13/19/20/
WTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVL
24/29/31/32/33/34/35/36/
VSALVLMAMVKRKDSRG
38/40/41/42/43/46/47/
48/49/53/55
Native TCR beta
constant region
Homo sapiens
(aa)
217
HIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRS
TCR 6/
MDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN
TCR 11
LNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
Native TCR alpha
constant region
Homo sapiens
(aa)
218
YIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRS
TCR 7/ TCR 14/ TCR
MDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN
15/ TCR 20/ TCR 36/
LNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
TCR 54 - Native TCR
alpha constant region
Homo sapiens
(aa)
219
ATGGAGAAGAATCCTTTGGCAGCCCCATTACTAATCCTCTGGTTTCATCTTGA
TCR 14 - Alpha
CTGCGTGAGCAGCATACTGAACGTGGAACAAAGTCCTCAGTCACTGCATGTT
Native
CAGGAGGGAGACAGCACCAATTTCACCTGCAGCTTCCCTTCCAGCAATTTTT
Homo sapiens
ATGCCTTACACTGGTACAGATGGGAAACTGCAAAAAGCCCCGAGGCCTTGTT
(nt)
TGTAATGACTTTAAATGGGGATGAAAAGAAGAAAGGACGAATAAGTGCCAC
TCTTAATACCAAGGAGGGTTACAGCTATTTGTACATCAAAGGATCCCAGCCT
GAAGACTCAGCCACATACCTCTGTGCCTCTCAAACTGGGGCAAACAACCTCT
TCTTTGGGACTGGAACGAGACTCACCGTTATTCCCTATATCCAGAACCCTGA
CCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGC
CTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTG
ATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAA
GAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAAC
GCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAA
GTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCT
AAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTG
GCCGGGTTTAATCTGCTCATGACGCTGCGGCTG
220
PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMR
TCR 4 - (E6)29 /
SMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT
TCR 5/ TCR 12 /TCR
NLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
8/
TCR 9/ TCR 13-
(E6)29 - Native TCR
alpha constant region
Homo sapiens
(aa)
221
MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQP
TCR 11 - Beta
GQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVV
Cysteine-modified
PWTRGGSTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Homo sapiens
TGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
(aa)
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG
222
MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLY
TCR 12/ TCR 55-
WYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAI
(E7)11 -alpha
YFCAVPSGATNKLIFGTGTLLAVQPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDS
native
QTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPE
Homo sapiens
DTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS
(aa)
S
223
MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHVSLF
TCR 3
WYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDS
Full sequence
AVYLCASSHLAGFTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKA
Native
TLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRL
Homo sapiens
RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCG
(aa)
FTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNF
SLLKQAGDVEENPGPMSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAV
TLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARK
SANLVISASQLGDSAMYFCAMREGRGFKTIFGAGTRLFVKANIQKPDPAVYQLR
DSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWS
NKSDFACANAFNNSIIPADTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRIL
LLKVAGFNLLMTLRLWSS
224
MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYW
TCR 4 - (E6)29
YRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAM
Full sequence
YLCASSPGGGNTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLV
Native
CLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSA
Homo sapiens
TFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVS
(aa)
YQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQA
GDVEENPGPMKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTISG
TDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAA
VYYCILLVIRGTSYGKLTFGQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLFTD
FDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS
IIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLR
LWSS
225
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 5- (E6)29 - TCR
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Full sequence
YLCASSPWGETHQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATL
Native
VCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVS
Homo sapiens
ATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQ
AGDVEENPGPMSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCT
YDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLV
ISASQLGDSAMYFCAMREGTGTSYGKLTFGQGTILTVHPNIQNPDPAVYQLRDS
KSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNK
SDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLL
KVAGFNLLMTLRLWSS
226
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 6
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Full sequence
CASTTRSSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Native
TGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQ
AGDVEENPGPMKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCT
YTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRI
ADTQTGDSAIYFCAESIRGFGNVLHCGSGTQVIVLPHIQNPDPAVYQLRDSKSSD
KSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFA
CANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAG
FNLLMTLRLWSS
227
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 7/ TCR 54-
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
(E7)11 -
CAITDRTNYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLA
Full sequence
TGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATF
Native
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSY
Homo sapiens
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQAG
(aa)
DVEENPGPMKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNY
TVSPFSNLRWYKQDTGRGPVSLTIMTFSENTKSNGRYTATLDADTKQSSLHITAS
QLSDSASYICVVSRDNYGQNFVFGPGTRLSVLPYIQNPDPAVYQLRDSKSSDKSV
CLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACAN
AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNL
LMTLRLWSS
228
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 8
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Full sequence
YLCASSLWGASTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATL
Native
VCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRV
Homo sapiens
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
ESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLL
KQAGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNC
SFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAA
SQPGDSATYLCAVRPLGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSKSSDKSV
CLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACAN
AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNL
LMTLRLWSS
229
MSLGLLCCGAFSLLWAGPVNAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMY
TCR 9 -
WYRQDPGMGLRLIHYSVGEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQT
Full sequence
SVYFCASSYFGTAYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKAT
Native
LVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLR
(aa)
VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF
Homo sapiens
TSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFS
(aa)
LLKQAGDVEENPGPMSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVT
LDCTYDTSDPSYGLFWYKQPSSGEMIFLIYQGSYDQQNATEGRYSLNFQKARKS
ANLVISASQLGDSAMYFCAMRTAGGTSYGKLTFGQGTILTVHPNIQNPDPAVYQ
LRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVA
WSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGF
RILLLKVAGFNLLMTLRLWSS
230
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 10
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Full sequence
YLCASSLSLTGNYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATL
Native
VCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVS
Homo sapiens
ATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQ
AGDVEENPGPMMISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAF
NCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIR
DSKLSDSATYLCVVNFPSRGAGGTSYGKLTFGQGTILTVHPNIQKPDPAVYQLR
DSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWS
NKSDFACANAFNNSIIPADTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRIL
LLKVAGFNLLMTLRLWSS
231
MLSLLLLLLGLGSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYRQQP
TCR 11
GQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVV
Full sequence
PWTRGGSTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Native
TGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQ
AGDVEENPGPMKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTIS
GTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDA
AVYYCILSAHSNSGYALNFGKGTSLLVTPHIQNPDPAVYQLRDSKSSDKSVCLFT
DFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNN
SIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTL
RLWSS
232
KLPQLCTEL
E6(18-26) peptide
233
TIHDIILECV
E6(29-38) peptide
234
FAFRDLCIV
E6(52-60) peptide
235
TLGIVCPI
E7(86-93) peptide
236
YMLDLQPET
E7(11-19) peptide
237
GTLGIVCPI
E7(85-93) peptide
238
LLMGTLGIV
E7(82-90) peptide
239
TLHEYMLDL
E7(7-15) peptide
240
X1X2X3X4X5X6X7
TCR alpha E6(29-38)
X1 = T, D, S, or N;
CDR1 consensus
X2 = I, or S;
X3 = S, D, N, Y, or A;
X4 = G, Q, P, or null;
X5 = T, S, F, or I;
X6 = D, Y, P, or Q;
X7 = Y, G, N, A, S, or Q
241
X1SX3X4X5X6
TCR alpha E7(11-19)
X1 = D or V;
CDR1 consensus
X3 = S, or P;
X4 = S or F;
X5 = T or S;
X6 = Y or N
242
MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLY
TCR 12/ TCR 55-
WYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAI
(E7)11 - Alpha
YFCAVPSGATNKLIFGTGTLLAVQPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDS
Cysteine-modified
QTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPE
Homo sapiens
DTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS
(aa)
S
243
X1X2X3X4X5X6X7
TCR alpha overall
X1 = T, D, N, S, or V;
CDR1 consensus
X2 = I or S;
X3 = S, D, A, P, N, or Y
X4 = G, Q, P, or null;
X5 = T, S, I, or F;
X6 = D, Y,Q, T, P, or S;
X7 = Y, G, N, A, S, or Q;
244
X1X2X3X4X5X6X7X8
TCR alpha E6(29-38)
X1 = G, Q, I, M, Y, or V;
CDR2 consensus
X2 = L, S, Q, T, or Y;
X3 = T, G, L, or S;
X4 = Y, S, N, A, or null;
X5 = null, A, or D;
X6 = null, E, Q, T, or S;
X7 = S, Q, R, L, or G;
X8 = N, V, or E;
245
X1X2X3X4X5X6X7
TCR alpha E7(11-19)
X1 = I or M;
CDR2 consensus
X2 = F or T;
X3 = S or F;
X4 = N or S;
X5 = M or E;
X6 = D or N;
X7 = M or T;
246
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 6, TCR 12 TCR
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
55 - (E7)11 Beta
CASTTRSSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Native
TGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG
247
X1X2X3X4X5X6X7X8
TCR alpha overall
X1 = G, Q, I, V, Y, or M;
CDR2 consensus
X2 = L, S, Q, Y, F, or T;
X3 = T, G, S, L, or F;
X4 = Y, S, N, A, or null;
X5 = null, A, or D;
X6 = null, E, Q, S, M, or T;
X7 = S, Q, R, G, D, L, or N;
X8 = N, E, M, T, or V
248
X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18
TCR alpha E6(29-38)
X1 = A, I, or V;
CDR3 consensus
X2 = M, L, S, or V;
X3 = R, L, Q, or N;
X4 = E, V, T, P, G, or F;
X5 = G, I, L, A, null, or P;
X6 = R, T, G, null, or S;
X7 = G, R, or null;
X8 = T, G, or null;
X9 = null or A;
X10 = null or G;
X11 = null or G;
X12 = null or T;
X13 = null or S;
X14 = G, Y, null, or N;
X15 = F, G, N, or T;
X16 = K or N, P;
X17 = T or L;
X18 = I, V, F or T
249
X1X2X3X4X5X6X7X8X9X10X11
TCR alpha E7(11-19)
X1 = A or V;
CDR3 consensus
X2 = E or V;
X3 = S or P
X4 = I, S, or R;
X5 = R, G, or D;
X6 = G, A, or N;
X7 = F, null, or Y;
X8 = G or T
X9 = N, T, or Q;
X10 = V, K or N;
X11 = L or F;
X12 = H, I, or V
250
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 6, TCR 12, TCR
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
55 - (E7)11 -Beta
CASTTRSSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Cysteine-modified
TGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG
251
X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18
TCR alpha overall
X1 = A, I, or V;
CDR3 consensus
X2 = M, L, V, E, or S;
X3 = R, L, N, Q, P, or S;
X4 = E, V, P, T, F, I, R, G, S, or A;
X5 = G, I, L, A, P, R, D, null, or H;
X6 = R, T, G, S, N, null, or A;
X7 = G, R, N, or null;
X8 = T, G, or null;
X9 = null or A;
X10 = null or G;
X11 = null or G;
X12 = null or T;
X13 = F, Y, S or null;
X14 = G, Y, null, or N;
X15 = F, G, T, N, Q, or Y;
X16 = K, P, V, N or A;
X17 = T, L, or F;
X18 = I, V, T, H, F, or N
252
X1X2HX4X5
TCR beta E6(29-38)
X1 = S or M;
CDR1 consensus
X2 = G, E, D, or N;
X4 = V, N, or E;
X5 = S, R, N, or Y;
253
MLLLLVPVLEVIFTLGGTRAQSVTQLDSHVSVSEGTPVLLRCNYSSSYSPSLFWY
TCR 13 - Alpha
VQHPNKGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYF
Native
CVVRGGKLIFGQGTELSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVS
Homo sapiens
QSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTI-MPS
(aa)
PESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
254
X1X2X3X4X5
TCR beta overall
X1 = S or M;
CDR1 consensus
X2 = G, E, D, N, or Q;
X3 = H or V;
X4 = V, N, E, L, or T;
X5 = S, R, N, Y, or M;
255
X1X2X3X4X5X6
TCR beta E6(29-38)
X1 = F or S;
CDR2 consensus
X2 = Q, Y, or V;
X3 = N, D, or G;
X4 = E or V;
X5 = A, K, or G;
X6 = Q, M, or T;
256
MLLLLVPVLEVIFTLGGTRAQSVTQLDSHVSVSEGTPVLLRCNYSSSYSPSLFWY
TCR 13 - Alpha
VQHPNKGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYF
Cysteine-modified
CVVRGGKLIFGQGTELSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVS
Homo sapiens
QSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFP
(aa)
SPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
257
X1X2X3GX5X6X7
TCR beta overall
X1 = F, S, or A;
CDR2 consensus
X2 = Q, Y, V, or N;
X3 = N, D, G, or Q;
X5 = E, V, N, or S;
X6 = A, K, G, or E;
X7 = Q, M, T, I, or A;
258
AS X3X4X5X6X7X8X9X10X11X12X13
TCR beta E6(29-38)
X3 = S or T
CDR3 consensus
X4 = H, P, L, F, or Y;
X5 = L, G, W, F, T, or S;
X6 = A, G, or L;
X7 = G, E, A, T, Q, or null;
X8 = F, G, T, R, or S;
X9 = T, N, H, R, E, or A;
X10 = G, T, Q, D, R, or Y;
X11 = E, P, T, or G;
X12 = L, A, Q, or Y;
X13 = F, H, Y, or T
259
AX2TX4RX6X7YX9X10X11
TCR beta E7(11-19)
X2 = S or I;
CDR3 consensus
X4 = T or D;
X6 = S or T;
X7 = S or N;
X9 = E or G;
X10 = Q or Y;
X11 = Y or T
260
MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHVSLF
TCR 13- Beta
WYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDS
Native
AVYLCASSPTGTERELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATL
Homo sapiens
VCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRV
(aa)
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
ESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG
261
X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15
TCR beta overall
X1 = A or S;
CDR3 consensus
X2 = S, I, or V;
X3 = S, T, or V;
X4 = H, P, L, Y, T, D, or F;
X5 = L, G, W, F, 5, T, or R;
X6 = A, G, L, S, or T;
X7 = G, E, A, T, R, Q, or null;
X8 = null or G;
X9 = null or G;
X10 = null, F, G, T, S, or R;
X11 = T, N, H, A, S, R, or E;
X12 = G, T, Q, D, Y, or R;
X13 = E, P, T, or G;
X14 = L, A, Q, or Y;
X15 = F, H, Y, or T
262
DIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAM
Mouse alpha constant
DSKSN GAIAWSNQTS FTCQDIFKETNATYPSSDVPCDATLTEKSF
Mus musculus
ETDMNLNFQN LSVMGLRILL LKVAGFNLLM TLRLWSS
(aa)
263
EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHS
Mouse beta constant
GVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPE
Mus musculus
GSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVS
(aa)
TLVVMAMVKR KNS
264
MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAF
HPV 16 E6
RDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLI
(aa)
RCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL
265
MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNI
HPV 16 E7
VTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP
(aa)
266
-PGGG-(SGGGG)n-P- wherein n is 5 or 6, P is proline, G is glycine and S is serine
Linker
(aa)
267
GSADDAKKDAAKKDGKS
Linker
(aa)
268
ESKYGPPCPPCP
spacer (IgG4hinge)
Homo sapiens
(aa)
269
GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT
spacer (IgG4hinge)
Homo sapiens
(nt)
270
ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES
Hinge-CH3 spacer
NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY
Homo sapiens
TQKSLSLSLGK
(aa)
271
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ
Hinge-CH2-CH3
FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
spacer
GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW
Homo sapiens
ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN
(aa)
HYTQKSLSLSLGK
272
RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQE
IgD-hinge-Fc
ERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWE
Homo sapiens
VAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQ
(aa)
RLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQR
EVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNA
SRSLEVSYVTDH
273
MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGD
tEGFR
LHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEI
artificial
IRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLF
(aa)
GTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGREC
VDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPH
CVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPK
IPSIATGMVGALLLLLVVALGIGLFM
274
LEGGGEGRGSLLTCGDVEENPGPR
T2A
Artificial
(aa)
275
FWVLVVVGGVLACYSLLVTVAFIIFWV
CD28 (amino acids
153-179 of Accession
No. P10747)
Homo sapiens
(aa)
276
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP
CD28 (amino acids
FWVLVVVGGVLACYSLLVTVAFIIFWV
114-179 of Accession
No. P10747)
Homo sapiens
(aa)
277
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
CD28 (amino acids
180-220 of P10747)
Homo sapiens
(aa)
278
RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
CD28 (LL to GG)
Homo sapiens
(aa)
279
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
4-1BB (amino acids
214-255 of Q07011.1)
Homo sapiens
(aa)
280
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK
CD3 zeta
NPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY
Homo sapiens
QGLSTATKDTYDALHMQALP PR
(aa)
281
RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN
CD3 zeta
PQEGLYNELQKDKMAEA YSEIGMKGER RRGKGHDGLY
Homo sapiens
QGLSTATKDTYDALHMQALP PR
(aa)
282
RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK
CD3 zeta
NPQEGLYNELQKDKMAEA YSEIGMKGER RRGKGHDGLY
Homo sapiens
QGLSTATKDTYDALHMQALP PR
(aa)
283
GEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMD
TCR 12/ TCR 55-
MKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVPSGATNKLIFGTGTLLA
(E7)11 alpha
VQPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLD
native
MRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFET
Homo sapiens
DTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
(aa)
284
GEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMD
TCR 12/ TCR 55-
MKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVPSGATNKLIFGTGTLLA
(E7)11 alpha
VQPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLD
Cysteine-modified
MRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFET
Homo sapiens
DTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
(aa)
285
EPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEIS
TCR 6, TCR 12, TCR
EKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASTTRSSYEQYFGPGTRLT
55 - (E7)11 beta
VTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEV
Native
HSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN
Homo sapiens
DEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYA
(aa)
VLVSALVLMAMVKRKDSRG
286
EPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEIS
TCR 6, TCR 12, TCR
EKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASTTRSSYEQYFGPGTRLT
55 - (E7)11 beta
VTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEV
Cysteine-modified
HSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEN
Homo sapiens
DEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYA
(aa)
VLVSALVLMAMVKRKDSRG
287
AQSVTQLDSHVSVSEGTPVLLRCNYSSSYSPSLFWYVQHPNKGLQLLLKYTSAA
TCR 13 - alpha
TLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCVVRGGKLIFGQGTELSVK
Native
PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMR
Homo sapiens
SMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT
(aa)
NLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
288
AQSVTQLDSHVSVSEGTPVLLRCNYSSSYSPSLFWYVQHPNKGLQLLLKYTSAA
TCR 13 - alpha
TLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCVVRGGKLIFGQGTELSVK
Cysteine-modified
PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMR
Homo sapiens
SMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT
(aa)
NLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
289
GAGVSQSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQNEA
TCR 13 - beta
QLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSPTGTERELFFGEGS
native
RLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNG
Homo sapiens
KEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGL
SENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKAT
LYAVLVSALVLMAMVKRKDSRG
290
GAGVSQSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQNEA
TCR 13 - beta
QLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSPTGTERELFFGEGS
Cysteine-modified
RLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNG
Homo sapiens
KEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGL
(aa)
SENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKAT
LYAVLVSALVLMAMVKRKDSRG
291
ILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNG
TCR 14 - alpha
DEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCASQTGANNLFFGTGTRLTV
native
IPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMR
Homo sapiens
SMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT
(aa)
NLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
292
ILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNG
TCR 14 - alpha
DEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCASQTGANNLFFGTGTRLTV
Cysteine-modified
IPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMR
Homo sapiens
SMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDT
(aa)
NLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
293
DVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDV
TCR 14 - beta
KMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASTFWGQRRTEAFFGQ
native
GTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWV
Homo sapiens
NGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFY
(aa)
GLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGK
ATLYAVLVSALVLMAMVKRKDF
294
DVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDV
TCR 14 - beta
KMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASTFWGQRRTEAFFGQ
Cysteine-modified
GTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWV
Homo sapiens
NGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQF
(aa)
YGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLG
KATLYAVLVSALVLMAMVKRKDF
295
GEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMD
TCR 12/ TCR 55-
MKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAVPSGATNKLIFGTGTLLA
alpha variable
VQP
Homo sapiens
(aa)
296
EPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEIS
TCR 6, TCR 12, TCR
EKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASTTRSSYEQYFGPGTRLT
55 - beta variable
VT
Homo sapiens
(aa)
297
AQSVTQLDSHVSVSEGTPVLLRCNYSSSYSPSLFWYVQHPNKGLQLLLKYTSAA
TCR 13 - alpha
TLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCVVRGGKLIFGQGTELSVK
variable
P
Homo sapiens
(aa)
298
GAGVSQSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQNEA
TCR 13 - beta variable
QLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSPTGTERELFFGEGS
Homo sapiens
RLTVL
(aa)
299
ILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNG
TCR 14 - alpha
DEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCASQTGANNLFFGTGTRLTV
variable
IP
Homo sapiens
(aa)
300
DVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDV
TCR 14 - beta variable
KMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASTFWGQRRTEAFFGQ
Homo sapiens
GTRLTVV
(aa)
301
AVPSGATNKLI
TCR 12/ TCR 55
CDR3 alpha
Homo sapiens
(aa)
302
SSYSPS
TCR 13
CDR1 alpha
Homo sapiens
(aa)
303
YTSAATLV
TCR 13
CDR2 alpha
Homo sapiens
(aa)
304
VVRGGKLI
TCR 13
CDR3 alpha
Homo sapiens
(aa)
305
ASSPTGTERELF
TCR 13
CDR3 alpha
Homo sapiens
(aa)
306
SSNFYA
TCR 14
CDR1 alpha
Homo sapiens
(aa)
307
MTLNGDE
TCR 14
CDR2 alpha
Homo sapiens
(aa)
308
ASQTGANNLF
TCR 14
CDR3 alpha
Homo sapiens
(aa)
309
ASTFWGQRRTEAF
TCR 14
CDR3 beta
Homo sapiens
(aa)
310
MLLLLVPVLEVIFTLGGTR
TCR 13 alpha
Signal sequence
Homo sapiens
(aa)
311
MEKNPLAAPLLILWFHLDCVSS
TCR 14 alpha
Signal sequence
Homo sapiens
(aa)
312
MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHVSLF
TCR 13 - Beta
WYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDS
Cysteine-modified
AVYLCASSPTGTERELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATL
Homo sapiens
VCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
(aa)
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
ESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG
313
MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYAL
TCR 14 - Alpha
HWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSA
Native
TYLCASQTGANNLFFGTGTRLTVIPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDS
Homo sapiens
QTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPE
(aa)
DTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS
S
314
MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYAL
TCR 14 - Alpha
HWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSA
Cysteine-modified
TYLCASQTGANNLFFGTGTRLTVIPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDS
Homo sapiens
QTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPE
(aa)
DTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS
S
315
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 14 - Beta
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Native
YLCASTFWGQRRTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATL
Homo sapiens
VCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVS
(aa)
ATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF
316
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 14 - Beta
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Cysteine-modified
YLCASTFWGQRRTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATL
Homo sapiens
VCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
(aa)
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF
317
NIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAM
Mouse Alpha Constant
DSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQN
Sequence
LSVMGLRILLLKVAGFNLLMTLRLWSS
Mus musculus
(aa)
318
MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGL
TCR 3 - Alpha
FWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSA
Native
MYFCAMREGRGFKTIFGAGTRLFVKANIQKPDPAVYQLRDSKSSDKSVCLFTDF
Homo sapiens
DSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSII
(aa)
PADTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRL
WSS
319
MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGL
TCR 3 - Alpha
FWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSA
Cysteine-modified
MYFCAMREGRGFKTIFGAGTRLFVKANIQKPDPAVYQLRDSKSSDKSVCLFTDF
Homo sapiens
DSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSII
(aa)
PADTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRL
WSS
320
MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHVSLF
TCR 3 - Beta
WYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDS
Native
AVYLCASSHLAGFTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKA
Homo sapiens
TLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRL
(aa)
RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCG
FTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG
321
MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHVSLF
TCR 3 - Beta
WYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDS
Cysteine-modified
AVYLCASSHLAGFTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKA
Homo sapiens
TLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRL
(aa)
RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCG
FTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG
322
MKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQ
TCR 4 - (E6)29 alpha
LPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILLVIR
Native
GTSYGKLTFGQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQ
Homo sapiens
SKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSP
(aa)
ESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
323
MKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQ
TCR 4 - (E6)29 alpha
LPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILLVIR
Cysteine-modified
GTSYGKLTFGQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQ
Homo sapiens
SKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSP
(aa)
ESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
324
MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYW
TCR 4 - (E6)29 Beta
YRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAM
Native
YLCASSPGGGNTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLV
Homo sapiens
CLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSA
(aa)
TFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVS
YQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF
325
MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYW
TCR 4 - (E6)29 Beta
YRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAM
Cysteine-modified
YLCASSPGGGNTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLV
Homo sapiens
CLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVS
(aa)
ATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF
326
MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGL
TCR 5 - (E6)29 - TCR
FWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSA
alpha
MYFCAMREGTGTSYGKLTFGQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLF
Native
TDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFN
Homo sapiens
NSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMT
(aa)
LRLWSS
327
MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGL
TCR 5 - (E6)29 - TCR
FWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSA
alpha
MYFCAMREGTGTSYGKLTFGQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLF
Cysteine-modified
TDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFN
Homo sapiens
NSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMT
(aa)
LRLWSS
328
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 5 - (E6)29 - TCR
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
beta
YLCASSPWGETHQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATL
Native
VCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVS
Homo sapiens
ATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF
329
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 5 - (E6)29 - TCR
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
beta
YLCASSPWGETHQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATL
Cysteine-modified
VCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
Homo sapiens
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF
330
MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLY
TCR 6 - Alpha
WYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAI
Native
YFCAESIRGFGNVLHCGSGTQVIVLPHIQNPDPAVYQLRDSKSSDKSVCLFTDFD
Homo sapiens
SQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIP
(aa)
EDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLW
SS
331
MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSSTYLY
TCR 6 - Alpha
WYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAI
Cysteine-modified
YFCAESIRGFGNVLHCGSGTQVIVLPHIQNPDPAVYQLRDSKSSDKSVCLFTDFD
Homo sapiens
SQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIP
(aa)
EDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLW
SS
332
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 6, TCR 12 - Beta
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Native
CASTTRSSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Homo sapiens
TGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATF
(aa)
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG
333
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 6, TCR 12 - Beta
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Cysteine-modified
CASTTRSSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Homo sapiens
TGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
(aa)
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG
334
MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLR
TCR 7/ TCR 54-
WYKQDTGRGPVSLTIMTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYI
(E7)11 - alpha
CVVSRDNYGQNFVFGPGTRLSVLPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDS
Native
QTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPE
Homo sapiens
DTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS
(aa)
S
335
MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLR
TCR 7/ TCR 54 -
WYKQDTGRGPVSLTIMTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYI
(E7)11 - alpha
CVVSRDNYGQNFVFGPGTRLSVLPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDS
Cysteine-modified
QTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPE
Homo sapiens
DTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS
(aa)
S
336
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 7/ TCR 54-
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
(E7)11 -Beta
CAITDRTNYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLA
Native
TGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF
337
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 7/ TCR 54-
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
(E7)11 -Beta
CAITDRTNYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLA
Cysteine-modified
TGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF
338
METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQW
TCR 8 - Alpha
FRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCA
Native
VRPLGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNV
Homo sapiens
SQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFP
(aa)
SPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
39
METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQW
TCR 8 - Alpha
FRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCA
Cysteine-modified
VRPLGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNV
Homo sapiens
SQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFF
(aa)
PSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
340
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 12/ TCR 55
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Full sequence
CASTTRSSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Native
TGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQ
AGDVEENPGPMKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCT
YTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRI
ADTQTGDSAIYFCAVPSGATNKLIFGTGTLLAVQPNIQNPDPAVYQLRDSKSSDK
SVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFAC
ANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGF
NLLMTLRLWSS
341
MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHVSLF
TCR 13
WYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDS
Full sequence
AVYLCASSPTGTERELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATL
Native
VCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRV
Homo sapiens
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
ESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLL
KQAGDVEENPGPMLLLLVPVLEVIFTLGGTRAQSVTQLDSHVSVSEGTPVLLRC
NYSSSYSPSLFWYVQHPNKGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTK
PSAHMSDAAEYFCVVRGGKLIFGQGTELSVKPNIQNPDPAVYQLRDSKSSDKSV
CLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACAN
AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNL
LMTLRLWSS
342
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 14
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Full sequence
YLCASTFWGQRRTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATL
Native
VCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVS
Homo sapiens
ATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQ
AGDVEENPGPMEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTC
SFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYI
KGSQPEDSATYLCASQTGANNLFFGTGTRLTVIPYIQNPDPAVYQLRDSKSSDKS
VCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACA
NAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFN
LLMTLRLWSS
343
RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQ
tEGFR
ELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITS
artificial
LGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATG
QVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQ
CHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKY
ADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGI
GLFM
344
VKQTLNFDLLKLAGDVESNPGP
F2A
345
ATNFSLLKQAGDVEENPGP
P2A
346
QCTNYALLKLAGDVESNPGP
E2A
347
ggaagcggcgccacaaacttctcactgctgaaacaggccggcgacgtggaggagaatcctggccca
TCR 49/ TCR 51/ TCR
52/ TCR 53/ TCR 55 -
P2A
Artificial (nt)
348
atatccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgatt
Human TCR alpha
ctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaag
constant (TRAC)
agcaacagtgctgtggcctggagcaacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacacct
NCBI Reference
tcttccccagcccaggtaagggcagctttggtgccttcgcaggctgtttccttgcttcaggaatggccaggttctgcccagagctc
Sequence:
tggtcaatgatgtctaaaactcctctgattggtggtctcggccttatccattgccaccaaaaccctctttttactaagaaacagtgag
NG_001332.3, TRAC
ccttgttctggcagtccagagaatgacacgggaaaaaagcagatgaagagaaggtggcaggagagggcacgtggcccagc
ctcagtctctccaactgagttcctgcctgcctgcctttgctcagactgtttgccccttactgctcttctaggcctcattctaagcccctt
ctccaagttgcctctccttatttctccctgtctgccaaaaaatctttcccagctcactaagtcagtctcacgcagtcactcattaaccc
accaatcactgattgtgccggcacatgaatgcaccaggtgttgaagtggaggaattaaaaagtcagatgaggggtgtgcccag
aggaagcaccattctagttgggggagcccatctgtcagctgggaaaagtccaaataacttcagattggaatgtgttttaactcagg
gttgagaaaacagctaccttcaggacaaaagtcagggaagggctctctgaagaaatgctacttgaagataccagccctaccaa
gggcagggagaggaccctatagaggcctgggacaggagctcaatgagaaaggagaagagcagcaggcatgagttgaatga
aggaggcagggccgggtcacagggccttctaggccatgagagggtagacagtattctaaggacgccagaaagctgttgatcg
gcttcaagcaggggagggacacctaatttgcttttcttttttttttttttttttttttttttttttgagatggagttttgctcttgttgcccaggct
ggagtgcaatggtgcatcttggctcactgcaacctccgcctcccaggttcaagtgattctcctgcctcagcctcccgagtagctg
agattacaggcacccgccaccatgcctggctaattttttgtatttttagtagagacagggtttcactatgttggccaggctggtctcg
aactcctgacctcaggtgatccacccgcttcagcctcccaaagtgctgggattacaggcgtgagccaccacacccggcctgctt
ttcttaaagatcaatctgagtgctgtacggagagtgggttgtaagccaagagtagaagcagaaagggagcagttgcagcagag
agatgatggaggcctgggcagggtggtggcagggaggtaaccaacaccattcaggtttcaaaggtagaaccatgcagggatg
agaaagcaaagaggggatcaaggaaggcagctggattttggcctgagcagctgagtcaatgatagtgccgtttactaagaaga
aaccaaggaaaaaatttggggtgcagggatcaaaactttttggaacatatgaaagtacgtgtttatactctttatggcccttgtcact
atgtatgcctcgctgcctccattggactctagaatgaagccaggcaagagcagggtctatgtgtgatggcacatgtggccaggg
tcatgcaacatgtactttgtacaaacagtgtatattgagtaaatagaaatggtgtccaggagccgaggtatcggtcctgccagggc
caggggctctccctagcaggtgctcatatgctgtaagttccctccagatctctccacaaggaggcatggaaaggctgtagttgttc
acctgcccaagaactaggaggtctggggtgggagagtcagcctgctctggatgctgaaagaatgtctgtttttccttttagaaagt
tcctgtgatgtcaagctggtcgagaaaagctttgaaacaggtaagacaggggtctagcctgggtttgcacaggattgcggaagt
gatgaacccgcaataaccctgcctggatgagggagtgggaagaaattagtagatgtgggaatgaatgatgaggaatggaaac
agcggttcaagacctgcccagagctgggtggggtctctcctgaatccctctcaccatctctgactttccattctaagcactttgagg
atgagtttctagcttcaatagaccaaggactctctcctaggcctctgtattcctttcaacagctccactgtcaagagagccagagag
agcttctgggtggcccagctgtgaaatttctgagtcccttagggatagccctaaacgaaccagatcatcctgaggacagccaag
aggttttgccttctttcaagacaagcaacagtactcacataggctgtgggcaatggtcctgtctctcaagaatcccctgccactcct
cacacccaccctgggcccatattcatttccatttgagttgttcttattgagtcatccttcctgtggtagcggaactcactaaggggcc
catctggacccgaggtattgtgatgataaattctgagcacctaccccatccccagaagggctcagaaataaaataagagccaag
tctagtcggtgtttcctgtcttgaaacacaatactgttggccctggaagaatgcacagaatctgtttgtaaggggatatgcacagaa
gctgcaagggacaggaggtgcaggagctgcaggcctcccccacccagcctgctctgccttggggaaaaccgtgggtgtgtc
ctgcaggccatgcaggcctgggacatgcaagcccataaccgctgtggcctcttggttttacagatacgaacctaaactttcaaaa
cctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagctgag
gtgaggggccttgaagctgggagtggggtttagggacgcgggtctctgggtgcatcctaagctctgagagcaaacctccctgc
agggtcttgcttttaagtccaaagcctgagcccaccaaactctcctacttcttcctgttacaaattcctcttgtgcaataataatggcc
tgaaacgctgtaaaatatcctcatttcagccgcctcagttgcacttctcccctatgaggtaggaagaacagttgtttagaaacgaag
aaactgaggccccacagctaatgagtggaggaagagagacacttgtgtacaccacatgccttgtgttgtacttctctcaccgtgt
aacctcctcatgtcctctctccccagtacggctctcttagctcagtagaaagaagacattacactcatattacaccccaatcctggc
tagagtctccgcaccctcctcccccagggtccccagtcgtcttgctgacaactgcatcctgttccatcaccatcaaaaaaaaactc
caggctgggtgcgggggctcacacctgtaatcccagcactttgggaggcagaggcaggaggagcacaggagctggagacc
agcctgggcaacacagggagaccccgcctctacaaaaagtgaaaaaattaaccaggtgtggtgctgcacacctgtagtccca
gctacttaagaggctgagatgggaggatcgcttgagccctggaatgttgaggctacaatgagctgtgattgcgtcactgcactcc
agcctggaagacaaagcaagatcctgtctcaaataataaaaaaaataagaactccagggtacatttgctcctagaactctaccac
atagccccaaacagagccatcaccatcacatccctaacagtcctgggtcttcctcagtgtccagcctgacttctgttcttcctcattc
cagatctgcaagattgtaagacagcctgtgctccctcgctccttcctctgcattgcccctcttctccctctccaaacagagggaact
ctcctacccccaaggaggtgaaagctgctaccacctctgtgcccccccggcaatgccaccaactggatcctacccgaatttatg
attaagattgctgaagagctgccaaacactgctgccaccccctctgttcccttattgctgcttgtcactgcctgacattcacggcag
aggcaaggctgctgcagcctcccctggctgtgcacattccctcctgctccccagagactgcctccgccatcccacagatgatgg
atcttcagtgggttctcttgggctctaggtcctgcagaatgttgtgaggggtttatttttttttaatagtgttcataaagaaatacatagt
attcttcttctcaagacgtggggggaaattatctcattatcgaggccctgctatgctgtgtatctgggcgtgttgtatgtcctgctgcc
gatgccttc
349
aggacctgaacaaggtgttcccacccgaggtcgctgtgtttgagccatcagaagcagagatctcccacacccaaaaggccaca
Human TCR beta
ctggtgtgcctggccacaggcttcttccccgaccacgtggagctgagctggtgggtgaatgggaaggaggtgcacagtgggg
constant 1 (TRBC1)
tcagcacagacccgcagcccctcaaggagcagcccgccctcaatgactccagatactgcctgagcagccgcctgagggtctc
NCBI Reference
ggccaccttctggcagaacccccgcaaccacttccgctgtcaagtccagttctacgggctctcggagaatgacgagtggaccc
Sequence:
aggatagggccaaacccgtcacccagatcgtcagcgccgaggcctggggtagagcaggtgagtggggcctggggagatgc
NG_001333.2, TRBC1
ctggaggagattaggtgagaccagctaccagggaaaatggaaagatccaggtagcagacaagactagatccaaaaagaaag
gaaccagcgcacaccatgaaggagaattgggcacctgtggttcattcttctcccagattctcagcccaacagagccaagcagct
gggtcccctttctatgtggcctgtgtaactctcatctgggtggtgccccccatccccctcagtgctgccacatgccatggattgcaa
ggacaatgtggctgacatctgcatggcagaagaaaggaggtgctgggctgtcagaggaagctggtctgggcctgggagtctg
tgccaactgcaaatctgactttacttttaattgcctatgaaaataaggtctctcatttattttcctctccctgctttctttcagactgtggct
ttacctcgggtaagtaagcccttccttttcctctccctctctcatggttcttgacctagaaccaaggcatgaagaactcacagacact
ggagggtggagggtgggagagaccagagctacctgtgcacaggtacccacctgtccttcctccgtgccaacagtgtcctacca
gcaaggggtcctgtctgccaccatcctctatgagatcctgctagggaaggccaccctgtatgctgtgctggtcagcgcccttgtg
ttgatggccatggtaagcaggagggcaggatggggccagcaggctggaggtgacacactgacaccaagcacccagaagtat
agagtccctgccaggattggagctgggcagtagggagggaagagatttcattcaggtgcctcagaagataacttgcacctctgt
aggatcacagtggaagggtcatgctgggaaggagaagctggagtcaccagaaaacccaatggatgttgtgatgagccttacta
tttgtgtggtcaatgggccctactactttctctcaatcctcacaactcctggctcttaataacccccaaaactttctcttctgcaggtca
agagaaaggatttctga
350
MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWY
TCR 15
RQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVY
Full sequence
FCASSLVGRSRTEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLV
Cysteine-modified
CLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVS
Homo sapiens
ATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSE
(aa)
SYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLK
QAGDVEENPGPMSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDC
TYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANL
VISASQLGDSAMYFCAMKPGGYNKLIFGAGTRLAVHPYIQNPDPAVYQLRDSKS
SDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSD
FACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKV
AGFNLLMTLRLWSS
351
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 16
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Full sequence
YLCASSLWGRSNQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATL
Cysteine-modified
VCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
Homo sapiens
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQ
AGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSF
TDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAAS
QPGDSATYLCAVRPANNNDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKS
VCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACA
NAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFN
LLMTLRLWSS
352
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 17
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Full sequence
YLCASSLWGRSNQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATL
Cysteine-modified
VCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
Homo sapiens
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQ
AGDVEENPGPMSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCT
YDTSDPSYGLFWYKQPSSGEMIFLIYQGSYDQQNATEGRYSLNFQKARKSANLV
ISASQLGDSAMYFCAMREGRGDKIIFGKGTRLHILPNIQNPDPAVYQLRDSKSSD
KSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFA
CANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAG
FNLLMTLRLWSS
353
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 18
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Full sequence
YLCASSFWGRSNSPLHFGNGTRLTVTEDLNKVFPPEVAVFEPSEAEISHTQKATL
Cysteine-modified
VCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
Homo sapiens
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQ
AGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSF
TDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAAS
QPGDSATYLCAEGNAGGTSYGKLTFGQGTILTVHPNIQNPDPAVYQLRDSKSSD
KSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFA
CANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAG
FNLLMTLRLWSS
354
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 19
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Full sequence
YLCASSSWGQSTGEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATL
Cysteine-modified
VCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
Homo sapiens
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
ESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLL
KQAGDVEENPGPMSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLD
CTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSAN
LVISASQLGDSAMYFCAMRENTGTASKLTFGTGTRLQVTLDIQNPDPAVYQLRD
SKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSN
KSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILL
LKVAGFNLLMTLRLWSS
355
MLLLLLLLGPGSGLGAVVSQHPSRVICKSGTSVKIECRSLDFQATTMFWYRQFPK
TCR 20
QSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSA
Full sequence
SSLARRSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLAT
Cysteine-modified
GFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFW
Homo sapiens
QNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQ
(aa)
GVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQAG
DVEENPGPMNMLTASLLRAVIASICVVSSMAQKVTQAQTEISVVEKEDVTLDCV
YETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTIT
ASQVVDSAVYFCALWTGANNLFFGTGTRLTVIPYIQNPDPAVYQLRDSKSSDKS
VCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACA
NAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFN
LLMTLRLWSS
356
MHRPRRPLHPVAPAMSIGLLCCVAFSLLWASPVNAGVTQTPKFQVLKTGQSMT
TCR 21
LQCAQDMNHNSMYWYRQDPGMGLRLIYYSASEGTTDKGEVPNGYNVSRLNKR
Full sequence
EFSLRLESAAPSQTSVYFCASRPWGNQNTEAFFGQGTRLTVVEDLNKVFPPEVA
Cysteine-modified
VFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQ
Homo sapiens
PALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI
(aa)
VSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVK
RKDFGSGATNFSLLKQAGDVEENPGPMSLSSLLKVVTASLWLGPGIAQKITQTQ
PGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEG
RYSLNFQKARKSANLVISASQLGDSAMYFCAMREGRVTGGGNKLTFGTGTQLK
VELNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLD
MRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFET
DTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
357
MGPGLLCWVLLCLLGAGPVDAGVTQSPTHLIKTRGQHVTLRCSPISGHKSVSWY
TCR 22
QQVLGQGPQFIFQYYEKEERGRGNFPDRFSARQFPNYSSELNVNALLLGDSALY
Full sequence
LCASSRTENYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCL
Cysteine-modified
ATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQAG
DVEENPGPMAQELGMQCQARGILQQMWGVFLLYVSMKMGGTTGQNIDQPTE
MTATEGAIVQINCTYQTSGFNGLFWYQQHAGEAPTFLSYNVLDGLEEKGRFSSF
LSRSKGYSYLLLKELQMKDSASYLCAVRARMDSNYQLIWGAGTKLIIKPDIQNP
DPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKS
NSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQN
LSVIGFRILLLKVAGFNLLMTLRLWSS
358
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 23
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Full sequence
YLCASSPWGQSNQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATL
Cysteine-modified
VCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
Homo sapiens
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQ
AGDVEENPGPMSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCT
YDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLV
ISASQLGDSAMYFCAMSPPGGSARQLTFGSGTQLTVLPDIQNPDPAVYQLRDSKS
SDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSD
FACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKV
AGFNLLMTLRLWSS
359
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 24
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Full sequence
YLCASSPFGRGSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATL
Cysteine-modified
VCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
Homo sapiens
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
ESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLL
KQAGDVEENPGPMSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLD
CTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSAN
LVISASQLGDSAMYFCAMREGRGDSWGKLQFGAGTQVVVTPDIQNPDPAVYQL
RDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAW
SNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRI
LLLKVAGFNLLMTLRLWSS
360
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 25
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Full sequence
YLCASSLWGQSNQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATL
Cysteine-modified
VCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
Homo sapiens
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
VSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQ
AGDVEENPGPMSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCT
YDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLV
ISASQLGDSAMYFCAMREGSLTGGGNKLTFGTGTQLKVELNIQNPDPAVYQLRD
SKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSN
KSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILL
LKVAGFNLLMTLRLWSS
361
MSLGLLCCGAFSLLWAGPVNAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMY
TCR 26
WYRQDPGMGLRLIHYSVGEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQT
Full sequence
SVYFCASSYYASGRNYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQK
Cysteine-modified
ATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSR
Homo sapiens
LRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC
(aa)
GFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATN
FSLLKQAGDVEENPGPMSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEA
VTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKAR
KSANLVISASQLGDSAMYFCAMRDARNNDMRFGAGTRLTVKPNIQNPDPAVYQ
LRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVA
WSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGF
RILLLKVAGFNLLMTLRLWSS
362
MHRPRRPLHPVAPAMSIGLLCCVAFSLLWASPVNAGVTQTPKFQVLKTGQSMT
TCR 27
LQCAQDMNHNSMYWYRQDPGMGLRLIYYSASEGTTDKGEVPNGYNVSRLNKR
Full sequence
EFSLRLESAAPSQTSVYFCASSEFGSLNEKLFFGSGTQLSVLEDLNKVFPPEVAVF
Cysteine-modified
EPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPA
Homo sapiens
LNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS
(aa)
AEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK
DFGSGATNFSLLKQAGDVEENPGPMKLVTSITVLLSLGIMGDAKTTQPNSMESN
EEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDR
KSSTLILHRATLRDAAVYYCILRVPPQSGGYQKVTFGTGTKLQVIPNIQNPDPAV
YQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAV
AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIG
FRILLLKVAGFNLLMTLRLWSS
363
MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMF
TCR 28
WYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSM
Full sequence
YLCASSLWGRSSGNTIYFGEGSWLTVVEDLNKVFPPEVAVFEPSEAEISHTQKAT
Cysteine-modified
LVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLR
Homo sapiens
VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF
(aa)
TSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLL
KQAGDVEENPGPMMISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATV
AFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISL
LIRDSKLSDSATYLCVVRGGGTSYGKLTFGQGTILTVHPNIQNPDPAVYQLRDSK
SSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKS
DFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLK
VAGFNLLMTLRLWSS
364
MSNQVLCCVVLCLLGANTVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMY
TCR 29
WYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAF
Full sequence
YLCASSPWGRATNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATL
Cysteine-modified
VCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
Homo sapiens
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
ESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLL
KQAGDVEENPGPMSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLD
CTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSAN
LVISASQLGDSAMYFCAMRLNTGTASKLTFGTGTRLQVTLDIQNPDPAVYQLRD
SKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSN
KSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILL
LKVAGFNLLMTLRLWSS
365
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 30
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Full sequence
CASSRQPSSGNTIYFGEGSWLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVC
Cysteine-modified
LATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSA
Homo sapiens
TFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVS
(aa)
YQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQA
GDVEENPGPMRLVARVTVFLTFGTIIDAKTTQPPSMDCAEGRAANLPCNHSTISG
NEYVYWYRQIHSQGPQYIIHGLKNNETNEMASLIITEDRKSSTLILPHATLRDTAV
YYCIVRGTSVLQGNEKLTFGTGTRLTIIPNIQNPDPAVYQLRDSKSSDKSVCLFTD
FDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS
IIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLR
LWSS
366
MGTRLLFWVAFCLLGADHTGAGVSQSPSNKVTEKGKDVELRCDPISGHTALYW
TCR 31
YRQSLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGGSVSTLTIQRTQQEDSAV
Full sequence
YLCASSRFLGSTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATL
Cysteine-modified
VCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRV
Homo sapiens
SATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTS
(aa)
ESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLL
KQAGDVEENPGPMAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQ
EGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSA
KHLSLHIVPSQPGDSAVYFCAASERGTYKYIFGTGTRLKVLANIQNPDPAVYQLR
DSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWS
NKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRIL
LLKVAGFNLLMTLRLWSS
367
MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWY
TCR 32
QQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYF
Full sequence
CASSVGGDHSDEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVC
Cysteine-modified
LATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSA
Homo sapiens
TFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSES
(aa)
YQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLK
QAGDVEENPGPMVLKFSVSILWIQLAWVSTQLLEQSPQFLSIQEGENLTVYCNSS
SVFSSLQWYRQEPGEGPVLLVTVVTGGEVKKLKRLTFQFGDARKDSSLHITAAQ
PGDTGLYLCAGGSNYKLTFGKGTLLTVNPNIQNPDPAVYQLRDSKSSDKSVCLF
TDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFN
NSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMT
LRLWSS
368
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 33
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Full sequence
CASTPRDTYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Cysteine-modified
TGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQ
AGDVEENPGPMMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASL
NCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLI
RDSQPSDSATYLCAVNAHHTGGFKTIFGAGTRLFVKANIQNPDPAVYQLRDSKS
SDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSD
FACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKV
AGFNLLMTLRLWSS
369
MGPGLLCWALLCLLGAGSVETGVTQSPTHLIKTRGQQVTLRCSSQSGHNTVSW
TCR 34
YQQALGQGPQFIFQYYREEENGRGNFPPRFSGLQFPNYSSELNVNALELDDSALY
Full sequence
LCASSSYAGSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVC
Cysteine-modified
LATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSA
Homo sapiens
TFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSES
(aa)
YQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLK
QAGDVEENPGPMKKLLAMILWLQLDRLSGELKVEQNPLFLSMQEGKNYTIYCN
YSTTSDRLYWYRQDPGKSLESLFVLLSNGAVKQEGRLMASLDTKARLSTLHITA
AVHDLSATYFCAVSGTYKYIFGTGTRLKVLANIQNPDPAVYQLRDSKSSDKSVC
LFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANA
FNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLL
MTLRLWSS
370
MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWY
TCR 35
QQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYF
Full sequence
CASTTSGDSSYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVC
Cysteine-modified
LATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSA
Homo sapiens
TFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSES
(aa)
YQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLK
QAGDVEENPGPMALQSTLGAVWLGLLLNSLWKVAESKDQVFQPSTVASSEGAV
VEIFCNHSVSNAYNFFWYLHFPGCAPRLLVKGSKPSQQGRYNMTYERFSSSLLIL
QVREADAAVYYCAVAGDYKLSFGAGTTVTVRANIQNPDPAVYQLRDSKSSDKS
VCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACA
NAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFN
LLMTLRLWSS
371
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 36
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Full sequence
CAMTGRSNYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCL
Cysteine-modified
ATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSAT
Homo sapiens
FWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQ
AGDVEENPGPMMISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAF
NCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIR
DSKLSDSATYLCVVNRDNYGQNFVFGPGTRLSVLPYIQNPDPAVYQLRDSKSSD
KSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFA
CANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAG
FNLLMTLRLWSS
372
MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHVSLF
TCR 37
WYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDS
Full sequence
AVYLCASSLLLGAYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKAT
Cysteine-modified
LVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLR
Homo sapiens
VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF
(aa)
TSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLL
KQAGDVEENPGPMKKLLAMILWLQLDRLSGELKVEQNPLFLSMQEGKNYTIYC
NYSTTSDRLYWYRQDPGKSLESLFVLLSNGAVKQEGRLMASLDTKARLSTLHIT
AAVHDLSATYFCAGYSGAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSD
KSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFA
CANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAG
FNLLMTLRLWSS
373
MGPGLLCWVLLCLLGAGSVETGVTQSPTHLIKTRGQQVTLRCSSQSGHNTVSW
TCR 38
YQQALGQGPQFIFQYYREEENGRGNFPPRFSGLQFPNYSSELNVNALELDDSALY
Full sequence
LCASSLVAGGETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVC
Cysteine-modified
LATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSA
Homo sapiens
TFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSES
(aa)
YQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLK
QAGDVEENPGPMVKMPGARRQSIMKRILGALLGLLSAQVCCVRGIQVEQSPPDL
ILQEGANSTLRCNFSDSVNNLQWFHQNPWGQLINLFYIPSGTKQNGRLSATTVA
TERYSLLYISSSQTTDSGVYFCAVGFNDMRFGAGTRLTVKPNIQNPDPAVYQLR
DSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWS
NKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRIL
LLKVAGFNLLMTLRLWSS
374
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 39
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Full sequence
CASTPRDRGKEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCL
Cysteine-modified
ATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQAG
DVEENPGPMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQE
PGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGYSSS
NDYKLSFGAGTTVTVRANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQS
KDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPE
SSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
375
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 40
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Full sequence
CAITARSSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLAT
Cysteine-modified
GFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFW
Homo sapiens
QNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQ
(aa)
GVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQAG
DVEENPGPMHTSTFQNRPQLFLLIWKKLVPGNPFRRSWMKREREMLLITSMLVL
WMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVF
LIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGRNNFNKFY
FGSGTKLNVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYI
TDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKL
VEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
376
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 41
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Full sequence
CASNPRDRVSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVC
Cysteine-modified
LATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSA
Homo sapiens
TFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSES
(aa)
YQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLK
QAGDVEENPGPMMISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVA
FNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLI
RDSKLSDSATYLCVVTFALTGGFKTIFGAGTRLFVKANIQNPDPAVYQLRDSKSS
DKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDF
ACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA
GFNLLMTLRLWSS
377
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 42
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Full sequence
CAKTSRSSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Cysteine-modified
TGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQ
AGDVEENPGPMHTSTFQNRPQLFLLIWKKLVPGNPFRRSWMKREREMLLITSML
VLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGH
PVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGPDNFN
KFYFGSGTKLNVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDS
DVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSC
DVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
378
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 43
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Full sequence
CASTPRDSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Cysteine-modified
TGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQ
AGDVEENPGPMMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASL
NCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLI
RDSQPSDSATYLCAVNVPTSGTYKYIFGTGTRLKVLANIQNPDPAVYQLRDSKSS
DKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDF
ACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA
GFNLLMTLRLWSS
379
MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWY
TCR 44
QQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYF
Full sequence
CASSGTPDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLAT
Cysteine-modified
GFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFW
Homo sapiens
QNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQ
(aa)
GVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQAG
DVEENPGPMAQELGMQCQARGILQQMWGVFLLYVSMKMGGTTGQNIDQPTE
MTATEGAIVQINCTYQTSGFNGLFWYQQHAGEAPTFLSYNVLDGLEEKGRFSSF
LSRSKGYSYLLLKELQMKDSASYLCAQYSGGYQKVTFGTGTKLQVIPNIQNPDP
AVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNS
AVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS
VIGFRILLLKVAGFNLLMTLRLWSS
380
MGTRLLFWVAFCLLGAYHTGAGVSQSPSNKVTEKGKDVELRCDPISGHTALYW
TCR 45
YRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAV
Full sequence
YLCASSLYLGTTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLV
Cysteine-modified
CLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVS
Homo sapiens
ATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSE
(aa)
SYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLK
QAGDVEENPGPMHTSTFQNRPQLFLLIWKKLVPGNPFRRSWMKREREMLLITSM
LVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGG
HPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGSSGA
GSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSK
DSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPES
SCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS
381
MGTRLLCWAALCLLGADHTGAGVSQTPSNKVTEKGKYVELRCDPISGHTALY
TCR 46
WYRQSLGQGPEFLIYFQGTGAADDSGLPNDRFFAVRPEGSVSTLKIQRTERGDSA
Full sequence
VYLCASSLYLGGSETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKAT
Cysteine-modified
LVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLR
Homo sapiens
VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF
(aa)
TSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFS
LLKQAGDVEENPGPMLLLLVPAFQVIFTLGGTRAQSVTQLDSQVPVFEEAPVEL
RCNYSSSVSVYLFWYVQYPNQGLQLLLKYLSGSTLVKGINGFEAEFNKSQTSFH
LRKPSVHISDTAEYFCAVSPSSGTYKYIFGTGTRLKVLANIQNPDPAVYQLRDSK
SSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKS
DFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLK
VAGFNLLMTLRLWSS
382
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 47
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Full sequence
CAMTGRTTYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Cysteine-modified
TGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQ
AGDVEENPGPMMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASL
NCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLI
RDSQPSDSATYLCAVNLLSGSARQLTFGSGTQLTVLPDIQNPDPAVYQLRDSKSS
DKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDF
ACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA
GFNLLMTLRLWSS
383
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 48
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Full sequence
CASTGRVSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Cysteine-modified
TGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQ
AGDVEENPGPMMKSLRVLLVILWLQLSWVWSQQKEVEQDPGPLSVPEGAIVSL
NCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISL
FIRDSQPSDSATYLCAMRIQGAQKLVFGQGTRLTINPNIQNPDPAVYQLRDSKSS
DKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDF
ACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA
GFNLLMTLRLWSS
384
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 49
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Full sequence
CASTPRYSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Cysteine-modified
TGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQ
AGDVEENPGPMMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASL
NCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLI
RDSQPSDSATYLCAVNIGTSGTYKYIFGTGTRLKVLANIQNPDPAVYQLRDSKSS
DKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDF
ACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA
GFNLLMTLRLWSS
385
MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWY
TCR 50
QQSLDQGLQFLIHYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYF
Full sequence
CASSATRDAYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCL
Cysteine-modified
ATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQAG
DVEENPGPMKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYT
DSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIAD
TQTGDSAIYFCAESPPGTYKYIFGTGTRLKVLANIQNPDPAVYQLRDSKSSDKSV
CLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACAN
AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNL
LMTLRLWSS
386
MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWY
TCR 51
RQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYF
Full sequence
CAIASRVSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA
Cysteine-modified
TGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATF
Homo sapiens
WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQ
AGDVEENPGPMMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASL
NCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLI
RDSQPSDSATYLCAVNMRGGGSNYKLTFGKGTLLTVNPNIQNPDPAVYQLRDS
KSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNK
SDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLL
KVAGFNLLMTLRLWSS
387
MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWY
TCR 52
QQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYF
Full sequence
CASSVGDLNNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCL
Cysteine-modified
ATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSAT
Homo sapiens
FWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESY
(aa)
QQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQ
AGDVEENPGPMVLKFSVSILWIQLAWVSTQLLEQSPQFLSIQEGENLTVYCNSSS
VFSSLQWYRQEPGEGPVLLVTVVTGGEVKKLKRLTFQFGDARKDSSLHITAAQP
GDTGLYLCAGARDYKLSFGAGTTVTVRANIQNPDPAVYQLRDSKSSDKSVCLFT
DFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNN
SIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTL
RLWSS
388
MGTSLLCWVVLGFLGTDHTGAGVSQSPRYKVTKRGQDVALRCDPISGHVSLYW
TCR 53
YRQALGQGPEFLTYFNYEAQQDKSGLPNDRFSAERPEGSISTLTIQRTEQRDSAM
Full sequence
YRCASSGSGTSGYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKAT
Cysteine-modified
LVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLR
Homo sapiens
VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGF
(aa)
TSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFS
LLKQAGDVEENPGPMASAPISMLAMLFTLSGLRAQSVAQPEDQVNVAEGNPLT
VKCTYSVSGNPYLFWYVQYPNRGLQFLLKYITGDNLVKGSYGFEAEFNKSQTSF
HLKKPSALVSDSALYFCAVRDFGSGTYKYIFGTGTRLKVLANIQNPDPAVYQLR
DSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWS
NKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRIL
LLKVAGFNLLMTLRLWSS
389
TCR 15 - Alpha Native Homo sapiens (nt)
390
TCR 15 - Beta Native Homo sapiens (nt)
391
TCR 15 Full sequence Native Homo sapiens (aa)
392
TCR 16 Full sequence Native Homo sapiens (aa)
393
TCR 17 Full sequence Native Homo sapiens (aa)
394
TCR 18 Full sequence Native Homo sapiens (aa)
395
TCR 19 Full sequence Native Homo sapiens (aa)
396
TCR 20 Full sequence Native Homo sapiens (aa)
397
TCR 21 Full sequence Native Homo sapiens (aa)
398
TCR 22 Full sequence Native Homo sapiens (aa)
399
TCR 23 Full sequence Native Homo sapiens (aa)
400
TCR 24 Full sequence Native Homo sapiens (aa)
401
TCR 25 Full sequence Native Homo sapiens (aa)
402
TCR 26 Full sequence Native Homo sapiens (aa)
403
TCR 27 Full sequence Native Homo sapiens (aa)
404
TCR 28 Full sequence Native Homo sapiens (aa)
405
TCR 29 Full sequence Native Homo sapiens (aa)
406
TCR 30 Full sequence Native Homo sapiens (aa)
407
TCR 31 Full sequence Native Homo sapiens (aa)
408
TCR 32 Full sequence Native Homo sapiens (aa)
409
TCR 33 Full sequence Native Homo sapiens (aa)
410
TCR 34 Full sequence Native Homo sapiens (aa)
411
TCR 35 Full sequence Native Homo sapiens (aa)
412
TCR 36 Full sequence Native Homo sapiens (aa)
413
TCR 37 Full sequence Native Homo sapiens (aa)
414
TCR 38 Full sequence Native Homo sapiens (aa)
415
TCR 39 Full sequence Native Homo sapiens (aa)
416
TCR 40 Full sequence Native Homo sapiens (aa)
417
TCR 41 Full sequence Native Homo sapiens (aa)
418
TCR 42 Full sequence Native Homo sapiens (aa)
419
TCR 43 Full sequence Native Homo sapiens (aa)
420
TCR 44 Full sequence Native Homo sapiens (aa)
421
TCR 45 Full sequence Native Homo sapiens (aa)
422
TCR 46 Full sequence Native Homo sapiens (aa)
423
TCR 47 Full sequence Native Homo sapiens (aa)
424
TCR 48 Full sequence Native Homo sapiens (aa)
425
TCR 49 Full sequence Native Homo sapiens (aa)
426
TCR 50 Full sequence Native Homo sapiens (aa)
427
TCR 51 Full sequence Native Homo sapiens (aa)
428
TCR 52 Full sequence Native Homo sapiens (aa)
429
TCR 53 Full sequence Native Homo sapiens (aa)
430
TCR 16 - Alpha Native Homo sapiens (nt)
431
TCR 16 - Beta Native Homo sapiens (nt)
432
TCR 15 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
433
TCR 16 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
434
TCR 17 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
435
TCR 18 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
436
TCR 19 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
437
TCR 20 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
438
TCR 21 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
439
TCR 22 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
440
TCR 23 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
441
TCR 24 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
442
TCR 25 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
443
TCR 26 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
444
TCR 27 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
445
TCR 28 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
446
TCR 29 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
447
TCR 30 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
448
TCR 31 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
449
TCR 32 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
450
TCR 33 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
451
TCR 34 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
452
TCR 35 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
453
TCR 36 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
454
TCR 37 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
455
TCR 38 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
456
TCR 39 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
457
TCR 40 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
458
TCR 41 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
459
TCR 42 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
460
TCR 43 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
461
TCR 44 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
462
TCR 45 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
463
TCR 46 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
464
TCR 47 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
465
TCR 48 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
466
TCR 49 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
467
TCR 50 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
468
TCR 51 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
469
TCR 52 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
470
TCR 53 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
471
TCR 54 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
472
TCR 55 Codon-optimized/ cysteine-modified full sequence Homo sapiens (nt)
473
TCR 15 - Alpha Native Homo sapiens (aa)
474
TCR 15 - Alpha Cysteine-modified Homo sapiens (aa)
475
TCR 15 - Alpha Native Homo sapiens (aa)
476
TCR 15 - Alpha Cysteine-modified Homo sapiens (aa)
477
TCR 15 Alpha variable region Homo sapiens (aa)
478
TCR 15 alpha CDR3 Homo sapiens (aa)
479
TCR 15 - Beta Native Homo sapiens (aa)
480
TCR 15 - Beta Cysteine-modified Homo sapiens (aa)
481
TCR 15 - Beta Native Homo sapiens (aa)
482
TCR 15 - Beta Cysteine-modified Homo sapiens (aa)
483
TCR 15 beta variable region Homo sapiens (aa)
484
TCR 15 Beta CDR1 Homo sapiens (aa)
485
TCR 15 Beta CDR2 Homo sapiens (aa)
486
TCR 15 Beta CDR3 Homo sapiens (aa)
487
TCR 15 - Beta signal peptide Homo sapiens (aa)
488
TCR 16 - Alpha Native Homo sapiens (aa)
489
TCR 16 - Alpha Cysteine-modified Homo sapiens (aa)
490
TCR 16 - Alpha Native Homo sapiens (aa)
491
TCR 16 - Alpha Cysteine-modified Homo sapiens (aa)
492
TCR 16 Alpha variable region Homo sapiens (aa)
493
TCR 16 alpha CDR3 Homo sapiens (aa)
494
TCR 16 - Beta Native Homo sapiens (aa)
495
TCR 16/ TCR 17 - Beta Cysteine-modified Homo sapiens (aa)
496
TCR 16/ TCR 17 - Beta Native Homo sapiens (aa)
497
TCR 16/ TCR 17 - Beta Cysteine-modified Homo sapiens (aa)
498
TCR 16/ TCR 17 - Beta variable region Homo sapiens (aa)
499
TCR 16/ TCR 17 Beta CDR3 Homo sapiens (aa)
500
TCR 17 - Alpha Native Homo sapiens (aa)
501
TCR 17 - Alpha Cysteine-modified Homo sapiens (aa)
502
TCR 17 - Alpha Native Homo sapiens (aa)
503
TCR 17 - Alpha Cysteine-modified Homo sapiens (aa)
504
TCR 17 Alpha variable region Homo sapiens (aa)
505
TCR 17 Alpha CDR3 Homo sapiens (aa)
506
TCR 18 - Alpha Native Homo sapiens (aa)
507
TCR 18 - Alpha Cysteine-modified Homo sapiens (aa)
508
TCR 18 - Alpha Native Homo sapiens (aa)
509
TCR 18 - Alpha Cysteine-modified Homo sapiens (aa)
510
TCR 18 Alpha variable region Homo sapiens (aa)
511
TCR 18 Alpha CDR3 Homo sapiens (aa)
512
TCR 18 - Beta Native Homo sapiens (aa)
513
TCR 18 - Beta Cysteine-modified Homo sapiens (aa)
514
TCR 18 - Beta Native Homo sapiens (aa)
515
TCR 18 - Beta Cysteine-modified Homo sapiens (aa)
516
TCR 18 Beta variable region Homo sapiens (aa)
517
TCR 18 Beta CDR3 Homo sapiens (aa)
518
TCR 19 - Alpha Native Homo sapiens (aa)
519
TCR 19 - Alpha Cysteine-modified Homo sapiens (aa)
520
TCR 19 - Alpha Native Homo sapiens (aa)
521
TCR 19 - Alpha Cysteine-modified Homo sapiens (aa)
522
TCR 19 Alpha variable region Homo sapiens (aa)
523
TCR 19 Alpha CDR3 Homo sapiens (aa)
524
TCR 19/ TCR 22/ TCR 23/ TCR 24/ TCR 25/ TCR 47 Native TCR alpha constant region Homo sapiens (aa)
525
TCR 19/ TCR 22/ TCR 23/ TCR 24/ TCR 25/ TCR 29/ TCR 47 Alpha constant region Homo sapiens (aa)
526
TCR 19 - Beta Native Homo sapiens (aa)
527
TCR 19 - Beta Cysteine-modified Homo sapiens (aa)
528
TCR 19 - Beta Native Homo sapiens (aa)
529
TCR 19 - Beta Cysteine-modified Homo sapiens (aa)
530
TCR 19 Beta variable region Homo sapiens (aa)
531
TCR 19 Beta CDR3 Homo sapiens (aa)
532
TCR 20 - Alpha Native Homo sapiens (aa)
533
TCR 20 - Alpha Cysteine-modified Homo sapiens (aa)
534
TCR 20 - Alpha Native Homo sapiens (aa)
535
TCR 20 - Alpha Cysteine-modified Homo sapiens (aa)
536
TCR 20 Alpha variable region Homo sapiens (aa)
537
TCR 20 Alpha CDR1 Homo sapiens (aa)
538
TCR 20 Alpha CDR2 Homo sapiens (aa)
539
TCR 20 Alpha CDR3 Homo sapiens (aa)
540
TCR 20 alpha signal peptide Homo sapiens (aa)
541
TCR 20 - Beta Native Homo sapiens (aa)
542
TCR 20 - Beta Cysteine-modified Homo sapiens (aa)
543
TCR 20 - Beta Native Homo sapiens (aa)
544
TCR 20 - Beta Cysteine-modified Homo sapiens (aa)
545
TCR 20 Beta variable region Homo sapiens (aa)
546
TCR 20 Beta CDR1 Homo sapiens (aa)
547
TCR 20 Beta CDR2 Homo sapiens (aa)
548
TCR 20 Beta CDR3 Homo sapiens (aa)
549
TCR 20 beta signal peptide Homo sapiens (aa)
550
TCR 21 - Alpha Native Homo sapiens (aa)
551
TCR 21 - Alpha Cysteine-modified Homo sapiens (aa)
552
TCR 21 - Alpha Native Homo sapiens (aa)
553
TCR 21 - Alpha Cysteine-modified Homo sapiens (aa)
554
TCR 21 Alpha variable region Homo sapiens (aa)
555
TCR 21 Alpha CDR3 Homo sapiens (aa)
556
TCR 21 - Beta Native Homo sapiens (aa)
557
TCR 21 - Beta Cysteine-modified Homo sapiens (aa)
558
TCR 21 - Beta Native Homo sapiens (aa)
559
TCR 21 - Beta Cysteine-modified Homo sapiens (aa)
560
TCR 21 Beta variable region Homo sapiens (aa)
561
TCR 21/ TCR 27 Beta CDR1 Homo sapiens (aa)
562
TCR 21/ TCR 27 Beta CDR2 Homo sapiens (aa)
563
TCR 21 Beta CDR3 Homo sapiens (aa)
564
TCR 21/ TCR 27 Beta signal peptide Homo sapiens (aa)
565
TCR 22 - Alpha Native Homo sapiens (aa)
566
TCR 22 - Alpha Cysteine-modified Homo sapiens (aa)
567
TCR 22 - Alpha Native Homo sapiens (aa)
568
TCR 22 - Alpha Cysteine-modified Homo sapiens (aa)
569
TCR 22 Alpha variable region Homo sapiens (aa)
570
TCR 22/ TCR 44 Alpha CDR1 Homo sapiens (aa)
571
TCR 22/ TCR 44 Alpha CDR2 Homo sapiens (aa)
572
TCR 22 Alpha CDR3 Homo sapiens (aa)
573
TCR 22/ TCR 44Alpha signal peptide Homo sapiens (aa)
574
TCR 22 - Beta Native Homo sapiens (aa)
575
TCR 22 - Beta Cysteine-modified Homo sapiens (aa)
576
TCR 22 - Beta Native Homo sapiens (aa)
577
TCR 22 - Beta Cysteine-modified Homo sapiens (aa)
578
TCR 22 Beta variable region Homo sapiens (aa)
579
TCR 22 Beta CDR1 Homo sapiens (aa)
580
TCR 22 Beta CDR2 Homo sapiens (aa)
581
TCR 22 Beta CDR3 Homo sapiens (aa)
582
TCR 22 Beta signal peptide Homo sapiens (aa)
583
TCR 23 - Alpha Native Homo sapiens (aa)
584
TCR 23 - Alpha Cysteine-modified Homo sapiens (aa)
585
TCR 23 - Alpha Native Homo sapiens (aa)
586
TCR 23 - Alpha Cysteine-modified Homo sapiens (aa)
587
TCR 23 Alpha variable region Homo sapiens (aa)
588
TCR 23 Alpha CDR3 Homo sapiens (aa)
589
TCR 23 - Beta Native Homo sapiens (aa)
590
TCR 23 - Beta Cysteine-modified Homo sapiens (aa)
591
TCR 23 - Beta Native Homo sapiens (aa)
592
TCR 23 - Beta Cysteine-modified Homo sapiens (aa)
593
TCR 23 Beta variable region Homo sapiens (aa)
594
TCR 23 Beta CDR3 Homo sapiens (aa)
595
TCR 24 - Alpha Native Homo sapiens (aa)
596
TCR 24 - Alpha Cysteine-modified Homo sapiens (aa)
597
TCR 24 - Alpha Native Homo sapiens (aa)
598
TCR 24 - Alpha Cysteine-modified Homo sapiens (aa)
599
TCR 24 Alpha variable region Homo sapiens (aa)
600
TCR 24 Alpha CDR3 Homo sapiens (aa)
601
TCR 24 - Beta Native Homo sapiens (aa)
602
TCR 24 - Beta Cysteine-modified Homo sapiens (aa)
603
TCR 24 - Beta Native Homo sapiens (aa)
604
TCR 24 - Beta Cysteine-modified Homo sapiens (aa)
605
TCR 24 Beta variable region Homo sapiens (aa)
606
TCR 24 Beta CDR3 Homo sapiens (aa)
607
TCR 25 - Alpha Native Homo sapiens (aa)
608
TCR 25 - Alpha Cysteine-modified Homo sapiens (aa)
609
TCR 25 - Alpha Native Homo sapiens (aa)
610
TCR 25 - Alpha Cysteine-modified Homo sapiens (aa)
611
TCR 25 Alpha variable region Homo sapiens (aa)
612
TCR 25 Alpha CDR3 Homo sapiens (aa)
613
TCR 25 - Beta Native Homo sapiens (aa)
614
TCR 25 - Beta Cysteine-modified Homo sapiens (aa)
615
TCR 25 - Beta Native Homo sapiens (aa)
616
TCR 25 - Beta Cysteine-modified Homo sapiens (aa)
617
TCR 25 Beta variable region Homo sapiens (aa)
618
TCR 25 Beta CDR3 Homo sapiens (aa)
619
TCR 26 - Alpha Native Homo sapiens (aa)
620
TCR 26 - Alpha Cysteine-modified Homo sapiens (aa)
621
TCR 26 - Alpha Native Homo sapiens (aa)
622
TCR 26 - Alpha Cysteine-modified Homo sapiens (aa)
623
TCR 26 Alpha variable region Homo sapiens (aa)
624
TCR 26 Alpha CDR3 Homo sapiens (aa)
625
TCR 26 - Beta Native Homo sapiens (aa)
626
TCR 26 - Beta Cysteine-modified Homo sapiens (aa)
627
TCR 26 - Beta Native Homo sapiens (aa)
628
TCR 26 - Beta Cysteine-modified Homo sapiens (aa)
629
TCR 26 Beta variable region Homo sapiens (aa)
630
TCR 26 Beta CDR3 Homo sapiens (aa)
631
TCR 26 - Native TCR beta constant region Homo sapiens (aa)
632
TCR 26 - TCR beta constant region Homo sapiens (aa)
633
TCR 27 - Alpha Native Homo sapiens (aa)
634
TCR 27 - Alpha Cysteine-modified Homo sapiens (aa)
635
TCR 27 - Alpha Native Homo sapiens (aa)
636
TCR 27 - Alpha Cysteine-modified Homo sapiens (aa)
637
TCR 27 Alpha variable region Homo sapiens (aa)
638
TCR 27 Alpha CDR3 Homo sapiens (aa)
639
TCR 27 - Beta Native Homo sapiens (aa)
640
TCR 27 - Beta Cysteine-modified Homo sapiens (aa)
641
TCR 27 - Beta Native Homo sapiens (aa)
642
TCR 27 - Beta Cysteine-modified Homo sapiens (aa)
643
TCR 27 Beta variable region Homo sapiens (aa)
644
TCR 27 Beta CDR3 Homo sapiens (aa)
645
TCR 28 - Alpha Native Homo sapiens (aa)
646
TCR 28 - Alpha Cysteine-modified Homo sapiens (aa)
647
TCR 28 - Alpha Native Homo sapiens (aa)
648
TCR 28 - Alpha Cysteine-modified Homo sapiens (aa)
649
TCR 28 Alpha variable region Homo sapiens (aa)
650
TCR 28 Alpha CDR3 Homo sapiens (aa)
651
TCR 28 - Beta Native Homo sapiens (aa)
652
TCR 28 - Beta Cysteine-modified Homo sapiens (aa)
653
TCR 28 - Beta Native Homo sapiens (aa)
654
TCR 28 - Beta Cysteine-modified Homo sapiens (aa)
655
TCR 28 Beta variable region Homo sapiens (aa)
656
TCR 28 Beta CDR3 Homo sapiens (aa)
657
TCR 29 - Alpha Native Homo sapiens (aa)
658
TCR 29 - Alpha Cysteine-modified Homo sapiens (aa)
659
TCR 29 - Alpha Native Homo sapiens (aa)
660
TCR 29 - Alpha Cysteine-modified Homo sapiens (aa)
661
TCR 29 Alpha variable region Homo sapiens (aa)
662
TCR 29 Alpha CDR3 Homo sapiens (aa)
663
TCR 29 - Beta Native Homo sapiens (aa)
664
TCR 29 - Beta Cysteine-modified Homo sapiens (aa)
665
TCR 29 - Beta Native Homo sapiens (aa)
666
TCR 29 - Beta Cysteine-modified Homo sapiens (aa)
667
TCR 29 Beta variable region Homo sapiens (aa)
668
TCR 29 Beta CDR1 Homo sapiens (aa)
669
TCR 29 Beta CDR2 Homo sapiens (aa)
670
TCR 29 Beta CDR3 Homo sapiens (aa)
671
TCR 29 Beta signal peptide Homo sapiens (aa)
672
TCR 30 - Alpha Native Homo sapiens (aa)
673
TCR 30 - Alpha Cysteine-modified Homo sapiens (aa)
674
TCR 30 - Alpha Native Homo sapiens (aa)
675
TCR 30 - Alpha Cysteine-modified Homo sapiens (aa)
676
TCR 30 Alpha variable region Homo sapiens (aa)
677
TCR 30 Alpha CDR1 Homo sapiens (aa)
678
TCR 30 Alpha CDR2 Homo sapiens (aa)
679
TCR 30 Alpha CDR3 Homo sapiens (aa)
680
TCR 30 Alpha signal peptide Homo sapiens (aa)
681
TCR 30 - Beta Native Homo sapiens (aa)
682
TCR 30 - Beta Cysteine-modified Homo sapiens (aa)
683
TCR 30 - Beta Native Homo sapiens (aa)
684
TCR 30 - Beta Cysteine-modified Homo sapiens (aa)
685
TCR 30 Beta variable region Homo sapiens (aa)
686
TCR 30 Beta CDR3 Homo sapiens (aa)
687
TCR 31 - Alpha Native Homo sapiens (aa)
688
TCR 31 - Alpha Cysteine-modified Homo sapiens (aa)
689
TCR 31 - Alpha Native Homo sapiens (aa)
690
TCR 31 - Alpha Cysteine-modified Homo sapiens (aa)
691
TCR 31 Alpha variable region Homo sapiens (aa)
692
TCR 31 Alpha CDR1 Homo sapiens (aa)
693
TCR 31 Alpha CDR2 Homo sapiens (aa)
694
TCR 31 Alpha CDR3 Homo sapiens (aa)
695
TCR 31 Alpha signal peptide Homo sapiens (aa)
696
TCR 31 - Beta Native Homo sapiens (aa)
697
TCR 31 - Beta Cysteine-modified Homo sapiens (aa)
698
TCR 31 - Beta Native Homo sapiens (aa)
699
TCR 31 - Beta Cysteine-modified Homo sapiens (aa)
700
TCR 31 Beta variable region Homo sapiens (aa)
701
TCR 31/ TCR 45/ TCR 46 Beta CDR1 Homo sapiens (aa)
702
TCR 31/ TCR 45 Beta CDR2 Homo sapiens (aa)
703
TCR 31 Beta CDR3 Homo sapiens (aa)
704
TCR 31/ TCR 32 Beta signal peptide Homo sapiens (aa)
705
TCR 32 - Alpha Native Homo sapiens (aa)
706
TCR 32 - Alpha Cysteine-modified Homo sapiens (aa)
707
TCR 32 - Alpha Native Homo sapiens (aa)
708
TCR 32 - Alpha Cysteine-modified Homo sapiens (aa)
709
TCR 32 Alpha variable region Homo sapiens (aa)
710
TCR 32/ TCR 52 Alpha CDR1 Homo sapiens (aa)
711
TCR 32/ TCR 52 Alpha CDR2 Homo sapiens (aa)
712
TCR 32 Alpha CDR3 Homo sapiens (aa)
713
TCR 32/ TCR 52 Alpha signal peptide Homo sapiens (aa)
714
TCR 32 - Beta Native Homo sapiens (aa)
715
TCR 32 - Beta Cysteine-modified Homo sapiens (aa)
716
TCR 32 - Beta Native Homo sapiens (aa)
717
TCR 32 - Beta Cysteine-modified Homo sapiens (aa)
718
TCR 32 Beta variable region Homo sapiens (aa)
719
TCR 32/ TCR 35/ TCR 44/ TCR 50/ TCR 52 Beta CDR1 Homo sapiens (aa)
720
TCR 32/ TCR 35/ TCR 44/ TCR 50/ TCR 52 Beta CDR2 Homo sapiens (aa)
721
TCR 32 Beta CDR3 Homo sapiens (aa)
722
TCR 33 - Alpha Native Homo sapiens (aa)
723
TCR 33 - Alpha Cysteine-modified Homo sapiens (aa)
724
TCR 33 - Alpha Native Homo sapiens (aa)
725
TCR 33 - Alpha Cysteine-modified Homo sapiens (aa)
726
TCR 33 Alpha variable region Homo sapiens (aa)
727
TCR 33/ TCR 43/ TCR 47/ TCR 49/ TCR 51 Alpha CDR1 Homo sapiens (aa)
728
TCR 33/ TCR 43/ TCR 47/ TCR 49/ TCR 51 Alpha CDR2 Homo sapiens (aa)
729
TCR 33 Alpha CDR3 Homo sapiens (aa)
730
TCR 33/ TCR 43/ TCR 47/ TCR 48/ TCR 49/ TCR 51 Alpha signal peptide Homo sapiens (aa)
731
TCR 33 - Beta Native Homo sapiens (aa)
732
TCR 33 - Beta Cysteine-modified Homo sapiens (aa)
733
TCR 33 - Beta Native Homo sapiens (aa)
734
TCR 33 - Beta Cysteine-modified Homo sapiens (aa)
735
TCR 33 Beta variable region Homo sapiens (aa)
736
TCR 33 Beta CDR3 Homo sapiens (aa)
737
TCR 34 - Alpha Native Homo sapiens (aa)
738
TCR 34 - Alpha Cysteine-modified Homo sapiens (aa)
739
TCR 34 - Alpha Native Homo sapiens (aa)
740
TCR 34 - Alpha Cysteine-modified Homo sapiens (aa)
741
TCR 34 Alpha variable region Homo sapiens (aa)
742
TCR 34/ TCR 37 Alpha CDR1 Homo sapiens (aa)
743
TCR 34/ TCR 37 Alpha CDR2 Homo sapiens (aa)
744
TCR 34 Alpha CDR3 Homo sapiens (aa)
745
TCR 34/ TCR 37 Alpha signal peptide Homo sapiens (aa)
746
TCR 34 - Beta Native Homo sapiens (aa)
747
TCR 34 - Beta Cysteine-modified Homo sapiens (aa)
748
TCR 34 - Beta Native Homo sapiens (aa)
749
TCR 34 - Beta Cysteine-modified Homo sapiens (aa)
750
TCR 34 Beta variable region Homo sapiens (aa)
751
TCR 34/ TCR 38 Beta CDR1 Homo sapiens (aa)
752
TCR 34/ TCR 38 Beta CDR2 Homo sapiens (aa)
753
TCR 34 Beta CDR3 Homo sapiens (aa)
754
TCR 34 Beta signal peptide Homo sapiens (aa)
755
TCR 35 - Alpha Native Homo sapiens (aa)
756
TCR 35 - Alpha Cysteine-modified Homo sapiens (aa)
757
TCR 35 - Alpha Native Homo sapiens (aa)
758
TCR 35 - Alpha Cysteine-modified Homo sapiens (aa)
759
TCR 35 Alpha variable region Homo sapiens (aa)
760
TCR 35 Alpha CDR1 Homo sapiens (aa)
761
TCR 35 Alpha CDR2 Homo sapiens (aa)
762
TCR 35 Alpha CDR3 Homo sapiens (aa)
763
TCR 35 Alpha signal peptide Homo sapiens (aa)
764
TCR 35 - Beta Native Homo sapiens (aa)
765
TCR 35 - Beta Cysteine-modified Homo sapiens (aa)
766
TCR 35 - Beta Native Homo sapiens (aa)
767
TCR 35 - Beta Cysteine-modified Homo sapiens (aa)
768
TCR 35 Beta variable region Homo sapiens (aa)
769
TCR 35 Beta CDR3 Homo sapiens (aa)
770
TCR 35/ TCR 44/ TCR 50/ TCR 52Beta signal peptide Homo sapiens (aa)
771
TCR 36 - Alpha Native Homo sapiens (aa)
772
TCR 36 - Alpha Cysteine-modified Homo sapiens (aa)
773
TCR 36 - Alpha Native Homo sapiens (aa)
774
TCR 36 - Alpha Cysteine-modified Homo sapiens (aa)
775
TCR 36 Alpha variable region Homo sapiens (aa)
776
TCR 36 Alpha CDR3 Homo sapiens (aa)
777
TCR 36 - Beta Native Homo sapiens (aa)
778
TCR 36 - Beta Cysteine-modified Homo sapiens (aa)
779
TCR 36 - Beta Native Homo sapiens (aa)
780
TCR 36 - Beta Cysteine-modified Homo sapiens (aa)
781
TCR 36 Beta variable region Homo sapiens (aa)
782
TCR 36 Beta CDR3 Homo sapiens (aa)
783
TCR 37 - Alpha Native Homo sapiens (aa)
784
TCR 37 - Alpha Cysteine-modified Homo sapiens (aa)
785
TCR 37 - Alpha Native Homo sapiens (aa)
786
TCR 37 - Alpha Cysteine-modified Homo sapiens (aa)
787
TCR 37 Alpha variable region Homo sapiens (aa)
788
TCR 37 Alpha CDR3 Homo sapiens (aa)
789
TCR 37 - Beta Native Homo sapiens (aa)
790
TCR 37 - Beta Cysteine-modified Homo sapiens (aa)
791
TCR 37 - Beta Native Homo sapiens (aa)
792
TCR 37 - Beta Cysteine-modified Homo sapiens (aa)
793
TCR 37 Beta variable region Homo sapiens (aa)
794
TCR 37 Beta CDR3 Homo sapiens (aa)
795
TCR 38 - Alpha Native Homo sapiens (aa)
796
TCR 38 - Alpha Cysteine-modified Homo sapiens (aa)
797
TCR 38 - Alpha Native Homo sapiens (aa)
798
TCR 38 - Alpha Cysteine-modified Homo sapiens (aa)
799
TCR 38 Alpha variable region Homo sapiens (aa)
800
TCR 38 Alpha CDR1 Homo sapiens (aa)
801
TCR 38 Alpha CDR2 Homo sapiens (aa)
802
TCR 38 Alpha CDR3 Homo sapiens (aa)
803
TCR 38 Alpha signal peptide Homo sapiens (aa)
804
TCR 38 - Beta Native Homo sapiens (aa)
805
TCR 38 - Beta Cysteine-modified Homo sapiens (aa)
806
TCR 38 - Beta Native Homo sapiens (aa)
807
TCR 38 - Beta Cysteine-modified Homo sapiens (aa)
808
TCR 38 Beta variable region Homo sapiens (aa)
809
TCR 38 Beta CDR3 Homo sapiens (aa)
810
TCR 38 Beta signal peptide Homo sapiens (aa)
811
TCR 39 - Alpha Native Homo sapiens (aa)
812
TCR 39 - Alpha Cysteine-modified Homo sapiens (aa)
813
TCR 39 - Alpha Native Homo sapiens (aa)
814
TCR 39 - Alpha Cysteine-modified Homo sapiens (aa)
815
TCR 39 Alpha variable region Homo sapiens (aa)
816
TCR 39/ TCR 40/ TCR 42/ TCR 45 Alpha CDR1 Homo sapiens (aa)
817
TCR 39 Alpha CDR2 Homo sapiens (aa)
818
TCR 39 Alpha CDR3 Homo sapiens (aa)
819
TCR 39 Alpha signal peptide Homo sapiens (aa)
820
TCR 39 - Beta Native Homo sapiens (aa)
821
TCR 39 - Beta Cysteine-modified Homo sapiens (aa)
822
TCR 39 - Beta Native Homo sapiens (aa)
823
TCR 39 - Beta Cysteine-modified Homo sapiens (aa)
824
TCR 39 Beta variable region Homo sapiens (aa)
825
TCR 39 Beta CDR3 Homo sapiens (aa)
826
TCR 40 - Alpha Native Homo sapiens (aa)
827
TCR 40 - Alpha Cysteine-modified Homo sapiens (aa)
828
TCR 40 - Alpha Native Homo sapiens (aa)
829
TCR 40 - Alpha Cysteine-modified Homo sapiens (aa)
830
TCR 40 Alpha variable region Homo sapiens (aa)
831
TCR 40 Alpha CDR2 Homo sapiens (aa)
832
TCR 40/ TCR 42 Alpha CDR3 Homo sapiens (aa)
833
Transmembrane-modified/cysteine modified mouse constant alpha Mus musculus (aa)
834
TCR 40/ TCR 42/ TCR 45 Alpha signal peptide Homo sapiens (aa)
835
TCR 40 - Beta Native Homo sapiens (aa)
836
TCR 40 - Beta Cysteine-modified Homo sapiens (aa)
837
TCR 40 - Beta Native Homo sapiens (aa)
838
TCR 40 - Beta Cysteine-modified Homo sapiens (aa)
839
TCR 40 Beta variable region Homo sapiens (aa)
840
TCR 40 Beta CDR3 Homo sapiens (aa)
841
TCR 41 - Alpha Native Homo sapiens (aa)
842
TCR 41 - Alpha Cysteine-modified Homo sapiens (aa)
843
TCR 41 - Alpha Native Homo sapiens (aa)
844
TCR 41 - Alpha Cysteine-modified Homo sapiens (aa)
845
TCR 41 Alpha variable region Homo sapiens (aa)
846
TCR 41 Alpha CDR3 Homo sapiens (aa)
847
TCR 41 - Beta Native Homo sapiens (aa)
848
TCR 41 - Beta Cysteine-modified Homo sapiens (aa)
849
TCR 41 - Beta Native Homo sapiens (aa)
850
TCR 41 - Beta Cysteine-modified Homo sapiens (aa)
851
TCR 41 Beta variable region Homo sapiens (aa)
852
TCR 41 Beta CDR3 Homo sapiens (aa)
853
TCR 42 - Alpha Native Homo sapiens (aa)
854
TCR 42 - Alpha Cysteine-modified Homo sapiens (aa)
855
TCR 42 - Alpha Native Homo sapiens (aa)
856
TCR 42 - Alpha Cysteine-modified Homo sapiens (aa)
857
TCR 42 Alpha variable region Homo sapiens (aa)
858
TCR 42 Alpha CDR3 Homo sapiens (aa)
859
TCR 42 - Beta Native Homo sapiens (aa)
860
TCR 42 - Beta Cysteine-modified Homo sapiens (aa)
861
TCR 42 - Beta Native Homo sapiens (aa)
862
TCR 42 - Beta Cysteine-modified Homo sapiens (aa)
863
TCR 42 Beta variable region Homo sapiens (aa)
864
TCR 42 Beta CDR3 Homo sapiens (aa)
865
TCR 43 - Alpha Native Homo sapiens (aa)
866
TCR 43 - Alpha Cysteine-modified Homo sapiens (aa)
867
TCR 43 - Alpha Native Homo sapiens (aa)
868
TCR 43 - Alpha Cysteine-modified Homo sapiens (aa)
869
TCR 43 Alpha variable region Homo sapiens (aa)
870
TCR 43 Alpha CDR3 Homo sapiens (aa)
871
TCR 43 - Beta Native Homo sapiens (aa)
872
TCR 43 - Beta Cysteine-modified Homo sapiens (aa)
873
TCR 43 - Beta Native Homo sapiens (aa)
874
TCR 43 - Beta Cysteine-modified Homo sapiens (aa)
875
TCR 43 Beta variable region Homo sapiens (aa)
876
TCR 43 Beta CDR3 Homo sapiens (aa)
877
TCR 44 - Alpha Native Homo sapiens (aa)
878
TCR 44 - Alpha Cysteine-modified Homo sapiens (aa)
879
TCR 44 - Alpha Native Homo sapiens (aa)
880
TCR 44 - Alpha Cysteine-modified Homo sapiens (aa)
881
TCR 44 Alpha variable region Homo sapiens (aa)
882
TCR 44 Alpha CDR3 Homo sapiens (aa)
883
TCR 44 - Beta Native Homo sapiens (aa)
884
TCR 44 - Beta Cysteine-modified Homo sapiens (aa)
885
TCR 44 - Beta Native Homo sapiens (aa)
886
TCR 44 - Beta Cysteine-modified Homo sapiens (aa)
887
TCR 44 Beta variable region Homo sapiens (aa)
888
TCR 44 Beta CDR3 Homo sapiens (aa)
889
TCR 44 Native TCR beta constant region Homo sapiens (aa)
890
TCR 44 TCR beta constant region Homo sapiens (aa)
891
TCR 45 - Alpha Native Homo sapiens (aa)
892
TCR 45 - Alpha Cysteine-modified Homo sapiens (aa)
893
TCR 45 - Alpha Native Homo sapiens (aa)
894
TCR 45 - Alpha Cysteine-modified Homo sapiens (aa)
895
TCR 45 Alpha variable region Homo sapiens (aa)
896
TCR 45 Alpha CDR3 Homo sapiens (aa)
897
TCR 45 - Beta Native Homo sapiens (aa)
898
TCR 45 - Beta Cysteine-modified Homo sapiens (aa)
899
TCR 45 - Beta Native Homo sapiens (aa)
900
TCR 45 - Beta Cysteine-modified Homo sapiens (aa)
901
TCR 45 Beta variable region Homo sapiens (aa)
902
TCR 45 Beta CDR3 Homo sapiens (aa)
903
TCR 45 Beta signal peptide Homo sapiens (aa)
904
TCR 46 - Alpha Native Homo sapiens (aa)
905
TCR 46 - Alpha Cysteine-modified Homo sapiens (aa)
906
TCR 46 - Alpha Native Homo sapiens (aa)
907
TCR 46 - Alpha Cysteine-modified Homo sapiens (aa)
908
TCR 46 Alpha variable region Homo sapiens (aa)
909
TCR 46 Alpha CDR1 Homo sapiens (aa)
910
TCR 46 Alpha CDR2 Homo sapiens (aa)
911
TCR 46 Alpha CDR3 Homo sapiens (aa)
912
TCR 46 Alpha signal peptide Homo sapiens (aa)
913
TCR 46 - Beta Native Homo sapiens (aa)
914
TCR 46 - Beta Cysteine-modified Homo sapiens (aa)
915
TCR 46 - Beta Native Homo sapiens (aa)
916
TCR 46 - Beta Cysteine-modified Homo sapiens (aa)
917
TCR 46 Beta variable region Homo sapiens (aa)
918
TCR 46 Beta CDR2 Homo sapiens (aa)
919
TCR 46 Beta CDR3 Homo sapiens (aa)
920
TCR 46 Beta signal peptide Homo sapiens (aa)
921
TCR 47 - Alpha Native Homo sapiens (aa)
922
TCR 47 - Alpha Cysteine-modified Homo sapiens (aa)
923
TCR 47 - Alpha Native Homo sapiens (aa)
924
TCR 47 - Alpha Cysteine-modified Homo sapiens (aa)
925
TCR 47 Alpha variable region Homo sapiens (aa)
926
TCR 47 Alpha CDR3 Homo sapiens (aa)
927
TCR 47 - Beta Native Homo sapiens (aa)
928
TCR 47 - Beta Cysteine-modified Homo sapiens (aa)
929
TCR 47 - Beta Native Homo sapiens (aa)
930
TCR 47 - Beta Cysteine-modified Homo sapiens (aa)
931
TCR 47 Beta variable region Homo sapiens (aa)
932
TCR 47 Beta CDR3 Homo sapiens (aa)
933
TCR 48 - Alpha Native Homo sapiens (aa)
934
TCR 48 - Alpha Cysteine-modified Homo sapiens (aa)
935
TCR 48 - Alpha Native Homo sapiens (aa)
936
TCR 48 - Alpha Cysteine-modified Homo sapiens (aa)
937
TCR 48 Alpha variable region Homo sapiens (aa)
938
TCR 48 Alpha CDR1 Homo sapiens (aa)
939
TCR 48 Alpha CDR2 Homo sapiens (aa)
940
TCR 48 Alpha CDR3 Homo sapiens (aa)
941
TCR 48 - Beta Native Homo sapiens (aa)
942
TCR 48 - Beta Cysteine-modified Homo sapiens (aa)
943
TCR 48 - Beta Native Homo sapiens (aa)
944
TCR 48 - Beta Cysteine-modified Homo sapiens (aa)
945
TCR 48 Beta variable region Homo sapiens (aa)
946
TCR 48 Beta CDR3 Homo sapiens (aa)
947
TCR 49 - Alpha Native Homo sapiens (aa)
948
TCR 49 - Alpha Cysteine-modified Homo sapiens (aa)
949
TCR 49 - Alpha Native Homo sapiens (aa)
950
TCR 49 - Alpha Cysteine-modified Homo sapiens (aa)
951
TCR 49 Alpha variable region Homo sapiens (aa)
952
TCR 49 Alpha CDR3 Homo sapiens (aa)
953
TCR 49 - Beta Native Homo sapiens (aa)
954
TCR 49 - Beta Cysteine-modified Homo sapiens (aa)
955
TCR 49 - Beta Native Homo sapiens (aa)
956
TCR 49 - Beta Cysteine-modified Homo sapiens (aa)
957
TCR 49 Beta variable region Homo sapiens (aa)
958
TCR 49 Beta CDR3 Homo sapiens (aa)
959
TCR 50 - Alpha Native Homo sapiens (aa)
960
TCR 50 - Alpha Cysteine-modified Homo sapiens (aa)
961
TCR 50 - Alpha Native Homo sapiens (aa)
962
TCR 50 - Alpha Cysteine-modified Homo sapiens (aa)
963
TCR 50 Alpha variable region Homo sapiens (aa)
964
TCR 50 Alpha CDR3 Homo sapiens (aa)
965
TCR 50 - Beta Native Homo sapiens (aa)
966
TCR 50 - Beta Cysteine-modified Homo sapiens (aa)
967
TCR 50 - Beta Native Homo sapiens (aa)
968
TCR 50 - Beta Cysteine-modified Homo sapiens (aa)
969
TCR 50 Beta variable region Homo sapiens (aa)
970
TCR 50 Beta CDR3 Homo sapiens (aa)
971
TCR 51 - Alpha Native Homo sapiens (aa)
972
TCR 51 - Alpha Cysteine-modified Homo sapiens (aa)
973
TCR 51 - Alpha Native Homo sapiens (aa)
974
TCR 51 - Alpha Cysteine-modified Homo sapiens (aa)
975
TCR 51 Alpha variable region Homo sapiens (aa)
976
TCR 51 Alpha CDR3 Homo sapiens (aa)
977
TCR 51 - Beta Native Homo sapiens (aa)
978
TCR 51 - Beta Cysteine-modified Homo sapiens (aa)
979
TCR 51 - Beta Native Homo sapiens (aa)
980
TCR 51 - Beta Cysteine-modified Homo sapiens (aa)
981
TCR 51 Beta variable region Homo sapiens (aa)
982
TCR 51 Beta CDR3 Homo sapiens (aa)
983
TCR 52 - Alpha Native Homo sapiens (aa)
984
TCR 52 - Alpha Cysteine-modified Homo sapiens (aa)
985
TCR 52 - Alpha Native Homo sapiens (aa)
986
TCR 52 - Alpha Cysteine-modified Homo sapiens (aa)
987
TCR 52 Alpha variable region Homo sapiens (aa)
988
TCR 52 Alpha CDR3 Homo sapiens (aa)
989
TCR 52 - Beta Native Homo sapiens (aa)
990
TCR 52 - Beta Cysteine-modified Homo sapiens (aa)
991
TCR 52 - Beta Native Homo sapiens (aa)
992
TCR 52 - Beta Cysteine-modified Homo sapiens (aa)
993
TCR 52 Beta variable region Homo sapiens (aa)
994
TCR 52 Beta CDR3 Homo sapiens (aa)
995
TCR 53 - Alpha Native Homo sapiens (aa)
996
TCR 53 - Alpha Cysteine-modified Homo sapiens (aa)
997
TCR 53 - Alpha Native Homo sapiens (aa)
998
TCR 53 - Alpha Cysteine-modified Homo sapiens (aa)
999
TCR 53 Alpha variable region Homo sapiens (aa)
1000
TCR 53 Alpha CDR1 Homo sapiens (aa)
1001
TCR 53 Alpha CDR2 Homo sapiens (aa)
1002
TCR 53 Alpha CDR3 Homo sapiens (aa)
1003
TCR 53 Alpha signal peptide Homo sapiens (aa)
1004
TCR 53 - Beta Native Homo sapiens (aa)
1005
TCR 53 - Beta Cysteine-modified Homo sapiens (aa)
1006
TCR 53 - Beta Native Homo sapiens (aa)
1007
TCR 53 - Beta Cysteine-modified Homo sapiens (aa)
1008
TCR 53 Beta variable region Homo sapiens (aa)
1009
TCR 53 Beta CDR2 Homo sapiens (aa)
1010
TCR 53 Beta CDR3 Homo sapiens (aa)
1011
TCR 53 Beta signal peptide Homo sapiens (aa)
1012
Mouse alpha constant Mus musculus (aa)
1013
Mouse beta constant Mus musculus (aa)
1014
Mouse alpha constant Mus musculus (aa)
1015
Mouse alpha constant Mus musculus (aa)
1016
Mouse beta constant Mus musculus (aa)
1017
Mouse beta constant Cysteine-substituted Mus musculus (aa)
1018
Mouse alpha constant Transmembrane modified Mus musculus (aa)
1019
TCR 17 - Alpha Native Homo sapiens (nt)
1020
TCR 17 - Beta Native Homo sapiens (nt)
1021
TCR 18 - Alpha Native Homo sapiens (nt)
1022
TCR 18 - Beta Native Homo sapiens (nt)
1023
TCR 19 - Alpha Native Homo sapiens (nt)
1024
TCR 19 - Beta Native Homo sapiens (nt)
1025
TCR 20 - Alpha Native Homo sapiens (nt)
1026
TCR 20 - Beta Native Homo sapiens (nt)
1027
TCR 21 - Alpha Native Homo sapiens (nt)
1028
TCR 21 - Beta Native Homo sapiens (nt)
1029
TCR 22 - Alpha Native Homo sapiens (nt)
1030
TCR 22 - Beta Native Homo sapiens (nt)
1031
TCR 23 - Alpha Native Homo sapiens (nt)
1032
TCR 23 - Beta Native Homo sapiens (nt)
1033
TCR 24 - Alpha Native Homo sapiens (nt)
1034
TCR 24 - Beta Native Homo sapiens (nt)
1035
TCR 25 - Alpha Native Homo sapiens (nt)
1036
TCR 25 - Beta Native Homo sapiens (nt)
1037
TCR 26 - Alpha Native Homo sapiens (nt)
1038
TCR 26 - Beta Native Homo sapiens (nt)
1039
TCR 27 - Alpha Native Homo sapiens (nt)
1040
TCR 27 - Beta Native Homo sapiens (nt)
1041
TCR 28 - Alpha Native Homo sapiens (nt)
1042
TCR 28 - Beta Native Homo sapiens (nt)
1043
TCR 29 - Alpha Native Homo sapiens (nt)
1044
TCR 29 - Beta Native Homo sapiens (nt)
1045
TCR 30 - Alpha Native Homo sapiens (nt)
1046
TCR 30 - Beta Native Homo sapiens (nt)
1047
Human TCR beta constant 2 (TRBC2) NCBI Reference Sequence: NG_001333.2, TRBC2
1048
TRAC gRNA targeting domain
1049
TCR 32 - Alpha Native Homo sapiens (nt)
1050
TCR 32 - Beta Native Homo sapiens (nt)
1051
TCR 33 - Alpha Native Homo sapiens (nt)
1052
TCR 33 - Beta Native Homo sapiens (nt)
1053
TRBC gRNA targeting domain
1054
TRBC target sequence Homo sapiens (nt)
1055
TCR 35 - Alpha Native Homo sapiens (nt)
1056
TCR 35 - Beta Native Homo sapiens (nt)
1057
TCR 36 - Alpha Native Homo sapiens (nt)
1058
TCR 36 - Beta Native Homo sapiens (nt)
1059
TCR 37 - Alpha Native Homo sapiens (nt)
1060
TCR 37 - Beta Native Homo sapiens (nt)
1061
TCR 38 - Alpha Native Homo sapiens (nt)
1062
TCR 38 - Beta Native Homo sapiens (nt)
1063
TCR 39 - Alpha Native Homo sapiens (nt)
1064
TCR 39 - Beta Native Homo sapiens (nt)
1065
TCR 40 - Alpha Native Homo sapiens (nt)
1066
TCR 40 - Beta Native Homo sapiens (nt)
1067
TCR 41 - Alpha Native Homo sapiens (nt)
1068
TCR 41 - Beta Native Homo sapiens (nt)
1069
TCR 42 - Alpha Native Homo sapiens (nt)
1070
TCR 42 - Beta Native Homo sapiens (nt)
1071
TCR 43 - Alpha Native Homo sapiens (nt)
1072
TCR 43 - Beta Native Homo sapiens (nt)
1073
TCR 44 - Alpha Native Homo sapiens (nt)
1074
TCR 44 - Beta Native Homo sapiens (nt)
1075
TCR 45 - Alpha Native Homo sapiens (nt)
1076
TCR 45 - Beta Native Homo sapiens (nt)
1077
TCR 46 - Alpha Native Homo sapiens (nt)
1078
TCR 46 - Beta Native Homo sapiens (nt)
1079
TCR 47 - Alpha Native Homo sapiens (nt)
1080
TCR 47 - Beta Native Homo sapiens (nt)
1081
TCR 48 - Alpha Native Homo sapiens (nt)
1082
TCR 48 - Beta Native Homo sapiens (nt)
1083
TCR 49 - Alpha Native Homo sapiens (nt)
1084
TCR 49 - Beta Native Homo sapiens (nt)
1085
TCR 50 - Alpha Native Homo sapiens (nt)
1086
TCR 50 - Beta Native Homo sapiens (nt)
1087
TCR 51 - Alpha Native Homo sapiens (nt)
1088
TCR 51 - Beta Native Homo sapiens (nt)
1089
TCR 52 - Alpha Native Homo sapiens (nt)
1090
TCR 52 - Beta Native Homo sapiens (nt)
1091
TCR 53 - Alpha Native Homo sapiens (nt)
1092
TCR 53 - Beta Native Homo sapiens (nt)
1093
TCR 54 - Alpha Native Homo sapiens (nt)
1094
TCR 54 - Beta Native Homo sapiens (nt)
1095
TCR 55 - Alpha Native Homo sapiens (nt)
1096
TCR 50/ TCR 54 P2A Artificial (nt)
1097
TCR 15 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1098
TCR 15 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1099
TCR 16 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1100
TCR 16 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1101
TCR 17 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1102
TCR 17 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1103
TCR 18 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1104
TCR 18 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1105
TCR 19 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1106
TCR 19 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1107
TCR 20 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1108
TCR 20 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1109
TCR 21 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1110
TCR 21 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1111
TCR 22 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1112
TCR 22 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1113
TCR 23 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1114
TCR 23 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1115
TCR 24 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1116
TCR 24 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1117
TCR 25 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1118
TCR 25 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1119
TCR 26 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1120
TCR 26 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1121
TCR 27 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1122
TCR 27 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1123
TCR 28 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1124
TCR 28 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1125
TCR 29 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1126
TCR 29 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1127
TCR 30 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1128
TCR 30 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1129
TCR 31 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1130
TCR 31 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1131
TCR 32 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1132
TCR 32 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1133
TCR 33 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1134
TCR 33 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1135
TCR 34 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1136
TCR 34 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1137
TCR 35 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1138
TCR 35 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1139
TCR 36 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1140
TCR 36 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1141
TCR 37 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1142
TCR 37 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1143
TCR 38 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1144
TCR 38 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1145
TCR 39 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1146
TCR 39 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1147
TCR 40 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1148
TCR 40 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1149
TCR 41 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1150
TCR 41 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1151
TCR 42 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1152
TCR 42 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1153
TCR 43 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1154
TCR 43 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1155
TCR 44 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1156
TCR 44 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1157
TCR 45 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1158
TCR 45 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1159
TCR 46 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1160
TCR 46 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1161
TCR 47 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1162
TCR 47 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1163
TCR 48 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1164
TCR 48 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1165
TCR 49 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1166
TCR 49 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1167
TCR 50 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1168
TCR 50 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1169
TCR 51 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1170
TCR 51 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1171
TCR 52 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1172
TCR 52 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1173
TCR 53 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1174
TCR 53 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1175
TCR 54 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1176
TCR 54 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1177
TCR 55 Codon-optimized/ cysteine-modified alpha Homo sapiens (nt)
1178
TCR 55 Codon-optimized/ cysteine-modified beta Homo sapiens (nt)
1179
TCR 15/ TCR 16/ TCR 17/ TCR 18/ TCR 19/ TCR 20/ TCR 21/ TCR22/ TCR 23/ TCR 24/ TCR 25/ TCR 26/ TCR
27/ TCR 28/ TCR 29/ TCR 30/ TCR 31/ TCR 32/ TCR 33/ TCR 34 P2A Artificial (nt)
1180
TCR 35/ TCR 36/ TCR 38/ TCR 40/ TCR 41/ TCR 42/ TCR 43/ TCR 44/ TCR 45/ TCR 46/ TCR 47/ TCR 48
P2A Artificial (nt)
1181
TCR 37/ TCR 39 P2A Artificial(nt)
1182
TRAC target sequence Homo sapiens (nt)
1183
TCR alpha E7(11-19) CDR3 consensus
1184
TCR alpha E7(11-19) CDR3 consensus
1185
TCR alpha E7(11-19) CDR3 consensus
1186
TCR alpha E7(11-19) CDR3 consensus
1187
TCR alpha E7(11-19) CDR3 consensus
1188
TCR alpha E7(11-19) CDR3 consensus
1189
TCR alpha E7(11-19) CDR3 consensus
1190
TCR alpha E7(11-19) CDR3 consensus
1191
TCR alpha E7(11-19) CDR1 consensus
1192
TCR alpha E7(11-19) CDR2 consensus
1193
TCR beta E7(11-19) CDR3 consensus
1194
TCR beta E7(11-19) CDR3 consensus
1195
TCR beta E7(11-19) CDR3 consensus
1196
TCR beta E7(11-19) CDR3 consensus
1197
TCR beta E7(11-19) CDR3 consensus
1198
TCR beta E7(11-19) CDR3 consensus
1199
TCR beta E7(11-19) CDR3 consensus
1200
TCR beta E7(11-19) CDR3 consensus
1201
TCR beta E7(11-19) CDR3 consensus
1202
TCR beta E7(11-19) CDR3 consensus
1203
TCR beta E7(11-19) CDR1 consensus
1204
TCR beta E7(11-19) CDR1 consensus
1205
TCR alpha E6(29-38) CDR3 consensus
1206
TCR alpha E6(29-38) CDR3 consensus
1207
TCR alpha E6(29-38) CDR3 consensus
1208
TCR alpha E6(29-38) CDR3 consensus
1209
TCR alpha E6(29-38) CDR1consensus
1210
TCR alpha E6(29-38) CDR2consensus
1211
TCR beta E6(29-38) CDR3 consensus
1212
TCR beta E6(29-38) CDR3 consensus
1213
TCR beta E6(29-38) CDR3 consensus
1214
TCR beta E6(29-38) CDR3 consensus
1215
TCR beta E6(29-38) CDR3 consensus
1216
TCR beta E6(29-38) CDR3 consensus
1217
TCR beta E6(29-38) CDR3 consensus
1218
TCR beta E6(29-38) CDR3 consensus
1219
TCR beta E6(29-38) CDR3 consensus
1220
TCR beta E6(29-38) CDR3 consensus
1221
TCR beta E6(29-38) CDR3 consensus
1222
TCR beta E6(29-38) CDR3 consensus
1223
TCR beta E6(29-38) CDR3 consensus
1224
TCR 31 - beta Native Homo sapiens (nt)
1225
TCR 31 - Alpha Native Homo sapiens (nt)
1226
TCR 34 - Alpha Native Homo sapiens (nt)
1227
TCR 34 - Beta Native Homo sapiens (nt)
1228
TCR 55 - Beta Native Homo sapiens (nt)
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16338452
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juno therapeutics, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Mar 30th, 2022 06:04PM
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Mar 30th, 2022 06:04PM
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Bristol-Myers Squibb
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:bmy
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Juno Therapeutics
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Oct 1st, 2019 12:00AM
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Nov 4th, 2015 12:00AM
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https://www.uspto.gov?id=US10428351-20191001
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Methods for transduction and cell processing
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Provided are methods, systems, and kits for cell processing, e.g., for therapeutic use, such as for adoptive cell therapy. The provided methods include transduction methods, in which cells and virus are incubated under conditions that result in transduction of the cells with a viral vector. The incubation in some embodiments is carried out in an internal cavity of a generally rigid centrifugal chamber, such as a cylindrical chamber made of hard plastic, the cavity of which may have a variable volume. The methods include other processing steps, including those carried out in such a chamber, including washing, selection, isolation, culture, and formulation. In particular, the disclosure relates to method providing advantages over available processing methods, such as available methods for large-scale processing. Such advantages include, for example, reduced cost, streamlining, increased efficacy, increased safety, and increased reproducibility among different subjects and conditions.
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10428351
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1. A transduction method, the method comprising incubating, in an internal cavity of a centrifugal chamber, an input composition comprising cells and viral particles containing a recombinant viral vector, wherein:
the centrifugal chamber comprises:
an end wall, a substantially rigid side wall extending from said end wall, and one or more opening, wherein at least a portion of said side wall surrounds said internal cavity and at least one of the one or more opening is capable of permitting intake of liquid or gas into said internal cavity and expression of liquid or gas from said cavity; and
a movable member capable of moving within the chamber to vary the internal volume of the internal cavity, whereby the internal cavity is a cavity of variable volume defined by said end wall, said substantially rigid side wall, and said movable member;
the centrifugal chamber is rotatable around an axis of rotation and is rotating around said axis of rotation during at least a portion of the incubation;
during at least a portion of the incubation in the chamber or during the rotation of the chamber, the liquid volume of the input composition occupies only a portion of the volume of the internal cavity of the chamber, the volume of the cavity during said at least a portion or during said rotation further comprising a gas; wherein prior to or during said incubation, effecting movement of the movable member and effecting intake of the gas into said internal cavity, thereby increasing the volume of the internal cavity of the chamber; and
wherein the method generates an output composition comprising a plurality of the cells transduced with the viral vector.
2. The method of claim 1, wherein said rotating comprises rotation at a relative centrifugal force at an internal surface of the side wall of the cavity and/or at a surface layer of the cells that is or is at least at or about 600 g.
3. The method of claim 1, wherein the at least a portion of the incubation during which the chamber is rotating is for a time that is between about 5 minutes and about 60 minutes, inclusive.
4. The method of claim 1, wherein:
the average liquid volume of said input composition present in said cavity during said incubation is no more than about 5 milliliters (mL) per square inch of the internal surface area of the cavity during said incubation.
5. The method of claim 1, wherein:
the number of said cells in said input composition is at or about the number of said cells sufficient to form a monolayer on the surface of said cavity during rotation of said centrifugal chamber at a force of at or about 1000 g or at or about 2000 g at an internal surface of the side wall and/or at a surface layer of the cells; and/or
the number of said cells in said input composition is no more than 2 times the number of said cells sufficient to form a monolayer on the surface of said cavity during rotation of said centrifugal chamber at a force of at or about 1000 g or at or about 2000 g at an internal surface of the side wall and/or at a surface layer of the cells; and/or
said input composition in the cavity comprises at least at or about 1×106 of said cells.
6. The method of claim 1, wherein said input composition comprises at least at or about 1 infectious unit (IU) of viral particles per one of said cells.
7. The method of claim 1, wherein:
the internal cavity of the centrifugal chamber has an internal surface area of at least at or about 1×109 μm2;
the number of cells in the input composition is at least 1×107 cells, and
the viral particles are present in the input composition at, at about, or at least 1 infectious unit (IU) per one of said cells.
8. The method of claim 7, wherein:
the liquid volume of the input composition is less than or equal to 200 mL; and/or
the liquid volume of the input composition is no more than 50% of the volume of the internal cavity during rotation.
9. The method of claim 7, wherein the volume of said gas is up to 200 mL.
10. The method of claim 7, wherein said rotation is at a relative centrifugal force at an internal surface of the side wall of the cavity or at a surface layer of the cells of at least at or about 600 g.
11. The method of claim 1, wherein:
the input composition comprises greater than or about 20 mL, and/or said input composition comprises at least 1×108 cells; and
said rotating conditions comprise a relative centrifugal force on a surface layer of the cells of greater than about 800 g.
12. The method of claim 11, wherein said incubation is carried out in a cavity of the centrifugal chamber and the number of said cells in said input composition is at or about the number of said cells sufficient to form a monolayer or a bilayer on the inner surface of said cavity during said rotation.
13. The method of claim 1, wherein a further portion of the incubation of the input composition is carried out outside of the centrifugal chamber and/or without rotation, said further portion carried out subsequent to the at least a portion carried out in the chamber and/or with rotation.
14. The method of claim 13, wherein:
the further incubation is carried out for a time that is no more than 24 hours;
the cells in the input composition have not been subjected to a temperature of greater than 30° C. for more than 24 hours; and/or
the further incubation is not performed in the presence of a stimulating agent.
15. The method of claim 1, wherein:
the output composition containing transduced cells comprises at least at or about 1×107 cells;
for an input composition comprising a virus at a ratio of about 1 or about 2 IU per cells, said method is capable of producing an output composition in which at least 10% of the cells in said output composition generated by the method comprise said recombinant viral vector and/or express a product of a recombinant nucleic acid comprised within said vector; or
among all the cells in said output composition that contain the recombinant viral vector or into which the viral vector is integrated, the average copy number of said recombinant viral vector is no more than about 10; or among the cells in the output composition, the average copy number of said vector is no more than about 2.
16. The method of claim 1, wherein the centrifugal chamber is integral to a closed system, said closed system comprising:
said chamber and at least one tubing line operably linked to at least one of the one or more opening via at least one connector, whereby liquid and gas are permitted to move between said cavity and said at least one tubing line in at least one configuration of said closed system; and
at least one container operably linked to the at least one tubing line, the connection permitting liquid and/or gas to pass between said at least one container and at least one of the one or more opening via the at least one tubing line.
17. The method of claim 16, wherein said at least one container comprises at least one input container comprising said viral vector particles and said cells, a waste container, a product container, and at least one diluent container, and said at least one tubing line comprises a series of tubing lines, wherein each of said containers is connected to said cavity via said series of tubing lines and at least one of the one or more opening.
18. The method of claim 17, wherein said method further comprises,
prior to and/or during said incubation, effecting intake of said input composition into said cavity, said intake comprising flowing of liquid from said at least one input container into said cavity through at least one of the one or more opening; and/or
prior to and/or during said incubation, providing or effecting intake of said gas into said cavity under sterile conditions, said intake being effected by (a) flow of said gas from a container that comprises said gas, (b) flow of said gas from an environment external to the closed system, via a microbial filter, or (c) flow of said gas from a syringe connected to the closed system at a syringe port.
19. The method of claim 17, wherein the closed system comprises:
at least one further container that comprises said gas for intake into the internal cavity prior to and/or during at least a point during said incubation; and/or
a microbial filter capable of taking in gas to the internal cavity of the centrifugal chamber; and/or
a syringe port for effecting intake of said gas.
20. The method of claim 18, wherein:
the effecting intake of the gas into the internal cavity of the centrifugal chamber is carried out simultaneously or together with the effecting intake of the input composition to the internal cavity of the centrifugal chamber; or
the effecting of the intake of the gas is carried out separately, either simultaneously or sequentially, from the effecting of the intake of the input composition into said cavity.
21. The method of claim 1, wherein the gas is air.
22. The method of claim 1, wherein the incubation is part of a continuous process, the method further comprising:
during at least a portion of said incubation, effecting continuous intake of said input composition into said cavity during rotation of the chamber; and
during a portion of said incubation, effecting continuous expression of liquid from said cavity through at least one of the one or more opening during rotation of the chamber.
23. The method of claim 1, wherein the incubation is part of a semi-continuous process and the method further comprises:
prior to said incubation, effecting intake of said input composition, and said gas, into said cavity through at least one of the one or more opening;
subsequent to said incubation, effecting expression of liquid and/or said gas from said cavity;
effecting intake of another input composition comprising cells and said viral particles containing a recombinant viral vector, and gas, into said internal cavity; and
incubating said another input composition in said internal cavity,
wherein the method generates another output composition comprising a plurality of cells of the another input composition that are transduced with said viral vector.
24. The method of claim 18, wherein the method further comprises:
effecting rotation of said centrifugal chamber prior to and/or during said incubation;
effecting expression of liquid from said cavity into said waste container following said incubation;
effecting expression of liquid from said at least one diluent container into said cavity via at least one of the one or more opening and effecting mixing of the contents of said cavity; and
effecting expression of liquid from said cavity into said product container, thereby transferring cells transduced with the viral vector into said product container.
25. The method of claim 1, further comprising:
(a) washing a biological sample comprising said cells in an internal cavity of the centrifugal chamber prior to said incubation; and/or
(b) isolating said cells from a biological sample, wherein at least a portion of the isolation step is performed in an internal cavity of the centrifugal chamber prior to said incubation; and/or
(c) stimulating cells prior to and/or during said incubation, said stimulating comprising exposing said cells to stimulating conditions, thereby inducing cells of the input composition to proliferate, wherein at least a portion of the step of stimulating cells is performed in an internal cavity of the centrifugal chamber.
26. The method of claim 1, wherein:
said cells in said input composition comprise suspension cells;
said cells in said input composition comprise white blood cells; and/or
said cells in said input composition comprise T cells or NK cells.
27. The method of claim 1, wherein during said incubation, said centrifugal chamber is located within a centrifuge and associated with a sensor, said sensor capable of monitoring the position of said movable member, and control circuitry capable of receiving and transmitting information from said sensor and causing movement of said movable member, intake and expression of liquid and/or gas to and from said cavity via one or more tubing lines, and rotation of said chamber via said centrifuge.
28. The method of claim 1, wherein said recombinant viral vector encodes a recombinant receptor, which is thereby expressed by cells of the output composition.
29. The method of claim 1, wherein:
the movable member is a piston; and/or
the movable member is capable of axially moving within the chamber to vary the internal volume of the cavity.
30. The method of claim 1, wherein:
the maximum total liquid volume of said input composition present in said cavity at any one time during said incubation is no more than 100 times the total volume of said cells in said cavity or the average liquid volume of the input composition over the course of the incubation is no more than 100 times the total volume of cells in the cavity, or
the liquid volume of the input composition is no more than 200 mL.
31. The method of claim 1, further comprising formulating cells transduced by the method in a pharmaceutically acceptable buffer in an internal cavity of the centrifugal chamber, thereby producing a formulated composition.
32. The method of claim 31, further comprising effecting expression of the formulated composition to one or a plurality of containers.
33. The method of claim 1, wherein said movement of said movable member is axial within said centrifugal chamber.
34. The method of claim 1, wherein said method comprises prior to said incubation, effecting intake of said input composition into said internal cavity through at least one of the one or more opening.
35. The method of claim 1, wherein the intake of said gas into said internal cavity is carried out under sterile conditions.
36. The method of claim 35, wherein the centrifugal chamber is integral to a closed system, and wherein the intake of gas is effected by (a) flow of said gas from a container that comprises said gas, (b) flow of said gas from an environment external to the closed system, via a microbial filter, or (c) flow of said gas from a syringe connected to the closed system at a syringe port.
37. The transduction method of claim 1, wherein movement of the movable member effects the intake of said gas into the internal cavity.
38. The transduction method of claim 35, wherein movement of the movable member effects the intake of said gas into the internal cavity.
39. The transduction method of claim 1, wherein the maximum liquid volume of said input composition present in said cavity at any one time during said incubation is no more than about 5 mL per square inch of the maximum internal surface area of the cavity.
40. The transduction method of claim 1, wherein the maximum liquid volume of said input composition present in said cavity at any one time during said incubation, or the average liquid volume over the course of the incubation, is no more than at or about 2 times the volume of a monolayer of said cells formed on the inner surface of said cavity during rotation of said chamber at a force of at or about 1000 g or at or about 2000 g at an internal surface of the side wall and/or at a surface layer of the cells.
41. The method of claim 1, wherein:
the internal cavity of the centrifugal chamber has an internal surface area of at least at or about 1×1010 μm2;
the number of cells in the input composition is at least 1×107 cells, and
the viral particles are present in the input composition at, at about, or at least 1 infectious unit (IU) per one of said cells.
42. The method of claim 1, wherein the liquid volume of said input composition present in said cavity during rotation of said centrifugal chamber per square inch of the internal surface area of the cavity is decreased compared to the absence of gas in the chamber.
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42
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S. provisional application No. 62/075,801 filed Nov. 5, 2014, entitled “Methods for Transduction and Cell Processing,” and U.S. provisional application No. 62/129,023 filed Mar. 5, 2015, entitled “Methods for Transduction and Cell Processing,” the contents of which are incorporated by reference in their entirety.
FIELD
The present disclosure relates to cell processing for therapeutic use, such as for adoptive cell therapy. The provided methods generally include transduction methods, in which cells and viral vector particles are incubated under conditions that result in transduction of the cells with a viral vector. The incubation may be carried out in an internal cavity of a generally rigid centrifugal chamber, such as a cylindrical chamber made of hard plastic. The methods include other processing steps, including those carried out in such a chamber, including washing, selection, isolation, culture, and formulation. In particular, the disclosure relates to method providing advantages over available processing methods, such as available methods for large-scale processing. Such advantages include, for example, reduced cost, streamlining, increased efficacy, increased safety, and increased reproducibility among different subjects and conditions.
BACKGROUND
Certain methods are available for cell processing, including large-scale methods and methods for use in preparation of cells for adoptive cell therapy. For example, methods for viral vector transfer, e.g., transduction, selection, isolation, stimulation, culture, washing, and formulation, are available. Available methods have not been entirely satisfactory. Improved methods are needed, for example, for large-scale processing, e.g., transduction, of cells for adoptive cell therapy. For example, methods are needed to improve efficiency and reproducibility, and to reduce time, cost, handling, complexity, and/or other parameters associated with such production. Among the provided embodiments are methods, systems, and kits addressing such needs.
SUMMARY
Provided are methods for cell processing, such as for transfer of viral vectors and/or immunoaffinity-based selection of cells. In some embodiments, the cells are for use in cell therapy, such primary cells prepared for autologous or allogeneic transfer, e.g., in adoptive cell therapy. The methods may include additional cell processing steps, such as cell washing, isolation, separation.
In some embodiments, the methods are carried out by incubating, in a vessel, such as an internal cavity of a centrifugal chamber, a composition (deemed an input composition), which contains cells and viral vector particles, the viral particles containing a recombinant viral vector, thereby generating an output composition that contains a plurality of the cells transduced with the viral vector. The centrifugal chamber typically is rotatable around an axis of rotation. The axis of rotation in some embodiments is vertical. The chamber typically includes an end wall, a side wall extending from the end wall, such as a substantially rigid side wall, and at least one opening, such as an inlet/outlet or an inlet and an outlet. At least a portion of the side wall generally surrounds the internal cavity. The at least one opening (e.g., the inlet/outlet or the inlet and the outlet) is capable of permitting intake of liquid into the internal cavity and expression of liquid from the cavity. The at least one opening in some embodiments is coaxial with the chamber and in some embodiments is in an end wall of the chamber. The side wall may be a curvilinear, e.g., cylindrical or generally cylindrical.
In some embodiments, the methods include incubating, in an internal cavity of a centrifugal chamber, an input composition containing cells and viral particles containing a recombinant viral vector, wherein said centrifugal chamber is rotatable around an axis of rotation and includes an end wall, a substantially rigid side wall extending from said end wall, and at least one opening, at least a portion of said side wall surrounding said internal cavity and said at least one opening being capable of permitting intake of liquid into said internal cavity and expression of liquid from said cavity, wherein the centrifugal chamber is rotating around said axis of rotation during at least a portion of the incubation and the method generates an output composition containing a plurality of the cells transduced with the viral vector.
In some embodiments, the centrifugal chamber further includes a movable member, such as a piston. In such embodiments, the internal cavity is generally one of variable volume, e.g., a cavity of variable volume defined by the end wall, the side wall, and the movable member, e.g., the piston, such that the movable member is capable of moving within the chamber (such as axially within the chamber) to vary the internal volume of the cavity. In some embodiments, liquid is moved in and out of the chamber alternatively by way of a pump, syringe, and/or motor, or other device for intake and expressing liquid or gas, which for example pulls liquid from the cavity and/or pushes liquid in, while the volume of the cavity itself remains constant.
In some embodiments, the methods include incubating, in an internal cavity of a centrifugal chamber, an input composition containing cells and a viral particle containing a recombinant viral vector, said centrifugal chamber being rotatable around an axis of rotation and comprising an end wall, a substantially rigid side wall extending from said end wall, and at least one opening, wherein at least a portion of said side wall surrounds said internal cavity and said at least one opening is capable of permitting intake of liquid into said internal cavity and expression of liquid from said cavity, wherein the centrifugal chamber is rotating around the axis of rotation during at least a portion of the incubation, the total liquid volume of said input composition present in said cavity during rotation of said centrifugal chamber is no more than about 5 mL per square inch of the internal surface area of the cavity and the method generates an output composition comprising a plurality of the cells transduced with the viral vector.
The chamber may comprise two end walls. In some such embodiments, one end wall together with other features defines the internal cavity, while the other is outside of the cavity. In some embodiments, the cavity is bound by both end walls.
The at least one opening may comprise: an inlet and an outlet, respectively capable of permitting said intake and expression, or a single inlet/outlet, capable of permitting said intake and said expression.
Typically, the incubation is carried out at least in part under rotation of the chamber, such as under centrifugal force or acceleration. Thus, the methods in some embodiments further include effecting rotation of the centrifugal chamber, such as around its axis of rotation, during at least a portion of the incubation.
In some of any such embodiments, said rotating includes rotation at a relative centrifugal force (RCF) at an internal surface of the side wall of the cavity and/or at a surface layer of the cells of greater than at or about 200 g, greater than at or about 300 g, or greater than at or about 500 g. In some of any such embodiments, said rotating includes rotation at a relative centrifugal force at an internal surface of the side wall of the cavity and/or at a surface layer of the cells that is: at or about 1000 g, 1500 g, 2000 g, 2100 g, 2200 g, 2500 g or 3000 g; or at least at or about 1000 g, 1500 g, 2000 g, 2100 g, 2200 g, 2500 g, or 3000 g. In some of any such embodiments, said rotating includes rotation at a relative centrifugal force at an internal surface of the side wall of the cavity and/or at a surface layer of the cells that is: between or between about 1000 and 3600, 1000 and 3200, 1000 and 2800, 1000 and 2000, 1000 and 1600, 1600 and 3600, 1600 and 3200, 1600 and 2800, 1600 and 2000, 2000 and 3600, 2000 and 3200, 2000 and 2800, 2800 and 3600, 2800 and 3200, 3200 and 3600, each inclusive; or at least at or about 2000 g, 2100, 2200 g, 2400 g, 2600 g, 2800 g, 3000 g, 3200 g or 3600 g; or at or about 2000 g, 2100 g, 2200 g, 2400 g, 2600 g, 2800 g, 3000 g, 3200 g or 3600 g.
In some of any such embodiments, the at least a portion of the incubation during which the chamber is rotating is for a time that is: greater than or about 5 minutes, such as greater than or about 10 minutes, greater than or about 15 minutes, greater than or about 20 minutes, greater than or about 30 minutes, greater than or about 45 minutes, greater than or about 60 minutes, greater than or about 90 minutes or greater than or about 120 minutes; or between or between about 5 minutes and 60 minutes, 10 minutes and 60 minutes, 15 minutes and 60 minutes, 15 minutes and 45 minutes, 30 minutes and 60 minutes or 45 minutes and 60 minutes, each inclusive.
In some embodiments, the input composition (or the number of cells) in the cavity during the incubation, e.g., at any one time or during the entire incubation, and/or processed by the methods, includes at or about or at least about 1×106, 5×106, 1×107, 5×107, 1×108 or 5×108 of the cells.
In some of any such embodiments, said input composition in the cavity contains at least at or about 1×107 of said cells, at least at or about 2×107 of said cells, 3×107 of said cells, at least at or about 4×107 of said cells, at least at or about 5×107 of said cells, at least at or about 6×107 of said cells, at least at or about 7×107 of said cells, at least at or about 8×107 of said cells, at least at or about 9×107 of said cells, at least at or about 1×108 of said cells, at least at or about 2×108 of said cells, at least at or about 3×108 of said cells or at least at or about 4×108 of said cells.
In some embodiments, the internal surface area of the cavity is at least at or about 1×109 μm2 or 1×1010 μm2, and/or the length of the side wall in the direction extending from the end wall is at least about 5 cm and/or at least about 8 cm; and/or the internal cavity has a radius of at least about 2 cm at least one cross-section.
In some embodiments, the input composition includes at least or about 1 infectious unit (IU) per one of the cells, at least or about 2 IU per one of the cells, at least or about 3 IU per one of the cells, at least or about 4 IU per one of the cells, at least or about 5 IU per one of the cells, at least or about 10 IU per one of the cells, at least or about 20 IU per one of the cells, at least or about 30 IU per one of the cells, at least or about 40 IU per one of the cells, at least or about 50 IU per one of the cells, or at least or about 60 IU per one of the cells. In some embodiments, the input composition includes at or about 1 infectious unit (IU) per one of the cells, at or about 2 IU per one of the cells, at or about 3 IU per one of the cells, at or about 4 IU per one of the cells, at or about 5 IU per one of the cells, at or about 10 IU per one of the cells, at or about 20 IU per one of the cells, at or about 30 IU per one of the cells, at or about 40 IU per one of the cells, at or about 50 IU per one of the cells, or at or about 60 IU per one of the cells.
In some embodiments, the average liquid volume or maximum liquid volume of the input composition, composition with viral vector particles and cells, and/or any liquid composition present in the cavity during the incubation is no more than about 10, 5, or 2.5 milliliters (mL) per square inch of the internal surface area of the cavity during the incubation. In some embodiments, the maximum total volume of such liquid composition present in the cavity at any one time during the incubation is no more than 2 times, no more than 10 times, no more than 100 times, no more than 500 times or no more than 1000 times the total volume of the cells. In some embodiments, the total volume of cells is the total volume of a pellet of the cells. In some embodiments, the total volume of cells is the volume of a monolayer of the cells, such as a monolayer of cells present on the internal surface in the cavity during rotation of the centrifugal chamber.
In some embodiments, the liquid volume of the input composition occupies all or substantially all of the volume of the internal cavity during at least a portion of the incubation. In other embodiments, during at least a portion of the incubation, the liquid volume of the input composition occupies only a portion of the volume of the internal cavity, the volume of the cavity during this at least a portion further comprising a gas, which is taken into the cavity, e.g., via said at least one opening or another opening, prior to or during the incubation.
In some of any such embodiments, the liquid volume of said input composition present in said cavity during said rotation is between or between about 0.5 mL per square inch of the internal surface area of the cavity (mL/sq.in) and 5 mL/sq.in, 0.5 mL/sq.in. and 2.5 mL/sq.in., 0.5 mL/sq.in. and 1 mL/sq.in., 1 mL/sq.in. and 5 mL/sq.in., 1 mL/sq.in. and 2.5 mL/sq.in. or 2.5 mL/sq.in. and 5 mL/sq.in.
In some of any such embodiments, the maximum total liquid volume of said input composition present in said cavity at any one time during said incubation is no more than 2 times, no more than 10 times, or no more than 100 times, the total volume of said cells in said cavity or the average volume of the input composition over the course of the incubation is no more than 2, 10, or 100 times the total volume of cells in the cavity.
In some of any such embodiments, the maximum volume of said input composition present in said cavity at any one time during said incubation or the average volume over the course of the incubation is no more than at or about 2 times, 10 times, 25 times, 50 times, 100 times, 500 times, or 1000 times the volume of a monolayer of said cells formed on the inner surface of said cavity during rotation of said chamber at a force of at or about 2000 g at an internal surface of the side wall.
In some of any such embodiments, the liquid volume of the input composition is no more than 20 mL, no more than 40 mL, no more than 50 mL, no more than 70 mL, no more than 100 mL, no more than 120 mL, no more than 150 mL or no more than 200 mL.
In some of any such embodiments, during at least a portion of the incubation in the chamber or during the rotation of the chamber, the liquid volume of the input composition occupies only a portion of the volume of the internal cavity of the chamber, the volume of the cavity during said at least a portion or during said rotation further comprising a gas, said gas taken into said cavity via said at least one opening, prior to or during said incubation.
In some embodiments, the centrifugal chamber includes a movable member, whereby intake of gas into the centrifugal chamber effects movement of the movable member to increase the volume of the internal cavity of the chamber, thereby decreasing the total liquid volume of said input composition present in said cavity during rotation of said centrifugal chamber per square inch of the internal surface area of the cavity compared to the absence of gas in the chamber.
In some embodiments, the number of cells in the cavity during the incubation is at or about the number of the cells sufficient to form a monolayer on the internal surface of the cavity during rotation of the centrifugal chamber at a force of at or about 2000 g and/or is no more than 1.5 times or 2 times such a number of the cells.
In some of any such embodiments, the number of said cells in said input composition is at or about the number of said cells sufficient to form a monolayer on the surface of said cavity during rotation of said centrifugal chamber at a force of at or about 2000 g at an internal surface of the side wall; and/or the number of said cells in said input composition is no more than 1.5 times or 2 times the number of said cells sufficient to form a monolayer on the surface of said cavity during rotation of said centrifugal chamber at a force of at or about 2000 g at an internal surface of the side wall.
In some embodiments, the centrifugation is for a duration of between 120 and 7200 seconds, such as between 120 and 3600 seconds, including values inclusive or within the range, such as whole-minute values inclusive or within the range.
In some embodiments, the methods include a) providing to an internal cavity of a centrifugal chamber that has an internal surface area of at least at or about 1×109 μm2 or at least at or about 1×1010 μm2:i) an input composition including cells and viral particles containing a recombinant viral vector, wherein: the number of cells in the input composition is at least 1×107 cells, and the viral particles are present in the input composition at least at or about 1 infectious unit (IU) per one of said cells, and the input composition contains a liquid volume that is less than the maximum volume of the internal cavity of the centrifugal chamber; and ii) gas, at a volume that is up to the remainder of the maximum volume of the internal cavity of the centrifugal chamber; and b) incubating the input composition, wherein at least a portion of the incubation is carried out in said internal cavity of said centrifugal chamber while effecting rotation of said centrifugal chamber; and wherein the method generates an output composition containing a plurality of the cells transduced with the viral vector.
In some embodiments, the number of cells is at least or about 50×106 cells; 100×106 cells; or 200×106 cells; and/or the viral particles are present at least 1.6 IU/cell, 1.8 IU/cell, 2.0 IU/cell, 2.4 IU/cell, 2.8 IU/cell, 3.2 IU/cell or 3.6 IU/cell, 4.0 IU/cell, 5.0 IU/cell, 6.0 IU/cell, 7.0 IU/cell, 8.0 IU/cell, 9.0 IU/cell or 10.0 IU/cell.
In some of any such embodiments, the liquid volume of the input composition is less than or equal to 200 mL, less than or equal to 100 mL or less than or equal to 50 mL or less than or equal to 20 mL. In some of any such embodiments, the volume of gas is up to 200 mL, up to 180 mL, up to 140 mL or up to 100 mL.
In some of any such embodiments, said rotation is at a relative centrifugal force at an internal surface of the side wall of the cavity or at a surface layer of the cells of at least at or about 1000 g, 1500 g, 2000 g, 2400 g, 2600 g, 2800 g, 3000 g, 3200 g or 3600 g.
In some embodiments, the methods are for large-scale processing.
In some embodiments, the composition in the cavity (e.g., input composition) includes at least 50 mL, at least 100 mL, or at least 200 mL, liquid volume, and/or at least or about 1 million cells per cm2 of the internal surface area of the cavity during at least a portion of said incubation.
In some embodiments, the maximum liquid volume of the input composition present in the cavity at any one time during said incubation is no more than at or about 2 times, 10 times, 25 times, 50 times, 100 times, 500 times, or 1000 times the volume of a monolayer of said cells formed on the inner surface of said cavity during rotation of said chamber, e.g., at a force, e.g., effective force, of at or about 2000 g.
In some embodiments, the rotation of the chamber during at least a portion of the incubation is at a force of greater than at or about 200 g, greater than at or about 300 g, or greater than at or about 500 g, such as greater than at or about 1000 g, 1500 g, 2000 g, 2500 g, 3000 g, or 3200 g, at an internal wall of the cavity of the centrifugal chamber and/or a layer, e.g., surface layer, of the cells. In some embodiments, the force is at least at or about 1000 g, 1500 g, 2000 g, or 2500 g, 3000 g or 3200 g. In some embodiments, the force is at or about 2100 g, 2200 g or 3000 g.
In some embodiments, the methods include incubating an input composition containing cells and viral particles containing a recombinant viral vector, at least a portion of said incubating being carried out under rotating conditions, thereby generating an output composition containing a plurality of the cells transduced with the viral vector, wherein said input composition contains greater than or about 20 mL, 50 mL, at least 100 mL, or at least 150 mL in volume, and/or said input composition comprises at least 1×108 cells; and said rotating conditions comprise a relative centrifugal force on a surface layer of the cells of greater than about 1500 g.
In some embodiments of the methods, at least 25% or at least 50% of said cells in the output composition are transduced with said viral vector; and/or at least 25% or at least 50% of said cells in the output composition express a product of a heterologous nucleic acid contained within said viral vector.
In some of any such embodiments, said incubation is carried out in a cavity of a centrifugal chamber and the number of said cells in said input composition is at or about the number of said cells sufficient to form a monolayer or a bilayer on the inner surface of said cavity during said rotation.
In some embodiments, said centrifugal chamber includes an end wall, a substantially rigid side wall extending from said end wall, and at least one opening, wherein at least a portion of said side wall surrounds said internal cavity and said at least one opening is capable of permitting intake of liquid into said internal cavity and expression of liquid from said cavity,
In some embodiments, said centrifugal chamber further includes a movable member and said internal cavity is a cavity of variable volume defined by said end wall, said substantially rigid side wall, and said movable member, said movable member being capable of moving within the chamber to vary the internal volume of the cavity.
In some of any such embodiments, the input composition in said cavity contains a liquid volume of at least 20 mL or at least 50 mL and at or about 1 million cells per cm2 of the internal surface area of the cavity during at least a portion of said incubation.
In some of any such embodiments, a further portion of the incubation is carried out outside of the centrifugal chamber and/or without rotation, said further portion carried out subsequent to the at least a portion carried out in the chamber and/or with rotation.
In some of any such embodiments, the at least a portion of the incubation carried out in the cavity of the centrifugal chamber and/or the further portion of the incubation is effected at or at about 37° C.±2° C.
In some of any such embodiments, the incubation further includes transferring at least a plurality of the cells to a container during said incubation and said further portion of the incubation is effected in the container. In some embodiments, the transferring is performed within a closed system, wherein the centrifugal chamber and container are integral to the closed system.
In some of any such embodiments, the incubation is carried out for a time between at or about 1 hour and at or about 96 hours, between at or about 4 hours and at or about 72 hours, between at or about 8 hours and at or about 48 hours, between at or about 12 hours and at or about 36 hours, between at or about 6 hours and at or about 24 hours, between at or about 36 hours and at or about 96 hours, inclusive; or the further portion of the incubation is carried out for a time between at or about 1 hour and at or about 96 hours, between at or about 4 hours and at or about 72 hours, between at or about 8 hours and at or about 48 hours, between at or about 12 hours and at or about 36 hours, between at or about 6 hours and at or about 24 hours, between at or about 36 hours and at or about 96 hours, inclusive.
In some of any such embodiments, the incubation or further portion of the incubation is carried out for a time that is no more than 48 hours, no more than 36 hours or no more than 24 hours; or the further portion of the incubation is carried out for a time that is no more than 48 hours, no more than 36 hours or no more than 24 hours.
In some of any such embodiments, the incubation is performed in the presence of a stimulating agent; and/or the further portion of the incubation is performed in the presence of a stimulating agent.
In some of any such embodiments, the incubation is carried out for a time that is no more than 24 hours; the cells in the composition have not been subjected to a temperature of greater than 30° C. for more than 24 hours; and/or the incubation is not performed in the presence of a stimulating agent.
In some of any such embodiments, the stimulating agent is an agent capable of inducing proliferation of T cells, CD4+ T cells and/or CD8+ T cells.
In some of any such embodiments, the stimulating agent is a cytokine selected from among IL-2, IL-15 and IL-7.
In some of any such embodiments, the output composition containing transduced cells contains at least 1×107 cells or at least 5×107 cells.
In some of any such embodiments, the output composition containing transduced cells contains at least 1×108 cells, 2×108 cells, 4×108 cells, 6×108, 8×108 cells or 1×109 cells.
In some of any such embodiments, the cells are T cells. In some embodiments, the T cells are unfractionated T cells, isolated CD4+ T cells and/or isolated CD8+ T cells.
In some of any such embodiments, the method results in integration of the viral vector into a host genome of one or more of the at least a plurality of cells and/or into a host genome of at least at or about 20% or at least at or about 30% or at least at or about 40% of the cells in the output composition.
In some of any such embodiments, at least 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of said cells in said input composition are transduced with said viral vector by the method; and/or at least 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of said cells in said output composition are transduced with said viral vector; and/or at least 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of said cells in said output composition express a product of a heterologous nucleic acid contained within said viral vector.
Particular embodiments include methods of transduction carried out by incubating an input composition comprising cells and viral vector particles under rotating conditions, whereby a plurality of the cells are inoculated for transduction with the viral vector, wherein the input composition includes a total volume greater than 50 mL, such as at least 100 mL, or at least 150 mL in volume, and/or said input composition comprises at least 1×108 cells; and the rotating conditions comprise centrifugal force of greater than about 1500 g. In some such embodiments, the incubation is carried out in a cavity of a centrifugal chamber and the number of said cells in said input composition is at or about the number of said cells sufficient to form a monolayer on the inner surface of the cavity during the rotation. In some such embodiments, at least 25% or at least 50% of said cells are transduced with the viral vector.
In some embodiments, the methods result in at least 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of said cells in said input composition being transduced with the viral vector, and/or produce an output composition in which at least 10%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the cells are transduced with the vector and/or express a recombinant product encoded by the vector. In some embodiments, transduction efficiency is expressed for a particular input amount or relative amount of virus. For example, in some embodiments, such efficiencies are achieved by the methods for an input composition comprising a virus at a ratio of about 1 or about 2 IU per cells.
In some embodiments, among all the cells in said output composition produced by the methods, the average copy number of the recombinant viral vector is no more than about 10, no more than about 5, no more than about 2.5, or no more than about 1.5. In some embodiments, among the cells in the output composition that contain the recombinant viral vector, the average copy number of the vector is no more than about 5, no more than about 2, no more than about 1.5, or no more than about 1.
In some of any such embodiments, among all the cells in said output composition that contain the recombinant viral vector or into which the viral vector is integrated, the average copy number of said recombinant viral vector is no more than about 10, no more than about 5, no more than about 2.5, or no more than about 1.5; or among the cells in the output composition, the average copy number of said vector is no more than about 2, no more than about 1.5, or no more than about 1.
In some embodiments, the centrifugal chamber is integral to a closed system, for example, where the closed system includes the chamber and at least one tubing line operably linked to the at least one opening via at least one connector, such that liquid and gas are permitted to move between said cavity and said at least one tubing line in at least one configuration of the system. The at least one tubing line typically includes a series of tubing lines. The at least one connector typically includes a plurality of connectors. The closed system may further include at least one container operably linked to the series of tubing lines, such that the at least one connection permits liquid and/or gas to pass between the at least one container and the at least one opening via the series of tubing lines.
The at least one connector may include one or more connectors selected from the group consisting of valves, luer ports, and spikes, e.g., a rotational valve, such as a stopcock or multirotational port, and/or an aseptic connector.
The at least one container may include one or more bags, vials, and/or syringes, and may include container(s) designated as a diluent container, a waste container, a product collection container, output container, and/or an input container.
In some embodiments, the at least one container includes at least one input container including the virus and/or cells (which may be a single input container comprising the virus and cells or two input containers comprising the virus and cells, respectively), a waste container, a product container, and at least one diluent or wash solution-containing container, each connected to said cavity via said series of tubing lines and the at least one opening.
In some of any such embodiments, at least one container further includes a container that contains a gas prior to and/or during at least a point during said incubation and/or the closed system further includes a microbial filter capable of taking in gas to the internal cavity of the centrifugal chamber and/or the closed system contains a syringe port for effecting intake of gas.
The methods in some embodiments further include, prior to and/or during the incubation, effecting intake of the input composition into said cavity. The intake may include flow of liquid from the at least one input container into the cavity through said at least one opening. The intake may include intake of virus from one input container and input of cells from another, to produce the input composition for incubation.
In some embodiments, the method includes, prior to and/or during said incubation, providing or effecting intake of gas into said cavity under sterile conditions, said intake being effected by (a) flow of gas from the container that includes gas, (b) flow of gas from an environment external to the closed system, via the microbial filter, or (c) flow of gas from a syringe connected to the system at the syringe port.
In some embodiments, the effecting intake of the gas into the internal cavity of the centrifugal chamber is carried out simultaneously or together with the effecting intake of the input composition to the internal cavity of the centrifugal chamber.
In some of any such embodiments, the input composition and gas are combined in a single container under sterile conditions outside of the chamber prior to said intake of said input composition and gas into the internal cavity of the centrifugal chamber.
In some embodiments, the effecting of the intake of the gas is carried out separately, either simultaneously or sequentially, from the effecting of the intake of the input composition into said cavity.
In some of any such embodiments, the intake of gas is effected by permitting or causing flow of the gas from a sterile closed container containing the gas, an external environment through a microbial filter, or a syringe containing said gas.
In some of any such embodiments, the gas is air.
In some embodiments of the provided process methods, the incubation is part of a continuous process, where the method further includes, during at least a portion of the incubation, effecting continuous intake of said input composition into the cavity, typically during rotation of the chamber, and during a portion of the incubation, effecting continuous expression (i.e. outtake) of liquid from said cavity through said at least one opening, typically during rotation of the chamber. The continuous intake and outtake in some embodiments occur simultaneously.
In some embodiments, the method includes during a portion of said incubation, effecting continuous intake of gas into said cavity during rotation of the chamber; and/or during a portion of said incubation, effecting continuous expression of gas from said cavity.
In some embodiments, the method includes the expression of liquid and the expression of gas from said cavity, where each is expressed, simultaneously or sequentially, into a different container.
In some of any such embodiments, at least a portion of the continuous intake and the continuous expression occur simultaneously.
In some embodiments, the incubation is part of a semi-continuous process, such as one in which the method further includes effecting intake of the input composition into the cavity through the at least one opening, conducting all or part of the incubation, such as the centrifugation, and then effecting expression of liquid from the cavity, and then repeating the process, whereby another input composition is taken in to the cavity, followed by centrifugation, followed by expression. The process can be iterative and include several more rounds of intake, processing, and expression.
In some of any such embodiments, the incubation is part of a semi-continuous process, the method further including prior to said incubation, effecting intake of said input composition, and optionally gas, into said cavity through said at least one opening; subsequent to said incubation, effecting expression of liquid and/or optionally gas from said cavity; effecting intake of another input composition including cells and said viral particles containing a recombinant viral vector, and optionally gas, into said internal cavity; and incubating said another input composition in said internal cavity, wherein the method generates another output composition containing a plurality of cells of the another input composition that are transduced with said viral vector.
In some of any such embodiments, said providing or said intake of the input composition into the cavity includes intake of a single composition including the cells and the viral particles containing the recombinant viral vector; or intake of a composition including the cells and a separate composition containing the viral particles containing the recombinant viral vector, whereby the compositions are mixed, effecting intake of the input composition.
The intake may include intake of a single composition containing the cells and the virus; or intake of a composition containing the cells and a separate composition containing the virus, whereby the compositions are mixed, effecting intake of the input composition. In some embodiments of the continuous or semi-continuous process, at least 1×108 cells or at least 1×109 cells or at least 1×1010 cells or more are processed in total, over the multiple rounds or continuous process.
In some embodiments, the method includes effecting rotation of the centrifugal chamber prior to and/or during said incubation and effecting expression of liquid from the cavity into said waste container following the incubation; effecting expression of liquid from the at least one diluent container into said cavity via the at least one opening and effecting mixing of the contents of the cavity; and effecting expression of liquid from said cavity into the product container, thereby transferring cells transduced with the viral vector into the product bag.
In some embodiments, the method further includes carrying out other processing steps, or at least a portion of one or more other processing steps, within the same chamber and/or closed system. In some embodiments, the one or more processing steps can includes processes in which the cells are isolated, such as separated or selected, stimulated, and formulated within the same chamber and/or closed system. In some cases, the one or more further processing steps also can include washing cells, suspending cells and/or diluting or concentrating cells, which can be carried out prior to or subsequent to any one or more of the processing steps for isolating, such as separating or selecting, stimulating, transducing and/or formulating the cells. In some embodiments, the one or more other processing steps can be carried out prior to, simultaneously with or subsequent to the incubation of cells with the viral vector particles in the methods of transduction. In some embodiments, the one or more further processing steps, or a portion of the one or more further processing steps, can be carried out in a cavity of a centrifugal chamber that is the same or different as a cavity of a centrifugal chamber employed in the incubation of cells with the viral vector particles.
Among the provided processing methods, including isolation, e.g. selection, methods, stimulation methods, formulation methods and other processing methods, are those carried out according to any of the embodiments as described above.
For example, in some embodiments, the method further includes (a) washing a biological sample (e.g., a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cells (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product) containing cells in a cavity of a chamber, prior to the incubation for isolating, e.g. selecting cells, and/or prior to the incubation for incubating cells with viral vector particles, (b) isolating, e.g. selecting, the cells from a sample (e.g., a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cells (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product) in a cavity prior to the incubation of such cells with viral vector particles and/or (c) stimulating cells in a cavity prior to and/or during the incubation of such cells with viral vector particles, e.g., by exposing cells to stimulating conditions, thereby inducing cells of the input composition to proliferate. In some embodiments, the isolating includes immunoaffinity-based selection.
In some of any such embodiments, the method includes (a) washing a biological sample containing said cells in an internal cavity of a centrifugal chamber prior to said incubation; and/or (b) isolating said cells from a biological sample, wherein at least a portion of the isolation step is performed in an internal cavity of a centrifugal chamber prior to said incubation; and/or (c) stimulating cells prior to and/or during said incubation, said stimulating including exposing said cells to stimulating conditions, thereby inducing cells of the input composition to proliferate, wherein at least a portion of the step of stimulating cells is performed in an internal cavity of a centrifugal chamber.
In some embodiments, the methods may further include isolation, e.g., selection, of the cells in the chamber, e.g., by immunoaffinity based selection. In some embodiments, the isolation, e.g. selection, of cells is carried out prior to the incubation of cells with the viral vector particles in the methods of transduction, whereby the isolated, such as selected, cells are the cells present in the input composition and/or incubated with the viral vector particles. In some embodiments, the isolation, e.g., selection, includes incubation of cells with a selection reagent, such as an immunoaffinity reagent. In some embodiments, at least a portion of the isolation, e.g. selection, step, such as incubation of cells with a selection reagent, e.g. an immunoaffinity reagent, is carried out in the cavity of a chamber, which, in some cases, can include rotation of the chamber, for example, for mixing of the reagent and cells.
In some embodiments, the methods may further include stimulating cells prior to, during and/or subsequent to the incubation of cells with the viral vector particles, in which at least all or a portion of the stimulation can be carried out in a cavity of a centrifugal chamber. In some embodiments, the stimulating conditions may include incubation of cells in the presence of an agent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex, such as a primary agent that specifically binds to a member of a TCR complex, e.g., CD3, and a secondary agent that specifically binds to a T cell costimulatory molecule, e.g., CD28, CD137 (4-1-BB), OX40, or ICOS, including antibodies such as those present on the surface of a solid support, such as a bead. In some embodiments, at least a portion of the stimulation, such as incubation of cells in the presence of a stimulating condition, is carried out in the cavity of a chamber, which, in some cases, can include rotation of the chamber, for example, for mixing of the reagent and cells.
In some of any such embodiments, the method includes formulating cells, such as cells produced or generated in accord with the provided methods, including cell transduced by the method, in a pharmaceutically acceptable buffer in an internal cavity of a centrifugal chamber, thereby producing a formulated composition. In some embodiments, the methods further include effecting expression of the formulated composition to one or a plurality of containers. In some embodiments, the methods include the effecting of expression of the formulated composition includes effecting expression of a number of the cells present in a single unit dose to one or each of said one or a plurality of containers.
In some of any such embodiments, each of said a cavity of a centrifugal chamber is the same or different as a cavity of a centrifugal employed in one or more of the other steps and/or in the process of incubating and/or rotating an input composition containing cells and viral particles.
In some of any such embodiments, each of said centrifugal chambers is integral to a closed system, said closed system including said chamber and at least one tubing line operably linked to the at least one opening via at least one connector, whereby liquid and gas are permitted to move between said cavity and said at least one tubing line in at least one configuration of said system.
The cells processed by the methods typically are primary cells, such as cells obtained from a subject, typically a human. The cells may be derived from a subject to which the therapy is to be administered, such as one having a disease or condition targeted by a recombinant molecule expressed by a vector transduced, e.g., a recombinant antigen receptor such as a chimeric antigen receptor or transgenic TCR. Alternatively, the cells may be from a different subject. Thus, the methods encompass processing for autologous and allogeneic transfer. The cells may include suspension cells, e.g., white blood cells, e.g., T cells, such as isolated CD8+ T cells, or isolated CD4+ T cells or subsets thereof, or NK cells.
In some embodiments, during the incubation, the centrifugal chamber is associated with a sensor, for example, a sensor capable of monitoring the position of the movable member, and control circuitry, such as circuitry capable of receiving and transmitting information to and from the sensor, causing movement of said movable member, and/or that is further associated with a centrifuge and thus is capable of causing rotation of the chamber during said incubation.
In some embodiments, the chamber contains the movable member and during the incubation is located within a centrifuge and associated with a sensor capable of monitoring the position of the movable member, and control circuitry capable of receiving and transmitting information from the sensor and causing movement of the movable member, intake and expression of liquid to and from said cavity via said one or more tubing lines, and rotation of the chamber via the centrifuge.
In some embodiments, the chamber, control circuitry, centrifuge, and/or sensor are housed within a cabinet, e.g., during the incubation.
In some embodiments of any of the viral transfer, e.g., transduction methods, the recombinant viral vector encodes a recombinant receptor, which is thereby expressed by cells of the output composition. In some embodiments, the recombinant receptor is a recombinant antigen receptor, such as a functional non-T cell receptor, e.g., a chimeric antigen receptor (CAR), or a transgenic T cell receptor (TCR). In some embodiments, the recombinant receptor is a chimeric receptor containing an extracellular portion that specifically binds to a ligand and an intracellular signaling portion containing an activating domain and a costimulatory domain.
In some of any such embodiments, the cells include primary human T cells obtained from a human subject and prior to the incubation with viral vector particles and/or prior to completion of the transduction and/or, where the method includes formulation, prior to the formulation, the primary human T cells have not been present externally to the subject at a temperature of greater than 30° C. for greater than 1 hour, greater than 6 hours, greater than 24 hours, or greater than 48 hours or prior to the incubation and/or prior to the completion of the transduction, and/or where the method includes formulation, prior to the formulation, the primary human T cells have not been incubated in the presence of an antibody specific for CD3 and/or an antibody specific for CD28 and/or a cytokine, for greater than 1 hour, greater than 6 hours, greater than 24 hours, or greater than 48 hours.
Provided herein are methods for isolation, e.g. selection, of cells including (a) incubating a selection reagent and primary cells in an internal cavity of a centrifugal chamber under mixing conditions, whereby a plurality of the primary cells bind to said selection reagent and (b) separating the plurality of the primary cells from another one or more of the primary cells based on binding to the selection reagent, thereby enriching the primary cells based on binding to the selection reagent, wherein the centrifugal chamber is rotatable around an axis of rotation and the internal cavity has a maximum volume of at least 50 mL, at least 100 mL, or at least 200 mL. In some embodiments, the methods for isolation, e.g. selection, occur in a closed system. In some embodiments, prior to the step of separating the plurality of cells, the cells incubated with the selection reagent, are expressed from or transferred out of the chamber, but maintained in the closed system. In some embodiments, optionally, subsequent to incubation with the selection reagent and prior to separating the cells, the method further includes one or more washing steps, which in some cases, can be performed in the cavity of the chamber in accord with the provided methods. In some embodiments, the step of separating the cells can be effecting using a solid support, such as using an immunoaffinity-column, including those for magnetic separation, which can be contained in the closed system.
Provided herein are methods for stimulation of cells, including incubating a stimulation agent and primary cells under conditions whereby the stimulation agent binds to a molecule expressed by a plurality of the primary cells and the plurality of the cells are activated or stimulated, wherein at least a portion of the incubation is carried out in an internal cavity of a centrifugal chamber under mixing conditions, where the centrifugal chamber is rotatable around an axis of rotation and the internal cavity has a maximum volume of at least 50 mL, at least 100 mL, or at least 200 mL.
In some embodiments, the methods of stimulation are performed as part of a process that includes transducing cells, whereby all or a part of such process is performed in a centrifugal chamber and/or as part of the same closed system. In some embodiments, the primary cells that are stimulated with a stimulation agent include or are cells obtained following isolation, e.g. selection, of cells from a biological sample, such as in accord with the provided methods. In some embodiments, at least a portion of the stimulation is carried out simultaneously or during the incubation of cells with the viral vector particles, such that the primary cells include or are cells present in the input composition and/or are cells in which transduction has occurred or is initiated. In some embodiments, at least a portion of the stimulation is carried out prior to the incubation of cells with the viral vector particles, such that the cells incubated with the viral vector particles are stimulated cells, which, in some cases, includes proliferating cells.
In some embodiments, at least a portion of the one of more other processing steps of the method, including isolation, e.g. selection, stimulation, washing and/or formulation, that is carried out in a chamber includes where the chamber includes an end wall, a substantially rigid side wall extending from said end wall, and at least one opening, wherein at least a portion of the side wall surrounds the internal cavity and the at least one opening is capable of permitting intake of liquid into the internal cavity and expression of liquid from the cavity.
Provided herein are compositions containing transduced cells produced by the methods of any of the above embodiments. In some of any such embodiments, the composition contains cells that are primary cells and/or human cells and/or include white blood cells, and/or T cells, and/or NK cells. In some of any such embodiments, the composition contains at least 5×107 cells, 1×108 cells, 2×108 cells, 4×108 cells, 6×108 cells, 8×108 cells or 1×109 cells. In some of any such embodiments, the composition contains a therapeutically effective number of cells for use in adoptive T cell therapy. In some of any such embodiments, the cells are T cells and subsequent to transduction, the cells in the composition are not subjected to cell expansion in the presence of a stimulating agent and/or the cells are not incubated at a temperature greater than 30° C. for more than 24 hours or the composition does not contain a cytokine or the composition does not contain a stimulating agent that specifically binds to CD3 or a TCR complex.
Provided herein are compositions containing at least 1×107 or at least 5×107 T cells, at least a plurality of which are transduced with a recombinant viral vector, where subsequent to transduction, the cells in the composition have not been subjected to cell expansion in the presence of a stimulating agent and/or the cells have not been incubated at a temperature greater than 30° C. for more than 24 hours and/or at least 30, 40, 50, 60, 70, or 80% of the T cells in the composition contain high surface expression of CD69 or TGF-beta-II. In some embodiments, the composition contains at least 1×108 cells, 2×108 cells, 4×108 cells, 6×108, 8×108 cells or 1×109 cells.
In some of any such embodiments, the T cells are unfractionated T cells, isolated CD8+ T cells, or isolated CD4+ T cells.
In some of any such embodiments, at least 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of said cells in said composition are transduced with the viral vector.
In some of any such embodiments, the viral vector encodes a recombinant receptor and transduced cells in the composition express the recombinant receptor. In some embodiments, the recombinant receptor is a recombinant antigen receptor. In some embodiments, the recombinant antigen receptor is a functional non-T cell receptor. In some embodiments, the functional non-T cell receptor is a chimeric antigen receptor (CAR). In some embodiments, the recombinant receptor is a chimeric receptor containing an extracellular portion that specifically binds to a ligand and an intracellular signaling portion containing an activating domain and a costimulatory domain. In some embodiments, the recombinant antigen receptor is a transgenic T cell receptor (TCR).
In some of any such embodiments, among all the cells in the composition, the average copy number of the recombinant viral vector is no more than about 10, no more than 8, no more than 6, no more than 4, or no more than about 2, or among the cells in the composition transduced with the recombinant viral vector, the average copy number of said vector is no more than about 10, no more than 8, no more than 6, no more than 4, or no more than about 2.
In some of any such embodiments, the composition contains a pharmaceutically acceptable excipient.
Provided herein are methods of treatment, including administering to a subject having a disease or condition the composition of any of the above embodiments. In some embodiments, the transduced T cells in the composition exhibit increased or longer expansion and/or persistence in the subject than transduced T cells in a composition in which, subsequent to transduction, the cells in the composition have been subjected to cell expansion in the presence of a stimulating agent and/or the cells have been incubated at a temperature greater than 30° C. for more than 24 hours.
In some of any such embodiments, the recombinant receptor, chimeric antigen receptor or transgenic TCR specifically binds to an antigen associated with the disease or condition. In some embodiments, the disease or condition is a cancer, an autoimmune disease or disorder, or an infectious disease.
Provided herein are compositions containing at least 1×107 cells and at least at or about 1 infectious unit (IU) per cell of viral particles containing a recombinant viral vector. In some embodiments, the cells contain at least or about 50×106 cells, 100×106 cells, or 200×106 cells, and/or said viral particles are present in the composition in an amount that is at least 1.6 IU/cell, 1.8 IU/cell, 2.0 IU/cell, 2.4 IU/cell, 2.8 IU/cell, 3.2 IU/cell, 3.6 IU/cell, 4.0 IU/cell, 5.0 IU/cell, 6.0 IU/cell, 7.0 IU/cell, 8.0 IU/cell, 9.0 IU/cell or 10.0 IU/cell.
In any of such embodiments, the liquid volume of the composition is less than or equal to 220 mL, less than or equal to 200 mL, less than or equal to 100 mL, less than or equal to 50 mL or less than or equal to 20 mL.
In some of any such embodiments, the cells are primary cells. In some of any such embodiments, the cells are human cells. In some of any such embodiments, the cells include suspension cells, the cells include white blood cells and/or the cells include T cells or NK cells. In some embodiments, the cells are T cells and the T cells are unfractionated T cells, isolated CD8+ T cells, or isolated CD4+ T cells.
In some of any such embodiments, the viral vector encodes a recombinant receptor. In some embodiments, the recombinant receptor is a recombinant antigen receptor. In some embodiments, the recombinant antigen receptor is a functional non-T cell receptor. In some embodiments, the functional non-T cell receptor is a chimeric antigen receptor (CAR). In some embodiments, the recombinant receptor is a chimeric receptor containing an extracellular portion that specifically binds to a ligand and an intracellular signaling portion containing an activating domain and a costimulatory domain. In some embodiments, the recombinant antigen receptor is a transgenic T cell receptor (TCR).
Provided herein are centrifugal chambers rotatable around an axis of rotation, including an internal cavity containing the composition of any of the above embodiments.
Provided herein are centrifugal chambers rotatable around an axis of rotation, containing an internal cavity containing (a) a composition containing at least 5×107 primary T cells transduced with a recombinant viral vector and/or (b) a composition containing at least 5×107 primary T cells and viral particles containing a recombinant viral vector.
In some of any such embodiments, the chamber further contains an end wall, a substantially rigid side wall extending from said end wall, and at least one opening, wherein at least a portion of said side wall surrounds said internal cavity and said at least one opening is capable of permitting intake of liquid into said internal cavity and expression of liquid from said cavity.
In some of any such embodiments, said composition in said cavity contains at least 1×108 cells, 2×108 cells, 4×108 cells, 6×108 cells, 8×108 cells or 1×109 of the cells.
In some of any such embodiments, the T cells are unfractionated T cells, isolated CD8+ T cells, or isolated CD4+ T cells.
In some of any such embodiments of the chamber, at least 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of said cells in said composition are transduced with a viral vector.
In some of any such embodiments of the chamber, the viral vector encodes a recombinant receptor and cells in the composition express the recombinant receptor. In some embodiments, the recombinant receptor is a recombinant antigen receptor. In some embodiments, the recombinant antigen receptor is a functional non-T cell receptor. In some embodiments, the functional non-T cell receptor is a chimeric antigen receptor (CAR). In some embodiments, the recombinant receptor is a chimeric receptor containing an extracellular portion that specifically binds to a ligand and an intracellular signaling portion containing an activating domain and a costimulatory domain. In some embodiments, the recombinant antigen receptor is a transgenic T cell receptor (TCR).
In some of any such embodiments of the chamber, among all the cells in the composition, the average copy number of said recombinant viral vector is no more than about 10, no more than 8, no more than 6, no more than 4, or no more than about 2 or among the cells in the composition transduced with the recombinant viral vector, the average copy number of said vector is no more than about 10, no more than 8, no more than 6, no more than 4, or no more than about 2.
Provided herein are centrifugal chambers rotatable around an axis of rotation, including an internal cavity containing the composition of any of the above embodiments. In some embodiments, the chamber further contains a volume of gas up to the maximum volume of the internal cavity of the chamber. In some embodiments, the gas is air.
In some of any such embodiments of the chamber, the chamber is rotatable around an axis of rotation and includes an end wall, a substantially rigid side wall extending from said end wall, and at least one opening, wherein at least a portion of said side wall surrounds said internal cavity and said at least one opening is capable of permitting intake of liquid into said internal cavity and expression of liquid from said cavity. In some embodiments the side wall is curvilinear. In some embodiments the side wall is generally cylindrical.
In some of any such embodiments of the chamber, said at least one opening includes an inlet and an outlet, respectively capable of permitting said intake and expression or said at least one opening includes a single inlet/outlet, capable of permitting said intake and said expression. In some of any such embodiments of the chamber, aid at least one opening is coaxial with the chamber and is located in the end wall.
In some of any such embodiments, the chamber further includes a movable member and said internal cavity is a cavity of variable volume defined by said end wall, said substantially rigid side wall, and said movable member, said movable member being capable of moving within the chamber to vary the internal volume of the cavity. In some embodiments, the movable member is a piston and/or the movable member is capable of axially moving within the chamber to vary the internal volume of the cavity.
In some of any such embodiments, the internal surface area of said cavity is at least at or about 1×109 μm2, the internal surface area of said cavity is at least at or about 1×1010 μm2, the length of said rigid wall in the direction extending from said end wall is at least about 5 cm, the length of said rigid wall in the direction extending from said end wall is at least about 8 cm and/or the cavity contains a radius of at least about 2 cm at least one cross-section.
In some of any such embodiments of the chamber, the liquid volume of said composition present in said cavity is between or between about 0.5 mL per square inch of the internal surface area of the cavity (mL/sq.in) and 5 mL/sq.in, 0.5 mL/sq.in. and 2.5 mL/sq.in., 0.5 mL/sq.in. and 1 mL/sq.in., 1 mL/sq.in. and 5 mL/sq.in., 1 mL/sq.in. and 2.5 mL/sq.in. or 2.5 mL/sq.in. and 5 mL/sq.in. In some of any such embodiments, the liquid volume of said composition present in said cavity is at least 0.5 mL/sq.in., 1 mL/sq.in., 2.5 mL/sq.in., or 5 mL/sq.in.
Provided herein are closed systems containing the centrifugal chamber of any of the above embodiments. In some of any such embodiments of the closed system, the centrifugal chamber is capable of rotation at a speed up to 8000 g, wherein the centrifugal chamber is capable of withstanding a force of 500, 1000, 1500, 2000, 2500, 3000 or 3200 g, without substantially yielding, bending, or breaking or otherwise resulting in damage of the chamber and/or while substantially holding a generally cylindrical shape under such force.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows transduction efficiency calculated as percentage of CD3+ T cells with surface expression of a chimeric antigen receptor (CAR) encoded by a viral vector, following incubation under various conditions as described in Example 1. FIG. 1B shows population doublings over a six-day period during the transduction study described in Example 1.
FIG. 2 shows transduction efficiency calculated as percentage of CD3+ T Cells with surface expression of a CAR encoded by a viral vector following incubation under the indicated conditions as described in Example 2.
FIG. 3 shows transduction efficiency calculated as percentage of CD3+ T Cells with surface expression of a CAR encoded by a viral vector following incubation under various conditions as described in Example 3.
FIG. 4 shows mean vector copy number (VCN) of a viral vector in indicated cell populations following transduction under various conditions as described in Example 4.
FIG. 5 provides a schematic representation of an embodiment of a closed system (processing kit) for use in embodiments of the provided methods. The depicted exemplary system includes a generally cylindrical centrifugal chamber (1), rotatable around an axis of rotation and including an end wall (13), a rigid side wall (14), and a piston (2), defining an internal cavity (7) of the chamber. The chamber further includes an inlet/outlet opening (6) to permit flow of liquid and gas in and out of the cavity in at least some configurations of the system. The opening (6) is operably linked with a series of tubing lines (3) and connectors, including stopcock valves (4) and various additional containers. Clamps (5) are also depicted.
FIG. 6A shows population doublings over a ten-day period during the study described in Example 6. FIG. 6B shows percent viability of cells over a ten-day period during the study described in Example 6.
FIG. 7 provides a schematic representation of an embodiment of a closed system (processing kit) for use in embodiments of the provided methods. The depicted exemplary system includes a generally cylindrical centrifugal chamber (1), rotatable around an axis of rotation and including an end wall (13), a rigid side wall (14), and a piston (2), defining an internal cavity (7) of the chamber. The chamber further includes an inlet/outlet opening (6) to permit flow of liquid and gas in and out of the cavity in at least some configurations of the system. The opening (6) is operably linked with a series of tubing lines (3) and connectors, including stopcock valves (4), various additional containers, and an air filter (15) coupled to a removable cap (16). Clamps (5) are also depicted.
FIG. 8A shows transduction efficiency calculated as percentage of CD3+ T Cells with surface expression of a CAR encoded by a viral vector following incubation under the indicated conditions as described in Example 8A. FIG. 8B shows transduction efficiency calculated as percentage of CD3+ T Cells with surface expression of a CAR encoded by a viral vector following incubation under the indicated conditions as described in Example 8B. FIG. 8C shows mean vector copy number (VCN) of a viral vector in indicated cell populations following transduction under various conditions as described in Example 8B.
FIG. 9A shows transduction efficiency calculated as percentage of CD3+ T Cells with surface expression of a CAR encoded by a viral vector following incubation under the indicated conditions as described in Example 9. FIG. 9B shows mean vector copy number (VCN) of a viral vector in indicated cell populations following transduction under various conditions as described in Example 9.
FIG. 10 shows transduction efficiency calculated as percentage of CD3+ T Cells with surface expression of a CAR encoded by a viral vector following incubation under the indicated conditions as described in Example 10.
FIG. 11 provides a schematic representation of an embodiment of a closed system (processing kit) for use in embodiments of the provided methods. The depicted exemplary system includes a generally cylindrical centrifugal chamber (1), rotatable around an axis of rotation and including an end wall (13), a rigid side wall (14), and a piston (2), defining an internal cavity (7) of the chamber. The chamber further includes an inlet/outlet opening (6) to permit flow of liquid and gas in and out of the cavity in at least some configurations of the system. The opening (6) is operably linked with a series of tubing lines (3) and connectors, including stopcock valves (4) and ports (18), and various additional containers, including a plurality of output bags (17). Clamps (5) are also depicted.
DETAILED DESCRIPTION
Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
I. Methods of Cell Processing and Associated Systems, Kits, and Devices
Provided are methods for processing cells, for example, to generate compositions of cells for use in adoptive cell therapy. The methods include those for transferring recombinant viral vectors to the cells, such as by viral transduction. The viral vectors generally encode recombinant molecules to be expressed in the cells, e.g., for use in cell therapy. Processing steps of the methods can also or alternatively include all or a portion of cell washing, dilution, selection, isolation, separation, cultivation, stimulation, packaging, and/or formulation. The methods generally allow for the processing, e.g., selection or separation and/or transduction, of cells on a large scale (such as in compositions of volumes greater than at or about 50 mL). One or more of the cell processing steps generally are carried out in the internal cavity of a centrifugal chamber, such as a substantially rigid chamber that is generally cylindrical in shape and rotatable around an axis of rotation, which can provide certain advantages compared to other available methods. In some embodiments, all processing steps are carried out in the same centrifugal chamber. In some embodiments, one or more processing steps are carried out in different centrifugal chambers, such as multiple centrifugal chambers of the same type.
The provided methods offer various advantages compared with available methods for cell processing, including for transduction and selection, particularly those for large-scale cell processing. Certain available methods have not been entirely satisfactory, for example, due to less than optimal efficacy, accuracy, reproducibility, cost and time expenditure, risk of error, complexity, and need for user handling and biosafety facilities. In some embodiments, the provided methods are suitable for large-scale and/or clinical-grade cell production, while still providing desirable features otherwise available only with small-scale production methods, and offering additional advantages not provided by available methods. For example, the methods for cell transduction and/or affinity-based selection offer advantages compared with available methods performed in flexible plastic bags or plastic multi-well plates.
In some embodiments, the centrifugal chamber and/or its internal cavity in which the cells are processed is surrounded or defined at least in part by rigid or substantially rigid material. Incubation in a cavity bound by such materials, such as hard plastic, permits centrifugation under certain conditions, such as forces higher than those that may be used with bags used in other large-scale cell processing methods. For example, in some embodiments, the chamber and cavity withstand centrifugation at a force, e.g., a relative centrifugal force, of least at or about 500 g, 1000 g, 1500 g, 2000 g, 2500 g, 3000 g or 3200 g, as measured for example at an internal or external wall of the chamber or cavity, or at one or more cell, such as layer of cells, without substantially yielding, bending, or breaking or otherwise resulting in damage of the chamber or cavity holding the cells, such that the chamber and/or cavity substantially hold their shape under such force.
Accordingly, the chamber and/or its internal cavity typically are surrounded by all or a portion of a rigid or semi-rigid side wall, such as one made of hard plastic, which holds its shape under the centrifugal force applied. The side wall generally is curvilinear, e.g., cylindrical or generally cylindrical, and typically extends from one or two end walls of the chamber, the internal side of one or both of which may also define the boundaries of the internal cavity. The end walls in some embodiments are also made of rigid materials, and in some embodiments may include more flexible materials. In some embodiments, while a wall is made of rigid material or substantially rigid material, it may nonetheless be lined and/or coated with flexible material and/or contain small portions which are more flexible, so long as the cavity as a whole maintains its overall shape during the conditions of the methods.
The centrifugal chamber generally is rotatable around an axis of rotation, and the cavity typically is coaxial with the chamber. In some embodiments, the centrifugal chamber further includes a movable member, such as a piston, which generally is capable of movement (e.g., axial movement) within the chamber, to vary the volume of the cavity. Thus, in particular embodiments, the internal cavity is bound by the side wall and end wall of the chamber and the movable member, and has a variable volume that may be adjusted by moving the movable member. The movable member may be made of rigid, substantially or generally rigid, flexible materials, or combinations thereof.
The chamber generally also includes one or more opening(s), such as one or more inlet, one or more outlet, and/or one or more inlet/outlet, which can permit intake and expression of liquid and/or gas to and from the cavity. In some cases, the opening can be an inlet/outlet where both intake and expression of the liquid and/or gas occurs. In some cases, the one or more inlets can be separate or different from the one or more outlets. The opening or openings may be in one of the end walls. In some embodiments, liquid and/or gas is taken into and/or expressed from the cavity by movement of the movable member to increase and/or decrease the cavity's volume. In other embodiments, liquid and/or gas may be taken into and/or expressed from the cavity through a tubing line or other channel that is or is placed in connection with the opening, for example, by placing the line or channel in connection with and control of a pump, syringe, or other machinery, which may be controlled in an automated fashion.
In some embodiments, the chamber is part of a closed system, such as a sterile system, having various additional components such as tubing lines and connectors and caps, within which processing steps occur. Thus, in some embodiments, the provided methods and/or steps thereof are carried out in a completely closed or semi-closed environment, such as a closed or semi-closed sterile system, facilitating the production of cells for therapeutic administration to subjects without the need for a separate sterile environment, such as a biosafety cabinet or room. The methods in some embodiments are carried out in an automated or partially automated fashion.
In some embodiments, the chamber is associated with a centrifuge, which is capable of effecting rotation of the chamber, such as around its axis of rotation. Rotation may occur before, during, and/or after the incubation in one or more of the processing steps. Thus, in some embodiments, one or more of the various processing steps is carried out under rotation, e.g., at a particular force. The chamber is typically capable of vertical or generally vertical rotation, such that the chamber sits vertically during centrifugation and the side wall and axis are vertical or generally vertical, with the end wall(s) horizontal or generally horizontal. One exemplary chamber is depicted within exemplary closed systems depicted in FIG. 5, FIG. 7 or FIG. 11.
The processing steps of the methods (e.g., the steps carried out in whole or in part in the chamber) may include any one or more of a number of cell processing steps, alone or in combination. In particular embodiments, the processing steps include transduction of the cells with viral vector particles containing a retroviral vector, such as one encoding a recombinant product for expression in the cells, where at least a part of the incubation with the viral vector particles is performed in the chamber to initiate transduction. The methods may further and/or alternatively include other processing steps, such as steps for the isolation, separation, selection, cultivation (e.g., stimulation of the cells, for example, to induce their proliferation and/or activation), washing, suspension, dilution, concentration, and/or formulation of the cells. In some embodiments, the method includes processing steps carried out in an order in which: cells, e.g. primary cells, are first isolated, such as selected or separated, from a biological sample; resulting isolated or selected cells are stimulated in the presence of a stimulation reagent; stimulated cells are incubated with viral vector particles for transduction; and transduced cells are formulated in a composition. In some embodiments, the stimulation is additionally or alternatively performed during at least a part of the incubation with the viral vector particles. In some cases, stimulation is additionally or alternatively carried out after incubation of cells with the viral vector particles. In some cases, the methods do not include a step of stimulating the cells. In some embodiments, the method can include one or more processing steps from among washing, suspending, diluting and/or concentrating cells, which can occur prior to, during or simultaneous with or subsequent to one or more of the isolation, such as separation or selection, stimulation, transduction and/or formulation steps. All or a portion of each of the processing steps may be performed in a closed system, such as in a centrifugal chamber. In aspects of the methods, the processes need not be performed in the same closed system, such as in the same centrifugal chamber, but can be performed under a different closed system, such as in a different centrifugal chamber; in some embodiments, such different centrifugal chambers are at the respective points in the methods placed in association with the same system, such as placed in association with the same centrifuge. In some embodiments, all processing steps are performed in a closed system, in which all or a portion of each one or more processing step is performed in the same or a different centrifugal chamber.
In some embodiments, the methods provide the ability to transduce the cells at a higher transduction efficiency compared with available methods, e.g., by carrying out all or a part of transduction at higher centrifugal forces/speeds, and/or by allowing easy, automated, and/or independent control or adjustment of various parameters, such as volume or amount of reagents, speed, and/or temperature. In some embodiments, the methods increase efficacy and/or reduce variability (increasing reproducibility), e.g., by streamlining and/or decreasing the number of user-interactions and/or handling steps, such as by providing automated or semi-automated control of the various steps.
In some embodiments, by virtue of carrying out one or more, e.g., all or a portion of all, of the processing steps within a closed system, such as a sterile closed system, the provided methods allow for the large-scale preparation of cells for clinical use without exposing the cells to non-sterile conditions and without the use of a separate sterile room or cabinet. In some embodiments, the cells are isolated, separated or selected, stimulated, transduced, washed, and formulated within the closed system, e.g., in an automated fashion. In some embodiments, the methods are advantageous in that they are streamlined, e.g., require fewer steps, less user handling or intervention, e.g., by being carried out in a single, closed system and/or in an automated fashion. For example, in some embodiments, the methods provide improvement over methods for processing cells for use in clinical applications, which may require transduction in bags in a centrifuge or plate, by mixing viral vector particles and cells at appropriate ratios in a biosafety cabinet, followed by transportation of the plate or bag to the centrifuge for transduction or other processing step, and additional steps that may also require handling. In some embodiments, the provided methods are less manual and/or labor-intensive compared to such available methods, requiring a reduced degree or quantity of handling and user interaction.
In some embodiments, the methods allow for a greater degree of process control compared with available methods. For example, the methods in some embodiments allow for the independent control of various parameters, e.g., in an automated fashion. For example, the methods may allow independent control of volume, amount, and/or concentration of various components and reagents used in and processed with the methods or various conditions used in one or more of the processes or methods. They generally permit control of the duration of one or more various steps of the methods, and/or the control of the ratio of cells in a particular incubation or composition, liquid volume, and/or surface area of the vessel being used for the processing, such as the chamber or cavity. The ability to control such parameters independently, particularly in an automated fashion and independently of one another, can allow a user to easily optimize and carry out the methods for individual conditions.
Also provided are systems, devices, and apparatuses for use with such methods, kits containing the same, and methods of use of the compositions and cells produced by the methods. For example, provided are methods of treatment and therapeutic use of the cells and compositions produced by the methods, such as in adoptive cell therapy. Also provided are pharmaceutical compositions and formulations for use in such therapies.
II. Centrifugal Chambers and Associated Systems and Devices
In some embodiments, all or part of one or more of the processing steps, such as the incubation with virus to initiate or effect transduction and/or incubation with beads for immunoaffinity-based separation and/or one or more other processing steps as described, is carried out in a centrifugal chamber. In particular, such steps and incubations generally are carried out in an internal cavity of such a chamber, which can be a same or different centrifugal chamber for each of the one or more processes.
The centrifugal chamber is generally capable of being rotated, e.g., by a centrifuge that may be associated with the chamber during the incubation. In some embodiments, the centrifuge chamber is rotatable around an axis of rotation, such as a vertical or generally or substantially vertical axis of rotation. In some embodiments, the centrifuge chamber includes an end wall and a side wall, at least a portion of which surrounds or encircles the internal cavity of the chamber. The centrifuge chamber generally also includes another end wall, from which the side wall extends in the opposite direction.
The internal cavity generally is bound on its outside by the internal sides of all or a portion of the end wall, all or a portion of the side wall, and all or a portion of another end wall of the chamber or another surface or object, such as a movable member within the chamber, such as a piston. The cavity in some aspects is hollow. In other aspects, a solid or hollow object is contained within part of the internal space of the cavity, such as a tube or channel.
In some aspects, the cavity is of variable volume, meaning that the total volume available within the cavity that may be occupied, e.g., by liquid or gas, may be varied, for example, by movement of the moveable member, e.g., a piston. In some embodiments, such movement is possible during various steps of the methods, such as during the incubation to initiate or effect the transduction or selection or steps subsequent and/or prior thereto. The movement in some embodiments may be effected in an automated fashion, such as by a pre-specified program run by virtue of circuitry and machinery associated with the chamber, such as sensors and motors sensing and controlling position of the movable member and other aspects of the process and circuitry for communicating between the sensors and one or more components.
The side wall of the chamber, or the portion thereof that surrounds the internal cavity of the chamber (and thus the shape of the cavity), typically is curvilinear, such as cylindrical, substantially cylindrical, or generally cylindrical. The term cylindrical is generally understood to those in the art to refer to a particular type of curvilinear surface, formed by the points at a fixed distance from a given line segment, deemed the axis of a cylindrical shape. “Generally cylindrical” refers to a shape or surface having a configuration that is approximately cylindrical in shape or structure, such as one that appears cylindrical to the eye or is nearly cylindrical, but allows for some degree of variability. For example, the term encompasses shapes and surfaces of which not every point is at the same distance from the axis, and permits some degree of contouring and/or tapering, so long as the shape or surface appears cylindrical and/or has a primarily cylindrical shape. It also encompasses shapes in which the majority of the shape is cylindrical, such as where the majority of an outer wall of the centrifuge chamber is cylindrical or substantially cylindrical in shape but relatively minor portions of it adopt another configuration, for example, tapering or contouring at or approaching one or more ends of the wall. In some embodiments, the portion of the side wall of the chamber that surrounds the cavity is cylindrical, whereas other portions of the wall may not be cylindrical.
In some embodiments, all or portions of the chamber and/or cavity are rigid or substantially rigid. For example, all or part of the side wall may be rigid or substantially rigid, for example to allow the chamber and cavity to withstand force, e.g., as applied during centrifugation at high speeds, for example, at a force (relative centrifugal force (RCF)) at the internal surface of the side wall of the cavity and/or at a surface layer of the cells of greater than at or about 200 g, greater than at or about 300 g, or greater than at or about 500 g, such as greater than at or about 600 g, 800 g, 1100 g, 1000 g, 1500 g, 1600 g, 2000 g, 2200 g, 2500 g, 3000 g or 3200 g; or at least at or about 600 g, 800 g, 1000 g, 1100 g, 1500 g, 1600 g 2000 g, 2200 g, 2500 g, 3000 g, or 3200 g, such as at or about 2100 g or 2200 g. In some embodiments, the RCF at the internal surface of the side wall of the cavity and/or at a surface layer of the cells is greater than at or about or is at or about 1100 g, 1200 g, 1400 g, 1600 g, 1800 g, 2000 g, 2200 g or more. In contrast, available methods for processing cells on a large scale, e.g., greater than 50 or 100 mL volume, using flexible bags, may only permit centrifugation at a relative centrifugal force of no more than 200 g, 500 g, or 1000 g. Thus, the provided methods can produce greater efficacy compared to such methods.
The term “relative centrifugal force” or RCF is generally understood to be the effective force imparted on an object or substance (such as a cell, sample, or pellet and/or a point in the chamber or other container being rotated), relative to the earth's gravitational force, at a particular point in space as compared to the axis of rotation. The value may be determined using well-known formulas, taking into account the gravitational force, rotation speed and the radius of rotation (distance from the axis of rotation and the object, substance, or particle at which RCF is being measured).
The object, particle, or location (or average thereof) at which RCF is expressed or determined in a given case may be specified. For example, an RCF value or approximate value or range in some context herein is given for a particular portion or location within the centrifugal chamber used in such methods, such as at the internal surface of the side wall of the chamber's cavity in which the cells are processed, such as at any point along the surface of the cylindrical side wall of the cavity or at the average radial distance thereof. Similarly, the RCF value may be given for a radial distance or average radial distance within another container, such as a bag, in which cells are processed, relative to the axis of rotation. In other embodiments, the RCF is given for the location of the sample or composition as a whole or at one or more particular cells or average or layer thereof, during the rotation. For example, the value may be the RCF at a surface layer of the cells in the chamber or other container during rotation, such as at the cell surface at the interface between a liquid in which the cells are being spun and the cells themselves.
In general, the RCF is calculated by the formula 1.119×10−5 (rpm)2r (or 1.12×10−5×(rpm)2*r), where r=the radius (i.e., the distance in cm of a given particle, object or substance from the axis of rotation), rpm=revolutions per minute. For example, in some embodiments, the RCF at the internal surface of the side wall of internal processing cavity in which cells are processed may be calculated using this formula, in which r is the distance between a point on the internal surface of the side wall and the axis of rotation. Alternatively, the RCF at a cell or surface layer of cells (such as the interface between the cell layer(s) and liquid during rotation) may be calculated using the formula, in which r is the distance between the cell, surface layer, and/or interface, or an average thereof. For example, in some embodiments, the radius (r) value for an RCF of the side wall may be based upon the mean of the maximum and minimum possible radii or all possible radii along the length of the side wall of the chamber. In some embodiments, the radius for an exemplary centrifugal chamber sold by Biosafe AG for use with the Sepax® system (e.g., A-200/F) is at or about 2.6 cm or at or about 2.7 cm. In such an exemplary chamber, the radius for determining RCF at the interface between the cell layer(s) and the liquid during rotation in such a chamber may be calculated by adding the exact or approximate radial distance between the internal side wall of the cavity and the chamber occupied by cells of the layer(s) during rotation. Such value may be calculated or approximated using known methods, for example, based on the diameter of one of the cells being processed and/or the average diameter among such cells, for example, during rotation of the chamber. Such value may be based on the full size of the cell but typically will take into account impact on the relative volume occupied by each cell of the rotation or force itself, which generally speaking will reduce such volume. In some examples, the approximated value is determined using the size of a nucleus of the cell (or average thereof).
Thus, RCF or average RCF during a particular spin in a particular chamber or device may be calculated for a given point or area based on the revolutions per minute (rpm) and the distance between that point and the axis of rotation using well-known methods. Revolutions per minute (rpm) may be determined for various devices and chambers using known methods, for example, using a tachometer appropriate for the particular device, system, or chamber. For example, in some embodiments a hand-held photo or laser tachometer may be used, e.g., in combination with reflective tape, in the case of a centrifuge, system, or device with a window from the environment to the chamber or cavity, such as the Sepax®, which is clear or otherwise permits the passage of light between the tachometer to the chamber. For opaque systems, other tachometers may be used such as vibrating reed type tachometers.
As is understood by those in the art, when used in the context of various vessels and containers, such as chambers, plates, tubes and bags, used in cell processing and centrifugation and materials thereof, rigid generally describes an object, portion thereof, or material which substantially holds its shape and/or volume when placed in an environment, such as under a degree of force, temperature, or other condition, in which one would ordinarily expect to be present in the course of using the object. For example, it is understood in the art that rigid centrifugal chambers and tubes such as those made of hard plastic are distinguishable from flexible vessels such as cell processing and cell culture bags, such as bags made of soft plastics and rubbers, e.g., fluoro ethylene propylene and similar materials, the shape of which changes when pressure is applied manually or by pulling in liquid or gas, causing the bag to expand. Thus, in some embodiments, rigid materials include hard plastic, metal, carbon fiber, composites, ceramics, and glass, and/or are distinguished from flexible materials such as soft rubber, silicone, and plastics used in making flexible bags, the shape and volume of which is easily changed by ordinary pressure, e.g., manual pressure or the filling of a vessel with liquid under ambient temperature or ordinary conditions.
For example, in some embodiments, the rigid centrifugal chamber and/or portion(s) or material(s) thereof, such as the rigid side wall or portion thereof that surrounds the central cavity, is able to hold its shape and/or volume and/or does not rupture or break in a way that it would no longer contain liquid or gas, under particular conditions. In some embodiments, such conditions include manual pressure, such as pressure capable of being applied by human hand. In some embodiments, such conditions include specified centrifugal forces, such as at a force (RCF), e.g., effective force, at the internal surface of the side wall of the cavity, of greater than at or about 200 g, greater than at or about 300 g, or greater than at or about 500 g, such as greater than at or about 1000 g, 1500 g, 2000 g, 2500 g, 3000 g or 3200 g; or at least at or about 1000 g, 1500 g, 2000 g, or 2500 g, 3000 g, or 3200 g, such as at or about 2100 or 2200 g. In some embodiments, the environment includes particular conditions, such as temperatures down to at or about −80° C. and/or up to physiological temperatures or temperatures at which cells remain viable, and/or higher, such as temperatures of 18° C. to 42° C., such as 22° C. to 39° C., for example at least 25° C.±2° C. or 37° C.±2° C.
As is understood in the art, describing an object as rigid or substantially rigid does not exclude the possibility that any change in shape or volume of an object or material would ever occur, such as under excessive or unexpected force. For example, under excessive force or extreme environmental conditions, such as those well outside those ordinarily used in connection with the transduction methods described herein.
The chamber generally includes at least one opening, such as an inlet, an outlet, and/or an inlet/outlet, to permit substances to pass between the cavity or other portion of the chamber and other spaces. For example, such opening(s) generally are included in at least one of the walls of the chamber. The chamber generally includes at least one inlet and at least one outlet, which in some embodiments may be the same opening (inlet/outlet), through which liquid and/or gas may be taken into and expressed from the cavity. The opening is generally associated with another environment via a channel, e.g., tubing line or system of tubing lines, in some embodiments, such as via one or more connectors.
In some embodiments, the chamber is included as part of and/or integral to a system, such as a closed or partially closed system, which further includes additional components, such as tubing lines, connectors, and containers. In some embodiments, the chamber is pre-connected to one or more of the additional components, directly and/or indirectly. Such a chamber may be provided as part of a pre-assembled kit, e.g., a kit packaged for single, sterile, use in connection with the provided methods. In some embodiments, various components are packaged separately, for example, to allow for custom configurations in which a user connects and arranges the components for a particular embodiment of the processing methods.
The components typically include at least one tubing line, and generally a set or system of tubing lines, and at least one connector. Exemplary connectors include valves, ports, spikes, welds, seals, and hose clamps. The connectors and/or other components may be aseptic, for example, to permit the entire process to be carried out in a closed, sterile system, which can eliminate or reduce the need for clean rooms, sterile cabinets, and/or laminar flow systems.
In some embodiments, the at least one tubing line includes a series of tubing lines. Tubing can be made of a plastic, such as polycarbonate, and may be of various sizes and/or volumes, generally designed to permit flow of the desired liquid/gas at the appropriate rate, and connection with the chamber and/or other components. The series of tubing lines generally allows for the flow of liquids and gases between the chamber and/or one or more components of the system, such as the other containers, facilitated in some aspects by connectors. In some embodiments, the system includes tubing lines connecting each of the various components to at least one other of the components, where liquid is permitted to flow between each of the containers, such as bags, and the chamber, which may be permitted or stopped by the configuration of various connectors, such as valves, and/or clamps.
In some aspects, the connectors are such that they may be placed in or directed to alternative configurations, respectively blocking, allowing, and/or directing the flow of fluids and gases through various components, such as between various containers and through certain tubing lines connecting various components, such as rotational and gate valves. In other embodiments, certain connectors and/or other components have a single configuration which permits, directs, or blocks passage of liquid or gas, such as seals, caps, and/or open ports or channels. Various components in the system may include valves, ports, seals, and clamps. Valves can include rotational valves, such as stopcocks, rotary valves, and gate valves. Valves can be arranged in a manifold array or as a single multiport rotational valve. Ports may include Luer ports or spike ports. Seals may include O-rings, gaskets, adhesive seals, and couplings. Clamps may include pinch clamps.
Other components of a system include containers capable of holding or storing liquids and/or gases. The containers can include bags, vials, boxes, syringes, bulbs, tanks, bottles, beakers, buckets, flasks, and tubing lines. Such components can hold compositions used in and produced by the methods, including byproducts and interim products and waste. Such compositions may include liquid, including buffers, growth media, transduction media, water, diluents, washes, and/or saline, and may also include the cells, virus, and/or other agents for use in the processing steps, such as transduction. The containers also may include waste containers, and containers holding one or more output product, such as a product containing cells selected and/or transduced by one or more processing steps of the methods herein.
In some embodiments of the systems, a plurality of containers can be sterilely connected at one or more positions on the tubing line of the system. The containers can be connected simultaneously and/or sequentially during methods of cell processing in the provided embodiments. In some embodiments, the containers are detachable or removable from the connectors, such that the containers can be removed from the system and/or replaced by another container at the same position for use with the system. In some embodiments, not all connector positions of a system are connected to a container, such that the system can contain empty connectors. In some such embodiments, a closed system is maintained by operation of one or more stopcocks, valves or clamps, either manually or automatically, to close communication between a tubing line and an empty connector, e.g. port. In some embodiments, a closed system is maintained by sealing or detaching an empty connector, e.g. port.
In some embodiments of the systems, such as the exemplary systems depicted in FIG. 5, FIG. 7 or FIG. 11, containers can be operably connected to tubing lines, such as through a connector, at positions corresponding to an Input Bag position, Diluent Bag 1 position, a Diluent Bag 2 position, a Waste Bag position, and/or an Output Bag position. With reference to the Figures, the designation of these positions is for exemplification only, and is not meant to limit the particular type of container or content of the container that can be connected at a position. Also, in embodiments of the provided methods, not all positions of the system, such as depicted in the Figures, need to be utilized in performing the processing steps of the provided methods. In some such embodiments, a tubing line servicing an empty connector, e.g. port, can be disengaged or closed by operation of a stopcock or valve. In some embodiments, an empty connector can be sealed or detached.
In some embodiments, the system, such as a closed system, is sterile. In some embodiments, all connections of components of the system, such as between tubing line and a container via a connector, are made under sterile conditions. In some embodiments, connections are made under laminar flow. In some embodiments, connections are made using a sterile connection device that produces sterile connections, such as sterile welds, between a tubing and a container. In some embodiments, a sterile connection device effects connection under thermal condition high enough to maintain sterility, such as temperatures of at least 200° C., such as at least 260° C. or 300° C.
In some embodiments, the system may be disposable, such as a single-use kit. In some embodiments, a single-use kit can be utilized in a plurality of cycles of a process or processes, such as at least 2, 3, 4, 5 or more times, for example, in processes that occur in a continuous or a semi-continuous manner. In some embodiments, the system, such as a single-use kit, is employed for processing of cells from a single patient.
Exemplary centrifugal chambers include those produced and sold by Biosafe SA, including those for use with the Sepax® and Sepax® 2 system, including an A-200/F and A-200 centrifugal chambers and various kits for use with such systems. Exemplary chambers, systems, and processing instrumentation and cabinets are described, for example, in U.S. Pat. Nos. 6,123,655, 6,733,433 and Published U.S. patent application, Publication No.: US 2008/0171951, and published international patent application, publication no. WO 00/38762, the contents of each of which are incorporated herein by reference in their entirety. Depending on the particular process (e.g. dilution, wash, transduction, formulation), it is within the level of a skilled artisan to choose a particular kit that is appropriate for the process. Exemplary kits for use with such systems include, but are not limited to, single-use kits sold by BioSafe SA under product names CS-430.1, CS-490.1, CS-600.1 or CS-900.2.
In some embodiments, the system comprises a series of containers, e.g., bags, tubing, stopcocks, clamps, connectors, and a centrifuge chamber. In some embodiments, the containers, such as bags, include one or more containers, such as bags, containing the cells to be transduced and the viral vector particles, in the same container or separate containers, such as the same bag or separate bags. In some embodiments, the system further includes one or more containers, such as bags, containing medium, such as diluent and/or wash solution, which is pulled into the chamber and/or other components to dilute, resuspend, and/or wash components and/or compositions during the methods. The containers can be connected at one or more positions in the system, such as at a position corresponding to an input line, diluent line, wash line, waste line and/or output line.
Exemplary systems for use in embodiments of the provided methods for carrying out one or more or all part of the process are depicted in FIG. 5, FIG. 7 and FIG. 11. In one exemplary embodiment as shown in FIG. 5, the centrifugal chamber (1) is at least generally cylindrical and is rotatable around an axis of rotation. The chamber includes an end wall (13) and a rigid side wall (14), and the movable member, which is a piston (2). The internal surfaces of the end wall (13), rigid side wall (14), and piston (2) collectively define the boundaries of the internal cavity (7) of the chamber. The cavity (7) is of variable volume and is coaxial with the chamber, and is designed to contain the liquid and/or gas that is included within the chamber during the processing steps. The piston (2) is axially movable within the chamber (1) to vary the volume of the internal cavity (7). The chamber further includes an inlet/outlet opening (6) to permit flow of liquid and gas in and out of the cavity in at least some configurations of the system. The opening (6) is operably linked with a series of tubing lines (3) and connectors, including stopcock valves (4), which are capable of controlling movement of fluid and/or gas between the various components of the system. The series of tubing lines (3) further are linked with various additional containers, which in the depicted configuration include bags labeled as Input Bag, Diluent Bags 1 and 2, a Waste Bag, and an Output Bag. Clamps (5) may be opened and closed to permit and block movement of fluid through the indicated portions of the series of tubing lines (3), permitting flow between various components of the system. In some embodiments, each container is operably connected to a tubing line via a port, such as a luer port or spike port. As an example, with reference to FIG. 5, at each point that a container is shown, in some aspects, the container is connected indirectly via a port.
While FIG. 5 shows connection of a container at each position or line, in an alternative embodiment, in some aspects, a container is not connected at each position or line of the system. In some embodiments of provided systems, a port is available at each position or line for connection, and a container is connected to all positions or lines or less than all positions or lines. In some embodiments, not all connector positions of a system are connected to a container, such that the system can contain empty connectors at each position or line.
In some embodiments, the system, such as the system shown in FIG. 5, can include a sterile or microbial filter. Exemplary of such a system is shown in FIG. 7, which depicts a filter (15). In some embodiments, the filter includes a filtration membrane having a pore size that blocks passage of microbial organisms, such as bacteria or viruses. In some embodiments, the pore size is between 0.1 μm to 0.45 μm, such as between 0.1 μm to 0.22 μm, such as about or 0.20 μm. In some embodiments, the membrane is composed of nitrocellulose (cellulose nitrate), cellulose acetate, regenerated cellulose, polyamide, polytetrafluorethylene (PTFE) or polyethersulfone (PES). In some embodiments, the filter includes a cap (16) to close or seal the membrane of the filter from exposure to the environment outside of the closed system. In some embodiments, the cap is closed or non-vented. In some embodiments, the cap is detachable. In some embodiments, the cap is fitted to the filter by a luer lock fitting. As described in more detail below, in some embodiments, the filter can be used to effect passage of gas, such as air, to and from the chamber of the system. In some such embodiments, the passage of air is maintained under sterile or microbial-free conditions.
In one embodiment, the Input Bag includes cells for processing by the provided methods, such as transduction. In one embodiment, Diluent Bag 1 includes viral vector particles containing the vector with which to transduce the cells. Thus, in some embodiments, the input composition containing the viral vector particles and cells is generated by effecting intake of fluid from the Input Bag and effecting intake of fluid from Diluent Bag 1. In some embodiments, Diluent Bag 2 contains wash solution. The Output Bag generally is designed to take in the cells following one or more of the processing steps, such as by transfer of the output composition from the cavity of the chamber to the Output Bag after incubation with viral vector particles. Thus, in some embodiments, the Output Bag contains transferred cells transduced with and/or in which transduction is initiated with the viral vector particles. In some embodiments, the processing comprises transduction of the cells with a viral vector.
In some embodiments, a multi-way manifold (17) can be used to operably connect one or a plurality of containers to the system via a plurality of ports (18) connected to a manifold of tubing lines. The multi-way manifold can contain a series of tubing lines that feed to the inlet/outlet of the chamber to permit flow between the chamber and the connected container or containers. In some such embodiments, the manifold connects a plurality of containers, such as at least 2, 3, 4, 5, 6, 7, 8 or more containers, at the same position or line on the system. In some embodiments, all ports of the multi-way manifold (17) are connected to a container, such as a bag. In some embodiments, less than all of the ports of the multi-way manifold (17) are connected to a container, such as a bag, such that a container is connected at less than the total number of port positions, for example less than 8, 7, 6, 5, 4, 3, or 2 containers, such as bags, are connected. In some embodiments, the tubing lines associated with the manifold can contain a clamp or stopcock, which can be opened or closed to control movement through the line into the container as necessary. The multi-way manifold (17) can be connected to any of the positions or lines available on the system, such to a position or line designated an input line, diluent line, wash line, waste line and/or output line.
In some embodiments, exemplary of a multi-way manifold (17) for linking a container or containers is shown in FIG. 11 for connecting one or a plurality of containers, such as one or a plurality of bags, for example one or a plurality of output Bags. As shown, in some embodiments, a multi-way manifold (17) can be connected to an output position or line, which includes a series of manifold tubing lines that each end with a connector, such as a port (18), for operable connection to a container, such as a bag. One or more of the ports, such as all of the ports or less than all of the ports, can be connected to a container. In one embodiment as exemplified in FIG. 11, up to 3 containers can be connected to each tubing line of the manifold via a port. In other embodiments, up to 1, 2, 3, 4, 5, 6, 7, or 8 containers, such as Output bags, can be connected at the output line. In some embodiments, one or a plurality of clamps (5) associated with tubing lines, such as the manifold tubing line, may be opened or closed to permit or control the movement of liquid into one or more of the plurality of Output Bags. In some embodiments, a single clamp can control movement of liquid into all Output Bags simultaneously. In some embodiments, the movement of liquid into each of the plurality of bags is separately regulated by a clamp operably connected to a tubing line associated with only one respective container, such that the movement of liquid into the respective container can be made separable from the movement of liquid into all other containers. In some embodiments, movement of liquid into each container, such as each bag, for example each Output bag, can be made to be sequential.
In some embodiments, the system is included with and/or placed into association with other instrumentation, including instrumentation to operate, automate, control and/or monitor aspects of the various processing steps performed in the system. This instrumentation in some embodiments is contained within a cabinet.
In some embodiments, the instrumentation includes a cabinet, which includes a housing containing control circuitry, a centrifuge, a cover, motors, pumps, sensors, displays, and a user interface. An exemplary device is described in U.S. Pat. Nos. 6,123,655, 6,733,433 and US 2008/0171951.
The control circuitry in some aspects monitors and communicates information and instructions to and from the other instrumentation and various components of the system. In some embodiments, the cabinet contains a user interface device, comprising a display and an input device, such as a keyboard, a mouse, or a touchscreen. The user interface displays information from the control circuitry, allows the user to stop and start a process or steps, such as to effect a transduction protocol. The interface may also prompt the user to input settings for variables used by the control circuitry during a process step, such as a transduction protocol. Such variables may include volume of various solutions to be added and/or removed from the various containers and/or the cavity of the chamber, time/duration of sedimentation, centrifugation, agitation, mixing, and/or other process steps, rotational force, piston movement, and/or program selection.
The instrumentation generally further includes a centrifuge, into which the centrifuge chamber is placed in order to effect rotation of the chamber. In some embodiments, the centrifuge chamber is engaged with a rotary drive unit on the centrifuge apparatus, such that the chamber is rotatable about an axis of rotation. In some embodiments, a cover closes on top of the centrifuge chamber and holds the chamber in place. In some embodiments, the cover includes two semi-circular disks that can rotate on a hinge. An exemplary centrifuge and cover are described in U.S. Pat. No. 6,123,655 or 6,733,433. The centrifuge locks the centrifuge chamber into place and rotates the centrifuge chamber by contacting the chamber's sides or ends.
In some embodiments, a sensor or an array of sensors in the centrifuge can measure the rotational speed of the centrifuge chamber, the position of the movable member, or the volume contained within the internal cavity. Sensors outside of the centrifuge can detect the color and flow rate of liquid and gas flowing to and from the centrifuge chamber. Sensors can also detect an empty tubing or centrifuge chamber. Sensors include optical sensors, such as those described in U.S. Pat. Nos. 6,123,655, 6,733,433 and US 2008/0171951. In some embodiments, the information from the sensor or sensors can be received by control circuitry. Based on the information transmitted, the control circuitry, in some embodiments, can effect changes to one or more of the rotational speed of the centrifuge chamber, the position of the movable member, the volume contained in the cavity, the orientation of one or more valves, ports, seals or clamps, and other processes of the centrifuge, chamber or system.
In some embodiments, the cabinet includes a motor or array of motors. The motors can communicate information with the control circuitry, which can operate or adjust the motors.
In some embodiments, the motor or array of motors can rotate the centrifuge chamber within the centrifuge. The control circuitry can start, stop, or adjust the speed of the motors rotating the centrifuge chamber within the centrifuge.
In some embodiments, the motors or array of motors can move the movable member within the centrifuge chamber. Moving the movable member varies the volume of the internal cavity, causing the intake or expression of liquid or gas to or from the internal cavity.
In some embodiments, the motors or array of motors can operate the valves, ports, seals, and clamps described herein. The control circuitry can cause the motors to open, close, or direct fluid to or from a container or the centrifuge chamber through the series of tubing.
In some embodiments, the motor or motors is an electrical motor, pneumatic motor or hydraulic motor. In some embodiments, the cabinet includes an electrical motor for operating some aspects and a pneumatic motor for operating other aspects. In some embodiments, the cabinet includes an electrical motor for centrifugation and a pneumatic motor for controlling movement of the movable member.
III. Transfer of Viral Nucleic Acids to Cells, e.g., by Transduction
In some embodiments, the processing step(s) of the methods include those for transfer of viral particles to the cells, such as viral vectors encoding recombinant products to be expressed in the cells. The viral vector particles generally include a genome containing recombinant nucleic acids such as transgenes encoding such products. In some embodiments, the viral vector particles encode a recombinant receptor, such as a chimeric antigen receptor (CAR), whereby transduction of cells can generate recombinant receptor (e.g. CAR)-expressing cells. Transfer of the nucleic acid from the viral vector to the cells may use any of a number of known methods. Transfer is typically by transduction. Alternative methods for transferring viral vectors to cells include transposons and/or electroporation. Such processing steps can be performed in a centrifugal chamber according to embodiments of the provided methods. In some embodiments, the centrifugal chamber is integral to a closed system, such that such processing steps are performed in a closed system.
The transfer is generally carried out by transduction. The methods for viral transfer, e.g., transduction, generally involve at least initiation of transduction by incubating in a centrifugal chamber an input composition comprising the cells to be transduced and viral vector particles containing the vector, under conditions whereby cells are transduced or transduction is initiated in at least some of the cells in the input composition, wherein the method produces an output composition comprising the transduced cells.
In some embodiments, the cells for transduction and/or transduced cells contain immune cells, such as T cells, for use in adoptive immunotherapy. In some embodiments, prior to the incubation of cells with viral vector particles, the cells for transduction are obtained by methods that include isolating, such as selecting, a particular subset of cells present in a biological sample. Methods related to isolation and selection of cells for transduction, and the resulting cells, are described below. In some embodiments, prior to initiation of the processes for transduction, T cells are activated, such as by cultivation and stimulation as described below. In some embodiments, one or more of all or a part of the steps related to isolation, e.g. selection, and activation also can be carried out in the cavity of a centrifugal chamber according to provided embodiments as described below.
In some embodiments, the viral vector particles used in aspects of the transduction method are any suitable for transduction of the cells, such as an immune cell, for example a T cell. In some embodiments, the viral vector particles are retroviral vector particles, such as lentiviral vector particles or gammaretroviral vector particles. In some such embodiments, the viral vector particle contains a genome comprising a recombinant nucleic acid, i.e. a recombinant viral vector. Exemplary of such viral vector particles are described below.
The input composition (the composition that contains the viral vector particles and cells during the transduction step) may further include one or more additional agents, such as those to promote transduction efficiency, such as polycations including protamine (e.g. protamine sulfate), hexadimethrine bromide (POLYBRENE®, Abbott Laboratories Corp), and CH-296 (RETRONECTIN®, Clontech). In some embodiments, the polycation can be present in the input composition at a final concentration of 1 μg/mL to 100 μg/mL, such as 5 μg/mL to 50 μg/mL. The composition may also include media, including cell culture medium including medium designed for culture of the cell type to be processed, such as hematopoietic stem cell medium, e.g., serum free medium.
In the provided methods, all or a part of the processing steps for transduction of cells can occur in the centrifugal chamber, such as under centrifugation or rotation. In some such embodiments, the input composition containing the cells and the viral vector particles are provided to or taken into the internal cavity of the centrifugal chamber. In some embodiments, the input composition is incubated under conditions comprising rotation of the centrifugal chamber. In some embodiments, the rotation can be effected at relative centrifugal forces greater than can be achieved using flexible plastic bags or plastic multi-well plates.
Greater transduction efficiency is achieved in some embodiments in part due to the ability of the methods to carry out the transduction at a greater relative centrifugal force (RCF) compared with other methods for processing cells on large scales. For example, certain available methods for processing cells on a large scale, e.g., greater than 50 or 100 mL volume, using flexible bags, may only permit centrifugation at a relative centrifugal force of no more than 200, 500, or 1000 g. By allowing centrifugation at greater acceleration or relative force, e.g., at or about or at least at or about 1000, 1500, 2000, 2100, 2200, 2500, 3000 g, 3200 g or 3600 g, the provided methods can improve or permit co-sedimentation of virus and cells in the composition during transduction, improving the rate of virus-to-cell interactions, thereby improving transduction.
The methods generally are capable of conducting the transduction on a large scale. Thus, the input composition incubated during the transduction and/or output composition may contain at least a certain volume and/or number of cells. In some embodiments, the liquid volume of the input composition, or the liquid volume during at least a point during the incubation, is at least or greater than about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL, or 500 mL. In some embodiments, the input composition, the transduced composition, and/or the total cells transduced by the methods include at least at or about 1×105, 1×106, 1×107, 1×108, 1×109, or 1×1010 cells. In some embodiments, for at least a portion of the incubation, the vessel in which the cells are transduced, e.g., the centrifugal chamber or cavity thereof, contains at least at or about 1×105, 1×106, 1×107, 1×108, 1×109, or 1×1010 cells. Such numbers and volume may also apply to other processing steps carried out in the system, e.g., in the cavity of the chamber, such as cell separation and/or washing steps.
In some embodiments, in describing the various processes steps in a cavity of the centrifugal chamber, including processes for transduction, such as preparation of the input composition, or other process as described in subsequent sections, reference to any volume is a target volume. In some embodiments, the exact volumes utilized in various steps (e.g. wash, dilution or formulation) can vary from a desired target volume, due to, in some aspects, dead volumes in a tubing line, priming of lines, sensitivity of a sensor, user control, and other factors associated with maintaining or monitoring a volume. The methods can permit precise control of volumes, such as by, in some aspects, inclusion of a sensor as part of the circuitry associated with the system. In some embodiments, volumes vary by no more than 10% of a desired target volume, such as no more than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. In some embodiments, volumes are within 2 mL or 3 mL of a target volume and/or vary by no more than 2 mL or 3 mL of a target volume.
In some embodiments, the processing steps are carried out by combining the cells and the viral vector particles to generate an input composition. In aspects of the method, the composition of cells and viral vector particles are prepared in a manner so that the resulting combined input composition has a low ratio of total liquid volume to internal surface area of the cavity of the centrifugal chamber. In some embodiments, the total liquid volume is sufficient to cover or just exceed a volume of cells present as a monolayer on the internal surface of the cavity after rotation of the centrifugal chamber, while minimizing the liquid thickness covering the cells. In some embodiments, reducing the liquid thickness can reduce the sedimentation time required for contacting of the viral vector particles with the cells because the viral vector particles have less of a distance to travel and/or are subjected to less resistance from the viscous medium.
In some embodiments, advantages such as improved transduction efficiency are due at least in part to the ability to use a relatively lower volume of liquid per volume of cells, cell number, or cell pellet size, during processes of transduction, such as during rotation, particularly compared with other methods for large-scale production.
In some embodiments, the liquid volume of the input composition (containing cells and viral vector particles) present in the vessel, e.g., cavity, during rotation is no more than about 0.5, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 milliliters (mL) per square inch of the internal surface area of the cavity during the rotation or the maximum internal surface area of the cavity.
In particular embodiments, the average liquid volume of the input composition present in the vessel, e.g., cavity, in which transduction is initiated, such as the average of the liquid volume of all processes performed in a cycle of the method, is no more than about 0.5, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 milliliters (mL) per square inch of the internal surface area of the cavity during the incubation or of the maximum internal surface area of the cavity. In some embodiments, the maximum liquid volume of the input composition (containing cells and viral vector particles) present in the vessel, e.g., cavity, in which transduction is initiated, such as the maximum of the liquid volume of all processes performed in a cycle of the method, is no more than about 0.5, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 milliliters (mL) per square inch of the internal surface area of the cavity of the centrifugal chamber. In some embodiments, the liquid volume, such as the liquid volume of the input composition, present in the vessel, e.g., cavity, during rotation is no more than 50%, such as no more than 40%, no more than 30%, no more than 20% or no more than 10% of the volume of the internal surface area of the cavity during rotation or the maximum internal surface area of the cavity. In some embodiments, the remainder of the volume can be gas, such as air.
In some embodiments, the total liquid volume of the input composition (containing cells and viral vector particles) in the centrifugal chamber during incubation, such as during rotation, is at least 5 mL or at least 10 mL but is no more than 220 mL, such as no more than 200 mL. In some embodiments, the liquid volume of the input composition during incubation, such as during rotation, is no more than 100 mL, 90 mL, 80 mL, 70 mL, 60 mL, 50 mL, 40 mL, 30 mL or 20 mL. In aspects of the provided method, the input composition is prepared at such a total volume to achieve a desired concentration, amount and/or ratio of cells and viral vector particles, such as described below.
In some embodiments, the methods permit the user to control the ratio of cells to surface of the cavity, e.g., by varying the volume of the cavity and/or number of cells added. In some embodiments, this allows reduction of the layer of cells (e.g., cell pellet) on the surface of the cavity compared to other methods, particularly those available for large-scale transduction under centrifugal force, such as those carried out in centrifuge bags. In some embodiments, the ability to control the thickness of the layer of cells in the cavity of the centrifugal chamber during the transduction can lead to increased transduction efficiency under otherwise comparable conditions and/or a lack of increased copy number with increased virus transduction efficiency.
In some embodiments, the cells in the provided methods are present in the cavity in at or about a single monolayer, or no more than at or about 1.5 or 2-fold more than a single monolayer, or not substantially thicker than a monolayer, during the incubation for transduction under centrifugal force. This reduction during centrifugation can facilitate and improve interactions between the virus and cells and avoid increases in viral copy number (VCN) which can occur particularly in the context of high relative virus or infectious units (IU), for example, when outer or upper layers of cells are preferentially transduced.
In some embodiments, the input composition contains at least 1 million cells per cm2 of the internal surface area of the cavity during at least a portion of said incubation, such as during rotation of the input composition in the centrifugal chamber. In some embodiments, the input composition contains at least 2 million cells per cm2, 3 million cells per cm2, 4 million cells per cm2, 5 million cells per cm2, 6 million cells per cm2, 7 million cells per cm2, 8 million cells per cm2, 9 million cells per cm2, 10 million cells per cm2 or 20 million cells per cm2 of the internal surface area of the cavity during at least a portion of said incubation, such as during rotation of the input composition in the centrifugal chamber. In some embodiments, the internal surface area of the cavity during at least a portion of said incubation, such as during rotation, is at least at or about 1×109 μm2 or is at least at or about 1×1010 μm2.
In some embodiments, the total number of cells in the input composition during at least a portion of said incubation, such as during rotation of the input composition in the centrifugal chamber, is at least 10×106 cells, 20×106 cells, 30×106 cells, 40×106 cells, 50×106 cells, 60×106 cells, 70×106 cells, 80×106 cells, 100×106 cells, 200×106 cells, 300×106 cells or 400×106 cells.
In some embodiments, processing steps in the closed cavity of a centrifugal system also can be used to process the cells, such as activated cells, prior to transduction. In some embodiments, the processing can include dilution or concentration of the cells to a desired concentration or number. In some embodiments, the processing steps can include a volume-reduction to thereby increase the concentration of cells as desired. In some embodiments, the processing includes exchange of a medium into a medium acceptable or desired for transduction.
In some embodiments, the input composition comprises a certain ratio of copies of the viral vector particles or infectious units (IU) thereof, per total number of cells (IU/cell) in the input composition or total number of cells to be transduced. For example, in some embodiments, the input composition includes at or about or at least at or about 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or 60 IU of the viral vector particles per one of the cells.
In certain embodiments, the ability to use a higher IU in the present methods provides advantages compared to other methods. Under otherwise identical conditions, use of a higher IU/cell ratio generally leads to a higher transduction efficiency, or does so up to a certain upper level of IU/cell at which the corresponding increase in efficiency may plateau. Nonetheless, with certain available methods, increasing the IU/cell and thus the transduction efficiency also leads to an increase in vector copy number (VCN), which can present safety risks and may not meet regulatory standards.
In some embodiments, with the provided methods, average VCN among transduced cells in the output composition, such as cells containing the viral vector or cells expressing a molecule encoded by the viral vector, does not increase with an increase in IU/cell in the input composition. In some embodiments, in the provided methods, the average VCN among transduced cells decreases with an increased IU/cell ratio in the input composition.
In some embodiments, the titer of viral vector particles is between or between about 1×106 IU/mL and 1×108 IU/mL, such as between or between about 5×106 IU/mL and 5×107 IU/mL, such as at least 6×106 IU/mL, 7×106 IU/mL, 8×106 IU/mL, 9×106 IU/mL, 1×107 IU/mL, 2×107 IU/mL, 3×107 IU/mL, 4×107 IU/mL, or 5×107 IU/mL.
In some embodiments, the input composition contains a concentration of viral vector particles during at least a portion of said incubation, such as during rotation of the input composition in the centrifugal chamber, that has a certain ratio of copies of the viral vector particles or infectious units (IU) thereof, per total number of cells (IU/cell) in the input composition or total number of cells to be transduced per total liquid volume of the input composition present during at least a portion of said incubation, such as during rotation, i.e. IU/cell/mL. In some embodiments, the input composition includes at least 0.01 IU, 0.05 IU, 0.1 IU, 0.5 IU or 0.1 IU of the viral vector particles per one of the cells per mL of the liquid volume of the input composition during at least a portion of said incubation, such as during rotation.
In some embodiments, the step of creating the input composition (cells and viral vector particles) can be performed in the centrifugal chamber. In some embodiments, the step of creating the input composition is performed outside the centrifugal chamber. Thus, the term “input composition” is not meant to imply that the entire composition is taken into the respective vessel, e.g., tube, bag, or cavity, at once, or to exclude the pulling in of parts of the composition from different vessels or lines. Input compositions may include those formed by pulling in two different compositions into the chamber's cavity and mixing the two, thereby creating the input composition.
The input composition may be taken into or otherwise transferred to the vessel in which the incubation, such as rotation, takes place from the same container or from more than one separate containers. For example, the input composition may be taken into the chamber by pulling in a composition containing the cells and another composition containing the viral vector particles, which may be done sequentially or simultaneously. Alternatively, the input composition containing the viral vector particles and cells is taken into the cavity or other vessel in which the transduction is to be carried out.
In some embodiments, where the transduction is carried out in the internal cavity of the centrifugal chamber, this is achieved by allowing only a certain portion of the cavity to include the liquid input composition. This may be achieved, for example, by pulling in air or gas into a portion of the cavity, and/or by including one or more solid object in a space within the cavity, such as an internal space. In some embodiments, this can minimize or reduce the total liquid volume of said input composition present in said cavity during incubation, such as during rotation, of said centrifugal chamber per square inch of the internal surface area of the cavity compared to the absence of gas in the cavity and/or absence of one or more solid objects in the space of the cavity. In this way, compared with other methods, in which diffusion of virus through a large volume of liquid compared to volume of cells may limit efficacy of transduction, the provided methods can be advantageous. Thus, whereas in some embodiments, the input composition occupies all or substantially all of the volume of the internal cavity during at least a portion of the incubation, in some embodiments, during at least a portion of the incubation, the input composition occupies only a portion of the volume of the internal cavity during said incubation.
In some such embodiments, the volume of the cavity during this at least a portion of the incubation may further include a gas taken into said cavity by the one or more opening, e.g., inlet, in the cavity, such as prior to or during said incubation. In some embodiments of the method, the air is sterilized or is sterile air. In some embodiments, the air is free of or substantially free of microbial contaminants or other potentially pathogenic agents.
In some embodiments, providing or taking in gas, such as air, can be effected in any manner that permits passage of air into the internal cavity of the centrifugal chamber, such as, in some aspects, without compromising the sterility of the closed system. In some embodiments, gas, such as air, can be added to a container under sterile conditions, and the container can be sterilely connected at a position on the system for transfer into the chamber. In some embodiments, the addition of gas, such as air, to the container, such as a bag, is effected under laminar flow conditions, such as in a biological safety cabinet or hood. In some such embodiments, the gas, such as air, is added to the container together with a liquid volume, such as a liquid volume containing a composition of cells and/or a liquid volume containing a composition of viral vector particles. Hence, in some embodiments, providing or taking in gas, such as air, into the internal cavity of the chamber occurs together or simultaneously with the providing or intake of one or both of the cells or viral vector particles that make up the input composition.
In some embodiments, the providing or taking in gas, such as air, into the chamber, is achieved using a syringe that can be attached to any luer lock associated with the system, and, that is operably connected to the internal cavity of the centrifugal chamber. In some embodiments, air is transferred into the syringe under sterile conditions, such as under laminar flow. In some embodiments, the syringe is a sterile syringe, such as, in some aspects, a syringe containing a movable plunger that is not exposed to the surrounding non-sterile environment. In some embodiments, the syringe contains a filter at its end to effect sterile transfer of gas, such as air, into the internal cavity of the chamber.
In some embodiments, providing or taking in gas, such as air, into the internal cavity of the chamber is achieved by the use of a filter operably connected to the internal cavity of the chamber via a sterile tubing line. In some such embodiments, the filter is a sterile or microbial filter as described with reference to an exemplary system, such as in some aspects, a filter as exemplified in FIG. 7. In some embodiments, a device is connected to the filter, such as via a luer lock connection, to transfer the air. In some such embodiments, the device is a syringe, pump, or other infusion device. In some embodiments, the gas is air, and the intake of air through the filter is directly from the surrounding environment. In some embodiments, the filter contains a cap, such as a non-vented cap, that is removable or detachable to control transfer of air into filter as desired.
Hence, in some embodiments, the methods include providing or taking in a liquid input composition and a volume of gas, such as air, into the internal cavity of the chamber. The volume of gas, such as air, that is provided or taken in is a function of the volume of composition containing cells and composition containing viral vector particles that make up the input composition. In some embodiments, the volume of gas is the difference between the total volume of the internal cavity and the liquid volume of the input composition. In some embodiments, the total volume of gas and liquid is no more than 200 mL, such that the volume of gas provided or taken in to the internal cavity is the difference between 200 mL and the liquid volume of the input composition (cells and viral vector particles).
In an exemplary aspect of the provided methods, the method of transduction includes providing to an internal cavity of a closed centrifugal chamber system, in which the internal cavity has a surface area of at least at or about 1×109 μm2 or at least at or about 1×1010 μm2, a composition containing at least or about 50×106 cells in a volume that is no more than 100 mL. In some embodiments, the cell composition contains at least or about 100×106 cells or at least or about 200×106 cells in a volume that is no more no more than 50 mL, 40 mL, 30 mL, 20 mL, 10 mL or 5 mL. In some embodiments, prior to providing the cells to the internal cavity, the composition of cells are diluted or concentrated to a volume of no more than 100 mL, such as no more no more than 50 mL, 40 mL, 30 mL, 20 mL, 10 mL or 5 mL. In addition to the cell composition, the method also includes providing, in some aspects, a composition containing viral vector particles in an amount that is at least 1 IU/cell in a volume so that the total liquid volume, including from the composition containing cells, is less than the maximum volume of the internal cavity of the centrifugal chamber, such as no more than 200 mL, thereby generating the input composition. In some embodiments, the composition containing viral vector particles is provided in an amount that is at least 1.6 IU/cell, 1.8 IU/cell, 2.0 IU/cell, 2.4 IU/cell, 2.8 IU/cell, 3.2 IU/cell or 3.6 IU/cell. In some embodiments, the total liquid volume of the input composition is less than 100 mL, less than 90 mL, less than 80 mL, less than 60 mL, less than 40 mL, less than 20 mL. Optionally, the method also can include providing gas, such as air up to the total volume of the internal cavity, for example, so that the total volume occupied in the internal cavity of the centrifugal chamber is up to or about 200 mL.
In some embodiments, the composition containing cells and composition containing viral vector particles, and optionally air, can be combined or mixed prior to providing the compositions to the cavity. In some embodiments, the composition containing cells and composition containing viral vector particles, and optionally air, are provided separately and combined and mixed in the cavity. In some embodiments, a composition containing cells, a composition containing viral vector particles, and optionally air, can be provided to the internal cavity in any order. In any of such some embodiments, a composition containing cells and viral vector particles is the input composition once combined or mixed together, whether such is combined or mixed inside or outside the centrifugal chamber and/or whether cells and viral vector particles are provided to the centrifugal chamber together or separately, such as simultaneously or sequentially.
In some embodiments, intake of the volume of gas, such as air, occurs prior to the incubation, such as rotation, in the transduction method. In some embodiments, intake of the volume of gas, such as air, occurs during the incubation, such as rotation, in the transduction method.
In some embodiments, the liquid volume of the cells or viral vector particles that make up the input composition, and optionally the volume of air, can be a predetermined volume. The volume can be a volume that is programmed into and/or controlled by circuitry associated with the system.
In some embodiments, intake of the input composition, and optionally gas, such as air, is controlled manually, semi-automatically and/or automatically until a desired or predetermined volume has been taken into the internal cavity of the chamber. In some embodiments, a sensor associated with the system can detect liquid and/or gas flowing to and from the centrifuge chamber, such as via its color, flow rate and/or density, and can communicate with associated circuitry to stop or continue the intake as necessary until intake of such desired or predetermined volume has been achieved. In some aspects, a sensor that is programmed or able only to detect liquid in the system, but not gas (e.g. air), can be made able to permit passage of gas, such as air, into the system without stopping intake. In some such embodiments, a non-clear piece of tubing can be placed in the line near the sensor while intake of gas, such as air, is desired. In some embodiments, intake of gas, such as air, can be controlled manually.
In aspects of the provided methods, the internal cavity of the centrifuge chamber is subjected to high speed rotation. In some embodiments, rotation is effected prior to, simultaneously, subsequently or intermittently with intake of the liquid input composition, and optionally air. In some embodiments, rotation is effected subsequent to intake of the liquid input composition, and optionally air. In some embodiments, rotation is by centrifugation of the centrifugal chamber at a relative centrifugal force at the inner surface of side wall of the internal cavity and/or at a surface layer of the cells of at or about or at least at or about 800 g, 1000 g, 1100 g, 1500, 1600 g, 1800 g, 2000 g, 2200 g, 2500 g, 3000 g, 3500 g or 4000 g. In some embodiments, rotation is by centrifugation at a force that is greater than or about 1100 g, such as by greater than or about 1200 g, greater than or about 1400 g, greater than or about 1600 g, greater than or about 1800 g, greater than or about 2000 g, greater than or about 2400 g, greater than or about 2800 g, greater than or about 3000 g or greater than or about 3200 g.
In some embodiments, the method of transduction includes rotation or centrifugation of the input composition, and optionally air, in the centrifugal chamber for greater than or about 5 minutes, such as greater than or about 10 minutes, greater than or about 15 minutes, greater than or about 20 minutes, greater than or about 30 minutes, greater than or about 45 minutes, greater than or about 60 minutes, greater than or about 90 minutes or greater than or about 120 minutes. In some embodiments, the input composition, and optionally air, is rotated or centrifuged in the centrifugal chamber for greater than 5 minutes, but for no more than 60 minutes, no more than 45 minutes, no more than 30 minutes or no more than 15 minutes.
In some embodiments, the method of transduction includes rotation or centrifugation of the input composition, and optionally air, in the centrifugal chamber for between or between about 10 minutes and 60 minutes, 15 minutes and 60 minutes, 15 minutes and 45 minutes, 30 minutes and 60 minutes or 45 minutes and 60 minutes, each inclusive, and at a force at the internal surface of the side wall of the internal cavity and/or at a surface layer of the cells of at least or greater than or about 1000 g, 1100 g, 1200 g, 1400 g, 1500 g, 1600 g, 1800 g, 2000 g, 2200 g, 2400 g, 2800 g, 3200 g or 3600 g.
In some embodiments, the method includes effecting expression from the internal cavity of the centrifugal chamber an output composition, which is the resulting composition of cells incubated with viral vector particles under conditions that include rotation or centrifugation in the centrifugal chamber in any of the above embodiments as described. In aspects of the method, the output composition includes cells transduced with, or in which transduction has been initiated with, a viral vector. In some embodiments, the expression of the output composition is to an output bag that is operably linked as part of a closed system with the centrifugal chamber. In some embodiments, expression of the output composition is subsequent to the rotation or centrifugation. In some embodiments, expression of the output composition is simultaneous with or partly simultaneous with the rotation or centrifugation, such as in a semi-continuous or continuous process.
In some embodiments, the gas, such as air, in the cavity of the chamber is expelled from the chamber. In some embodiments, the gas, such as air, is expelled to a container that is operably linked as part of the closed system with the centrifugal chamber. In some embodiments, the container is a free or empty container. In some embodiments, the air, such as gas, in the cavity of the chamber is expelled through a filter that is operably connected to the internal cavity of the chamber via a sterile tubing line. In some embodiments, the air is expelled using manual, semi-automatic or automatic processes. In some embodiments, air is expelled from the chamber prior to, simultaneously, intermittently or subsequently with expressing the output composition containing incubated cells and viral vector particles, such as cells in which transduction has been initiated or cells have been transduced with a viral vector, from the cavity of the chamber.
In some embodiments, the transduction and/or other incubation is performed as or as part of a continuous or semi-continuous process. In some embodiments, a continuous process involves the continuous intake of the cells and viral vector particles, e.g., the input composition (either as a single pre-existing composition or by continuously pulling into the same vessel, e.g., cavity, and thereby mixing, its parts), and/or the continuous expression or expulsion of liquid, and optionally expelling of gas (e.g. air), from the vessel, during at least a portion of the incubation, e.g., while centrifuging. In some embodiments, the continuous intake and continuous expression are carried out at least in part simultaneously. In some embodiments, the continuous intake occurs during part of the incubation, e.g., during part of the centrifugation, and the continuous expression occurs during a separate part of the incubation. The two may alternate. Thus, the continuous intake and expression, while carrying out the incubation, can allow for a greater overall volume of sample to be processed, e.g., transduced.
In some embodiments, the incubation is part of a continuous process, the method including, during at least a portion of the incubation, effecting continuous intake of said input composition into the cavity during rotation of the chamber and during a portion of the incubation, effecting continuous expression of liquid and, optionally expelling of gas (e.g. air), from the cavity through the at least one opening during rotation of the chamber.
In some embodiments, the semi-continuous incubation is carried out by alternating between effecting intake of the composition into the cavity, incubation, expression of liquid from the cavity and, optionally expelling of gas (e.g. air) from the cavity, such as to an output container, and then intake of a subsequent (e.g., second, third, etc.) composition containing more cells and other reagents for processing, e.g., viral vector particles, and repeating the process. For example, in some embodiments, the incubation is part of a semi-continuous process, the method including, prior to the incubation, effecting intake of the input composition into the cavity through said at least one opening, and subsequent to the incubation, effecting expression of fluid from the cavity; effecting intake of another input composition comprising cells and the viral vector particles into said internal cavity; and incubating the another input composition in said internal cavity under conditions whereby said cells in said another input composition are transduced with said vector. The process may be continued in an iterative fashion for a number of additional rounds. In this respect, the semi-continuous or continuous methods may permit production of even greater volume and/or number of cells.
In some embodiments, a portion of the transduction incubation is performed in the centrifugal chamber, which is performed under conditions that include rotation or centrifugation.
In some embodiments, the method includes an incubation in which a further portion of the incubation of the cells and viral vector particles is carried out without rotation or centrifugation, which generally is carried out subsequent to the at least portion of the incubation that includes rotation or centrifugation of the chamber. In some such embodiments, the further incubation is effected under conditions to result in integration of the viral vector into a host genome of one or more of the cells. It is within the level of a skilled artisan to assess or determine if the incubation has resulted in integration of viral vector particles into a host genome, and hence to empirically determine the conditions for a further incubation. In some embodiments, integration of a viral vector into a host genome can be assessed by measuring the level of expression of a recombinant protein, such as a heterologous protein, encoded by a nucleic acid contained in the genome of the viral vector particle following incubation. A number of well-known methods for assessing expression level of recombinant molecules may be used, such as detection by affinity-based methods, e.g., immunoaffinity-based methods, e.g., in the context of cell surface proteins, such as by flow cytometry. In some examples, the expression is measured by detection of a transduction marker and/or reporter construct. In some embodiments, nucleic acid encoding a truncated surface protein is included within the vector and used as a marker of expression and/or enhancement thereof.
In some embodiments, the further incubation is carried out in the centrifuge chamber, but without rotation. In some embodiments, the further incubation is carried out outside of the centrifuge chamber. In some embodiments, the further incubation is effected at temperatures greater than room temperature, such as greater than or greater than about 25° C., such as generally greater than or greater than about 32° C., 35° C. or 37° C. In some embodiments, the further incubation is effected at a temperature of at or about 37° C.±2° C., such as at a temperature of at or about 37° C. In some embodiments, the further incubation is for a time between or about between 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours or 12 hours and 24 hours, inclusive.
In some embodiments, the further incubation occurs in a closed system. In some embodiments, after expression of the output composition from the chamber, such as into a container (e.g. bag), the container containing the output composition is incubated for a further portion of time. In some embodiments, the container, such as bag, is incubated at a temperature of at or about 37° C.±2° C. for a time between or about between 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours or 12 hours and 24 hours, inclusive.
In some embodiments, the methods effect transduction of a certain number or percentage of the cells in the input and/or output (transduced) composition, or subset thereof. For example, in some embodiments, at least 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the total cells (or of a particular target cell type, such as T cells) in the input composition and/or in the output (e.g., transduced) composition, are transduced with said viral vector and/or express the recombinant gene product encoded thereby. In some embodiments, the methods of transduction result in an output composition in which at least 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the total cells, such as T cells, in the composition are transduced with the viral vector and/or express the recombinant gene product encoded thereby.
In some embodiments, the methods are capable of achieving such at least a particular transduction efficiency under certain conditions. For example, in some embodiments, where the input composition includes the virus and cells at a ratio of from or from about 1 infectious unit (IU) per one of the cells to 10 IU per one of the cells, such as at or about 1 infectious units (IU) per one of the cells, or at or about 2 IU per one of the cells, at or about 5 IU per one of the cells, or at or about 10 IU per one of the cells, the method is capable of producing a transduced composition in which at least 10%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the cells in said transduced composition generated by the method comprise, e.g., have been transduced with, the recombinant viral vector. Transduction of the cells may be detected by detecting the presence of recombinant nucleic acid, e.g., transgene, included in the vector or product thereof in the cell. In some embodiments, the product is detected on the surface of the cell, indicating the cell has been successfully transduced. In some embodiments, detection of transduction involves detection of a transduction marker, such as another transgene or product included for the purposes of marking transduced cells, and/or other selection marker.
In some embodiments, the output composition resulting from the transduction methods includes a particular average or mean number of copies of the transduced vector per cell (vector copy number (VCN)). VCN may be expressed in terms of the number of copies in a single cell. Alternatively, it may be expressed as an average number over a total cell population or composition, such as the output or transduced composition (including any non-transduced cells within the composition, which would not include any copies of the vector). Alternatively, VCN may be expressed in terms of average copy number only among the transduced cells. In some embodiments, among all the cells in the transduced or output composition produced by the methods, the average VCN is no more than at or about 10, 5, 4, 2.5, 1.5, or 1. In some embodiments, among the cells in the transduced or output composition that contain the recombinant viral vector or express the recombinant gene product, the average VCN is no more than at or about 4, 3, 2, 2.5, 1.5, or 1.
Also provided are compositions produced by any of the above methods. In some embodiments, the compositions contain at least 1×107 cells or 5×107 cells, such as at least 1×108 cells, 2×108 cells, 4×108 cells, 6×108, 8×108 cells or 1×109 cells, in which at least a plurality of cells are transduced with the recombinant viral vector. In some embodiments, the cells are T cells.
In some embodiments, by practice of the methods provided herein, it is possible to produce an output composition containing a plurality of transduced cells in high number, such as, in some aspects, a number that can achieve a therapeutically effective dosage of T cells for use in adoptive immunotherapy. In some embodiments, this can be achieved not only because of the ability to transduce cells on a large scale, but also, in some aspects, by repeating the process in a continuous or semi-continuous manner.
In contrast, existing methods in the art in which transduction is performed on a smaller scale, such as in plates, requires large scale expansion of the cells after transduction in order to achieve numbers of cells necessary to obtain a therapeutically effective dosage. Expansion of cells, such as T cells, with one or more stimulating agents can activate the cells and/or alter the phenotype of the cells, such as by resulting in the generation of effector cells with an exhausted T cell phenotype. For example, activation or stimulation of T cells can result in a change in differentiation or activation state of T cells that may result and/or lead to reduced persistence in vivo when genetically engineered cells are administered to a subject. Among changes in differentiation state that may occur include, in some cases, loss of a naïve phenotype, loss of memory T cell phenotypes, and/or the generation of effector cells with an exhausted T cell phenotype. Exhaustion of T cells may lead to a progressive loss of T cell functions and/or in depletion of the cells (Yi et al. (2010) Immunology, 129:474-481). T cell exhaustion and/or the lack of T cell persistence is a barrier to the efficacy and therapeutic outcomes of adoptive cell therapy; clinical trials have revealed a correlation between greater and/or longer degree of exposure to the antigen receptor (e.g. CAR)-expressing cells and treatment outcomes.
In some embodiments, in the methods provided herein it is not necessary to stimulate and/or activate cells subsequent to transduction to the same extent as is necessary in other known methods in the art. In some embodiments, subsequent to transduction, the cells in the composition are not subject to expansion in the presence of a stimulating agent (e.g. a cytokine, such as IL-2) and/or are not incubated at a temperature greater than or about 30° C. or greater than or about 37° C. for more than 24 hours. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the T cells in the composition and/or transduced T cells in the output composition comprise high surface expression of CD69 or TGF-beta-II. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the T cells or transduced T cells in the composition comprise no surface expression of CD62L and/or comprise high expression of CD25, ICAM, GM-CSF, IL-8 and/or IL-2.
In some embodiments, engineered cells, such as cells transduced with the viral vectors encoding recombinant products to be expressed in the cells, of the output composition produced by the above method, or by a method that includes a further processing step, such as to generate a formulated composition, exhibit increased persistence when administered in vivo to a subject. In some embodiments, the persistence of a provided cells, such as receptor, e.g., CAR, -expressing cells, in the subject upon administration is greater as compared to that which would be achieved by alternative methods of transduction, such as those involving administration of cells genetically engineered by methods involving smaller scale transduction in which T cells are activated and/or stimulated to expand prior to and/or subsequent to transduction to achieve a number of cells that is a therapeutically effective dose. For example, in some aspects, the persistence of provided cells, such as cells produced by the provided methods, is greater as compared to that which would be achieved by administration of a population of genetically engineered recombinant receptor (e.g. CAR)-expressing in which at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% have a lower level of expression of CD69 or TGF-beta II. In some embodiments, the persistence of provided cells, such as cells produced by the provided methods, is greater compared to that which would be achieved by administration of a population of genetically engineered recombinant receptor (e.g. CAR)-expressing in which at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% exhibit surface expression of CD62L and/or comprise low surface expression of CD25, ICAM, GM-CSF, IL-8 and/or IL-2.
In some embodiments, the persistence is increased at least or about at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more.
In some embodiments, the degree or extent of persistence of administered cells can be detected or quantified after administration to a subject. For example, in some aspects, quantitative PCR (qPCR) is used to assess the quantity of cells expressing the recombinant receptor (e.g., CAR-expressing cells) in the blood or serum or organ or tissue (e.g., disease site) of the subject. In some aspects, persistence is quantified as copies of DNA or plasmid encoding the receptor, e.g., CAR, per microgram of DNA, or as the number of receptor-expressing, e.g., CAR-expressing, cells per microliter of the sample, e.g., of blood or serum, or per total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or T cells per microliter of the sample. In some embodiments, flow cytometric assays detecting cells expressing the receptor generally using antibodies specific for the receptors also can be performed. Cell-based assays may also be used to detect the number or percentage of functional cells, such as cells capable of binding to and/or neutralizing and/or inducing responses, e.g., cytotoxic responses, against cells of the disease or condition or expressing the antigen recognized by the receptor. In any of such embodiments, the extent or level of expression of another marker associated with the recombinant receptor (e.g. CAR-expressing cells) can be used to distinguish the administered cells from endogenous cells in a subject.
In some embodiments, by minimizing T cell activation and/or stimulation, the provided embodiments can result in genetically engineered T cells that are more potent for use in adoptive immunotherapy methods, due, in some aspects, to increased persistence. In some embodiments, the increased potency and/or increased persistence of the provided cells, such as cells produced by any of the provided methods, permits methods of administering cells at lower dosages. Such methods can minimize toxicity that can occur from adoptive immunotherapy methods.
Other Cell Processing Events
In some embodiments, in addition to and/or alternatively to the transduction steps, the processing methods of the provided methods include other processing steps and methods, such as for the isolation, separation, selection, cultivation (e.g., stimulation of the cells, for example, to induce their proliferation and/or activation), washing, suspension, dilution, concentration, and/or formulation of the cells. In some embodiments, at least a portion of one or more other processing steps and/or at least a portion of a plurality of the steps are carried out in whole or in part within the cavity of a centrifugal chamber, such as the same or different centrifugal chamber as used in the methods of transduction. In some embodiments, all or a portion of such one or more other processing steps are carried out in the closed system containing a centrifugal chamber, such as in a sterile closed system.
In some embodiments, the methods include one or more of (a) washing a biological sample containing cells (e.g., a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cells (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product) in a cavity of a chamber, (b) isolating, e.g. selecting, from the sample a desired subset or population of cells (e.g., CD4+ or CD8+ T cells) in a cavity of a chamber, for example, by incubation of cells with a selection or immunoaffinity reagent for immunoaffinity-based separation; c) incubating the isolated, such as selected cells, with viral vector particles, such as in accord with methods described above and d) formulating the transduced cells, such as in a pharmaceutically acceptable buffer, cryopreservative or other suitable medium. In some embodiments, the methods can further include (e) stimulating cells in a cavity of a chamber by exposing cells to stimulating conditions, thereby inducing cells to proliferate. In some embodiments, the step of stimulating the cells is performed prior to, during and/or subsequent to the incubation of cells with viral vector particles. In some embodiments, one or more further step of washing or suspending step, such as for dilution, concentration and/or buffer exchange of cells, can also be carried out prior to or subsequent to any of the above steps.
Thus, in some embodiments, the methods carry out one, more, or all steps in the preparation of cells for clinical use, e.g., in adoptive cell therapy, without exposing the cells to non-sterile conditions and without the need to use a sterile room or cabinet. In some embodiments of such a process, the cells are isolated, separated or selected, stimulated, transduced, washed, and formulated, all within a closed system. In some embodiments, the methods are carried out in an automated fashion. In some embodiments, one or more of the steps is carried out apart from the centrifugal chamber system.
Samples
In some embodiments, the processing steps include isolation of cells or compositions thereof from biological samples, such as those obtained from or derived from a subject, such as one having a particular disease or condition or in need of a cell therapy or to which cell therapy will be administered. In some aspects, the subject is a human, such as a subject who is a patient in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
In some aspects, the sample is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
In some embodiments, isolation of the cells or populations includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components. In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples may contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets.
In some embodiments, the provided methods include processing, in whole or in part, one or more of the samples in a closed system, such as in a centrifugal chamber. In some embodiments, the processing step can involve washing of the sample, e.g., blood cell-containing sample, from the subject, e.g., to remove the plasma fraction and/or replacing the cells in an appropriate buffer or media for subsequent processing steps and/or performing a density-based cell separation methods, such as in the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient. Exemplary of such processing steps can be performed using a centrifugal chamber in conjunction with one or more systems associated with a cell processing system, such as a centrifugal chamber produced and sold by Biosafe SA, including those for use with the Sepax® or Sepax 2® cell processing systems.
Affinity-Based Selection
The processing steps (e.g., carried out in the centrifugal chamber) may include isolation of cells from mixed populations and/or compositions, such as using one of various selection steps including density-based or other physical property-based separation methods and affinity-based selection. In some embodiments, the methods include selection in which all or a portion of the selection is carried out in the internal cavity of the centrifugal chamber, for example, under centrifugal rotation. In some embodiments, incubation of cells with selection reagents, such as immunoaffinity-based selection reagents, is performed in a centrifugal chamber. Such methods can offer certain advantages compared to other available selection methods.
For example, immunoaffinity-based selection can depend upon a favorable energetic interaction between the cells being separated and the molecule specifically binding to the marker on the cell, e.g., the antibody or other binding partner on the solid surface, e.g., particle. In certain available methods for affinity-based separation using particles such as beads, particles and cells are incubated in a container, such as a tube or bag, while shaking or mixing, with a constant cell density-to-particle (e.g., bead) ratio to aid in promoting energetically favored interactions. Such approaches may not be ideal for use with large-scale production, for example, in that they may require use of large volumes in order to maintain an optimal or desired cell-to-particle ratio while maintaining the desired number of cells. Accordingly, such approaches can require processing in batch mode or format, which can require increased time, number of steps, and handling, increasing cost and risk of user error.
In some embodiments, by conducting such selection steps or portions thereof (e.g., incubation with antibody-coated particles, e.g., magnetic beads) in the cavity of the centrifugal chamber, the user is able to control certain parameters, such as volume of various solutions, addition of solution during processing and timing thereof, which can provide advantages compared to other available methods. For example, the ability to decrease the liquid volume in the cavity during the incubation can increase the concentration of the particles (e.g. bead reagent) used in the selection, and thus the chemical potential of the solution, without affecting the total number of cells in the cavity. This in turn can enhance the pairwise interactions between the cells being processed and the particles used for selection. In some embodiments, carrying out the incubation step in the chamber, e.g., when associated with the systems, circuitry, and control as described herein, permits the user to effect agitation of the solution at desired time(s) during the incubation, which also can improve the interaction.
In some embodiments, at least a portion of the selection step is performed in a centrifugal chamber, which includes incubation of cells with a selection reagent. In some aspects of such processes, a volume of cells is mixed with an amount of a desired affinity-based selection reagent that is far less than is normally employed when performing similar selections in a tube or container for selection of the same number of cells and/or volume of cells according to manufacturer's instructions. In some embodiments, an amount of selection reagent or reagents that is/are no more than 5%, no more than 10%, no more than 15%, no more than 20%, no more than 25%, no more than 50%, no more than 60%, no more than 70% or no more than 80% of the amount of the same selection reagent(s) employed for selection of cells in a tube or container-based incubation for the same number of cells and/or the same volume of cells according to manufacturer's instructions is employed.
The incubation with a selection reagent or reagents, e.g., as part of selection methods which may be performed in the chamber cavity, include using one or more selection reagents for selection of one or more different cell types based on the expression or presence in or on the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method using a selection reagent or reagents for separation based on such markers may be used. In some embodiments, the selection reagent or reagents result in a separation that is affinity- or immunoaffinity-based separation. For example, the selection in some aspects includes incubation with a reagent or reagents for separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
In some embodiments, for selection, e.g., immunoaffinity-based selection of the cells, the cells are incubated in the cavity of the chamber in a composition that also contains the selection buffer with a selection reagent, such as a molecule that specifically binds to a surface marker on a cell that it desired to enrich and/or deplete, but not on other cells in the composition, such as an antibody, which optionally is coupled to a scaffold such as a polymer or surface, e.g., bead, e.g., magnetic bead, such as magnetic beads coupled to monoclonal antibodies specific for CD4 and CD8. In some embodiments, as described, the selection reagent is added to cells in the cavity of the chamber in an amount that is substantially less than (e.g. is no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the amount) as compared to the amount of the selection reagent that is typically used or would be necessary to achieve about the same or similar efficiency of selection of the same number of cells or the same volume of cells when selection is performed in a tube with shaking or rotation. In some embodiments, the incubation is performed with the addition of a selection buffer to the cells and selection reagent to achieve a target volume with incubation of the reagent of, for example, 10 mL to 200 mL, such as at least or about at least or about or 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL or 200 mL. In some embodiments, the selection buffer and selection reagent are pre-mixed before addition to the cells. In some embodiments, the selection buffer and selection reagent are separately added to the cells. In some embodiments, the selection incubation is carried out with periodic gentle mixing condition, which can aid in promoting energetically favored interactions and thereby permit the use of less overall selection reagent while achieving a high selection efficiency.
In some embodiments, the total duration of the incubation with the selection reagent is from or from about 5 minutes to 6 hours, such as 30 minutes to 3 hours, for example, at least or about at least 30 minutes, 60 minutes, 120 minutes or 180 minutes.
In some embodiments, the incubation generally is carried out under mixing conditions, such as in the presence of spinning, generally at relatively low force or speed, such as speed lower than that used to pellet the cells, such as from or from about 600 rpm to 1700 rpm (e.g. at or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm), such as at an RCF at the sample or wall of the chamber or other container of from or from about 80 g to 100 g (e.g. at or about or at least 80 g, 85 g, 90 g, 95 g, or 100 g). In some embodiments, the spin is carried out using repeated intervals of a spin at such low speed followed by a rest period, such as a spin and/or rest for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, such as a spin at approximately 1 or 2 seconds followed by a rest for approximately 5, 6, 7, or 8 seconds.
In some embodiments, such process is carried out within the entirely closed system to which the chamber is integral. In some embodiments, this process (and in some aspects also one or more additional step, such as a previous wash step washing a sample containing the cells, such as an apheresis sample) is carried out in an automated fashion, such that the cells, reagent, and other components are drawn into and pushed out of the chamber at appropriate times and centrifugation effected, so as to complete the wash and binding step in a single closed system using an automated program.
In some embodiments, after the incubation and/or mixing of the cells and selection reagent and/or reagents, the incubated cells are subjected to a separation to select for cells based on the presence or absence of the particular reagent or reagents. In some embodiments, the further selection is performed outside of the centrifugal chamber. In some embodiments, the separation is performed in the same closed system in which the centrifugal chamber is present and in which the incubation of cells with the selection reagent was performed. In some embodiments, after incubation with the selection reagents, incubated cells, including cells in which the selection reagent has bound, are expressed from the centrifugal chamber, such as transferred out of the centrifugal chamber, into a system for immunoaffinity-based separation of the cells. In some embodiments, the system for immunoaffinity-based separation is or contains a magnetic separation column. In some embodiments, prior to separation, one or more other processing steps can be performed in the chamber, such as washing.
Such separation steps can be based on positive selection, in which the cells having bound the reagents, e.g. antibody or binding partner, are retained for further use, and/or negative selection, in which the cells having not bound to the reagent, e.g., antibody or binding partner, are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types. In any of such examples, at least a portion of the further selection or selection steps is performed in a centrifugal chamber, which includes incubation of cells with a selection reagent, as described above.
For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some embodiments, such cells are selected by incubation with one or more antibody or binding partner that specifically binds to such markers. In some embodiments, the antibody or binding partner can be conjugated, such as directly or indirectly, to a solid support or matrix to effect selection, such as a magnetic bead or paramagnetic bead. For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander, and/or ExpACT® beads).
In some embodiments, the process steps further include negative and/or positive selection of the incubated and cells, such as using a system or apparatus that can perform an affinity-based selection. In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (markerhigh) on the positively or negatively selected cells, respectively.
In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.
In embodiments, memory T cells are present in both CD62L+ and CD62L− subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L−CD8+ and/or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.
In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.
CD4+ T helper cells may be sorted into naïve, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+, or CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L− and CD45RO−.
In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher© Humana Press Inc., Totowa, N.J.).
In some aspects, the incubated sample or composition of cells to be separated is incubated with a selection reagent containing small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS® beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.
In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. Many well-known magnetically responsive materials for use in magnetic separation methods are known, e.g., those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 also may be used.
The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody—or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.
In some aspects, separation is achieved in a procedure in which the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.
In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, Calif.). Magnetic Activated Cell Sorting (MACS), e.g., CliniMACS systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.
In some embodiments, the processing steps include expression from the centrifugal chamber of cells incubated with one or more selection reagents. In some embodiments, the cells can be expressed subsequent to and/or continuous with one or more washing steps, which can, in some aspects, be performed in the centrifugal chamber.
In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.
Freezing and Cryopreservation
In some embodiments, the cells, such as selected cells, are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively.
In some embodiments, the cells, such as selected cells, can be transferred to cryopreservation media using a centrifugal chamber in conjunction with one or more systems associated with a cell processing system, such as a centrifugal chamber produced and sold by Biosafe SA, including those for use with the Sepax® or Sepax 2® cell processing systems. In some embodiments, transfer to cryopreservation medium is associated with one or more processing steps that can involve washing of the sample, e.g., selected cell sample, such as to remove the selection media and/or replacing the cells in an appropriate cryopreservation buffer or media for subsequent freezing.
In some embodiments, the cells are frozen, e.g., cryopreserved, either before, during, or after said methods for processing. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. The cells are generally then frozen to −80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.
Cultivation and Stimulation
In some embodiments, the processing steps (e.g., those carried out in the chamber and/or closed system) include cultivation, stimulation and/or activation of cells, such as by incubation and/or culture of cells. For example, in some embodiments, provided are methods for stimulating the isolated cells, such as selected cell populations. In some embodiments, the processing steps include incubation of a composition containing the cells, such as selected cells, where at least a portion of the incubation is in a centrifugal chamber and/or other vessel, e.g., under stimulating conditions. The incubation may be prior to or in connection with genetic engineering, such as genetic engineering resulting from embodiments of the transduction method described above. In some embodiments, the stimulation results in activation and/or proliferation of the cells, for example, prior to transduction.
In some embodiments, the processing steps include incubations of cells, such as selected cells, in which the incubation steps can include culture, cultivation, stimulation, activation, and/or propagation of cells. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
In some embodiments, the conditions for stimulation and/or activation can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell, such as agents suitable to deliver a primary signal, e.g., to initiate activation of an ITAM-induced signal, such as those specific for a TCR component, and/or an agent that promotes a costimulatory signal, such as one specific for a T cell costimulatory receptor, e.g., anti-CD3, anti-CD28, or anti-41-BB, for example, bound to solid support such as a bead, and/or one or more cytokines. Among the stimulating agents are anti-CD3/anti-CD28 beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander, and/or ExpACT® beads). Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium. In some embodiments, the stimulating agents include IL-2 and/or IL-15, for example, an IL-2 concentration of at least about 10 units/mL. In some embodiments, incubation is carried out in accordance with techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, the T cells are expanded by adding to the composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.
In some embodiments, the stimulating conditions generally include a temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.
In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigen-specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.
In some embodiments, at least a portion of the incubation with one or more stimulating conditions or stimulatory agents, such as any described above, is performed in a centrifugal chamber. In some embodiments, at least a portion of the incubation performed in a centrifugal chamber includes mixing with a reagent or reagents to induce stimulation and/or activation. In some embodiments, cells, such as selected cells, are mixed with a stimulating condition or stimulatory agent in the centrifugal chamber. In some aspects of such processes, a volume of cells is mixed with an amount of one or more stimulating conditions or agents that is far less than is normally employed when performing similar stimulations in a cell culture plate or other system.
In some embodiments, the stimulating agent is added to cells in the cavity of the chamber in an amount that is substantially less than (e.g. is no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the amount) as compared to the amount of the stimulating agent that is typically used or would be necessary to achieve about the same or similar efficiency of selection of the same number of cells or the same volume of cells when selection is performed without mixing in a centrifugal chamber, e.g. in a tube or bag with periodic shaking or rotation. In some embodiments, the incubation is performed with the addition of an incubation buffer to the cells and stimulating agent to achieve a target volume with incubation of the reagent of, for example, 10 mL to 200 mL, such as at least or about at least or about or 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL or 200 mL. In some embodiments, the incubation buffer and stimulating agent are pre-mixed before addition to the cells. In some embodiments, the incubation buffer and stimulating agent are separately added to the cells. In some embodiments, the stimulating incubation is carried out with periodic gentle mixing condition, which can aid in promoting energetically favored interactions and thereby permit the use of less overall stimulating agent while achieving stimulating and activation of cells.
In some embodiments, the total duration of the incubation with the stimulating agent is from or from about 1 hour and 72 hours, 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours or 12 hours and 24 hours, such as at least or about at least 6 hours, 12 hours, 18 hours, 24 hours, 36 hours or 72 hours. In some cases, the total duration of the incubation in the centrifugal chamber is from or from about 5 minutes to 6 hours, such as 30 minutes to 3 hours, for example, at least or about at least 30 minutes, 60 minutes, 120 minutes or 180 minutes. In some cases, a further portion of the incubation can be performed outside of the centrifugal chamber.
In some embodiments, the incubation generally is carried out under mixing conditions, such as in the presence of spinning, generally at relatively low force or speed, such as speed lower than that used to pellet the cells, such as from or from about 600 rpm to 1700 rpm (e.g. at or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm), such as at an RCF at the sample or wall of the chamber or other container of from or from about 80 g to 100 g (e.g. at or about or at least 80 g, 85 g, 90 g, 95 g, or 100 g). In some embodiments, the spin is carried out using repeated intervals of a spin at such low speed followed by a rest period, such as a spin and/or rest for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, such as a spin at approximately 1 or 2 seconds followed by a rest for approximately 5, 6, 7, or 8 seconds.
In some embodiments, cells are incubated in a centrifugal chamber with a cell stimulating agent or agents that is/are a cell-binding agent, such as an antigen-binding reagent, such as antibody, that is able to induce intracellular signaling and/or cell proliferation. In some embodiments, cells are incubated with, including mixed with, anti-CD3/anti-CD28 beads in a centrifugal chamber according to aspects of processes in the provided methods.
In some embodiments, the processing steps include expression from the centrifugal chamber of cells incubated, such as mixed with, one or more stimulatory conditions or stimulating agents. In some embodiments, one or more other additional processing steps can be performed in the chamber, such as washing, which can be prior to, subsequent to and/or continuous with the stimulating incubation. In some embodiments, washing is performed prior to stimulation, such as on selected or thawed cells, to remove and replace media with a medium suitable for stimulation and cultivation of cells.
In some embodiments, expressed cells from the centrifugal chamber that have been incubated, such as mixed with, one or more stimulatory conditions or stimulating agents, are further incubated outside of the chamber. In some embodiments, the further incubation is effected at temperatures greater than room temperature, such as greater than or greater than about 25° C., such as generally greater than or greater than about 32° C., 35° C. or 37° C. In some embodiments, the further incubation is effected at a temperature of at or about 37° C.±2° C., such as at a temperature of at or about 37° C. In some embodiments, the further incubation is for a time between or about between 12 hours and 96 hours, such as at least or at least about 12 hours, 24 hours, 36 hours, 48 hours, 72 hours or 96 hours.
In some embodiments, the further incubation occurs in a closed system. In some embodiments, after expression from the chamber of the cells incubated, such as mixed, with one or stimulatory conditions or stimulating agents, such as into a container (e.g. bag), the container containing the cells is incubated for a further portion of time. In some embodiments, the container, such as bag, is incubated at a temperature of at or about 37° C.±2° C. for a time between or about between 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours or 12 hours and 24 hours, inclusive.
Formulation
In some embodiments, the process steps (e.g. carried out in the centrifugal chamber and/or closed system) may include formulation of cells, such as formulation of genetically engineered cells resulting from the provided transduction processing steps and/or one or more other processing steps as described. In some embodiments, the provided methods associated with formulation of cells include processing transduced cells, such as cells transduced and/or expanded using the processing steps described above, in a closed system, such as in or associated with a centrifugal chamber.
In some embodiments, the cells are formulated in a pharmaceutically acceptable buffer, which may, in some aspects, include a pharmaceutically acceptable carrier or excipient. In some embodiments, the processing includes exchange of a medium into a medium or formulation buffer that is pharmaceutically acceptable or desired for administration to a subject. In some embodiments, the processing steps can involve washing the transduced and/or expanded cells to replace the cells in a pharmaceutically acceptable buffer that can include one or more optional pharmaceutically acceptable carriers or excipients. Exemplary of such pharmaceutical forms, including pharmaceutically acceptable carriers or excipients, can be any described below in conjunction with forms acceptable for administering the cells and compositions to a subject. The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount.
In some embodiments, the formulation buffer contains a cryopreservative. In some embodiments, the cell are formulated with a cyropreservative solution that contains 1.0% to 30% DMSO solution, such as a 5% to 20% DMSO solution or a 5% to 10% DMSO solution. In some embodiments, the cryopreservation solution is or contains, for example, PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. In some embodiments, the cryopreservative solution is or contains, for example, at least or about 7.5% DMSO. In some embodiments, the processing steps can involve washing the transduced and/or expanded cells to replace the cells in a cryopreservative solution.
In some embodiments, the processing can include dilution or concentration of the cells to a desired concentration or number, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof. In some embodiments, the processing steps can include a volume-reduction to thereby increase the concentration of cells as desired. In some embodiments, the processing steps can include a volume-addition to thereby decrease the concentration of cells as desired.
In some embodiments, the processing includes adding a volume of a formulation buffer to transduced and/or expanded cells. In some embodiments, the volume of formulation buffer is from or from about 10 mL to 1000 mL, such as at least or about at least or about or 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL or 1000 mL.
Exemplary of such processing steps can be performed using a centrifugal chamber in conjunction with one or more systems or kits associated with a cell processing system, such as a centrifugal chamber produced and sold by Biosafe SA, including those for use with the Sepax® or Sepax 2® cell processing systems.
In some embodiments, the method includes effecting expression from the internal cavity of the centrifugal chamber a formulated composition, which is the resulting composition of cells formulated in a formulation buffer, such as pharmaceutically acceptable buffer, in any of the above embodiments as described. In some embodiments, the expression of the formulated composition is to a container, such as a bag that is operably linked as part of a closed system with the centrifugal chamber. In some embodiments, the container, such as bag, is connected to a system at an output line or output position as exemplified in the exemplary systems depicted in FIG. 5 or FIG. 7.
In some embodiments, the closed system, such as associated with a cell processing system, such as centrifugal chamber, includes a multi-port output kit containing a multi-way tubing manifold associated at each end of a tubing line with a port to which one or a plurality of containers can be connected for expression of the formulated composition. In some aspects, a desired number or plurality of output containers, e.g., bags, can be sterilely connected to one or more, generally two or more, such as at least 3, 4, 5, 6, 7, 8 or more of the ports of the multi-port output. For example, in some embodiments, one or more containers, e.g., bags can be attached to the ports, or to fewer than all of the ports. Thus, in some embodiments, the system can effect expression of the output composition into a plurality of output bags.
In some aspects, cells can be expressed to the one or more of the plurality of output bags in an amount for dosage administration, such as for a single unit dosage administration or multiple dosage administration. For example, in some embodiments, the output bags may each contain the number of cells for administration in a given dose or fraction thereof. Thus, each bag, in some aspects, may contain a single unit dose for administration or may contain a fraction of a desired dose such that more than one of the plurality of output bags, such as two of the output bags, or 3 of the output bags, together constitute a dose for administration.
Thus, the containers, e.g., bags, generally contain the cells to be administered, e.g., one or more unit doses thereof. The unit dose may be an amount or number of the cells to be administered to the subject or twice the number (or more) of the cells to be administered. It may be the lowest dose or lowest possible dose of the cells that would be administered to the subject.
In some embodiments, each of the containers, e.g., bags, individually comprises a unit dose of the cells. Thus in some embodiments, each of the containers comprises the same or approximately or substantially the same number of cells. In some embodiments, the unit dose includes less than about 1×108, less than about 5×107, less than about 1×106 or less than about 5×105 of cells, per kg of the subject to be treated and/or from which the cells have been derived. In some embodiments, each unit dose contains at least or about at least 1×106, 2×106, 5×106, 1×107, 5×107, or 1×108 engineered cells, total cells, T cells, or PBMCs. In some embodiments, the volume of the formulated cell composition in each bag is 10 mL to 100 mL, such as at least or about at least 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL or 100 mL.
In some embodiments, one or more of the plurality of output bags can be used for testing, such as for assessing transduction efficiency. For example, the transduction efficiency in some aspects may be assessed by measuring the level of expression of a recombinant protein, such as a heterologous protein, encoded by a nucleic acid contained in the genome of the viral vector particle following transduction using embodiments of the provided methods. Thus, in some embodiments, the expression level of recombinant molecules may be assessed by any of a number of well-known methods such as detection by affinity-based methods, e.g., immunoaffinity-based methods, e.g., in the context of cell surface proteins, such as by flow cytometry. In some aspects, the cells contained in one or more of the plurality of containers, e.g., bags, is tested for the expression level of recombinant molecules by detection of a transduction marker and/or reporter construct. In other embodiments, expression is assessed using a nucleic acid encoding a truncated surface protein included within the vector as a marker.
In some embodiments, all or substantially all of a plurality of containers to which cells are expressed contain the same number of cells and in the same or substantially the same concentration. In some embodiments, prior to expressing cells into one of a plurality of containers, the tubing lines are primed.
IV. Cells and Compositions
Among the cells to be used in the methods, such as the processing steps, e.g., the transfer of viral nucleic acids, e.g., transduction, are cells, cell populations, and compositions.
The cells generally are mammalian cells, and typically are human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs. In some aspects, the cells are cells of the immune system, such as cells of innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, and re-introducing them into the same subject, before or after cryopreservation, which, in some aspects, can be achieved in a closed system using one or more of the provided processing steps.
Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naïve T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
V. Viral Vector Particles, Viral Vectors, and Encoded Recombinant Products
The transduction methods generally involve transduction with viral vectors, such as those encoding recombinant products to be expressed in the cells. The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Vectors include viral vectors, such as retroviral vectors, for example lentiviral or gammaretroviral vectors, having a genome carrying another nucleic acid and capable of inserting into a host genome for propagation thereof.
In some embodiments, a viral vector is transferred to a cell in a vehicle that is a viral vector particle, which includes a virion that encapsulates and/or packages a viral vector genome. In some such embodiments, the genome of the viral vector typically includes sequences in addition to the nucleic acid encoding the recombinant molecule that allow the genome to be packaged into the virus particle.
In some embodiments, the viral vector contains a recombinant nucleic acid, such as a nucleic acid encoding a recombinant and/or heterologous molecule, such as a recombinant or heterologous protein. In some embodiments, such as in aspects of the provided methods, transduction with the viral vectors produces an output composition, cells of which have been transduced and express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cells being transduced and/or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.
In some embodiments, recombinant nucleic acids are transferred into cells using recombinant virus or viral vector particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into cells, such as T cells, using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557.
In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
The viral vectors generally include recombinant nucleic acids such as transgenes encoding recombinant products to be expressed by the cells. Recombinant products include recombinant receptors, including antigen receptors such as functional non-TCR antigen receptors, e.g., chimeric antigen receptors (CARs), and other antigen-binding receptors such as transgenic T cell receptors (TCRs). Also among the receptors are other chimeric receptors.
Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in international patent application publication numbers WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the antigen receptors include a CAR as described in U.S. Pat. No. 7,446,190, and those described in International Patent Application Publication No.: WO/2014055668 A1. Examples of the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190, 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190, and 8,389,282. The chimeric receptors, such as CARs, generally include an extracellular antigen binding domain, such as a portion of an antibody molecule, generally a variable heavy (VH) chain region and/or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment.
In some embodiments, the antibody portion of the recombinant receptor, e.g., CAR, further includes at least a portion of an immunoglobulin constant region, such as a hinge region, e.g., an IgG4 hinge region, and/or a CH1/CL and/or Fc region. In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgG1. In some aspects, the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. Exemplary spacers, e.g., hinge regions, include those described in international patent application publication number WO2014031687. In some examples, the spacer is or is about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain.
This antigen recognition domain generally is linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and/or signal via another cell surface receptor. Thus, in some embodiments, the antigen-binding component (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD5, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and/or transmembrane domain(s).
Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
The receptor, e.g., the CAR, generally includes at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the antigen-binding portion is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR or other chimeric receptor includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25 or CD16.
In some embodiments, upon ligation of the CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptors to initiate signal transduction following antigen receptor engagement.
In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.
T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.
In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.
In some embodiments, the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR includes both the activating and costimulatory components.
In some embodiments, the activating domain is included within one CAR, whereas the costimulatory component is provided by another CAR recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see WO2014/055668). In some aspects, the cells include one or more stimulatory or activating CAR and/or a costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than the one associated with and/or specific for the disease or condition whereby an activating signal delivered through the disease-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.
In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.
In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.
In some embodiments, the CAR or other antigen receptor further includes a marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor, such as a truncated version of a cell surface receptor, such as truncated EGFR (tEGFR). In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. See WO2014031687.
In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.
In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self” by the immune system of the host into which the cells will be adoptively transferred.
In some embodiments, the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive transfer and encounter with ligand.
In some cases, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR is one that includes multiple costimulatory domains of different costimulatory receptors.
In some embodiments, the chimeric antigen receptor includes an extracellular portion containing an antibody or antibody fragment. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment includes an scFv and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain includes a signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some aspects, the transmembrane domain contains a transmembrane portion of CD28. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.
The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided receptors and other polypeptides, e.g., linkers or peptides, may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
The recombinant receptors, such as CARs, expressed by the cells administered to the subject generally recognize or specifically bind to a molecule that is expressed in, associated with, and/or specific for the disease or condition or cells thereof being treated. Upon specific binding to the molecule, e.g., antigen, the receptor generally delivers an immunostimulatory signal, such as an ITAM-transduced signal, into the cell, thereby promoting an immune response targeted to the disease or condition. For example, in some embodiments, the cells express a CAR that specifically binds to an antigen expressed by a cell or tissue of the disease or condition or associated with the disease or condition.
In some contexts, overexpression of a stimulatory factor (for example, a lymphokine or a cytokine) may be toxic to a subject. Thus, in some contexts, the viral vector introduces into the cell gene segments that cause the cells to be susceptible to negative selection in vivo, such as upon administration in adoptive immunotherapy. For example, in some aspects, following transduction of the cells with such gene segments, the cells are eliminated as a result of a change in the in vivo condition of the subject to which they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell II:223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).
Among additional nucleic acids that may be included in the viral vector for transduction and expression in the cells are those encoding products that improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization, and/or improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.
VI. Therapeutic Methods and Compositions
In some aspects, the products of the methods are used in methods of treatment, e.g., therapeutic methods, such as for administrating the cells and compositions to subjects in adoptive cell therapy. Also provided are such methods and uses of cells processed and produced by the methods, and pharmaceutical compositions and formulations for use therein. The provided methods generally involve administering the cells or compositions, e.g., output composition and/or formulated compositions, to subjects.
In some embodiments, the cells express recombinant receptors, such as CARs, or other antigen receptors, such as transgenic TCRs, e.g., those transferred in the transduction methods provided herein. Such cells generally are administered to subjects having a disease or condition specifically recognized by the receptor. In one embodiment, the cells express a recombinant receptor or a chimeric receptor, such as an antigen receptor, e.g. a CAR or a TCR, that specifically binds to a ligand associated with the disease or condition or expressed by a cell or tissue thereof. For example, in some embodiments, the receptor is an antigen receptor and the ligand is an antigen specific for and/or associated with the disease or condition. The administration generally effects an improvement in one or more symptoms of the disease or condition and/or treats or prevents the disease or condition or a symptom thereof. Among the diseases, conditions, and disorders are tumors, including solid tumors, hematologic malignancies, and melanomas, and including localized and metastatic tumors, infectious diseases, such as infection with a virus or other pathogen, e.g., HIV, HCV, HBV, CMV, and parasitic disease, and autoimmune and inflammatory diseases. In some embodiments, the disease or condition is a tumor, cancer, malignancy, neoplasm, or other proliferative disease or disorder. Such diseases include but are not limited to leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), ALL, non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B cell lymphoma, B cell malignancies, cancers of the colon, lung, liver, breast, prostate, ovarian, skin, melanoma, bone, and brain cancer, ovarian cancer, epithelial cancers, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin lymphoma, cervical carcinoma, colorectal cancer, glioblastoma, neuroblastoma, Ewing sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and/or mesothelioma.
In some embodiments, the disease or condition is an infectious disease or condition, such as, but not limited to, viral, retroviral, bacterial, and protozoal infections, immunodeficiency, Cytomegalovirus (CMV), Epstein-Ban virus (EBV), adenovirus, BK polyomavirus. In some embodiments, the disease or condition is an autoimmune or inflammatory disease or condition, such as arthritis, e.g., rheumatoid arthritis (RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Grave's disease, Crohn's disease, multiple sclerosis, asthma, and/or a disease or condition associated with transplant.
In some embodiments, antigen associated with the disease or disorder that is targeted by the cells or compositions is selected from the group consisting of orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, 0EPHa2, ErbB2, 3, or 4, FBP, fetal acethycholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1), a cyclin, such as cyclin A1 (CCNA1), and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens.
In some embodiments, the cells or compositions are administered in an amount that is effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Thus, in some embodiments, the methods of administration include administration of the cells and compositions at effective amounts. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined.
As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.
As used herein, “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.
“Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided cells and compositions are used to delay development of a disease or to slow the progression of a disease.
As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, cells that suppress tumor growth reduce the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the cells.
An “effective amount” of an agent, e.g., a pharmaceutical formulation, cells, or composition, in the context of administration, refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.
A “therapeutically effective amount” of an agent, e.g., a pharmaceutical formulation or cells, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered.
A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.
In some embodiments, cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, and following isolation and processing the cells are administered to the same subject.
In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.
The cells can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.
For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.
In some embodiments, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents includes a cytokine, such as IL-2, for example, to enhance persistence.
Once the cells are administered to the subject (e.g., human), the biological activity of the cell populations in some aspects is measured by any of a number of known methods. Parameters to assess include specific binding of the cells to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells also can be measured by assaying expression and/or secretion of certain cytokines, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load. In some aspects, toxic outcomes, persistence and/or expansion of the cells, and/or presence or absence of a host immune response, are assessed.
In certain embodiments, the cells are modified in any number of ways, such that their therapeutic or prophylactic efficacy is increased. For example, the engineered CAR or TCR expressed by the population can be conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating compounds, e.g., the CAR or TCR, to targeting moieties is known in the art. See, for instance, Wadwa et al., J. Drug Targeting 3: 111 (1995), and U.S. Pat. No. 5,087,616.
Also provided are pharmaceutical compositions or formulations for use in such methods, which in some embodiments are formulated in connection with the provided processing methods, such as in the closed system in which other processing steps are carried out, such as in an automated or partially automated fashion.
In some embodiments, the cells and compositions are administered to a subject in the form of a pharmaceutical composition or formulation, such as a composition comprising the cells or cell populations and a pharmaceutically acceptable carrier or excipient.
The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
The pharmaceutical compositions in some embodiments additionally comprise other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the agents are administered in the form of a salt, e.g., a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p-toluenesulphonic acid.
A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
In some aspects, the choice of carrier is determined in part by the particular cell and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).
Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.
The cells and compositions may be administered using standard administration techniques, formulations, and/or devices. Administration of the cells can be autologous or heterologous. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyoi (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.
Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
Among the processing steps may include formulating such compositions.
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include “consisting” and/or “consisting essentially of” aspects and variations.
Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be constcued as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.
The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.
As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
As used herein, a statement that a cell or population of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.
As used herein, a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.
VII. Exemplary Embodiments
Among the embodiments provided herein are:
1. A transduction method, comprising incubating, in an internal cavity of a centrifugal chamber, an input composition comprising cells and viral particles containing a recombinant viral vector, wherein
said centrifugal chamber is rotatable around an axis of rotation and comprises an end wall, a substantially rigid side wall extending from said end wall, and at least one opening, at least a portion of said side wall surrounding said internal cavity and said at least one opening being capable of permitting intake of liquid into said internal cavity and expression of liquid from said cavity;
the centrifugal chamber is rotating around said axis of rotation during at least a portion of the incubation; and
the method generates an output composition comprising a plurality of the cells transduced with the viral vector.
2. A transduction method, comprising incubating, in an internal cavity of a centrifugal chamber, an input composition comprising cells and a viral particle containing a recombinant viral vector,
said centrifugal chamber being rotatable around an axis of rotation and comprising an end wall, a substantially rigid side wall extending from said end wall, and at least one opening, wherein at least a portion of said side wall surrounds said internal cavity and said at least one opening is capable of permitting intake of liquid into said internal cavity and expression of liquid from said cavity, wherein:
the centrifugal chamber is rotating around the axis of rotation during at least a portion of the incubation;
the total liquid volume of said input composition present in said cavity during rotation of said centrifugal chamber is no more than about 5 mL per square inch of the internal surface area of the cavity; and
the method generates an output composition comprising a plurality of the cells transduced with the viral vector.
3. The method of embodiment 1 or embodiment 2, wherein said rotating comprises rotation at a relative centrifugal force (RCF) at an internal surface of the side wall of the cavity and/or at a surface layer of the cells of greater than at or about 200 g, greater than at or about 300 g, or greater than at or about 500 g.
4. The method of any of embodiments 1-3, wherein said rotating comprises rotation at a relative centrifugal force at an internal surface of the side wall of the cavity and/or at a surface layer of the cells that is:
at or about 600 g, 800 g, 1000 g, 1100 g, 1600 g, 2000 g, 2100 g, 2200 g, 2500 g or 3000 g; or
at least at or about 600 g, 800 g, 1000 g, 1100 g, 1600 g, 2000 g, 2100 g, 2200 g, 2500 g or 3000 g.
5. The method of any of embodiments 1-4, wherein said rotating comprises rotation at a relative centrifugal force at an internal surface of the side wall of the cavity and/or at a surface layer of the cells that is between or between about 500 g and 2500 g, 500 g and 2000 g 500 g and 1600 g, 500 g an 1000 g, 600 g and 1600 g, 600 g and 1000 g, 1000 g and 2000 g or 1000 g and 1600 g, each inclusive.
6. The method of any of embodiments 1-5, wherein the at least a portion of the incubation during which the chamber is rotating is for a time that is:
greater than or about 5 minutes, greater than or about 10 minutes, greater than or about 15 minutes, greater than or about 20 minutes, greater than or about 30 minutes, greater than or about 45 minutes, greater than or about 60 minutes, greater than or about 90 minutes or greater than or about 120 minutes; or
between or between about 5 minutes and 60 minutes, 10 minutes and 60 minutes, 15 minutes and 60 minutes, 15 minutes and 45 minutes, 30 minutes and 60 minutes or 45 minutes and 60 minutes, each inclusive.
7. The transduction method of any of embodiments 1-6, wherein said centrifugal chamber further comprises a movable member and said internal cavity is a cavity of variable volume defined by said end wall, said substantially rigid side wall, and said movable member, said movable member being capable of moving within the chamber to vary the internal volume of the cavity.
8. The method of any of embodiments 1-7, wherein said side wall is curvilinear.
9. The method of embodiment 8, wherein said side wall is generally cylindrical.
10. The method of any of embodiments 7-9, wherein:
the movable member is a piston; and/or
the movable member is capable of axially moving within the chamber to vary the internal volume of the cavity.
11. The method of any of embodiments 1-10, wherein
said at least one opening comprises an inlet and an outlet, respectively capable of permitting said intake and expression; or
said at least one opening comprises a single inlet/outlet, capable of permitting said intake and said expression.
12. The method of any of embodiments 1-11, wherein said at least one opening is coaxial with the chamber and is located in the end wall.
13. The method of any of embodiments 1-12, wherein:
the internal surface area of said cavity is at least at or about 1×109 μm2;
the internal surface area of said cavity is at least at or about 1×1010 μm2;
the length of said rigid wall in the direction extending from said end wall is at least about 5 cm;
the length of said rigid wall in the direction extending from said end wall is at least about 8 cm; and/or
the cavity comprises a radius of at least about 2 cm at least one cross-section.
14. The method of any of embodiments 1-13, wherein:
the average liquid volume of said input composition present in said cavity during said incubation is no more than about 5 milliliters (mL) per square inch of the internal surface area of the cavity during said incubation;
the maximum liquid volume of said input composition present in said cavity at any one time during said incubation is no more than about 5 mL per square inch of the maximum internal surface area of the cavity;
the average liquid volume of said input composition present in said cavity during said incubation is no more than about 2.5 milliliters (mL) per square inch of the internal surface area of the cavity during said incubation; or
the maximum liquid volume of said input composition present in said cavity at any one time during said incubation is no more than about 2.5 mL per square inch of the maximum internal surface area of the cavity.
15. The method of any of embodiments 1-14, wherein the liquid volume of said input composition present in said cavity during said rotation is between or between about 0.5 mL per square inch of the internal surface area of the cavity (mL/sq.in) and 5 mL/sq.in, 0.5 mL/sq.in. and 2.5 mL/sq.in., 0.5 mL/sq.in. and 1 mL/sq.in., 1 mL/sq.in. and 5 mL/sq.in., 1 mL/sq.in. and 2.5 mL/sq.in. or 2.5 mL/sq.in. and 5 mL/sq.in.
16. The method of any of embodiments 1-15, wherein:
the number of said cells in said input composition is at or about the number of said cells sufficient to form a monolayer on the surface of said cavity during rotation of said centrifugal chamber at a force of at or about 1000 g or at or about 2000 g at an internal surface of the side wall and/or at a surface layer of the cells; and/or
the number of said cells in said input composition is no more than 1.5 times or 2 times the number of said cells sufficient to form a monolayer on the surface of said cavity during rotation of said centrifugal chamber at a force of at or about 1000 g or at or about 2000 g at an internal surface of the side wall and/or at a surface layer of the cells.
17. The method of any of embodiments 1-16, wherein
said input composition in the cavity comprises at least at or about 1×106 of said cells; or
said input composition in the cavity comprises at least at or about 5×106 of said cells; or
said input composition in the cavity comprises at least at or about 1×107 of said cells; or
said input composition in the cavity comprises at least at or about 1×108 of said cells.
18. The method of any of embodiments 1-17, wherein said input composition in the cavity comprises at least at or about 1×107 of said cells, at least at or about 2×107 of said cells, at least at or about 3×107 of said cells, at least at or about 4×107 of said cells, at least at or about 5×107 of said cells, at least at or about 6×107 of said cells, at least at or about 7×107 of said cells, at least at or about 8×107 of said cells, at least at or about 9×107 of said cells, at least at or about 1×108 of said cells, at least at or about 2×108 of said cells, at least at or about 3×108 of said cells or at least at or about 4×108 of said cells.
19. The method of any of embodiments 1-18, wherein:
said input composition comprises at least at or about 1 infectious unit (IU) of viral particles per one of said cells, at least at or about 2 IU per one of said cells, at least at or about 3 IU per one of said cells, at least at or about 4 IU per one of said cells, at least at or about 5 IU per one of said cells, at least at or about 10 IU per one of said cells, at least at or about 20 IU per one of said cells, at least at or about 30 IU per one of said cells, at least at or about 40 IU per one of said cells, at least at or about 50 IU per one of said cells, or at least at or about 60 IU per one of said cells; or
said input composition comprises at or about 1 infectious unit (IU) of viral particles per one of said cells, at or about 2 IU per one of said cells, at or about 3 IU per one of said cells, at or about 4 IU per one of said cells, at or about 5 IU per one of said cells, at or about 10 IU per one of said cells, at or about 20 IU per one of said cells, at or about 30 IU per one of said cells, at or about 40 IU per one of said cells, at or about 50 IU per one of said cells, or at or about 60 IU per one of said cells.
20. The method of any of embodiments 1-19, wherein:
the maximum total liquid volume of said input composition present in said cavity at any one time during said incubation is no more than 2 times, no more than 10 times, or no more than 100 times, the total volume of said cells in said cavity or the average volume of the input composition over the course of the incubation is no more than 2, 10, or 100 times the total volume of cells in the cavity.
21. The method of any of embodiments 1-20, wherein the maximum volume of said input composition present in said cavity at any one time during said incubation or the average volume over the course of the incubation is no more than at or about 2 times, 10 times, 25 times, 50 times, 100 times, 500 times, or 1000 times the volume of a monolayer of said cells formed on the inner surface of said cavity during rotation of said chamber at a force of at or about 1000 g or at or about 2000 g at an internal surface of the side wall and/or at a surface layer of the cells.
22. The method of any of embodiments 1-21, wherein the liquid volume of the input composition is no more than 20 mL, no more than 40 mL, no more than 50 mL, no more than 70 mL, no more than 100 mL, no more than 120 mL, no more than 150 mL or no more than 200 mL.
23. The method of any of embodiments 1-22, wherein the input composition occupies all or substantially all of the volume of the internal cavity during at least a portion of said incubation.
24. The method of any of embodiments 1-23, wherein, during at least a portion of the incubation in the chamber or during the rotation of the chamber, the liquid volume of the input composition occupies only a portion of the volume of the internal cavity of the chamber, the volume of the cavity during said at least a portion or during said rotation further comprising a gas, said gas taken into said cavity via said at least one opening, prior to or during said incubation.
25. The method of embodiment 24, wherein the centrifugal chamber comprises a movable member, whereby intake of gas into the centrifugal chamber effects movement of the movable member to increase the volume of the internal cavity of the chamber, thereby decreasing the total liquid volume of said input composition present in said cavity during rotation of said centrifugal chamber per square inch of the internal surface area of the cavity compared to the absence of gas in the chamber.
26. A method of transduction, comprising:
a) providing to an internal cavity of a centrifugal chamber that has an internal surface area of at least at or about 1×109 μm2 or at least at or about 1×1010 μm2:
i) an input composition comprising cells and viral particles comprising a recombinant viral vector, wherein:
the number of cells in the input composition is at least 1×107 cells, and
the viral particles are present in the input composition at least at or about 1 infectious unit (IU) per one of said cells, and
the input composition comprises a liquid volume that is less than the maximum volume of the internal cavity of the centrifugal chamber; and
ii) gas at a volume that is up to the remainder of the maximum volume of the internal cavity of the centrifugal chamber; and
b) incubating the input composition, wherein at least a portion of the incubation is carried out in said internal cavity of said centrifugal chamber while effecting rotation of said centrifugal chamber; and
wherein the method generates an output composition comprising a plurality of the cells transduced with the viral vector.
27. The method of embodiment 26, wherein:
the number of cells is at least at or about 50×106 cells; 100×106 cells; or 200×106 cells; and/or
the viral particles are present at least 1.6 IU/cell, 1.8 IU/cell, 2.0 IU/cell, 2.4 IU/cell, 2.8 IU/cell, 3.2 IU/cell or 3.6 IU/cell, 4.0 IU/cell, 5.0 IU/cell, 6.0 IU/cell, 7.0 IU/cell, 8.0 IU/cell, 9.0 IU/cell or 10.0 IU/cell.
28. The method of embodiment 26 or embodiment 27, wherein:
the liquid volume of the input composition is less than or equal to 200 mL, less than or equal to 100 mL, less than or equal to 50 mL or less than or equal to 20 mL; and/or
the liquid volume of the input composition is no more than 50%, no more than 40%, no more than 30%, no more than 20%, or no more than 10% of the volume of the internal surface area of the cavity during rotation or the maximum internal surface area of the cavity.
29. The method of any of embodiments 26-28, wherein the volume of gas is up to 200 mL, up to 180 mL, up to 140 mL or up to 100 mL.
30. The method of any of embodiments 26-29, wherein said rotation is at a relative centrifugal force at an internal surface of the side wall of the cavity or at a surface layer of the cells of at least at or about 600 g, 800 g, 1000 g, 1100 g, 1500 g, 1600 g, 2000 g, 2400 g, 2600 g, 2800 g, 3000 g, 3200 g or 3600 g.
31. A method of transduction, comprising incubating an input composition comprising cells and viral particles comprising a recombinant viral vector, at least a portion of said incubating being carried out under rotating conditions, thereby generating an output composition comprising a plurality of the cells transduced with the viral vector, wherein:
said input composition comprises greater than or about 20 mL, 50 mL, at least 100 mL, or at least 150 mL in volume, and/or said input composition comprises at least 1×108 cells; and
said rotating conditions comprise a relative centrifugal force on a surface layer of the cells of greater than about 800 g or greater than about 1000 g or greater than about 1500 g.
32. The method of embodiment 31, wherein:
at least 25% or at least 50% of said cells in the output composition are transduced with said viral vector; and/or
at least 25% or at least 50% of said cells in the output composition express a product of a heterologous nucleic acid comprised within said viral vector.
33. The method of embodiment 31 or embodiment 32, wherein said incubation is carried out in a cavity of a centrifugal chamber and the number of said cells in said input composition is at or about the number of said cells sufficient to form a monolayer or a bilayer on the inner surface of said cavity during said rotation.
34. The method of embodiment 33, wherein said centrifugal chamber comprises an end wall, a substantially rigid side wall extending from said end wall, and at least one opening, wherein at least a portion of said side wall surrounds said internal cavity and said at least one opening is capable of permitting intake of fluid into said internal cavity and expression of fluid from said cavity.
35. The method of embodiment 34, wherein said centrifugal chamber further comprises a movable member and said internal cavity is a cavity of variable volume defined by said end wall, said substantially rigid side wall, and said movable member, said movable member being capable of moving within the chamber to vary the internal volume of the cavity.
36. The method of any of embodiments 1-30 or 33-35, wherein the input composition in said cavity comprises a liquid volume of at least 20 mL or at least 50 mL and at or about 1 million cells per cm2 of the internal surface area of the cavity during at least a portion of said incubation.
37. The method of any of embodiments 1-36, wherein a further portion of the incubation is carried out outside of the centrifugal chamber and/or without rotation, said further portion carried out subsequent to the at least a portion carried out in the chamber and/or with rotation.
38. The method of any of embodiments 1-37, wherein the at least a portion of the incubation carried out in the cavity of the centrifugal chamber and/or the further portion of the incubation is effected at or at about 37° C.±2° C.
39. The method of embodiment 37 or embodiment 38, wherein the incubation further comprises transferring at least a plurality of the cells to a container during said incubation and said further portion of the incubation is effected in the container.
40. The method of embodiment 39, wherein the transferring is performed within a closed system, wherein the centrifugal chamber and container are integral to the closed system.
41. The method of any of embodiments 37-40, wherein:
the incubation is carried out for a time between at or about 1 hour and at or about 96 hours, between at or about 4 hours and at or about 72 hours, between at or about 8 hours and at or about 48 hours, between at or about 12 hours and at or about 36 hours, between at or about 6 hours and at or about 24 hours, between at or about 36 hours and at or about 96 hours, inclusive; or
the further portion of the incubation is carried out for a time between at or about 1 hour and at or about 96 hours, between at or about 4 hours and at or about 72 hours, between at or about 8 hours and at or about 48 hours, between at or about 12 hours and at or about 36 hours, between at or about 6 hours and at or about 24 hours, between at or about 36 hours and at or about 96 hours, inclusive.
42. The method of any of embodiments 37-41, wherein:
the incubation is carried out for a time that is no more than 48 hours, no more than 36 hours or no more than 24 hours; or
the further portion of the incubation is carried out for a time that is no more than 48 hours, no more than 36 hours or no more than 24 hours.
43. The method of any of embodiments 37-41, wherein:
the incubation is performed in the presence of a stimulating agent; and/or
the further portion of the incubation is performed in the presence of a stimulating agent.
44. The method of any of embodiments 37-41, wherein:
the incubation is carried out for a time that is no more than 24 hours;
the cells in the composition have not been subjected to a temperature of greater than 30° C. for more than 24 hours; and/or
the incubation is not performed in the presence of a stimulating agent.
45. The method of embodiment 43 or embodiment 44, wherein the stimulating agent is an agent capable of inducing proliferation of T cells, CD4+ T cells and/or CD8+ T cells.
46. The method of any of embodiments 43-45, wherein the stimulating agent is a cytokine selected from among IL-2, IL-15 and IL-7.
47. The method of any of embodiments 1-46, wherein the output composition containing transduced cells comprises at least at or about 1×107 cells or at least at or about 5×107 cells.
48. The method of embodiment 47, wherein the output composition containing transduced cells comprises at least at or about 1×108 cells, 2×108 cells, 4×108 cells, 6×108, 8×108 cells or 1×109 cells.
49. The method of embodiment 47 or embodiment 48, wherein the cells are T cells.
50. The method of embodiment 49, wherein the T cells are unfractionated T cells, isolated CD4+ T cells and/or isolated CD8+ T cells.
51. The method of any of embodiments 1-50, wherein the method results in integration of the viral vector into a host genome of one or more of the at least a plurality of cells and/or into a host genome of at least at or about 20% or at least at or about 30% or at least at or about 40% of the cells in the output composition.
52. The method of any of embodiments 1-51, wherein:
at least 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of said cells in said input composition are transduced with said viral vector by the method; and/or
at least 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of said cells in said output composition are transduced with said viral vector; and/or
at least 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of said cells in said output composition express a product of a heterologous nucleic acid comprised within said viral vector.
53. The method of any of embodiments 1-52, wherein, for an input composition comprising a virus at a ratio of about 1 or about 2 IU per cells, said method is capable of producing an output composition in which at least 10%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of the cells in said output composition generated by the method comprise said recombinant viral vector and/or express a product of a recombinant nucleic acid comprised within said vector.
54. The method of any of embodiments 1-53, wherein:
among all the cells in said output composition that contain the recombinant viral vector or into which the viral vector is integrated, the average copy number of said recombinant viral vector is no more than about 10, no more than about 5, no more than about 2.5, or no more than about 1.5; or
among the cells in the output composition, the average copy number of said vector is no more than about 2, no more than about 1.5, or no more than about 1.
55. The method of any of embodiments 1-30 and 33-54, wherein the centrifugal chamber is integral to a closed system, said closed system comprising said chamber and at least one tubing line operably linked to the at least one opening via at least one connector, whereby liquid and gas are permitted to move between said cavity and said at least one tubing line in at least one configuration of said system.
56. The method of embodiment 55, wherein:
said at least one tubing line comprises a series of tubing lines;
said at least one connector comprises a plurality of connectors; and
said closed system further comprises at least one container operably linked to said series of tubing lines, the connection permitting liquid and/or gas to pass between said at least one container and said at least one opening via the series of tubing lines.
57. The method of embodiment 55 or 56, wherein said at least one connector comprises a connector selected from the group consisting of valves, luer ports, and spikes.
58. The method of any of embodiments 55-57, wherein said at least one connector comprises a rotational valve.
59. The method of embodiment 58, wherein said rotational valve is a stopcock or multirotational port.
60. The method of any of embodiments 55-59, wherein said at least one connector comprises an aseptic connector.
61. The method of any of embodiments 56-60, wherein said at least one container comprises a container selected from the group consisting of bags, vials, and syringes.
62. The method of any of embodiments 56-61, wherein said at least one container comprises a diluent container, a waste container, a product collection container, and/or an input product container.
63. The method of any of embodiments 56-62, wherein:
said at least one container comprises at least one input container comprising said viral vector particles and said cells, a waste container, a product container, and at least one diluent container, each connected to said cavity via said series of tubing lines and said at least one opening.
64. The method of embodiment 63, wherein said method further comprises, prior to and/or during said incubation, effecting intake of said input composition into said cavity, said intake comprising flowing of liquid from said at least one input container into said cavity through said at least one opening.
65. The method of any of embodiments 56-64, wherein at least one container further comprises a container that comprises a gas prior to and/or during at least a point during said incubation and/or the closed system further comprises a microbial filter capable of taking in gas to the internal cavity of the centrifugal chamber and/or the closed system contains a syringe port for effecting intake of gas.
66. The method of embodiment 65, wherein the method comprises, prior to and/or during said incubation, providing or effecting intake of gas into said cavity under sterile conditions, said intake being effected by (a) flow of gas from the container that comprises gas, (b) flow of gas from an environment external to the closed system, via the microbial filter, or (c) flow of gas from a syringe connected to the system at the syringe port.
67. The method of embodiment 66, wherein the effecting intake of the gas into the internal cavity of the centrifugal chamber is carried out simultaneously or together with the effecting intake of the input composition to the internal cavity of the centrifugal chamber.
68. The method of embodiment 66 or embodiment 67, wherein the input composition and gas are combined in a single container under sterile conditions outside of the chamber prior to said intake of said input composition and gas into the internal cavity of the centrifugal chamber.
69. The method of embodiment 68, wherein the effecting of the intake of the gas is carried out separately, either simultaneously or sequentially, from the effecting of the intake of the input composition into said cavity.
70. The method of any of embodiments 66-69, wherein the intake of gas is effected by permitting or causing flow of the gas from a sterile closed container comprising the gas, an external environment through a microbial filter, or a syringe comprising said gas.
71. The method of any of embodiments 24-70, wherein the gas is air.
72. The method of any of embodiments 1-71, wherein the incubation is part of a continuous process, the method further comprising:
during at least a portion of said incubation, effecting continuous intake of said input composition into said cavity during rotation of the chamber; and
during a portion of said incubation, effecting continuous expression of liquid from said cavity through said at least one opening during rotation of the chamber.
73. The method of embodiment 72, further comprising:
during a portion of said incubation, effecting continuous intake of gas into said cavity during rotation of the chamber; and/or
during a portion of said incubation, effecting continuous expression of gas from said cavity.
74. The method of embodiment 73, wherein the method comprises the expression of liquid and the expression of gas from said cavity, where each is expressed, simultaneously or sequentially, into a different container.
75. The method of any of embodiments 72-74, wherein at least a portion of the continuous intake and the continuous expression occur simultaneously.
76. The method of any of embodiments 1-75, wherein the incubation is part of a semi-continuous process, the method further comprising:
prior to said incubation, effecting intake of said input composition, and optionally gas, into said cavity through said at least one opening;
subsequent to said incubation, effecting expression of liquid and/or optionally gas from said cavity;
effecting intake of another input composition comprising cells and said viral particles containing a recombinant viral vector, and optionally gas, into said internal cavity; and
incubating said another input composition in said internal cavity,
wherein the method generates another output composition comprising a plurality of cells of the another input composition that are transduced with said viral vector.
77. The method of any of embodiments 64-76, wherein said providing or said intake of the input composition into the cavity comprises:
intake of a single composition comprising the cells and the viral particles containing the recombinant viral vector; or
intake of a composition comprising the cells and a separate composition comprising the viral particles containing the recombinant viral vector, whereby the compositions are mixed, effecting intake of the input composition.
78. The method of embodiment 64-77, wherein the method further comprises:
effecting rotation of said centrifugal chamber prior to and/or during said incubation;
effecting expression of liquid from said cavity into said waste container following said incubation;
effecting expression of liquid from said at least one diluent container into said cavity via said at least one opening and effecting mixing of the contents of said cavity; and
effecting expression of liquid from said cavity into said product container, thereby transferring cells transduced with the viral vector into said product container.
79. The method of any of embodiments 1-78, further comprising:
(a) washing a biological sample comprising said cells in an internal cavity of a centrifugal chamber prior to said incubation; and/or
(b) isolating said cells from a biological sample, wherein at least a portion of the isolation step is performed in an internal cavity of a centrifugal chamber prior to said incubation; and/or
(c) stimulating cells prior to and/or during said incubation, said stimulating comprising exposing said cells to stimulating conditions, thereby inducing cells of the input composition to proliferate, wherein at least a portion of the step of stimulating cells is performed in an internal cavity of a centrifugal chamber.
80. The method of embodiment 79, wherein said isolating comprises carrying out immunoaffinity-based selection.
81. The method of embodiment 79 or 80, wherein said stimulating conditions comprise the presence of an agent capable of activating one or more intracellular signaling domains of one or more components of a TCR complex.
82. The method of embodiment 81, wherein said agent comprises a primary agent that specifically binds to a member of a TCR complex and a secondary agent that specifically binds to a T cell costimulatory molecule.
83. The method of embodiment 82, wherein the primary agent specifically binds to CD3; and/or
the costimulatory molecule is selected from the group consisting of CD28, CD137 (4-1-BB), OX40, or ICOS.
84. The method of embodiment 83, wherein said primary and secondary agents comprise antibodies and/or are present on the surface of a solid support.
85. The method of any of embodiments 79-84, wherein said biological sample in (a) and/or in (b) is or comprises a whole blood sample, a buffy coat sample, a peripheral blood mononuclear cells (PBMC) sample, an unfractionated T cell sample, a lymphocyte sample, a white blood cell sample, an apheresis product, or a leukapheresis product.
86. The method of any of embodiments 1-85, further comprising formulating cells transduced by the method in a pharmaceutically acceptable buffer in an internal cavity of a centrifugal chamber, thereby producing a formulated composition.
87. The method of embodiment 86, further comprising effecting expression of the formulated composition to one or a plurality of containers.
88. The method of embodiment 87, wherein the effecting of expression of the formulated composition comprises effecting expression of a number of the cells present in a single unit dose to one or each of said one or a plurality of containers.
89. The method of any of embodiments 79-88, wherein each of said a cavity of a centrifugal chamber is the same or different as a cavity of a centrifugal employed in one or more of the other steps and/or in the process of incubating and/or rotating an input composition containing cells and viral particles.
90. The method of any of embodiments 79-89, wherein each of said centrifugal chambers is integral to a closed system, said closed system comprising said chamber and at least one tubing line operably linked to the at least one opening via at least one connector, whereby liquid and gas are permitted to move between said cavity and said at least one tubing line in at least one configuration of said system.
91. The method of any of embodiments 1-90, wherein said cells in said input composition are primary cells.
92. The method of any of embodiments 1-91, wherein:
said cells in said input composition comprise suspension cells;
said cells in said input composition comprise white blood cells; and/or
said cells in said input composition comprise T cells or NK cells.
93. The method of any of embodiments 1-92, wherein said cells in said input composition are unfractionated T cells, isolated CD8+ T cells, or isolated CD4+ T cells.
94. The method of any of embodiments 1-93, wherein said cells in said input composition are human cells.
95. The method of any of embodiments 7-94, wherein, during said incubation, said centrifugal chamber is associated with a sensor, said sensor capable of monitoring the position of said movable member, and control circuitry, said circuitry capable of receiving and transmitting information to and from said sensor and causing movement of said movable member, said control circuitry further associated with a centrifuge capable of causing rotation of said chamber during said incubation.
96. The method of any of embodiments 7-95, wherein said chamber comprises said movable member and during said incubation, said centrifugal chamber is located within a centrifuge and associated with a sensor, said sensor capable of monitoring the position of said movable member, and control circuitry capable of receiving and transmitting information from said sensor and causing movement of said movable member, intake and expression of liquid and/or gas to and from said cavity via said one or more tubing lines, and rotation of said chamber via said centrifuge.
97. The method of embodiment 95 or embodiment 96, wherein said chamber, said control circuitry, said centrifuge, and said sensor are housed within a cabinet during said incubation.
98. The method of any of embodiments 1-97, wherein said recombinant viral vector encodes a recombinant receptor, which is thereby expressed by cells of the output composition.
99. The method of embodiment 98, wherein said recombinant receptor is a recombinant antigen receptor.
100. The method of embodiment 99, wherein said recombinant antigen receptor is a functional non-T cell receptor.
101. The method of embodiment 100, wherein said functional non-T cell receptor is a chimeric antigen receptor (CAR).
102. The method of embodiment 99, wherein said recombinant antigen receptor is a transgenic T cell receptor (TCR).
103. The method of embodiment 99, wherein said recombinant receptor is a chimeric receptor comprising an extracellular portion that specifically binds to a ligand and an intracellular signaling portion containing an activating domain and a costimulatory domain.
104. The method of any of embodiments 1-103, wherein:
the cells comprise primary human T cells obtained from a human subject; and
prior to said incubation and/or prior to completion of said transduction and/or, where the method includes formulation, prior to the formulation, the primary human T cells have not been present externally to the subject at a temperature of greater than 30° C. for greater than 1 hour, greater than 6 hours, greater than 24 hours, or greater than 48 hours; or
prior to said incubation and/or prior to the completion of the transduction, and/or where the method includes formulation, prior to the formulation, the primary human T cells have not been incubated in the presence of an antibody specific for CD3 and/or an antibody specific for CD28 and/or a cytokine, for greater than 1 hour, greater than 6 hours, greater than 24 hours, or greater than 48 hours.
105. A method for selection, the method comprising:
(a) incubating a selection reagent and primary cells in an internal cavity of a centrifugal chamber under mixing conditions, whereby a plurality of the primary cells bind to said selection reagent; and
(b) separating said plurality of said primary cells from another one or more of the primary cells based on binding to the selection reagent,
thereby enriching the primary cells based on binding to the selection reagent,
wherein said centrifugal chamber is rotatable around an axis of rotation and said internal cavity has a maximum volume of at least 50, at least 100, or at least 200 mL.
106. A method for stimulation of cells, the method comprising incubating a stimulation agent and primary cells under conditions whereby the stimulation agent binds to a molecule expressed by a plurality of the primary cells and said plurality of the cells are activated or stimulated, wherein
at least a portion of the incubation being carried out in an internal cavity of a centrifugal chamber under mixing conditions,
said centrifugal chamber is rotatable around an axis of rotation; and
said internal cavity has a maximum volume of at least 50, at least 100, or at least 200 mL.
107. The method of embodiment 105 or embodiment 106, wherein the chamber further comprises an end wall, a substantially rigid side wall extending from said end wall, and at least one opening, wherein at least a portion of said side wall surrounds said internal cavity and said at least one opening is capable of permitting intake of liquid into said internal cavity and expression of liquid from said cavity.
108. A composition, comprising transduced cells produced by the method of any of embodiments 1-107.
109. The composition of embodiment 108, wherein said cells:
are primary cells; and/or
are human cells; and/or
comprise white blood cells; and/or
comprise T cells; and/or
comprise NK cells.
110. The composition of embodiment 108 or embodiment 109, wherein the composition comprises at least at or about 5×107 cells, 1×108 cells, 2×108 cells, 4×108 cells, 6×108, 8×108 cells or 1×109 cells.
111. The composition of any of embodiments 106-110, wherein the composition comprises a therapeutically effective number of cells for use in adoptive T cell therapy.
112. The composition of any of embodiments 106-111, wherein:
the cells are T cells; and
subsequent to transduction, the cells in the composition are not subjected to cell expansion in the presence of a stimulating agent and/or the cells are not incubated at a temperature greater than 30° C. for more than 24 hours or the composition does not contain a cytokine or the composition does not contain a stimulating agent that specifically binds to CD3 or a TCR complex.
113. A composition, comprising at least 1×107 or at least 5×107 cells T cells, at least a plurality of which are transduced with a recombinant viral vector or express a recombinant or engineered antigen receptor, wherein:
subsequent to transduction, the cells in the composition have not been subjected to cell expansion in the presence of a stimulating agent; and/or
subsequent to transduction, the cells have not been incubated at a temperature greater than 30° C. for more than 24 hours.
114. A composition, comprising at least 1×107 or at least 5×107 primary human T cells, at least a plurality of which are transduced with a recombinant viral vector or express a recombinant or engineered antigen receptor, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the T cells in the composition comprise high expression of CD69 and/or TGF-beta-II.
115. The composition of embodiment 114, wherein said at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the T cells in the composition comprise no surface expression of CD62L and/or comprise high expression of CD25, ICAM, GM-CSF, IL-8 and/or IL-2.
116. The composition of any of embodiments 113-115, wherein said composition comprises at least 1×108 cells, 2×108 cells, 4×108 cells, 6×108, 8×108 cells or 1×109 cells.
117. The composition of any of embodiments 109-116, wherein said T cells are unfractionated T cells, isolated CD8+ T cells, or isolated CD4+ T cells.
118. The composition of any of embodiments 108-117, wherein at least 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of said cells in said composition are transduced with the viral vector.
119. The composition of any of embodiments 108-118, wherein:
the viral vector encodes a recombinant receptor; and
transduced cells in the composition express the recombinant receptor.
120. The composition of embodiment 119, wherein said recombinant receptor is a recombinant antigen receptor.
121. The composition of embodiment 120, wherein said recombinant antigen receptor is a functional non-T cell receptor.
122. The composition of embodiment 121, wherein said functional non-T cell receptor is a chimeric antigen receptor (CAR).
123. The composition of any of embodiments 119-122, wherein said recombinant receptor is a chimeric receptor comprising an extracellular portion that specifically binds to a ligand and an intracellular signaling portion containing an activating domain and a costimulatory domain.
124. The composition of embodiment 120, wherein said recombinant antigen receptor is a transgenic T cell receptor (TCR).
125. The composition of any of embodiments 110-124, wherein:
among all the cells in the composition, the average copy number of said recombinant viral vector is no more than about 10, no more than 8, no more than 6, no more than 4, or no more than about 2; or
among the cells in the composition transduced with the recombinant viral vector, the average copy number of said vector is no more than about 10, no more than 8, no more than 6, no more than 4, or no more than about 2.
126. The composition of any of embodiments 110-125, comprising a pharmaceutically acceptable excipient.
127. An article of manufacture comprising a container or plurality of containers, the container or the plurality of containers collectively containing a composition according to any of embodiments 113-126.
128. The article of manufacture of embodiment 127, wherein the container or plurality of containers comprises two or more or three or more bags and the composition further comprises a pharmaceutically acceptable excipient.
129. A method of treatment, the method comprising administering to a subject having a disease or condition the composition of any of embodiments 110-126.
130. The method of embodiment 129, wherein the transduced T cells in the composition exhibit increased or longer expansion and/or persistence in the subject than transduced T cells in a composition in which, subsequent to transduction, the cells in the composition have been subjected to cell expansion in the presence of a stimulating agent and/or the cells have been incubated at a temperature greater than 30° C. for more than 24 hours.
131. The method of embodiment 129 or embodiment 130, wherein the recombinant receptor, chimeric antigen receptor or transgenic TCR specifically binds to an antigen associated with the disease or condition.
132. The method of any of embodiments 129-131, wherein the disease or condition is a cancer, and autoimmune disease or disorder, or an infectious disease.
133. A composition, comprising:
at least 1×107 cells; and
at least at or about 1 infectious unit (IU) per cell of viral particles comprising a recombinant viral vector.
134. The composition of embodiment 133, wherein:
said cells comprise at least or about 50×106 cells; 100×106 cells; or 200×106 cells; and/or
said viral particles are present in the composition in an amount that is at least 1.6 IU/cell, 1.8 IU/cell, 2.0 IU/cell, 2.4 IU/cell, 2.8 IU/cell, 3.2 IU/cell, 3.6 IU/cell, 4.0 IU/cell, 5.0 IU/cell, 6.0 IU/cell, 7.0 IU/cell, 8.0 IU/cell, 9.0 IU/cell or 10.0 IU/cell.
135. The composition of embodiment 133 or embodiment 134, wherein the liquid volume of the composition is less than or equal to 220 mL, less than or equal to 200 mL, less than or equal to 100 mL, less than or equal to 50 mL or less than or equal to 20 mL.
136. The composition of any of embodiments 133-135, wherein said cells are primary cells.
137. The composition of any of embodiments 133-136, wherein said cells are human cells.
138. The composition of any of embodiments 133-137, wherein:
said cells comprise suspension cells;
said cells comprise white blood cells; and/or
said cells comprise T cells or NK cells.
139. The composition of embodiment 138, wherein said cells are T cells and the T cells are unfractionated T cells, isolated CD8+ T cells, or isolated CD4+ T cells.
140. The composition of any of embodiments 133-139, wherein the viral vector encodes a recombinant receptor.
141. The composition of embodiment 140, wherein said recombinant receptor is a recombinant antigen receptor.
142. The composition of embodiment 141, wherein said recombinant antigen receptor is a functional non-T cell receptor.
143. The composition of embodiment 142, wherein said functional non-T cell receptor is a chimeric antigen receptor (CAR).
144. The composition of any of embodiments 140-143, wherein said recombinant receptor is a chimeric receptor comprising an extracellular portion that specifically binds to a ligand and an intracellular signaling portion containing an activating domain and a costimulatory domain.
145. The composition of embodiment 141, wherein said recombinant antigen receptor is a transgenic T cell receptor (TCR).
146. A centrifugal chamber rotatable around an axis of rotation, said chamber comprising an internal cavity comprising the composition of any of embodiments 110-126.
147. A centrifugal chamber rotatable around an axis of rotation, said chamber comprising an internal cavity comprising: (a) a composition containing at least 5×107 primary T cells transduced with a recombinant viral vector and/or (b) a composition containing at least 5×107 primary T cells and viral particles containing a recombinant viral vector.
148. The centrifugal chamber of embodiment 146 or 147, said chamber further comprising an end wall, a substantially rigid side wall extending from said end wall, and at least one opening, wherein at least a portion of said side wall surrounds said internal cavity and said at least one opening is capable of permitting intake of liquid into said internal cavity and expression of liquid from said cavity.
149. The centrifugal chamber of embodiment 147 or 148, wherein said composition in said cavity comprises at least 1×108 cells, 2×108 cells, 4×108 cells, 6×108, 8×108 cells or 1×109 of the cells.
150. The centrifugal chamber of embodiment 147 or embodiment 148, wherein said T cells are unfractionated T cells, isolated CD8+ T cells, or isolated CD4+ T cells.
151. The centrifugal chamber of any of embodiments 147-150, wherein at least 2.5%, at least 5%, at least 6%, at least 8%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% of said cells in said composition are transduced with a viral vector.
152. The centrifugal chamber of any of embodiments 147-151, wherein:
the viral vector encodes a recombinant receptor; and
cells in the composition express the recombinant receptor.
153. The centrifugal chamber of embodiment 151, wherein said recombinant receptor is a recombinant antigen receptor.
154. The centrifugal chamber of embodiment 153, wherein said recombinant antigen receptor is a functional non-T cell receptor.
155. The centrifugal chamber of embodiment 154, wherein said functional non-T cell receptor is a chimeric antigen receptor (CAR).
156. The centrifugal chamber of any of embodiments 151-155, wherein said recombinant receptor is a chimeric receptor comprising an extracellular portion that specifically binds to a ligand and an intracellular signaling portion containing an activating domain and a costimulatory domain.
157. The centrifugal chamber of embodiment 153, wherein said recombinant antigen receptor is a transgenic T cell receptor (TCR).
158. The centrifugal chamber of any of embodiments 147-157, wherein:
among all the cells in the composition, the average copy number of said recombinant viral vector is no more than about 10, no more than 8, no more than 6, no more than 4, or no more than about 2; or
among the cells in the composition transduced with the recombinant viral vector, the average copy number of said vector is no more than about 10, no more than 8, no more than 6, no more than 4, or no more than about 2.
159. A centrifugal chamber rotatable around an axis of rotation, said chamber comprising an internal cavity comprising the composition of any of embodiments 133-145.
160. The centrifugal chamber of embodiment 159, further comprising a volume of gas up to the maximum volume of the internal cavity of the chamber.
161. The centrifugal chamber of embodiment 160, wherein said gas is air.
162. The centrifugal chamber of any of embodiments 146-161, said chamber being rotatable around an axis of rotation and comprising an end wall, a substantially rigid side wall extending from said end wall, and at least one opening, wherein at least a portion of said side wall surrounds said internal cavity and said at least one opening is capable of permitting intake of liquid into said internal cavity and expression of liquid from said cavity.
163. The centrifugal chamber of any of embodiments 146-162, wherein said side wall is curvilinear.
164. The centrifugal chamber of embodiment 163, wherein said side wall is generally cylindrical.
165. The centrifugal chamber of any of embodiments 162-164, wherein
said at least one opening comprises an inlet and an outlet, respectively capable of permitting said intake and expression; or
said at least one opening comprises a single inlet/outlet, capable of permitting said intake and said expression.
166. The centrifugal chamber of any of embodiments 162-165, wherein said at least one opening is coaxial with the chamber and is located in the end wall.
167. The centrifugal chamber of any of embodiments 162-166, wherein said centrifugal chamber further comprises a movable member and said internal cavity is a cavity of variable volume defined by said end wall, said substantially rigid side wall, and said movable member, said movable member being capable of moving within the chamber to vary the internal volume of the cavity.
168. The centrifugal chamber of embodiment 167, wherein:
the movable member is a piston; and/or
the movable member is capable of axially moving within the chamber to vary the internal volume of the cavity.
169. The centrifugal chamber of any of embodiments 162-168, wherein:
the internal surface area of said cavity is at least at or about 1×109 μm2;
the internal surface area of said cavity is at least at or about 1×1010 μm2;
the length of said rigid wall in the direction extending from said end wall is at least about 5 cm;
the length of said rigid wall in the direction extending from said end wall is at least about 8 cm; and/or
the cavity comprises a radius of at least about 2 cm at least one cross-section.
170. The centrifugal chamber of any of embodiments 159-169, wherein the liquid volume of said composition present in said cavity is between or between about 0.5 mL per square inch of the internal surface area of the cavity (mL/sq.in) and 5 mL/sq.in, 0.5 mL/sq.in. and 2.5 mL/sq.in., 0.5 mL/sq.in. and 1 mL/sq.in., 1 mL/sq.in. and 5 mL/sq.in., 1 mL/sq.in. and 2.5 mL/sq.in. or 2.5 mL/sq.in. and 5 mL/sq.in.
171. The centrifugal chamber of any of embodiments 159-169, wherein the liquid volume of said composition present in said cavity is at least 0.5 mL/sq.in., 1 mL/sq.in., 2.5 mL/sq.in., or 5 mL/sq.in.
172. A closed system, comprising the centrifugal chamber of any of embodiments 147-158 and 162-171.
173. The closed system of embodiment 172, further comprising a multi-way manifold operably connected to one or a plurality of containers.
174. The closed system, comprising the centrifugal chamber of any of embodiments 159-171.
175. The closed system of embodiment 174, further comprising a sterile filter.
176. The closed system of any of embodiments 172-175, wherein the centrifugal chamber is capable of rotation at a speed up to 8000 g, wherein the centrifugal chamber is capable of withstanding a force of up to 500 g, 600 g, 1000 g, 1100 g, 1200 g, 1400 g, 1500 g, 1600 g, 2000 g, 2500 g, 3000 g or 3200 g, without substantially yielding, bending, or breaking or otherwise resulting in damage of the chamber and/or while substantially holding a generally cylindrical shape under such force.
177. The method of embodiment 49, wherein at least at or about 30, 40, 50, 60, 70, 80, or 80% of the T cells in the output composition comprise high expression of CD69 and/or TGF-beta-II.
178. The method of embodiment 177, wherein said at least 30, 40, 50, 60, 70, 80, or 80% of the T cells in the composition comprise no surface expression of CD62L and/or comprise high expression of CD25, ICAM, GM-CSF, IL-8 and/or IL-2.
179. A method comprising
washing primary human cells; and
incubating said cells with a selection reagent under agitation conditions whereby at least a plurality of the human cells are specifically bound by the selection reagents,
wherein said washing and incubating are carried out within a closed, sterile system and at least in part in an internal cavity of a centrifugal chamber integral to the closed, sterile system.
180. The method of embodiment 179, wherein the method steps are carried out in an automated fashion based on input from a user that the method should be initiated, resulting in completion of the method steps.
181. The method of embodiment 105, 179 or 180, wherein the incubation under mixing conditions comprises effecting rotation of the chamber for at least a portion thereof.
182. The method of embodiment 181, wherein the effecting rotation for at least a portion thereof comprises effecting rotation at a plurality of periods during the incubation, said plurality of periods being separated by one or more periods of rest, at which the chamber is not rotated.
183. The method of embodiment 182, wherein one or more or all of the plurality of periods of effecting rotation is for a time that is or is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, such as 1 or 2 seconds and/or one or more or all of the one or more periods of rest is for a time that is or is about 3, 4, 5, 6, 7, 8, 9, or 10 or 15 seconds, such as 4, 5, 6, or 7 seconds.
184 The method of any of embodiments 105 or 179-183, wherein the incubation under mixing conditions is carried out for at least or approximately 10, 15, 20, 30, or 45 minutes, such as at or about 30 minutes.
EXAMPLES
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1: Viral Transduction of Primary Human T Cells in a Centrifugal Chamber
This example demonstrates transduction of isolated primary human T cells with a recombinant viral vector encoding a chimeric antigen receptor (CAR), with transduction initiated under centrifugal force in a substantially rigid cylindrical centrifugal chamber, according to an embodiment provided herein. T cells were isolated via positive selection from a human apheresis product sample.
The resulting cells were activated using an anti-CD3/CD28 reagent. For initiation of transduction, the cells were incubated with a viral particle containing a viral vector genome encoding an anti-CD19 CAR under various conditions following activation.
Under one set of conditions (“Sepax”), transduction was initiated by incubating the cells in a cavity of a centrifuge chamber (Biosafe SA, A200), under centrifugation in a Sepax® 2 processing unit (Biosafe SA). A 50 mL liquid composition containing 50×106 of the isolated cells was combined in a 300 mL transfer pack with 50 mL liquid stock containing the viral vector particles. Using the Sepax® system to move the piston of the chamber, the composition was pulled into the cavity of the centrifuge chamber. 100 mL air also was pulled in, thereby increasing the volume of the cavity to 200 mL and resulting in a decrease in the ratio of the volume of liquid in the cavity to the internal surface area of the cavity. The chamber was spun by ramping up to a speed of at approximately 4600 rpm on the Sepax® 2 unit, corresponding to a relative g force (relative centrifugal force (RCF)) at the internal side wall of the processing cavity of the chamber of approximately 600. The duration of the spin at this speed was 60 minutes.
For another set of conditions (“VueLife”), a composition containing 25×106 cells and the same stock of viral vector particles at a 1:1 volumetric ratio were incubated in 50 mL in a centrifuge bag, in a CI-50 centrifuge adapter, and spun at an approximate relative centrifugal force (RCF) on the cells of approximately 1000 g for 60 minutes. A bag with a smaller volume compared to the centrifuge chamber was used in order to permit centrifugation at a relative centrifugal force on the cell of 1000 g. Controls included an “untransduced” sample (same cell concentration incubated for the same time in a 24-well plate without virus without centrifugation and a “no-spin” control (“0xg”) sample (same cell/virus concentration incubated for the same time in the same plate without centrifugation). Under each set of conditions, a polycation was included. Following spin (or comparable “no-spin” incubation), the compositions were incubated for 24 hours at 37 degrees C. to complete transduction.
The cells were expanded and transduction efficiency for each of the respective conditions was calculated on Day 6 post-isolation as percentage of CD3+ T Cells with surface expression of the encoded CAR (as detected by flow cytometry using an antibody specific for the CAR). The results are shown in FIG. 1A. As shown, greater transduction efficiency was observed following the initiation of transduction by incubating cells in the cavity of the centrifuge chamber under rotation, as compared to in the centrifuge bag (VueLife®) and controls. FIG. 1B shows cell expansion (as indicated by number of population doublings) over the six-day period.
Example 2: Transduction of Primary Human T Cells in a Centrifugal Chamber at Different Ratios of Liquid Volume to Surface Area
Transduction efficiency following initiation of transduction in the centrifuge chamber was assessed under various conditions, using the same number of cells and infectious units of virus, and different ratios of liquid volume to internal surface area of the chamber's cavity. Cells were generally prepared and stimulated as described in Example 1. All transduction initiation conditions used an IU:cell ratio of 2:1 and a total number of 100×106 cells.
For a first sample (“5.1 mL/sq.in.,” referring to the 5.1:1 mL of liquid per square inch of internal cavity surface used in this condition), 100×106 cells, in a liquid volume of 100 mL, were combined with 100 mL of a liquid composition containing the viral vector particles. For a second sample (“2.5 mL/sq.in.,” referring to 2.5 mL of liquid per square inch of cavity surface used in this condition), 50 mL of a liquid composition with the same number of cells was combined with 50 mL of a liquid composition containing the viral vector particles. In each case, a polycation was included for a final concentration during centrifugation of 10 μg/mL. The respective liquid compositions (and for the second sample, 100 mL of air) were drawn into and incubated in the liquid-holding cavity of the chamber. In each case, the chamber was spun (by ramping up) in the Sepax® 2 processing unit at an rpm of approximately 4600, corresponding to an RCF at the internal cavity side wall of approximately 600 g for 60 minutes. The samples then were incubated for an additional 24 hours at 37 degrees, for completion of transduction.
The cells were expanded and transduction efficiency calculated on Day 6 post-isolation, by determining the percentage of CD3+ T Cells with surface expression of the CAR, detected as described above. The results are shown in FIG. 2. As shown, for initiation of transduction in the centrifugal chamber using the same number of cells and infectious units of virus, a greater transduction efficiency was observed when using a lower ratio of liquid volume of the composition in the cavity to the internal surface area of the cavity.
Example 3: Transduction of Primary Human T Cells in a Centrifugal Chamber
Another study compared transduction efficiency under various conditions, including transduction in a centrifugal chamber according to embodiments of the provided methods, using various ratios of liquid volume to cavity surface area. Human T cells were isolated from an apheresis product and stimulated as described above.
Following the stimulation, 80×106 cells were incubated under varying conditions, including for transduction with a viral vector encoding a CAR. A polycation was included in all samples.
For conditions under which transduction was initiated in the centrifuge chamber, 80×106 cells were incubated with virus containing the vector in the cavity of the chamber, at a ratio of 2 IU virus per cell. The incubation was carried out while centrifuging the chamber using the Sepax® 2 Processing system at an RCF at the internal side wall of the cavity of approximately 600 g for 60 minutes. Under one set of conditions (“Sepax (0.1 IU/cell/mL),” with 0.5 mL liquid volume per square inch of internal cavity surface), for centrifugation, the cells and virus were pulled into the cavity of the chamber in a total liquid volume of 20 mL; 180 mL of air also was pulled into the cavity. Under another set of conditions (“Sepax (0.01 IU/cell/mL),” with 5.1 mL liquid volume per square inch of internal cavity surface), the same number of cells and infectious units of virus were pulled in in a 200 mL liquid volume.
Under separate conditions, “1000 g in plate,” transduction of cells was initiated in the presence of virus (2 IU/cell) in a 24-well plate, with centrifugation at an RCF on the cells of approximately 1000 g for 60 minutes. An “untransduced” negative control (incubation in a 24-well plate without virus or centrifugation) and a “no spin” control (incubation with virus at a ratio of 2 infectious units (IU) per cell without centrifugation in the same 24-well plate) also were used. Cells were incubated for 24 hours at 37 degrees C. to complete transduction.
Cells were expanded and transduction efficiency for each sample calculated as percent of CD3+ cells expressing the CAR on their surface, as described in Examples 1 and 2. The results are shown in FIG. 3. As shown, transduction was observed following initiation of transduction under rotation in the centrifuge chamber and in the 24-well plate as compared to the control conditions. For transduction initiation in the centrifuge chamber, greater transduction efficiency was observed with a lower ratio of liquid volume to internal surface area of the chamber cavity.
Example 4: Assessment of Vector Copy Number (VCN) Following Transduction in a Centrifugal Chamber
Copy number of the integrated viral vector (VCN) was assessed following transduction initiated under certain conditions in the study described in Example 3. VCN per cell was determined for the SGF-derived retroviral vector by real-time quantitative PCR (RT-qPCR). Mean VCN was determined by qPCR specific for viral genome among all cells in the composition following transduction (“VCN/cell”), and separately among the transduced cells (cells expressing the transgene) alone (“VCN/CAR”). The results are presented in FIG. 4. In the graph shown in FIG. 4, the label “Sepax 20” refers to a 20 mL liquid volume used in the chamber cavity during transduction initiation; the results so-labeled are from the same study and condition labeled as “Sepax (0.1 IU/cell/mL)” in Example 3 (for which transduction efficiency was determined to be 25%). Similarly, the label “Sepax 200” refers to a 200 mL liquid volume used in the chamber cavity during transduction initiation; the results so-labeled are from the same study and conditions labeled as “Sepax (0.01 IU/cell/mL)” in Example 3 (for which the transduction efficiency was determined to be 7%). As shown, when transduction was initiated by initiating transduction of cells in the cylindrical, substantially rigid centrifuge chamber under rotation, increased transduction efficiency was not associated with increased vector copy number. In this study, the conditions producing increased transduction efficiency also produced decreased mean vector copy number per cell.
Example 5: Transduction Using a Sepax® 2 Processing System
In an exemplary process, T cells are transduced with a viral vector particle in an automated fashion in a centrifugal chamber integral to a single-use system and the Sepax® 2 processing system (Biosafe SA). The chamber is integral to a sterile, disposable closed system, which is a single-use processing kit sold by Biosafe SA for use in regenerative medicine. The kit is configured to include a series of tubing lines connecting the chamber to a series of containers, with a general configuration shown in FIG. 5 and/or FIG. 7. The chamber (1) includes an end wall (13) including an inlet/outlet opening (6), a rigid side wall (14), and a piston (2), which collectively define an internal cavity (7) of the chamber. The system is configured to include various containers labeled: Output Bag, Waste Bag, Input Bag, and two diluent bags (Diluent Bag 1 and Diluent Bag 2), and various connectors, including stopcocks, and valves. Clamps (5) are included for blocking flow between different portions of the system via the tubing lines. In some embodiments, the system includes a male luer lock sterile filter (15) with female luer lock cap (16), through which gas, e.g., air, may be drawn in a sterile manner, when the cap is released/removed. The system is placed in association with the Sepax® 2 processing unit, including a centrifuge and cabinet for housing components.
In the exemplary process, the Input Bag contains a composition containing the cells to be transduced. Diluent Bag 1 contains viral vector particles, polycation, and medium. In some embodiments, air is included in the bag with the vector particles. For example, in an alternative embodiment, a container with air and/or additional medium may be connected at this position instead of and/or in addition to the virus composition.
A user indicates to the processing unit via a user interface that a new program is to be run and inputs various parameters into the system, including an Initial Volume (between 20-900 mL), a Final Volume (between 20-220 mL), an Intermediate Volume (between 10-100 mL), a Dilution Volume (between 50-220 mL), a g-force (between 100-1600 or between 200-3200 g) (RCF at the internal side wall of the processing cavity of the chamber)), and a Sedimentation Time (between 120 and 3600 seconds). The user indicates to the system that the process should be initiated, inputs identification information for the subject from which cells are derived, and indicates to the system that input is complete, which prompts the system to carry out a test of the closed system kit.
With all respective stopcock valves in the closed position, clamps blocking movement of fluid between the tubing lines and Diluent Bag 1, Waste Bag, Input Bag, and Output Bag (for collection of the product containing the cells), respectively, are opened and an automated program initiated by communication with the system by the user.
In response, the system causes, in an automated fashion, movement of liquid and/or gas between the various components of the closed system by causing opening and closing of the valves and movement of the piston to vary the volume of the cavity. It causes repositioning of a stopcock to permit flow between the Input Bag and the internal cavity of the chamber, via the inlet/outlet opening and lowering of the piston within the centrifugal chamber, thereby increasing the volume of the cavity and drawing a volume (the user-defined Initial Volume) of the composition of cells and viral vector particles from the Input bag to the processing cavity, via an inlet/outlet in the end wall of the chamber.
The system prompts the centrifuge to spin the chamber for 120 seconds at 500 g, prompts purging of 20 mL volume from the cavity into the Input Bag to rinse it, and drawing of the volume back into the cavity. The system prompts the centrifuge to spin the chamber for 180 seconds at 500 g, causing sedimentation. The system repositions the stopcocks to permit flow of fluid and/or gas between the cavity and the Waste Bag and effects extraction of fluid from the cavity into the Waste Bag, leaving the user-defined Intermediate Volume in the cavity.
The system causes rotation of the stopcock to block movement of fluid between the tubing and the waste bag. The system causes intake of viral vector particle-containing liquid composition and, if applicable, air (collectively, at the user-defined Diluent Volume) from Diluent Bag 1 to the cavity of the chamber. These steps collectively effect intake of an input composition containing cells to be transduced and viral vector particles and in some cases, air, into the cavity. In some embodiments, the total volume of the cavity is 200 mL, for example, including 200 mL liquid volume or including less than 200 mL liquid volume and the remainder of the cavity volume including air.
Centrifugation of the chamber is carried out for the user-defined Sedimentation Time at the user-defined g-force, resulting in initiation of transduction of cells in the input composition with viral vector particles. In an alternative embodiment, a volume of air and/or medium is pulled into the cavity from another bag at the position of Diluent Bag 1 and 2, prior to centrifugation. In an alternative embodiment, air is drawn in prior to centrifugation through the luer lock filter (15), e.g., by the user opening the clamp (5) blocking movement of fluid between the filter (15) and tubing lines and the cavity (7) and releasing the female cap (16), allowing air remaining in the chamber to pass in through the filter from the environment. In some embodiments, the movement of air is automated by the system, for example, based on an additional user-defined air input volume inputted into the system and the user indicating to the system that air may be taken in at the defined air volume. In some embodiments, air, if present, is released through the tubing line and uncapped filter (15) by a similar process following centrifugation.
When prompted by the system, the user closes the clamp blocking movement of fluid between Diluent Bag 1 and the tubing lines and opens the clamp blocking movement of fluid between Diluent Bag 2 and the tubing lines. The system causes movement of fluid from Diluent Bag 2 to the processing cavity by opening of the appropriate stopcock valve and movement of the piston to draw into the cavity a volume of fluid from Diluent Bag 2 equal to the amount required to result in a total liquid volume in the chamber equal to the user-defined Final Volume. The system then causes mixture of the fluid in the cavity for 60 seconds and then transfer of the fluid in the internal cavity to the Output Bag, which thereby contains an output composition with cells to which viral particles have bound and/or infected with the viral vector. These cells then are generally incubated for completion of transduction, for example, at 37 degrees C., for example, for 24 hours.
Example 6: Assessment of Cell Growth and Viability at Different Centrifugal Forces
The effect of centrifugal force on cells during the centrifugation used to initiate transduction of cells in a chamber according to certain provided embodiments was assessed. Cell expansion and cell viability were assessed upon exposure to different centrifugal forces.
T cells were isolated and stimulated essentially as described in Example 1. At day 4, various, each individually containing the cells, were pulled into a cavity of a centrifuge chamber in a Sepax® 2 processing unit (Biosafe SA) and subjected to centrifugation at various centrifugal forces. Specifically, samples were spun for 60 minutes in a chamber (A-200F) integral to a single-use kit using the Sepax® 2 processing system at approximately 4600 rpm, approximately 6000 rpm, and approximately 7400 rpm, respectively), which achieved an RCF at the internal surface of the side wall of the cavity of approximately 600 g 1000 g, and 1600 g, respectively. As a control, a sample of the cells was separately pulled into the centrifuge cavity, but not spun (0 g condition). In each case, after the spin (or incubation with no spin), the cells were incubated at 37° C., 5% CO2, through day 10. At various points throughout the process, cell expansion (population doublings as compared with cell number at day 0) and viability were monitored. Specifically, these measurements were taken at days 0, 3, 4, 5, 6, 7, and 10. The results are shown in FIG. 6.
As shown in FIG. 6A and FIG. 6B, respectively, centrifugation at the various speeds was observed to have no substantial effect on cell expansion (“population doublings”) or viability over the 10 days. The results demonstrate that the T cells could tolerate centrifugation at relative centrifugal forces of at least up to or about 1600 g, as measured at the side wall of the cavity of the chamber, corresponding to approximately the same average force on the cells at the cell surface:liquid interface, under conditions used for transduction initiation in embodiments provided herein, without detectable substantial changes in expansion or viability.
Example 7: Transduction Process Step Using Transduction Initiation in Generally Cylindrical Centrifugal Chamber
This example describes the general parameters of a transduction process step that was used in the studies described in Examples 8-10. Transduction of cells with a recombinant viral vector encoding a chimeric antigen receptor (CAR) was initiated in a centrifugal chamber according to provided embodiments.
CD4+/CD8+ T cells were isolated via positive selection from a human apheresis product sample. The isolated cells were cryopreserved and thawed at 37° C. The thawed cells were activated using CD3/CD28 beads in the presence IL-2 (100 IU/mL) for 72 hours at 37° C. prior to initiation of transduction. In some cases, various aspects of the apheresis preparation, isolation, and/or activation steps also were carried out in the cavity of a centrifugal chamber according to provided embodiments, in association with the Sepax® 2 system, e.g., as described in Example 11.
In preparation for transduction, a centrifugal processing chamber (1) (A-200F), integral to a sterile, single-use disposable kit sold by Biosafe SA for regenerative medicine use, essentially as depicted in FIG. 7, was placed in association with a Sepax® 2 processing unit, which thus could provide to the chamber centrifugal force and axial displacement (permitting control of the dimensions of the internal cavity). (U.S. Pat. No. 6,733,433).
To initiate transduction, the following steps were carried out.
To generate a composition containing viral vector particles for sterile mixing with the activated cells in the centrifuge chamber, complete media (containing serum free hematopoietic cell medium supplemented with 5% human serum, and IL-2, and a polycation in an amount sufficient for a final concentration during transduction initiation of 10 μg/mL), viral vector particles at the indicated number or relative number of infectious units (IU) (for example, 1.8 IU/cell or 3.6 IU/cell), and, where applicable, air, were aseptically transferred to a centrifuge bag, which ultimately would be sterilely connected with the kit at a Diluent Bag position for intake as described below.
A culture bag containing the activated cells was sterilely connected to the single-use disposable kit via tubing line at the position of the “Input Bag” shown in FIG. 5 and FIG. 7. An automated “dilution” protocol was run on the Sepax® 2 processing unit. Thereby, through movement of the piston, the desired number of cells (as indicated in individual studies described, for example, 50×106, 100×106 or 200×106 cells) was transferred from the culture bag to a product bag at the “Output Bag” position shown in FIG. 5 and FIG. 7, by way of the chamber cavity.
To generate an input composition with both cells and virus (and where applicable, air) for intake into the chamber and transduction, the product bag containing the desired number of activated cells then was sterilely connected at the Input Bag position as shown in FIG. 5 and FIG. 7. The centrifuge bag containing the viral particles, media, and optionally air was sterilely connected at the position of Diluent Bag 1 shown in the figures. An automated Wash cycle was run on the Sepax® 2, facilitating drawing in of the composition containing the cells into the cavity of the chamber, spinning of the composition on the Sepax® 2 at an approximate RCF at the internal wall of the cavity of 500 g to pellet the cells, and removal of the appropriate volume of liquid required to achieve a desired volume, e.g., 10 mL. The contents of the centrifuge bag at the Diluent Bag 1 position, including the viral particles, media, polycation, and where applicable, air, then was drawn into the cavity of the chamber with the cells. This process thus effected a volume-reduction of the cell composition and combined the volume-reduced cells with the virus-containing composition and, where applicable, air. The resulting 200 mL volume (containing the cells, virus, and optionally, air) then was transferred into a centrifuge bag in the “Output Bag” position of the kit as shown in FIG. 5 and FIG. 7.
To initiate transduction, the centrifuge bag containing 200 mL of the virus, cells, and air where applicable then was removed and sterilely connected at the Input Bag position of the kit. A bag containing complete media was sterilely connected to the kit at a “Diluent Bag” position. A cell culture bag was sterilely connected to the system at the “Output Bag” position. The user indicated via the interface that an automated protocol should be run on the system for initiation of transduction. Specifically, the program caused transfer of the 200 mL volume containing cells, virus, and where indicated, air, via the tubing lines to the cavity of the chamber by movement of the piston. The program continued with centrifugation of the contents in the cavity of the chamber (total volume 200 mL) at the indicated force, to initiate transduction of cells with the viral vector particles. In some embodiments, a hand-held laser tachometer was used to verify revolutions per minute (rpm) at various set points on the Sepax® unit using known methods. Except where specifically indicated, the spin was carried out at the indicated speed for 1 hour (3600 s), with additional ramp-up and ramp-down time. Following the spin and when prompted by the system, the user closed the clamp permitting movement of fluid between the cavity and the Input Bag and opened the clamp blocking movement of fluid between the cavity and product bag at the Output Bag position. Upon input from the user, the program continued by effecting movement of liquid from the chamber to the output bag.
Where applicable, for expulsion of air, when prompted by the system, the user opened the clamp blocking movement of fluid between the chamber cavity and the filter, and the program caused expression of air via the filter.
A dilution program then was run on the Sepax® 2 system, with the clamp blocking movement of fluid between the diluent bag with the media and the chamber opened and the program causing movement (by opening of stopcock(s) and movement of the piston) of the appropriate amount of liquid from that bag to the chamber, mixing for 60 seconds, and then transfer of the fluid from the processing cavity to the output bag, the appropriate amount being that needed to achieve a user-defined Final Volume of 200 mL, given the presence of air during centrifugation, if any.
The culture bag in the Output Bag position thereby contained an output composition with cells containing bound viral particles and/or inoculated with the viral genome. The cells then were incubated in the bag for ˜24 hours at 37 degrees C., 5% CO2, for completion of transduction. During the transduction initiation and completion, viral vector particles inoculated cells and their genomes became integrated into the cellular genomes, as indicated by the various measures for transduction efficiency and copy number in the individual examples.
Example 8: Transduction Initiation in a Centrifugal Chamber with Constant Volume and Viral Particle Number and Different Cell Concentrations
Compositions with various cell numbers and infectious units (IU) of viral particles, in constant liquid volume, were subjected to the transduction process described in Example 7. In each case, prior to transduction initiation, cells were collected, washed, isolated, cryopreserved, and activated as described in Example 11.
Example 8A
Transduction initiation and further culture to complete transduction were carried out as described in Example 7, with the following specifics.
Under two separate conditions in two separate studies, the transduction initiation process was carried out in a 70 mL total liquid volume (the remaining 130 mL volume of the cavity during spin containing air). The separate conditions were carried out on 200×106 cells and 100×106 cells, respectively. The same total number of units of viral vector particles containing vectors encoding anti-CD19 CAR were used, resulting in 1.8 IU/cell and 3.6 IU/cell for the two conditions, respectively. During the transduction program, the 3600 second spin was at approximately 7400 rpm, corresponding to an RCF of approximately 1600 g at the side wall of the processing cavity.
After the ˜24-hour incubation, the cells were expanded in a Bioreactor System with perfusion. Transduction efficiency for the respective compositions was calculated on Day 6 as percentage of CD3+ T Cells with surface expression of the encoded CAR, detected as described in Example 1, and was compared to an untransduced population of cells as a control (“untransduced”). The results are shown in FIG. 8A. As shown, with the same total volume and total number of infectious units during incubation under rotation, a greater transduction efficiency was observed for the condition using a smaller number of 100×106 cells in the cavity during the incubation.
Example 8B
In another study, transduction initiation and further culture to complete transduction were carried out as described in Example 7, with the following specifics.
Under three separate conditions, the transduction initiation process was carried out in a 70 mL total liquid volume (the remaining 130 mL volume of the cavity during spin containing air). The separate conditions were carried out on 200×106 cells, 100×106 cells, and 50×106 cells, respectively. The same total number of units of viral vector particles containing vectors encoding anti-CD19 CAR were used, which was the number of units needed to result in 1.8 IU/cell for the condition with 200×106 cells. During the transduction program, the 3600 second spin was carried out on the Sepax® 2 system at approximately 7400 rpm, corresponding to an RCF of approximately 1600 g at the side wall of the processing cavity.
After the ˜24-hour incubation, the cells were expanded in a Bioreactor System with perfusion. Transduction efficiency for the respective compositions was calculated on Day 6 as percentage of CD3+ T Cells with surface expression of the encoded CAR, detected as described in Example 1, and was compared to an untransduced population of cells as a control (“untransduced”). The results are shown in FIG. 8B. As shown, with the same total volume and total number of infectious units during incubation under rotation, a greater transduction efficiency was observed for the condition using a smaller number of 100×106 cells in the cavity during the incubation.
Vector copy number (VCN) also was assessed in transduced cells at day 6, as described in Example 4, with mean VCN determined among the transduced cells (cells containing SGF+ viral vector nucleic acid in their genome). An untransduced cell control (“PD UnTD”) and positive control cells (“2 Copy Positive Control”) also were assessed. The results are presented in FIG. 8C.
Example 9: Transduction Initiation in Centrifugal Chamber with Various Volumes and Units of Viral Vector Particles
Compositions with increasing numbers of infectious units (IU) of viral particles and liquid volumes (with constant number (100×106) of cells) were subjected to the transduction process described in Example 7, with the following specifics. In each case, prior to transduction initiation, cells were collected, washed, isolated, cryopreserved, and activated as described in Example 11.
In the process described in Example 7, the composition containing the viral particles, media, and air that was drawn in from the Diluent Bag position for combining with the cells via the dilution protocol, included 60, 90, and 120 mL liquid volumes, respectively, for the different conditions (with the 10 mL cell-containing composition, resulting in 70, 100, and 130 mL liquid volume, respectively, for the individual conditions), with the remaining of the 200 mL total volume pulled into the chamber for spinning being comprised of air. Each of these liquid volumes included 6×106 IU viral vector particles per mL of liquid volume, resulting in an increasing IU and IU/cell for each condition. The speed for the 3600 second spin was carried out at approximately 7400 rpm, corresponding to an RCF of approximately 1600 g at the internal wall of the cavity on the Sepax® unit. An untransduced (“mock”) control also was used.
After the ˜24-hour incubation, the cells were expanded in a Bioreactor System with perfusion. Transduction efficiency for the respective compositions was calculated on Day 6 as percentage of CD3+ T Cells with surface expression of the encoded CAR, detected as described in Example 1. The results are shown in FIG. 9A. As shown, for the same number of cells, an increasing amount of virus with corresponding increase in volume resulted in an increased transduction efficiency in this study.
Mean vector copy number (VCN) per transduced cell (cells expressing the transgene) also was determined at day 6 for each condition by real-time quantitative PCR (RT-qPCR) as described in Example 4. The results are presented in FIG. 9B.
Example 10: Effect of Centrifugation Time on Transduction Efficiency Using a Centrifugal Chamber
100×106 cells were subjected to transduction as described in Example 7, with various durations used for the incubation under centrifugation. Specifically, the total liquid volume used for the centrifugation in the processing cavity of the chamber was 70 mL (with the remaining 130 mL of the 200 mL total volume composed of air). Viral vector particles containing a vector encoding an anti-CD19 CAR were included in this volume at a ratio of 3.6 IU/cell. In each case, prior to transduction initiation, cells were collected, washed, isolated, cryopreserved, and activated as described in Example 11.
The spin for initiation of transduction was carried out at approximately 7400 rpm, corresponding to an approximately a 1600 g relative centrifugal force on the inner side wall of the processing chamber. The duration of the spin at this speed was 10 minutes for one condition and 60 minutes for the other.
After the ˜24-hour incubation, the cells were expanded in a Bioreactor System with perfusion. Transduction efficiency for the respective compositions was calculated on Day 6 as percentage of CD3+ T Cells with surface expression of the encoded CAR detected as described in Example 1. An untransduced control also was assessed (“untransduced”). The results are shown in FIG. 10. As shown, in this study, greater transduction efficiency was observed following initiation of transduction of 100×106 cells in the processing cavity of the centrifuge chamber under centrifugation for 60 minutes as compared to 10 minutes.
Example 11: Preparation of Genetically Engineered Cells
This example describes an exemplary process which has been carried out to prepare, from a biological sample, genetically engineered T cells transduced with a nucleic acid encoded by a viral vector, according to certain embodiments provided herein. As described in individual examples, prior to the transduction steps carried out in studies described in various examples herein, some of the steps of this process were carried out, for example, collection, wash, cryopreservation, selection, and activation steps, as described in this example.
Various steps of the process were carried out within the processing cavity of a centrifugal chamber having a rigid, generally cylindrical side wall and a piston capable of moving within the chamber to vary the volume of the cavity (the processing cavity of a Sepax® centrifuge chamber contained within a single-use kit). Specifically, steps carried out in the chamber included cell washing, dilution/buffer-exchange, steps for affinity-based selection (e.g., incubation with immunospecific binding agents), transduction initiation, formulation, and steps for activation/expansion (e.g., incubation with stimulatory agent(s)).
1. Sample Collection and Leukapheresis
A human leukapheresis sample enriched in mononuclear cells was obtained from a whole blood sample from a subject using a leukapheresis collection system. The leukapheresis sample was stored sealed at 2-8° C., for no more than about 48 hours.
2. Leukapheresis Wash
The leukapheresis sample was sterilely transferred to a transfer pack. Cells of the leukapheresis sample were washed and resuspended in a buffer for use in affinity-based selection, the buffer containing PBS, EDTA, and human serum albumin. The wash was carried out within a sterile, single-use disposable kit sold by Biosafe SA for use in regenerative medicine, which included a centrifugal chamber (1), essentially as depicted in FIG. 7. The transfer pack containing the cells and a bag containing the buffer were sterilely connected to the kit, which was placed in association with a Sepax® 2 processing unit. The wash and resuspension were carried out using a standard cell wash protocol on the unit, with the cells retained in the processing cavity (7) of the centrifuge chamber at the end of the protocol, for subsequent incubation with reagents for affinity-based selection (see 3).
3. Affinity-Based Selection
For positive, immunoaffinity-based selection of T cells, the same automated program was continued to incubate the washed cells in the selection buffer with magnetic beads coupled to monoclonal antibodies specific for CD4 and CD8. The incubation was carried out at room temperature in the same centrifugal chamber (1) in which the cells were retained after the wash (see 2) described above. Specifically, the beads were mixed in selection buffer in a transfer pack, which then was sterilely connected at a Diluent Bag position of the single-use kit used for the wash step. A program was run on the Sepax® 2 unit which caused the bead mixture and selection buffer to be drawn into the chamber with the washed cells, and the contents of the chamber (total liquid volume 100 mL) to be mixed for 30 minutes, via a semi-continuous process. The mixing was carried out with repeated intervals, each including short duration (approximately 1 second) centrifugation at low speed (approximately 1700 rpm), followed by a short rest period (approximately 6 seconds).
At the end of the program, the Sepax® 2 unit caused pelleting of the cells and expulsion of excess buffer/beads into a bag at the Waste Bag position, washing of the pelleted cells, and resuspension in selection buffer. The wash was carried out on the Sepax® at an RCF at the internal wall of the cavity of approximately 200 g, for 180 seconds. The program caused the washed cells to be collected into a transfer pack placed at the Output Bag position in the exemplary kit shown in FIG. 7, the contents of which could be transferred via tubing lines to a column for magnetic separation, within a closed system. Thus, the cell wash and incubation with the affinity-based selection reagent was carried out entirely within the same closed, sterile system, by passing liquid and cells to and from the cavity of the centrifugal chamber. The ability to control and adjust liquid volumes and to mix the cells under rotation in the chamber allowed use of substantially less of the selection reagent per cell processed as compared to incubation in a tube with shaking or rotation.
The cells then were passed from the transfer pack, through a closed, sterile system of tubing lines and a separation column, in the presence of a magnetic field using standard methods, to separate cells that had bound to the CD4- and/or CD8-specific reagents. These magnetically-labeled cells then were collected in a transfer pack for further processing.
4. Cryopreservation
The transfer pack with the labeled, selected cells was sterilely-connected to a single-use disposable kit sold by Biosafe AS for regenerative medicine for use with the Sepax® 2 system. The kit was essentially as shown in FIG. 7, except that two ports, as opposed to one, were present at the position to which the Output Bag is attached in the exemplary system shown in FIG. 7, with a collection bag sterilely connected at each port; two ports, as opposed to one, were present at the position to which the Input Bag is connected in FIG. 7; and a single port, as opposed to two, were present at the position of Diluent Bags 1 and 2 in FIG. 7. A standard wash cycle was carried out on the Sepax® 2 unit to reduce the volume of the washed cells. A bag with cryomedia was sterilely connected to the kit and a dilution protocol run twice to transfer the cryomedia to the cell composition and expel the resulting composition into the two output cryopreservation bags. The cells in the cryopreservation bags were cryopreserved and stored in liquid nitrogen until further use.
5. Thaw and Activation
Cryopreserved cells for were thawed. The thawed cells were activated using an anti-CD3/CD28 reagent(s), generally at 37° C., for a period of time as indicated for individual studies. Prior to incubation with the reagent, the cells were washed and resuspended in complete media using the Sepax® 2 system, using a standard cell washing program and in a kit essentially as shown in FIG. 7. In the same kit, the cells were combined with the anti-CD3/28 reagent(s) in the cavity of the chamber by mixing with intervals of low-speed centrifugation and rest as described for bead incubation for selection for 30 minutes at room temperature. Following the incubation, the incubated material was transferred via the Sepax® 2 unit into an output cell culture bag, which then was incubated at 37° C. for the remainder of the activation period.
6. Transduction
Transduction was carried out in the centrifugal chamber integral to the kit, placed in association with the Sepax® processing unit, as described in Example 7, with specific details given in particular examples.
7. Expansion
In some cases, following transduction, cells were further incubated, generally at 37 degrees C., to allow for expansion.
8. Wash, Formulation
In some cases, the expanded and/or transduced cells were further washed, diluted, and/or formulated for testing, storage, and/or administration. In some examples, expanded and/or transduced cells were washed in the chamber integral to a single-use kit for use with the Sepax® 2 system, for example as described for cryopreservation. In some cases, a bag containing washed cells was sterilely connected to a kit such as shown in FIG. 7, or such a kit with a plurality of ports available for connection of containers, e.g., bags, at the Output Bag position shown in FIG. 7.
One example of such a multi-port output kit is shown in FIG. 11, which shows a plurality of ports (17), to one or more of which may be connected a container, such as a bag, for collection of output composition. The connection may be by sterile welding of the desired number of containers, depending for example, on the desired number of unit dosage form of the cells to produce by a given method. To generate the kit shown in FIG. 11, a multi-way tubing manifold with a plurality of ports (in the example shown in FIG. 11, eight) was sterilely welded to an output line of a single-use kit sold by Biosafe AS for regenerative medicine use. A desired number of plurality of output bags were sterilely connected to one or more, generally two or more, of these ports. In some examples, such bags were attached to fewer than all the ports. Clamps (5) were placed on the tubing lines preventing movement of fluid into the individual bags until desired. A bag containing the desired liquid, such as formulation, assay, and/or cryopreservation media, was sterilely connected to the kit and a dilution protocol run on the Sepax® 2 unit a plurality of times, with the user opening and closing the respective clamps leading to the appropriate number of bags, thereby generating an output composition in the desired formulation, split into the desired number of bags. In some embodiments, a single unit dose of cells was collected in each of the respected bags, in a formulation for administration to a subject, such as the subject from which the leukapheresis product was derived.
The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.
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14932660
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juno therapeutics, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Mar 30th, 2022 06:04PM
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Mar 30th, 2022 06:04PM
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Bristol-Myers Squibb
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:bmy
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Juno Therapeutics
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Dec 17th, 2019 12:00AM
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Oct 20th, 2015 12:00AM
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https://www.uspto.gov?id=US10507219-20191217
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Methods and compositions for dosing in adoptive cell therapy
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Provided are methods for administering multiple doses of cells, such as T cells, to subjects for cell therapy. Also provided are compositions and articles of manufacture for use in the methods. The cells generally express recombinant receptors such as chimeric receptors, e.g., chimeric antigen receptors (CARs) or other transgenic receptors such as T cell receptors (TCRs). The methods generally involve administering a first and at least one consecutive dose of the cells. Timing of the doses relative to one another, and/or size of the doses, in some embodiments provide various advantages such as lower or reduced toxicity and improved efficacy, for example, due to increased exposure of the subject to the administered cells. In some embodiments, the first dose is a relatively low dose, such as one that reduces tumor or disease burden, thereby improving the efficacy of consecutive or subsequent doses, and the consecutive dose is a consolidating dose.
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10507219
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1. A method of treatment, comprising:
administering a consecutive dose of T cells expressing a chimeric antigen receptor (CAR) that binds CD19 to a subject having a hematologic malignancy expressing CD19 and that has been previously administered a first dose of T cells expressing a CAR that binds CD19, said first dose comprising no more than about 1×106 of the CAR-expressing cells per kilogram body weight of the subject, no more than about 1×108 of the CAR-expressing cells, or no more than about 1×108 of the CAR-expressing cells/m2 of the subject, wherein:
the CAR expressed by the cells in the first dose and the CAR expressed by the cells of the consecutive dose each, individually, comprises (i) an antigen binding domain that binds CD19; and (ii) an intracellular signaling region comprising an immunoreceptor tyrosine-based activation motif (ITAM) and a T cell co-stimulatory signaling domain; and
the consecutive dose of cells is administered at a point in time that is at least or more than about 14 days after and less than about 28 days after initiation of the administration of the first dose of cells.
2. The method of treatment of claim 1, further comprising administering to the subject the first dose of cells.
3. The method of claim 1, wherein, at the time of the administration of the consecutive dose:
(i) the serum level in the subject of a factor indicative of cytokine release syndrome (CRS) is no more than about 50 times that in the subject immediately prior to the administration of the first dose; and/or
(ii) the subject does not exhibit grade 3 or higher neurotoxicity; and/or
(iii) a CRS-related outcome or symptom of neurotoxicity in the subject has reached a peak level and begun to decline following the administration of the first dose; and/or
(iv) the subject does not exhibit a detectable humoral or cell-mediated immune response against the CAR expressed by the cells of said first dose.
4. The method of claim 1, wherein prior to the administration of the first dose of cells, said subject had not received a dose of cells expressing the CAR expressed by the cells in the first dose.
5. The method of claim 1, wherein the CAR expressed by the cells in the first dose and the CAR expressed by the cells in the consecutive dose contain the same antigen-binding domain.
6. The method of claim 1, wherein:
the first and/or consecutive dose of cells comprises cells in an amount sufficient for reduction in burden of the hematologic malignancy in the subject; and/or
the administration of the consecutive dose leads to a reduction in burden of the hematologic malignancy in the subject.
7. The method of claim 1, wherein:
if at a time just prior to initiation of administration of the consecutive dose of cells the subject exhibits morphologic disease, the consecutive dose comprises less than or about the same number of CAR-expressing cells as the number of CAR-expressing cells in the first dose; and
if at a time just prior to initiation of administration of the consecutive dose of cells the subject exhibits minimum residual disease, the consecutive dose comprises an increased number of CAR-expressing cells as compared to the first dose.
8. The method of claim 1, wherein the consecutive dose comprises an increased number of CAR-expressing cells as compared to the first dose.
9. The method of claim 1, wherein the number of CAR-expressing cells administered in the consecutive dose comprises between about 2×106 per kilogram (kg) body weight and about 6×106/kg, inclusive.
10. The method of claim 9, wherein the number of cells administered in the first dose is between about 0.5×106 cells/kg body weight of the subject and 3×106 cells/kg.
11. The method of claim 1, further comprising administering a chemotherapeutic agent prior to the administration of the consecutive dose of cells.
12. The method of claim 1, wherein the subject has been previously treated with a chemotherapeutic agent prior to administration of the first dose and/or prior to the administration of the consecutive dose.
13. The method of claim 11, wherein the chemotherapeutic agent comprises an agent selected from the group consisting of cyclophosphamide and fludarabine.
14. The method of claim 11, wherein the chemotherapeutic agent comprises a combination of cyclophosphamide and fludarabine.
15. The method of claim 12, wherein the chemotherapeutic agent comprises an agent selected from the group consisting of cyclophosphamide and fludarabine.
16. The method of claim 12, wherein the chemotherapeutic agent comprises a combination of cyclophosphamide and fludarabine.
17. The method of claim 1, wherein:
the subject has been previously treated with a chemotherapeutic agent prior to the initiation of administration of the first dose; or
the subject has been previously treated with a chemotherapeutic agent subsequently to the initiation of the administration of the first dose, and prior to the initiation of administration of the consecutive dose.
18. The method of claim 2, further comprising administering a chemotherapeutic agent prior to the administration of the first dose of cells.
19. The method of claim 1, wherein the hematologic malignancy is a leukemia or lymphoma.
20. The method of claim 1, wherein the hematologic malignancy is acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or non-Hodgkin lymphoma (NHL).
21. The method of claim 1, wherein the number of CAR-expressing cells administered in the first dose is at or about or no more than at or about 1×106 per kilogram of the subject and/or the number of CAR-expressing cells administered in the consecutive dose is at or about 3×106 per kilogram of the subject.
22. The method of claim 1, wherein the number of cells administered in the consecutive dose comprises 1×106 CAR-expressing cells or at least 1×106 CAR-expressing cells.
23. The method of claim 1, wherein:
the number of cells administered in the first dose is between 1×106 and 1×108 CAR-expressing cells, inclusive.
24. The method of claim 1, wherein the number of cells administered in the consecutive dose comprises 1×107 CAR-expressing cells or at least 1×107 CAR-expressing cells.
25. The method of claim 1, wherein the number of cells administered in the consecutive dose comprises 1×108 CAR-expressing cells or at least 1×108 CAR-expressing cells.
26. The method of claim 8, wherein:
the number of cells administered in the first dose is between 1×106 and 1×108 CAR-expressing cells.
27. The method of claim 26, wherein the number of cells administered in the consecutive dose comprises 1×107 CAR-expressing cells or at least 1×107 CAR-expressing cells.
28. The method of claim 26, wherein the number of cells administered in the consecutive dose comprises 1×108 CAR-expressing cells or at least 1×108 CAR-expressing cells.
29. The method of claim 1, wherein the T cell co-stimulatory signaling domain is a signaling domain of 4-1BB or CD28.
30. The method of claim 1, wherein the CAR expressed by the cells in the consecutive dose is identical to the CAR expressed by the cells in the first dose.
31. A method of treatment, comprising:
administering to a subject having a hematologic malignancy expressing CD19 a first dose of T cells expressing a chimeric antigen receptor (CAR) that binds CD19, wherein the first dose comprises no more than about 1×106 of the CAR-expressing cells per kilogram body weight of the subject, no more than about 1×108 of the CAR-expressing cells, or no more than about 1×108 of the CAR-expressing cells/m2 of the subject, and is an amount sufficient to reduce burden of the hematologic malignancy in the subject; and
after administering the first dose of cells, administering to the subject a consecutive dose of T cells expressing a (CAR) that binds CD19, wherein the consecutive dose of cells is administered at a time point that is at least or more than about 14 days after and less than about 28 days after initiation of the administration of the first dose of cells and the consecutive dose of cells comprises an increased number of CAR-expressing cells as compared to the first dose,
and wherein the CAR expressed by the cells in the first dose and the CAR expressed by the cells of the consecutive dose each, individually, comprises (i) an antigen binding domain that binds to CD19; and (ii) an intracellular signaling region comprising an immunoreceptor tyrosine-based activation motif (ITAM) and a T cell co-stimulatory signaling domain.
32. The method of claim 31, wherein the number of CAR-expressing cells in the consecutive dose or the number of CAR-expressing cells per kilogram administered in the consecutive dose is at least at or about 2 times greater than the number of CAR-expressing cells or CAR-expressing cells per kilogram administered in the first dose.
33. The method of claim 31, wherein the tumor hematologic malignancy is a leukemia or a lymphoma.
34. The method of claim 31, wherein the hematologic malignancy is an acute lymphoblastic leukemia (ALL), a chronic lymphocytic leukemia (CLL), or a non-Hodgkin lymphoma (NHL).
35. The method of claim 31, wherein the T cell co-stimulatory signaling domain is a signaling domain of 4-1BB or CD28.
36. The method of claim 31, wherein:
the number of cells administered in the first dose is between about 0.5×106 cells/kg body weight of the subject and 3×106 cells/kg; and
the number of CAR-expressing cells administered in the consecutive dose comprises between about 2×106 per kilogram (kg) body weight and about 6×106/kg, inclusive.
37. The method of claim 32, wherein the number of cells administered in the first dose is between 1×106 and 1×108 CAR-expressing cells, inclusive.
38. The method of claim 31, wherein the CAR expressed by the cells in the consecutive dose is identical to the CAR expressed by the cells in the first dose.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S. provisional application No. 62/066,279 filed Oct. 20, 2014, entitled “Methods and Compositions for Dosing in Adoptive Cell Therapy,” U.S. provisional application No. 62/162,647, filed May 15, 2015, entitled “Methods and Compositions for Dosing in Adoptive Cell Therapy,” U.S. provisional application No. 62/168,710, filed May 29, 2015, entitled “Methods and Compositions for Dosing in Adoptive Cell Therapy,” and U.S. provisional application No. 62/215,732, filed Sep. 28, 2015, entitled “Methods and Compositions for Dosing in Adoptive Cell Therapy,” the contents of which are incorporated by reference in their entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042001000seqlist.txt, created Oct. 20, 2015, which is 16 kilobytes in size. The information in electronic format of the Sequence Listing is incorporated by reference in its entirety.
FIELD
The present disclosure relates to adoptive cell therapy involving the administration of multiple doses of cells, and methods, compositions, and articles of manufacture for use in the same. The cells generally express recombinant receptors such as chimeric receptors, e.g., chimeric antigen receptors (CARs) or other transgenic receptors such as T cell receptors (TCRs). Features of the methods, including the timing of the doses and numbers of cells administered, provide various advantages, such as lower toxicity and/or improved efficacy, for example, due to increased exposure of the subject to the administered cells. In some embodiments, the first dose involves a relatively lower number of cells compared with dosage amounts administered in other methods. In some embodiments, the first dose reduces tumor or disease burden, thereby improving the efficacy of consecutive or subsequent doses. In some embodiments, the timing of a consecutive dose is designed to minimize risk of toxicity and/or host immune response to the cells by the subject, thereby improving persistence and efficacy.
BACKGROUND
Various methods are available for adoptive cell therapy using engineered cells expressing recombinant receptors, such as chimeric antigen receptor (CARs). Improved methods are needed, for example, to reduce the risk of toxicity and/or to increase efficacy, for example, by increasing exposure of the subject to the administered cells, for example, by improving expansion and/or persistence of the administered cells. Provided are methods, compositions, and articles of manufacture that meet such needs.
SUMMARY
Provided are methods of for administering to subjects cells expressing genetically engineered (recombinant) cell surface receptors in adoptive cell therapy, for example, to treat diseases and/or conditions in the subjects. The methods generally involve administering multiple doses of such cells, and/or administering a consecutive dose to a subject having been previously treated with a prior (e.g., first) dose of such cells. In some embodiments, the doses are a first dose and one or more consecutive doses. In some embodiments, the first dose is a relatively low dose and/or is a conditioning or debulking dose and/or the consecutive dose(s) is a consolidating dose(s). Also provided are cells, compositions, and articles of manufacture for use in such methods. In some embodiments, the recombinant receptors are genetically engineered antigen receptors, such as functional non-TCR antigen receptors, e.g., chimeric antigen receptors (CARs) and other recombinant antigen receptors such as transgenic T cell receptors (TCRs). Also among the receptors are other recombinant chimeric receptors, such as those containing an extracellular portion that specifically binds to a ligand or receptor or other binding partner and an intracellular signaling portion, such as the intracellular signaling portion of a CAR. The doses in some embodiments include a relatively low first dose.
In some embodiments, the methods involve (a) administering to a subject with a disease or condition a first dose of cells expressing a recombinant receptor (e.g., chimeric antigen receptor (CAR)), the first dose containing the cells; and (b) administering to the subject a consecutive dose of recombinant receptor-expressing (e.g., CAR-expressing) cells. In other embodiments, e.g., for providing consolidating treatment, the methods are carried out by administering to the subject the consecutive dose or doses as in (b), to a subject that has been previously administered the first dose as in (a).
In some embodiments, the methods involve (a) administering to a subject having a disease or condition a first dose of cells expressing a recombinant receptor (e.g., chimeric antigen receptor (CAR)). In some embodiments, the first dose contains no more than about 1×106 of the cells per kilogram body weight of the subject, no more than about 1×108 of the cells, and/or no more than about 1×108 of the cells/m2 of the subject. In some embodiments, the methods further involve (b) administering to the subject a consecutive dose of cells expressing a recombinant receptor (e.g., CAR) at a time point that is at least or more than about 14 days after and less than about 28 days after initiation of the administration in (a).
In some embodiments, at the time of the administration in (b) the serum level in the subject of a factor indicative of cytokine release syndrome (CRS) is less than about 10 times, less than about 25 times, and/or less than about 50 times that in the subject immediately prior to said administration in (a). In some embodiments, the subject does not exhibit grade 3 or higher neurotoxicity. In some embodiments, a CRS-related outcome or symptom of neurotoxicity in the subject following the administration of the first dose has reached a peak level and begun to decline following the administration in (a). In some embodiments, the subject does not exhibit a detectable humoral or cell-mediated immune response against the receptor (e.g., CAR) expressed by the cells of said first dose.
In some embodiments, the methods further involve the administration of additional consecutive or subsequent doses, such that a first and multiple consecutive doses are administered, e.g., in accordance with the dosing amounts and timing schedules as specified for the first and consecutive doses. In some embodiments, the first of one or more subsequent doses is administered at a time that is at least or greater than 14 days after the initiation of the administration of the consecutive dose. In some embodiments, the administration of the first, consecutive, and subsequent doses includes administering at least three of the doses within at or about 28 days. In some embodiments, the consecutive dose is administered at about day 14 following the initiation of administration of the first dose, and an additional consecutive or subsequent dose is administered at day 28 following the initiation of administration of the first dose. In some embodiments, additional subsequent doses are administered at day 42 and/or day 56 following the initiation of administration of the first dose.
In some embodiments, the first dose is administered in an amount sufficient to reduce burden of the disease or condition in the subject. In some embodiments, the first dose is a low dose, such as a debulking dose, such as a dose that is lower than that required to eradicate the disease or condition but that may effect a reduction in burden or bulk of such disease or condition. In some embodiments, the administration of the first dose does not induce severe cytokine release syndrome (CRS) in the subject. In some embodiments, administration of the first dose does not induce CRS in the subject. In some embodiments, based on clinical data, administration of the first dose does not induce severe CRS in a majority of subjects. In some embodiments, the administration of the first dose does not induce CRS encompassing a combination of (1) persistent fever (fever of at least 38 degrees Celsius for at least three days) and (2) a serum level of C reactive protein (CRP) of at least at or about 20 mg/dL, and/or does not induce CRS encompassing hypotension requiring the use of two or more vasopressors or respiratory failure requiring mechanical ventilation.
In some embodiments, administration of the first dose does not induce grade 3 or higher neurotoxicity in the subject. In some embodiments, based on clinical data, administration of the first dose does not induce grade 3 or higher neurotoxicity in a majority of subjects. In some embodiments, symptoms associated with a clinical risk of neurotoxicity and/or grade 3 or higher neurotoxicity include confusion, delirium, expressive aphasia, obtundation, myoclonus, lethargy, altered mental status, convulsions, seizure-like activity, seizures (optionally as confirmed by electroencephalogram [EEG]), elevated levels of beta amyloid (Aβ), elevated levels of glutamate, and elevated levels of oxygen radicals.
In some embodiments, the first dose is lower than a dose that would cause CRS or severe CRS in the subject. In some embodiments, the first dose comprises no more than about 1×106 of the cells per kilogram body weight of the subject, no more than 5×105 of the cells per kilogram body weight of the subject, no more than about 1×108 of the cells, or no more than about 1×108 of the cells/m2 of the subject.
In some embodiments, the consecutive dose(s) is administered at a time at which a clinical risk for neurotoxicity, cytokine-release syndrome (CRS), macrophage activation syndrome, or tumor lysis syndrome, is not present or has passed or has subsided following the administration of the first (or prior) dose. In some embodiments, the consecutive dose is administered at a time at which a biochemical readout evidencing CRS, neurotoxicity, macrophage activation syndrome, or tumor lysis syndrome, is not present or has passed or has subsided following said administration of the first (or prior) dose. In some embodiments, the consecutive dose(s) is administered at a time at which a serum level of a factor indicative of cytokine-release syndrome (CRS) or neurotoxicity in the subject is less than about 10 times, less than about 25 times, and/or less than about 50 times the serum level of the indicator in the subject immediately prior to said administration of the first dose. In some embodiments, the consecutive dose(s) is administered at a time after a neurotoxicity and/or CRS-related outcome or serum factor indicating CRS in the subject has reached a peak level and begun to decline following said administration, such as where at the time of the administration of the consecutive dose, the level of a CRS-related outcome or serum factor indicative of CRS is no more than 50% of the peak level, is no more than 20% of the peak level, or is no more than 5% of the peak level, or is at or about the level immediately prior to the administration of the first dose.
In some embodiments, the subject does not exhibit cytokine release syndrome (CRS), does not exhibit severe CRS, does not exhibit neurotoxicity, does not exhibit severe neurotoxicity, or does not exhibit neurotoxicity above grade 3 following administration of the first dose and/or following the administration of the consecutive dose.
Among the CRS-related outcomes are fever, hypotension, hypoxia, neurologic disturbances, or a serum level of an inflammatory cytokines, such as interferon gamma (IFNγ), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFα), IL-6, IL-10, IL-1β, IL-8, IL-2, MIP-1, Flt-3L, fracktalkine, and IL-5, and C reactive protein (CRP). Among the factors, e.g., serum factors, indicative of CRS are inflammatory cytokines such as IFNγ, GM-CSF, TNFα, IL-6, IL-10, IL-1β, IL-8, IL-2, MIP-1, Flt-3L, fracktalkine, and IL-5, and CRP.
In some aspects, the time of administering the consecutive dose(s) is further one at which the subject does not exhibit an immune response, e.g., does not exhibit a detectable adaptive host immune response specific for the receptor (e.g., CAR) expressed by the cells of said first (or prior) dose.
In some embodiments, the time between the administration of the first dose, e.g., the initiation of the administration of the first or prior dose, and the initiation of the administration of the consecutive dose (e.g., the initiation of the administration of the consecutive dose) is greater than about 4 days, e.g., greater than about 5, 6, 7, 8, or 9 days, e.g., greater than about 20 days, e.g., between about 9 and about 35 days, between about 14 and about 28 days, between 15 and 27 days, or between 17 days and about 21 days; and/or at or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 days. In some embodiments, administration of the consecutive dose (e.g., initiation thereof) is more than about 14 days after and less than about 28 days after administration of the first or prior dose (e.g., initiation thereof). In some embodiments, the administration of the consecutive dose is initiated 21 days following the initiation of the first dose. In some embodiments, the time between administration of the first and the consecutive dose (e.g., initiation thereof) or prior and next consecutive dose is greater than about 14 days and less than about 28 days, such as between 15 and 27 days, such as about 21 days. In some embodiments, the time between administration of the first and the consecutive dose (e.g., initiation thereof) is about 17 days.
In some embodiments, at the time of the administration of the consecutive dose, the serum level in the subject of a factor indicative of CRS is less than about 10 times, less than about 25 times, and/or less than about 50 times that in the subject immediately prior to said administration of the first dose; and/or a CRS-related outcome in the subject following said administration of said first dose has reached a peak level and begun to decline following the administration in (a); and/or the subject does not exhibit a detectable humoral or cell-mediated immune response against the receptor (e.g., CAR) expressed by the cells of said first dose. In some embodiments, at the time of the administration in (b) or of the consecutive dose, the serum level of said factor indicative of CRS is no more than ten times the level immediately prior to the administration in (a) or of the first dose.
In some embodiments, the subject has not received a dose of cells expressing the receptor (e.g., CAR) that is expressed by the cells in the first dose prior to the administration in (a). In some embodiments, the receptor (e.g., the CAR) expressed by the cells in the consecutive dose contains at least one immunoreactive epitope present in the receptor (e.g., CAR) expressed by the cells in the first dose. In some embodiments, the receptor in the cells of the consecutive dose is identical or substantially identical to the receptor (e.g. the CAR) expressed by the cells in the first dose. In some embodiments, the receptor expressed by the cells of the first dose specifically binds to an antigen expressed by a cell or tissue of the disease or condition or associated with the disease or condition. In some embodiments, the receptor expressed by the cells of the consecutive dose binds to the same antigen, e.g., to the same epitope, and/or contains the same antigen-binding domain as that in the first dose.
In some embodiments, the disease or condition is a tumor. In some embodiments, it is a cancer, malignancy, neoplasm, or other proliferative disease or disorder, such as leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), ALL, non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B cell lymphoma, B cell malignancies, cancers of the colon, lung, liver, breast, prostate, ovarian, skin, melanoma, bone, and brain cancer, ovarian cancer, epithelial cancers, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin lymphoma, cervical carcinoma, colorectal cancer, glioblastoma, neuroblastoma, Ewing sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and/or mesothelioma. In some embodiments, the disease or condition is a leukemia or lymphoma. In some embodiments, the disease or condition is acute lymphoblastic leukemia. In some embodiments, the disease or condition is non-Hodgkin lymphoma (NHL).
In some embodiments, the disease is a cancer and the subject does not exhibit morphologic disease at the time of initiation of the administration of the consecutive dose. In some embodiments, the disease is a leukemia or lymphoma and the subject does not exhibit greater than 5% blast cells in the bone marrow at the time of the administration of the consecutive dose. In some embodiments, the subject exhibits detectable molecular disease and/or minimum residual disease at the time of the administration of the consecutive dose.
In some embodiments, administration of the first dose leads to a reduction in burden of the disease or condition in the subject, for example, as indicated by a reduction in one or more factors indicative of disease burden following said administration of the first dose, e.g., following the administration in (a). In some embodiments, at the time of the administration in (b) or of the consecutive dose, the subject has not relapsed and/or the one or more factors indicative of disease burden have not increased following the initial reduction experienced after the first dose. In some embodiments, the consecutive dose of cells contains cells in an amount sufficient for reduction in burden of a disease or condition in the subject. In some embodiments, the administration of the consecutive dose leads to a further reduction in burden of the disease or condition in the subject. In some embodiments, administration of the consecutive dose leads to a reduction in burden of the disease or condition in the subject as compared with immediately prior to initiation of the administration of the consecutive dose. In some embodiments, the method reduces burden of the disease or condition to a greater degree and/or for a greater period of time as compared to a method with an alternative dosing regimen wherein the subject is administered the cells in the first dose and the cells in the consecutive dose in a single dose. The reduction in burden and/or further reduction in burden may comprise a reduction in total number of cells, e.g., tumor cells, of the disease in the subject, in an organ of the subject, in a tissue of the subject, or in a bodily fluid of the subject. The reduction may comprise a reduction in molecular detection by flow cytometry or quantitative PCR, mass or volume of a tumor, and/or a reduction in number and/or extent of metastases. In some embodiments, the reduction comprises improvement in survival of the subject, e.g., increased time of survival or incident-free, progression-free, or relapse-free survival.
In some embodiments, the disease or condition persists following the administration of the first dose and/or the administration of the first dose is not sufficient to eradicate the disease or condition in the subject. In some embodiments, the administration of said consecutive dose leads to a reduction in burden of the disease or condition in the subject as compared with immediately prior to initiation of the administration of the consecutive dose.
In some embodiments, the methods reduce burden of the disease or condition to a greater degree and/or for a greater period of time as compared to a method comprising an alternative dosing regimen wherein the subject is administered the cells in (a) (or of the first dose) and the cells in (b) (or of the consecutive dose) in a single dose. In some embodiments, the area under the curve (AUC) of recombinant receptor-expressing (e.g., CAR-expressing) cells over time, and/or the duration of detectable receptor-expressing cells in the subject following the administration of the consecutive dose (or the administration in (b)) is greater as compared to that achieved via a method comprising an alternative dosing regimen wherein the subject is administered the cells in (a) (or the first dose) and the cells in (b) (or the consecutive dose) as a single dose. In some embodiments, the method results in a maximum concentration or number of receptor-expressing (e.g., CAR-expressing) cells in the blood of the subject of at least at or about 10 receptor-expressing cells per microliter, at least at or about 100 receptor-expressing cells per microliter, at least 50% of the total number of peripheral blood mononuclear cells (PBMCs), at least at least about 1×105 receptor-expressing cells, or at least 5,000 copies of recombinant receptor-encoding (e.g., CAR-encoding) DNA per micrograms DNA, or at least 10,000 copies of recombinant receptor-encoding DNA per micrograms of DNA. In some embodiments, at day 90 following the initiation of the administration in (a) or of the first dose, recombinant receptor-expressing (e.g., CAR-expressing) cells are detectable in the blood or serum of the subject, e.g., the blood of the subject contains at least 10%, at least 20%, at least 40%, or at least 50%, receptor (e.g., CAR)-expressing cells, at least 10 receptor (e.g., CAR)-expressing cells per microliter or at least 1×104 receptor (e.g., CAR)-expressing cells.
In some embodiments, the AUC for blood concentration of receptor-expressing (e.g., CAR-expressing) cells over time following the administration in (a) or of the first dose is greater as compared to that achieved via a method comprising an alternative dosing regimen wherein the subject is administered the cells in (a) or of the first dose and the cells in (b) or of the second dose as a single dose.
In some embodiments, a CRS-related outcome in the subject at day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 following the administration in (b) or of the consecutive dose is not detectable or is reduced as compared to a method comprising an alternative dosing regimen wherein the subject is administered the cells in (b) or the consecutive dose without having been administered the first dose.
In some embodiments, the AUC for a serum level of a factor indicative of CRS over time in the subject following the administration in (b) or the consecutive dose is lower as compared to that of a method comprising an alternative dosing regimen wherein the subject is administered the cells in (b) or the consecutive dose without having been administered the first dose.
In some embodiments, the subject has been treated with a therapeutic agent targeting the tumor prior to the administration of the first dose. In some aspects, the subject is refractory or non-responsive to said therapeutic agent at the time of the administration of the first dose and/or the consecutive dose. In some embodiments, subsequent to administration in (a) or the first dose and before said administration in (b) or the consecutive dose, or prior to administration in (a) or the first dose, the methods further include assessing a serum level of a factor indicative of CRS, a factor indicative of disease burden, and/or an indicator of a host anti-recombinant receptor (e.g., anti-CAR) immune response in the subject, such as a humoral or cell-mediated immune response. In some such embodiments, the factor indicative of disease burden detected is or comprises a total number of cells of the disease in the subject, in an organ of the subject, in a tissue of the subject, or in a bodily fluid of the subject, molecular detection by flow cytometry or quantitative PCR, mass or volume of a solid tumor, or number or extent of metastases.
In some embodiments, the methods include assessing a factor indicative of disease burden prior to administration of the consecutive dose, and based on the result of the assessment, determining the consecutive dose of cells to be administered to the subject. In some embodiments, if the assessment determines that the subject has morphologic disease, the subject is administered a consecutive dose containing less than or about the same number of recombinant receptor- (e.g., CAR-) expressing cells as the number of recombinant receptor- (e.g., CAR-) expressing cells in the first dose. In some embodiments, if the assessment determines that the subject has minimal residual disease, the subject is administered a consecutive dose containing an increased number of receptor- (e.g., CAR-) expressing cells as compared to the first dose. In some embodiments, the consecutive dose comprises about the same number of recombinant receptor-expressing (e.g., CAR-expressing) cells as the number in the first dose. In some embodiments, the number of receptor- (e.g., CAR-) expressing cells per kilogram administered in the consecutive dose is less than or about less than or is the same or about the same as the number of receptor- (e.g., CAR-) expressing cells per kilogram administered in the first dose. In other embodiments, the number of receptor-expressing (e.g., CAR-expressing) cells per kilogram administered in the consecutive dose is greater than the number of receptor-expressing (e.g., CAR-expressing) cells per kilogram administered in the first dose. In some embodiments, the consecutive dose comprises an increased number of such cells as compared to the first dose, such as at least 2-fold, 5-fold, or 10-fold greater than the number in the first dose. In some embodiments, the number of receptor-expressing (e.g., CAR-expressing) cells per kilogram administered in the consecutive dose is at least at or about 2 times or at or about 3 times greater than the number of receptor-expressing (e.g., CAR-expressing) cells per kilogram administered in the first dose.
In some embodiments, the receptor-expressing (e.g., CAR-expressing) cells in the first dose expand in the subject following administration of the first dose and/or following the administration of the consecutive dose. In some embodiments, the expansion is evidenced by an increase in serum CRP level following the administration of the first dose and/or consecutive dose as compared to just prior to the administration. In some embodiments, the expansion is evidenced by an increase in a level of receptor- (e.g., CAR-) encoding nucleic acid in the serum, as measured by qPCR, following the administration of the first dose and/or consecutive dose as compared to just prior to the administration. In some embodiments, the increase is at least 1, 2, or 3-fold.
In some embodiments, the first and/or consecutive dose is not a split does. For example, in some embodiments, the cells of the first dose are administered in a single pharmaceutical composition comprising the cells of the first dose and/or the cells of the consecutive dose are administered in a single pharmaceutical composition comprising the cells of the consecutive dose. In other embodiments, the first and/or consecutive dose is a split dose, for example, where the cells of the first dose are administered in a plurality of compositions, collectively comprising the cells of the first dose, over a period of no more than three days; and/or the consecutive dose is a split dose, where the cells of the consecutive dose are administered in a plurality of compositions, collectively comprising the cells of the consecutive dose, over a period of no more than three days.
In some embodiments, the methods include administering a consecutive dose of cells expressing a recombinant receptor (e.g., a chimeric antigen receptor (CAR)) to a subject previously administered a first dose of cells expressing a CAR. In some embodiments, the consecutive dose of cells is administered at a time point that is at least or more than about 14 days after and less than about 28 days after initiation of the first dose. In some embodiments, the number of receptor- (e.g., CAR-) expressing cells administered in the consecutive dose is increased as compared to the first dose.
In some embodiments, the number of cells administered in the first dose is between about 0.5×106 cells/kg body weight of the subject and 3×106 cells/kg, between about 0.75×106 cells/kg and 2.5×106 cells/kg or between about 1×106 cells/kg and 2×106 cells/kg, each inclusive.
In some embodiments, the number of cells administered in the consecutive dose of receptor- (e.g., CAR-) expressing cells is between about 2×106 cells per kilogram (cells/kg) body weight and about 6×106 cells/kg, between about 2.5×106 cells/kg and about 5.0×106 cells/kg, or between about 3.0×106 cells/kg and about 4.0×106 cells/kg, each inclusive.
In some embodiments, the number of receptor- (e.g., CAR-) expressing cells administered in the first dose is at or about or no more than at or about 1×106 per kilogram of the subject and/or the number of receptor- (e.g., CAR-) expressing cells administered in the consecutive dose is at or about 3×106 per kilogram of the subject.
In some embodiments, the methods further include administering a chemotherapeutic agent prior to the administration in (a) or prior to the first dose and/or prior to the administration in (b) or prior to the consecutive dose. In some embodiments, the subject has been previously treated with a chemotherapeutic agent prior to the administration in (a). In some embodiments, the chemotherapeutic agent is or comprises a conditioning chemotherapy, which reduces burden of the disease or condition in the subject prior to the first dose and/or consecutive dose. In some embodiments, the chemotherapeutic agent is cyclophosphamide, fludarabine, and/or a combination thereof. In some embodiments, the administration of the chemotherapeutic agent includes administration of a chemotherapeutic agent prior to the administration of the first dose and optionally not prior to the administration of the consecutive dose. In some embodiments, the chemotherapeutic agent is administered between 2 and 5 days prior to the administration of the first dose. In some embodiments, the chemotherapeutic agent is administered between 2 and 5 days prior to the administration of the second dose. In some embodiments, the chemotherapeutic agent is administered between 2 and 5 days prior to the administration first dose and between 2 and 5 days prior to the administration of the second dose. In some embodiments, the chemotherapeutic agent is administered at a dose of between at or about 1 g/m2 of the subject and at or about 3 g/m2 of the subject.
In some embodiments, the subject has received cryoreductive chemotherapy prior to the administration or the first dose. In some embodiments, the method further includes the administration of cryoreductive chemotherapy prior to the administration of the first dose.
Also provided are cells and compositions for use and uses of cells and compositions for treating a disease or condition in a subject, such as a tumor or cancer, where said cells contain recombinant receptor- (e.g., chimeric antigen receptor (CAR)-) expressing cells for treatment of a disease or condition in a subject or for the manufacture of a medicament for treatment of a disease or condition in a subject previously treated with recombinant receptor-expressing (e.g., CAR-expressing) cells. In some embodiments, the compositions or cells for use or medical uses are for use 14 to 28 days after the previous treatment. In some embodiments, the compositions or cells for use are formulated for administration of a consecutive dose in an amount sufficient for reduction in burden of a disease or condition in the subject having been previously treated with the recombinant receptor-expressing (e.g., CAR-expressing) cells.
In some embodiments of such medical uses, the compositions or cells are for use that includes administering to a subject having the disease or condition a first dose of cells expressing the receptor (e.g., CAR). In some embodiments, the first dose contains no more than about 1×106 of the cells per kilogram body weight of the subject, no more than about 1×108 of the cells, and/or no more than about 1×108 of the cells/m2 of the subject. In some embodiments, the compositions or cells are for use that includes administering to the subject a consecutive dose of cells expressing a receptor (e.g., CAR) at a time point that is at least or more than about 14 days after and less than about 28 days after initiation of the administration of the first dose.
In some embodiments, the cells for use are cells expressing a recombinant receptor (e.g., chimeric antigen receptor (CAR)) are for use in methods of treating a disease in a subject previously treated with receptor- (e.g., CAR-) expressing cells. In some embodiments, the cells are for use between about 14 and 28 days after the previous treatment. In some embodiments, the cells for use are formulated for administration of a consecutive dose in an amount sufficient for reduction in burden of a disease or condition in the subject having been previously treated with the receptor- (e.g., CAR-) expressing cells.
In some embodiments of any such compositions or cells for use or medical uses, the subject does not exhibit morphologic disease and/or the subject does not exhibit greater than 5% blast cells in the bone marrow.
In some embodiments, the compositions or cells are for use in a method including administering to a subject having the disease or condition a first dose of cells expressing the receptor (e.g., CAR). In some embodiments, the first dose contains no more than about 1×106 of the cells per kilogram body weight of the subject, no more than about 1×108 of the cells, and/or no more than about 1×108 of the cells/m2 of the subject. In some embodiments, the cells are for use in a method that includes administering to the subject a consecutive dose of cells expressing a receptor (e.g., CAR) at a time point that is at least or more than about 14 days after and less than about 28 days after initiation of said administration of the first dose.
In some embodiments, the compositions or cells for use are formulated for administration in an amount that does not induce severe CRS in the subject or does not induce CRS in the subject. In some embodiments, the cells for use are formulated for administration in an amount that does not induce grade 3 or higher neurotoxicity in the subject. In some embodiments, the cells for use are formulated for administration in an amount that, based on clinical data, does not induce severe CRS in a majority of subjects so-treated. In some embodiments, the cells for use are formulated for administration in an amount that, based on clinical data, does not induce grade 3 or higher neurotoxicity in a majority of subjects so-treated.
In some embodiments, the use of cells expressing a receptor (e.g., chimeric antigen receptor (CAR)) are for manufacture of a medicament for the treatment of a disease or condition in a subject includes cells that are formulated and/or packaged for administration to the subject in a first and a consecutive dose. In some embodiments, the treatment includes administering the cells to the subject in a first and a consecutive dose, where the first dose includes no more than about 1×106 of the cells per kilogram body weight of the subject, no more than about 1×108 of the cells, and/or no more than about 1×108 of the cells/m2 of the subject.
In some embodiments of any such compositions or cells for use or medical uses, the consecutive dose is for administration at a time point that is at least or more than about 14 days after and less than about 28 days after initiation of the first administration. In some embodiments, the consecutive dose is for administration at a time point at which the serum level in the subject of a factor indicative of cytokine release syndrome (CRS) is less than about 10 times, less than about 25 times, and/or less than about 50 times that in the subject immediately prior to said first administration. In some embodiments, the consecutive dose is for administration at a time point at which the subject does not exhibit grade 3 or higher neurotoxicity. In some embodiments, the consecutive dose is for administration at a time point at which a CRS-related outcome or symptom of neurotoxicity in the subject following said administration of said first dose has reached a peak level and begun to decline following the first administration. In some embodiments, the consecutive dose is for administration at a time point at which the subject does not exhibit a detectable humoral or cell-mediated immune response against the receptor (e.g., CAR) expressed by the cells of said first dose.
In some embodiments, the cells for use are formulated and/or packaged for administration to the subject in a first and a consecutive dose and/or the treatment includes administering the cells to the subject in a first and a consecutive dose. In some embodiments, the first dose contains no more than about 1×106 of the cells per kilogram body weight of the subject, no more than about 1×108 of the cells, and/or no more than about 1×108 of the cells/m2 of the subject.
In some embodiments, the use includes where the first and consecutive administrations include administering the cells in one or more unit dose, each unit dose comprising about between 5×107 of the cells and about 5×108 cells, about between 5×107 of the cells and about 2.5×108 cells or about between 2.5×108 cells and 4×108 cells. In some embodiments, the cells are formulated in a unit dose comprising no more than about 5×107 cell, no more than about 1×108 cells, no more than about 2×108 of the cells, no more than about 2.5×108 of the cells, no more than about 3.0×108 of the cells or no more than about 4×108 of the cells.
In some embodiments, the cells or use includes where the first administration comprises administering a single unit dose. In some embodiments, the cells or use includes where the consecutive administration comprises administration of two or more unit doses. In some embodiments, the cells or use includes where the consecutive administration comprises administration a single unit dose.
In some embodiments, the cells composition for use or use includes where the disease or condition is a tumor or a cancer. In some embodiments, the cells or composition for use or use includes where the tumor or cancer is leukemia or lymphoma. In some embodiments, the composition or cells are for use in treating acute lymphoblastic leukemia. In some embodiments, the composition or cells are for use in treating non-Hodgkin lymphoma (NHL).
In some embodiments, the cells, composition, or use includes where the consecutive dose is formulated for administration of less than or about the same number of recombinant receptor-expressing (e.g., CAR-expressing) cells as the number of recombinant receptor-expressing (e.g., CAR-expressing) cells in the previous dose. In some embodiments, the composition containing the cells of the consecutive dose is formulated for administration of an increased number of recombinant receptor-expressing (e.g., CAR-expressing) cells as compared to the first dose or previous dose.
Also provided are articles of manufacture for carrying out the methods. In some embodiments, the article of manufacture includes a plurality of containers, e.g., sealable containers, each individually comprising a unit dose of cells expressing the recombinant receptor, e.g., chimeric antigen receptor (CAR), for administration to the subject, packaging material, and/or a label or package insert.
In some embodiments, the unit dose comprises the amount of cells to be given in the lowest dose in the methods, such as the size of the first dose. In some embodiments, the unit dose includes no more than about 1×108 of the cells, no more than about 5×107 of the cells, no more than about 1×106 of the cells per kg of the subject, or no more than about 5×105 of the cells per kg of the subject.
In some embodiments, the label or package insert includes instructions for administering a plurality of the unit doses to the subject, for example, by administering a certain number of such unit doses, e.g., one unit dose, in administration of a first dose, and then administering a consecutive dose including one or a plurality of the unit doses. In some embodiments, the instructions specify carrying out a first administration, said first administration comprising delivering one of said unit doses to the subject, and carrying out a consecutive administration, said consecutive administration comprising administering one or a plurality of said unit doses to the subject. In some embodiments, they specify that the consecutive administration is to be carried out at a time between about 15 and about 27 days following said first administration. In some embodiments, the instructions specify that the consecutive administration is to be carried out at a time after which it has been determined that a serum level of a factor indicative of cytokine-release syndrome (CRS) in the subject is less than about 10 times, less than about 25 times, and/or less than about 50 times the serum level of said indicator in the subject immediately prior to said previous dose, or at a time after which it has been determined that an indicator of CRS has peaked and is declining, such as is at less than at or about 40%, 30%, 20%, or 10% of the peak value, and/or that the subject does not exhibit a detectable adaptive host immune response specific for the receptor (e.g., CAR) expressed by the cells of the previous dose. In some embodiments, the containers are or comprise flexible cell infusion bags. In some embodiments, the cells to be administered and/or in the containers or unit doses are for autologous transfer. Thus, in some embodiments, the cells have been derived from the subject to which they are to be administered. In some embodiments, the methods are for allogeneic administration. In some embodiments, the label and/or packaging material further includes an identifier specific to the subject, indicating that the cells were derived from the subject and/or should be administered to the subject specifically.
In some embodiments, the cells are primary cells, such as primary human immune cells, e.g., PBMCs, T cells, and/or NK cells. In some embodiments, the cells comprise CD8+ and/or CD4+ T cells. In some embodiments, the T cells are autologous to the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows disease responses of subjects with morphological or molecular disease (disease burden at time of treatment initiation) treated with a single infusion of varying doses of CAR-expressing T cells. MRD−CR=no minimal residual disease, complete remission; MRD+CR=minimal residual disease, complete remission; NR=not responsive
FIG. 1B shows the presence (Y) or absence (N) of severe cytokine release syndrome (CRS) in subjects with morphological or molecular disease (disease burden at time of treatment initiation) treated with a single infusion of varying doses of CAR-expressing T cells.
FIG. 1C shows the presence (Y) or absence (N) of severe neurotoxicity in subjects with morphological or molecular disease (disease burden at time of treatment initiation) treated with a single infusion of varying doses of CAR-expressing T cells.
FIG. 2 shows peak C reactive protein (CRP) levels in subjects exhibiting morphological or molecular disease at the time of administration of a first dose (Infusion=#1) (left-hand panel) and in subjects exhibiting morphological or molecular disease at the time of administration of a consecutive dose (Infusion=#2) (right-hand panel) of CAR-expressing T cells. Numbers and text/abbreviations (e.g., “MRD−CR”, “MRD+CR,” etc.) shown next to individual data points reflect patient numbers and disease burden following the indicated infusion. MRD+=minimum residual disease; MRD−=no minimum residual disease; CR=complete remission.
FIG. 3A depicts surface expression, as detected by flow cytometry, of PD-1, PD-L1, and PD-L2 on a population of T cells gated for positive surface expression of CD4 and an anti-CD19 chimeric antigen receptor (CAR) (gating strategy shown in top panel), following incubation for 24 hours under various conditions (media, K562-tCD19, K562-tROR1, aCD3/aCD28), as described in Example 8.
FIG. 3B depicts surface expression, as detected by flow cytometry, of PD-1, PD-L1, and PD-L2 on a population of T cells gated for positive surface expression of CD4 and negative surface expression of an anti-CD19 chimeric antigen receptor (CAR) (gating strategy shown in top panel), following incubation for 24 hours under various conditions (media, K562-tCD19, K562-tROR1, aCD3/aCD28), as described in Example 8.
FIG. 4A depicts surface expression, as detected by flow cytometry, of PD-1, PD-L1, and PD-L2 on a population of T cells gated for positive surface expression of CD8 and an anti-CD19 chimeric antigen receptor (CAR) (gating strategy shown in top panel), following incubation for 24 hours under various conditions (media, K562-tCD19, K562-tROR1, aCD3/aCD28), as described in Example 8.
FIG. 4B depicts surface expression, as detected by flow cytometry, of PD-1, PD-L1, and PD-L2 on a population of T cells gated for positive surface expression of CD8 and negative surface expression for an anti-CD19 chimeric antigen receptor (CAR) (gating strategy shown in top panel), following incubation for 24 hours under various conditions (media, K562-tCD19, K562-tROR1, aCD3/aCD28), as described in Example 8.
FIG. 5A shows the degree of neurotoxicity (Grade 0-2, Grade 3, Grade 4-5) observed in subjects treated with a single infusion of CAR-expressing T cells. Data are plotted as tumor burden (percent marrow blasts) vs. number of CAR-expressing T cells (CD8+/EGFR+, top panel; or CD4+/EGFR+, bottom panel) per μL of peripheral blood.
FIG. 5B shows the requirement for care in the intensive care unit (ICU) in subjects treated with a single infusion of CAR-expressing T cells. Data are plotted as tumor burden (percent marrow blasts) vs. number of CD8+/EGFR+ (top panel) or CD4+/EGFR+ (bottom panel) cells per μL of peripheral blood.
FIG. 6A shows the number of CD8+CAR-expressing T cells (CD8+/EGFR+) per μL of peripheral blood of subjects treated with a single infusion of 2×105 or 2×106 CAR-expressing T cells over a 28 day period following the infusion, as measured by flow cytometry. Prior to the infusion, subjects were pre-conditioned with 2 g/m2 cyclophosphamide with or without 3 doses of 100 mg/m2 etoposide (No Flu), or were treated with 60 mg/kg (˜2 g/m2) cyclophosphamide and 3 to 5 doses of 25 mg/m2 fludarabine (Flu).
FIG. 6B shows the number of CD4+CAR-expressing T cells (CD4+/EGFR+) per μL of peripheral blood of subjects treated with a single infusion of 2×105 or 2×106 CAR-expressing T cells over a 28 day period following the infusion, as measured by flow cytometry. Prior to the infusion, subjects were pre-conditioned with 2 g//m2 cyclophosphamide with or without 3 doses of 100 mg/m2 etoposide (No Flu), or were treated with 60 mg/kg (˜2 g/m2) cyclophosphamide and 3 to 5 doses of 25 mg/m2 fludarabine (Flu).
FIG. 6C shows percent disease-free survival curves for subjects treated with a single infusion of 2×105 or 2×106 CAR-expressing T cells. Prior to the infusion, subjects were pre-conditioned with 2 g/m2 cyclophosphamide with or without 3 doses of 100 mg/m2 etoposide (No Flu), or were treated with 60 mg/kg (˜2 g/m2) cyclophosphamide and 3 to 5 doses of 25 mg/m2 fludarabine (Flu).
FIG. 7A shows the number (top panel) and percent (bottom panel) of CD8+CAR-expressing T cells (CD8+/EGFR+) per μL of peripheral blood of subjects treated with a single infusion of 2×107 CAR-expressing T cells over a 28 day period following the infusion, as measured by flow cytometry. Prior to the infusion, subjects were pre-conditioned with 2-4 g/m2 cyclophosphamide with or without 3 doses of 100-200 mg/m2 etoposide (No Flu), or were treated with 30-60 mg/kg (˜1-2 g/m2) cyclophosphamide and 3 to 5 doses of 25 mg/m2 fludarabine (Cy/Flu).
FIG. 7B shows the number (top panel) and percent (bottom panel) of CD4+ CAR-expressing T cells (CD4+/EGFR+) per μL of peripheral blood of subjects treated with a single infusion of 2×107 CAR-expressing T cells over a 28 day period following the infusion, as measured by flow cytometry. Prior to the infusion, subjects were pre-conditioned with 2-4 g/m2 cyclophosphamide with or without 3 doses of 100-200 mg/m2 etoposide (No Flu), or were treated with 30-60 mg/kg (˜1-2 g/m2) cyclophosphamide and 3 to 5 doses of 25 mg/m2 fludarabine (Cy/Flu).
DETAILED DESCRIPTION
I. Methods of Treatment with Cells Expressing Recombinant Receptors
Provided are methods, compositions, and articles of manufacture for use in cell therapy, for the treatment of diseases or conditions including various tumors. The methods involve administering engineered cells expressing recombinant receptors designed to recognize and/or specifically bind to molecules associated with the disease or condition and result in a response, such as an immune response against such molecules upon binding to such molecules. The receptors may include chimeric receptors, e.g., chimeric antigen receptors (CARs), and other transgenic antigen receptors including transgenic T cell receptors (TCRs).
In particular, the methods involve administering one or more consecutive doses of cells to subjects having received a first dose, and/or administering the first and one or more consecutive doses. The doses generally are administered in particular amounts and according to particular timing parameters. In some embodiments, the methods generally involve administering a first dose of cells, thereby reducing disease burden, followed by a consecutive dose of cells, administered during a particular time window with respect to the first dose, or the administration of the consecutive dose to a subject having received such a first dose. The first dose is often a relatively low dose. In some embodiments, additional consecutive doses then are administered, for example, within the same or a similar window of time with respect to the consecutive dose. In some embodiments, the number of cells administered and timing of the multiple doses are designed to improve one or more outcomes, such as to reduce the likelihood or degree of toxicity to the subject, improve exposure of the subject to and/or persistence of the administered cells, and/or improve therapeutic efficacy. Also provided are articles of manufacture containing the cells and designed for administration following such dosing regimens.
In some embodiments, the provided methods are based on observations herein that increased exposure of the subject to the administered cells (e.g., increased number of cells or duration over time) can improve efficacy and therapeutic outcomes in adoptive cell therapy. Preliminary analysis conducted following the administration of different CD19-targeting CAR-expressing T cells to subjects with various CD19-expressing cancers in multiple clinical trials revealed a correlation between greater and/or longer degree of exposure to the CAR-expressing cells and treatment outcomes. Such outcomes included patient survival and remission, even in individuals with severe tumor burden.
Yet delivering high initial doses of the recombinant immunostimulatory cells does not necessarily increase exposure. Particularly in the context of high disease burden (and thus higher amounts of antigen), administering large doses does not necessarily enhance efficacy and can lead to increased or rapid expansion of the cells and result in toxicity. Certain reports have indicated a lack of correlation between dose and toxicity. See Park et al, Molecular Therapy 15(4):825-833 (2007). On the other hand, higher initial doses can promote toxic outcomes such as cytokine release syndrome (CRS), particularly in the context of high disease burden. Moreover, high doses do not necessarily translate to increased persistence of the administered cells, and thus do not necessarily increase exposure over time. See Park et al, Molecular Therapy 15(4):825-833 (2007). Likewise, administering cells in the context of high disease burden, often present at the outset of treatment, can lead to exhaustion of the transferred cells, thereby reducing clinical efficacy. See Davila & Brentjens, Hematol Oncol Clin North Am. 27(2):341-53 (2013).
Administering subsequent doses may not be effective, particularly following relapse and/or where the subject has mounted an immune response specific for the cells or the recombinant receptors the express. Improved methods are needed to increase cell exposure over time while avoiding toxic outcomes.
The provided methods offer advantages over other approaches aimed at addressing the risk of toxic outcomes and/or improving efficacy. Many such approaches have focused, for example, on targeting downstream effects of toxicity, such as by cytokine blockade, and/or delivering agents such as high-dose steroids which can also eliminate or impair the function of administered cells. Many of these other approaches also do not prevent other forms of toxicity such as neurotoxicity, which can be associated with adoptive cell therapy. On the other hand, administering relatively low doses of cells (e.g. CAR-expressing cells) may decrease the risk but may not be completely effective. Delivery of subsequent doses of cells, for example, after relapse following an initial administration, also has not been entirely satisfactory. Such dosing approaches can lead to limited or ineffective responses of the subsequent dose, due to host immune responses mounted against the initial dose.
Provided are methods involving the administration of consecutive doses of cell therapy in a way that minimizes risk of toxicity and maximizes efficacy. As observed herein, following administration of different types of CD19-targeting CAR T cells to human subjects with B cell cancers, initial doses equal to or below 1×106 cells per kilogram body weight were effective in reducing tumor burden. These low doses also were associated with less frequent severe CRS, neurotoxicity, and other adverse events compared with higher doses. In some embodiments, the provided methods involve the safe initial administration of such low doses, followed by consecutive dose(s) for increased exposure, with dose amounts and timing designed to avoid impairment of efficacy by host immune responses while minimizing risk of toxicity
In some cases, if desired or need in a particular disease or context, the provided methods involve a consecutive dose of cells administered at an increased number, and hence at a higher dose, than the first dose of cells. As shown herein, in some aspects, administration of higher doses of cells is advantageous compared to lower doses. In some embodiments, administration of higher doses of cells, such as doses greater than or equal to 2.5×106 cells/kg or higher, are associated with an increased overall survival in subjects, particularly in subjects that exhibit morphological disease prior to treatment. In some embodiments, however, these higher doses can be associated with toxic outcomes, and in particular with neurotoxicity, such as severe neurotoxicity, for example grade 3 neurotoxicity or higher, particularly in subjects with morphological disease prior to treatment. In some cases, the toxic outcome following administration of high doses of recombinant receptor-expressing (e.g., CAR-expressing) cells is not observed, or is not observed to as great of an extent, when subjects with a reduced disease burden are treated, such as subjects with minimal residual disease having molecularly detectable disease. In some embodiments, methods of first administering a low dose of recombinant receptor-expressing (e.g., CAR-expressing) cells can reduce the disease burden in subjects, such as from morphological disease status to minimal residual disease, so that subsequent administration of a higher dose of recombinant receptor-expressing (e.g., CAR-expressing) cells in subjects is less likely to cause toxic outcomes in a majority of subjects treated. In some embodiments, the provided methods avoid the problems of toxicity associated with administration with higher doses, while maximizing the efficacy of treatment that can occur upon administration with higher numbers of recombinant receptor-expressing (e.g., CAR-expressing) cells.
In some embodiments, the methods include administering an initial dose of recombinant receptor-expressing (e.g., CAR-expressing) cells that can expand in the presence of disease-associated antigens and reduce disease burden but without the same degree of toxic outcomes that may be associated with a higher dose. In some aspects, the first dose is a low dose, such as a dose of less than at or about or no more than at or about 1×106 cells per kilogram body weight of the subject (cells/kg), 0.5×106 cells/kg or 1×105 cells/kg. In some aspects, the first dose is in an amount or number of cells that has been observed not to cause toxic outcomes in the disease or condition or subject, such as, in some embodiments, CRS or neurotoxicity. In some embodiments, the first dose is an amount or number of cells that has been associated with such outcomes, such as based on clinical data, in only a relatively small percentage of patients, such as no more than 50, 40, 25, 20, 15, 10, 5, or fewer percent of subjects.
In some embodiments, the first dose is generally also large enough to be effective in reducing disease burden. In some cases, the first dose is large enough to reduce disease burden if the cell dose is sufficient to expand in vivo and debulk disease. In some embodiments, the cells of the first dose thereby debulk or reduce disease burden, e.g., tumor size, without effecting severe unwanted outcomes. In some cases, the first dose is an amount of cells that is effective to reduce tumor burden, such as by reducing disease from a morphological setting to minimum residual disease and/or clinical or complete remission. In some aspects, the initial dose is a low dose. In some aspects, for example, in the context of relatively low disease burden, the first dose may be higher.
In some aspects, a risk-adapted dosing regime is used for determining the appropriate number or amount or relative number or amount of cells or recombinant receptor-expressing (e.g., CAR-expressing) cells in the dose. For example, in some aspects, prior to infusion of recombinant receptor-expressing (e.g., CAR-expressing) cells, the disease burden of the subject is determined and, based on the disease burden, a first dose of recombinant receptor-expressing (e.g., CAR-expressing) cells (e.g. low or high dose) is selected that can minimize toxicity and maximize efficacy, for example, based on observations described above and elsewhere herein in which higher doses of recombinant receptor-expressing cells (e.g., CAR-expressing, such as CAR-expressing T cells) can be associated with toxic outcomes, such as severe neurotoxicity, in subjects having morphological disease burden, which are not necessarily observed, or are not observed to as great of an extent, in subjects with relatively lower disease burden. For example, subjects with a high marrow tumor burden prior to treatment, which, in some cases, can be associated with a greater CAR T-cell expansion following treatment, were observed to have a higher risk of developing severe neurotoxicity that may require ICU care.
In some embodiments, in order to maximize efficacy in subjects that are not, or are less, susceptible to a toxic outcome following in infusion of recombinant receptor-expressing (e.g., CAR-expressing) cells, e.g. CAR-T cell infusion, (such as to subjects that do not exhibit morphologic disease (i.e. have non-morphologic disease) or do not exhibit substantial morphologic disease), a relatively higher dose of cells, such as greater than 1×106 cells/kg, 2×106 cells/kg, 5×106 cells/kg or 1×107 cells/kg, can be administered. Conversely, in some aspects, subjects that are determined to have a relatively higher disease burden, such as by the presence of morphologic disease or substantial morphologic disease, can be administered a relatively lower dose of cells than administered to subjects that do not exhibit morphologic disease or do not exhibit substantial morphologic disease, such as a dose of less than or equal to or about 1×106 cell/kg or less than or equal to or about 0.5×106 cells/kg. In some embodiments, one or more further consecutive doses can be administered, which, optionally, can be administered based on the extent or degree of disease burden as described above or alternatively be at a fixed dose regardless of disease burden. In some embodiments, a subject exhibits morphologic disease or substantial morphological disease burden if there are greater than or equal to or about 5% blasts present in the bone marrow. In some embodiments, a subject with higher relative disease burden, such has high marrow tumor burden, has greater than, equal to or greater than about 10% blasts in the bone marrow or greater than, equal to or greater than about 20% blasts in the bone marrow.
In some embodiments, a subject is assessed for disease burden prior to treatment, and, if the subject exhibits less than 5% bone marrow blast cells, the subject can be administered a dose of recombinant receptor-expressing (e.g., CAR-expressing, such as CAR-expressing T cells) of greater than 1×106 cells/kg, 2×106 cells/kg, 5×106 cells/kg or 1×107 cells/kg, or if the subject exhibits greater than or equal to 5% bone marrow blast cells, the subject can be administered a dose of recombinant receptor-expressing (e.g., CAR-expressing, such as CAR-expressing T cells) of less than or equal to or about 1×106 cell/kg or 0.5×106 cells/kg. In some embodiments, a subject is assessed for disease burden prior to treatment, and, if the subject exhibits less than 10% bone marrow blast cells, the subject can be administered a dose of recombinant receptor-expressing (e.g., CAR-expressing, such as CAR-expressing T cells) of greater than 1×106 cells/kg, 2×106 cells/kg, 5×106 cells/kg or 1×107 cells/kg, or if the subject exhibits greater than or equal to 10% bone marrow blast cells, the subject can be administered a dose of recombinant receptor-expressing (e.g., CAR-expressing, such as CAR-expressing T cells) of less than or equal to or about 1×106 cell/kg or 0.5×106 cells/kg. In some embodiments, a subject is assessed for disease burden prior to treatment, and, if the subject exhibits less than 20% bone marrow blast cells, the subject can be administered a dose of recombinant receptor-expressing (e.g., CAR-expressing, such as CAR-expressing T cells) of greater than 1×106 cells/kg, 2×106 cells/kg, 5×106 cells/kg or 1×107 cells/kg, or if the subject exhibits greater than or equal to 20% bone marrow blast cells, the subject can be administered a dose of recombinant receptor-expressing (e.g., CAR-expressing, such as CAR-expressing T cells) of less than or equal to or about 1×106 cell/kg or 0.5×106 cells/kg. In some embodiments, the disease burden is assessed and a risk-adapted dose is selected prior to the first dose of recombinant receptor-expressing (e.g., CAR-expressing, such as CAR-expressing T cells). In some embodiments, the disease burden is assessed and a risk-adapted dose is selected prior to one or more consecutive doses of recombinant receptor-expressing (e.g., CAR-expressing, such as CAR-expressing T cells).
In some embodiments, a consecutive dose of recombinant receptor-expressing (e.g., CAR-expressing) cells is administered to the subject at a time after administration of the first or initial dose of cells in which it is likely that tumor burden of the subject has been reduced by the first dose. In some embodiments, it is not necessary that the tumor burden actually be reduced in all subjects prior to administration of the consecutive dose, but that tumor burden is reduced on average in subjects treated, such as based on clinical data, in which a majority of subjects treated with such a first dose exhibit a reduced tumor burden, such as at least 50%, 60%, 70%, 80%, 90%, 95% or more of subjects treated with the first or initial dose exhibit a reduced tumor burden. Generally at a point in time after disease burden has been reduced by the first dose or is likely to have been reduced by the first dose, a consecutive dose is administered to the subject, thereby further reducing and/or eliminating disease or a symptom or outcome thereof or preventing expansion or progression thereof. The context of reduced disease burden at the time of the consecutive administration in some aspects reduces the likelihood of exhaustion of the transferred cells, thereby improving efficacy. The consecutive dose may be the same, lower, or a higher dose as compared with the first dose. In some embodiments, multiple consecutive doses are administered after a first dose.
In some embodiments, the consecutive dose of recombinant receptor-expressing (e.g., CAR-expressing) cells is administered at a dose that is higher than the first dose so that an increased number of recombinant receptor-expressing (e.g., CAR-expressing) cells is administered to the subject by the consecutive dose. In some embodiments, a higher dose of recombinant receptor-expressing (e.g., CAR-expressing) cells is one that can promote an increased response or efficacy, such as improved or greater reduction in tumor burden and/or an improved or greater overall survival time of the subject compared to that achieved by administration of a lower dose or number of cells. In some embodiments, because administration of the first dose of cells can reduce tumor burden in the subject, administration of the consecutive dose at a higher number of cells can avoid or minimize CRS and/or neurotoxicity in the subject after administration of the consecutive dose that can otherwise occur in subjects with morphological disease.
In some embodiments, prior to administration of the consecutive dose, the subject is one that exhibits non-morphological disease, such as molecularly detectable disease and/or minimal residual disease. In some embodiments, a subject can be assessed for tumor burden after administration of the first dose and prior to administration of the consecutive dose to confirm that tumor burden has been reduced compared to tumor burden present prior to treatment with the first dose. In some embodiments, if assessment of the subject indicates tumor burden is reduced and/or that the subject exhibits non-morphological disease, for example molecularly detectable disease and/or minimal residual disease, the provided methods include administering a consecutive dose of recombinant receptor-expressing (e.g., CAR-expressing) cells that is higher than the first or initial dose. In some embodiments, if assessment of the subject indicates tumor burden is not reduced and/or the subject exhibits morphological disease, the provided methods include administering a consecutive dose of recombinant receptor-expressing (e.g., CAR-expressing) cells that is the same as or less than the first or initial dose.
In some embodiments, the timing of the consecutive dose(s) in relation to the first and/or one another is designed to reduce the risk of unwanted toxic outcomes and promote maximum efficacy. In some embodiments, the consecutive dose, such as the same, lower or higher consecutive dose, is administered at a time at which disease burden remains reduced in the subject or reduced in subjects on average, such as based on clinical data, but at which the risk of CRS and/or neurotoxicity remain low. In some embodiments, methods are provided in which CRS and/or neurotoxicity in a subject can be prevented, minimized or reduced by first administering a low dose of CAR+ T cells to reduce disease or tumor burden to non-morphological disease, for example to achieve minimal residual disease or molecular detectable disease status, followed by administration of the consecutive dose of recombinant receptor-expressing (e.g., CAR-expressing) cells.
In some embodiments, a consecutive dose is generally given at a point in time relative to the first or previous dose at which the risk of a toxic outcome or symptom or biochemical indicator thereof—such as CRS or neurotoxicity, macrophage activation syndrome, or tumor lysis syndrome—is at or below an acceptable level. For example, the consecutive dose may be administered after a toxic outcome has peaked and is declining or has declined below an acceptable level following the initial dose. Thus, in some embodiments, the consecutive dose is administered at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 days following the initiation of the first or prior dose, or greater than about 14 or 15 days or 21 days following the initiation of the first or prior dose. In some embodiments, the appropriate timing is determined by monitoring and/or assessing the presence of one or more symptoms or outcomes associated with the toxic event and delivering the consecutive dose after determining that the symptom or outcome is at or below an acceptable level.
In some embodiments, the timing of the consecutive dose is such that it avoids a reduction in efficacy that may otherwise be induced upon administration of a subsequent dose by a host immune response that has been mounted following administration of a first dose. In some aspects, the consecutive dose is administered prior to the development of a host immune response, e.g., adaptive or specific, e.g., humoral or cell-mediated, immune response against the administered cells and/or recombinant receptor they express, and/or before such a response is detectable, e.g., by one or more specified detection methods. The consecutive dose or doses generally are administered at a time at which a host adaptive immune response against the cells is not detected, has not become established, and/or has not reached a certain level or degree or stage. In this regard, the methods are advantageous compared to providing subsequent doses at the time of relapse, which generally is after an anti-transgene response has developed. In some embodiments, the consecutive dose is administered before or within about 28 days or 35 days following the first or prior dose, or before about 24, 25, 26, or 27 days following the initiation of the first or prior dose.
Thus, the provided methods in some embodiments involve administering one or more consecutive doses after debulking or reducing disease burden with a first dose, after a window of risk for toxicity, but before the mounting of an immune response. In this environment and with the appropriate timing, the consecutive dose can safely and effectively provide immune surveillance, clearing or preventing expansion or metastasis of residual disease cells, whether measurable or not by standard or research grade analytical methods. Thus, in some embodiments, the consecutive dose is a disease-consolidating dose.
In particular embodiments, the first dose includes the cells in an amount sufficient to reduce burden of the disease or condition in the subject and the consecutive dose is administered a time at which a serum level of a factor indicative of cytokine-release syndrome (CRS) in the subject is no more than 10 or no more than 25 times the serum level of the indicator in the subject immediately prior to the administration of the first dose, and/or at a time after a CRS-related outcome in the subject has reached a peak level and begun to decline following administration of the first dose, and at which the subject does not exhibit a detectable adaptive host immune response specific for the recombinant receptor expressed by the cells of the first dose.
In particular embodiments, the first dose contains fewer than at or about 1×106 of the cells per kilogram body weight of the subject, fewer than at or about 1×108 of the cells, and/or fewer than at or about 1×108 of the cells/m2 of the subject, and the consecutive dose is administered at a time point that is more than about 14 days after and less than about 28 days after initiation of the administration of the first dose. In some embodiments, the consecutive dose is administered at or about 14 days, 15 days, 16, days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days following the first or prior dose. In particular embodiments, the consecutive dose is administered at about 17 days or at about 21 days following the first or prior dose.
Thus, the provided methods offer advantages over single-dose administrations, which can lead to severe toxicity, unwanted outcomes, and/or lower efficacy, particularly in the context of high disease burden. They also can be advantageous over methods administering subsequent dose(s) too soon following an initial dose—increasing the risk of unwanted side effects—or too late, e.g., after establishment of an immune response to a previous dose. The provided methods extend exposure to therapeutic cells, improving durability and extent of clinical response and/or patient survival, while reducing toxic outcomes. Also provided are compositions and articles of manufacture providing doses for use in such methods.
II. Administration of Cells in Adoptive Cell Therapy
The provided methods generally involve administering multiple doses of cells expressing recombinant receptors, such as CARs, or other antigen receptors, such as transgenic TCRs, to subjects having a disease or condition specifically recognized by the receptors. The administrations generally effect an improvement in one or more symptoms of the disease or condition and/or treat or prevent the disease or condition or symptom thereof.
As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human. In some embodiments, the subject has been treated with a therapeutic agent targeting the disease or condition, e.g. the tumor, prior to administration of the first dose and/or prior to the administration of the consecutive dose. In some aspects, the subject is refractory or non-responsive to the other therapeutic agent.
In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.
In some embodiments, the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk for relapse, such as at a high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.
In some aspects, the subject has not received prior treatment with another therapeutic agent. In some embodiments, the subject has not received a dose of cells expressing a CAR prior to the administration of the first dose and/or has not received a dose of cells expressing the CAR or other receptor expressed by such cells or expressing any recombinant receptor targeting the same molecule or antigen. In some aspects, the subject has not received a dose of cells expressing the CAR of the first dose prior to the administration of the first dose.
Among the diseases, conditions, and disorders are tumors, including solid tumors, hematologic malignancies, and melanomas, and including localized and metastatic tumors, infectious diseases, such as infection with a virus or other pathogen, e.g., HIV, HCV, HBV, CMV, and parasitic disease, and autoimmune and inflammatory diseases. In some embodiments, the disease or condition is a tumor, cancer, malignancy, neoplasm, or other proliferative disease or disorder. Such diseases include but are not limited to leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), acute-lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B cell lymphoma, B cell malignancies, cancers of the colon, lung, liver, breast, prostate, ovarian, skin, melanoma, bone, and brain cancer, ovarian cancer, epithelial cancers, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin lymphoma, cervical carcinoma, colorectal cancer, glioblastoma, neuroblastoma, Ewing sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and/or mesothelioma. In some embodiments, the subject has acute-lymphoblastic leukemia (ALL). In some embodiments, the subject has non-Hodgkin's lymphoma.
In some embodiments, the size or timing of the doses is determined as a function of the particular disease or condition in the subject. It is within the level of a skilled artisan to empirically determine the size or timing of the doses for a particular disease in view of the provided description. In some cases, for example, subjects with non-Hodgkin's lymphoma (NHL) can be less responsive or only partially responsive to treatment with recombinant receptor-expressing cells (e.g., CAR-expressing cells, such as CAR-expressing T cells) than subjects with acute-lymphoblastic leukemia (ALL). In some cases, a lower dose of cells administered to a subject with NHL may not reduce tumor burden in a subject, thereby increasing the risk of toxic outcomes, such as CRS and/or neurotoxicity, in a subject when administered a consecutive dose. In some embodiments, methods are provided in which subjects with NHL are administered a consecutive dose that is the same or less than the dose of recombinant receptor-expressing (e.g., CAR-expressing) cells administered in the first dose, thereby minimizing or reducing risk of toxic side effects that can occur upon administration of the consecutive dose.
In some embodiments, the disease or condition is a tumor or a cancer and the subject has a large tumor burden prior to the administration of the first dose, such as a large solid tumor or a large number or bulk of disease-associated, e.g., tumor or cancer cells. In some aspects, the subject has a high number of metastases and/or widespread localization of metastases. In some aspects, the initial tumor burden in the subject is low and the subject has few metastases.
In some embodiments, the size or timing of the doses is determined by the initial disease burden, such as tumor burden, in the subject. For example, in some cases, whereas the subject generally is administered a relatively low number of cells in the first dose, in context of lower disease burden, such as molecular detectable disease and/or minimal residual disease, the initial dose may be higher. In other cases, in subjects exhibiting a higher disease burden after administration of the first dose, the consecutive dose may be the same as or lower than the first dose. In some embodiments, subjects are assessed for disease burden using methods as described herein, such as methods that assess blasts in bone marrow or molecular disease by flow cytometry or qPCR methods.
In some embodiments, the disease or condition is an infectious disease or condition, such as, but not limited to, viral, retroviral, bacterial, and protozoal infections, immunodeficiency, Cytomegalovirus (CMV), Epstein-Ban virus (EBV), adenovirus, BK polyomavirus. In some embodiments, the disease or condition is an autoimmune or inflammatory disease or condition, such as arthritis, e.g., rheumatoid arthritis (RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Grave's disease, Crohn's disease, multiple sclerosis, asthma, and/or a disease or condition associated with transplant.
In some embodiments, the antigen associated with the disease or disorder is selected from the group consisting of orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, 0EPHa2, ErbB2, 3, or 4, FBP, fetal acethycholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1), a cyclin, such as cyclin A1 (CCNA1), and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens.
As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.
As used herein, “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.
“Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided cells and compositions are used to delay development of a disease or to slow the progression of a disease.
As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, cells that suppress tumor growth reduce the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the cells.
An “effective amount” of an agent, e.g., a pharmaceutical formulation, cells, or composition, in the context of administration, refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.
A “therapeutically effective amount” of an agent, e.g., a pharmaceutical formulation or cells, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered. In some embodiments, the provided methods involve administering the cells and/or compositions at effective amounts, e.g., therapeutically effective amounts.
A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. In the context of lower tumor burden, the prophylactically effective amount in some aspects will be higher than the therapeutically effective amount.
Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.
In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, and following isolation and processing are administered to the same subject.
In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.
The cells can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration of the cells. In some embodiments, it is administered by multiple bolus administrations of the cells, for example, over a period of no more than 3 days, or by continuous infusion administration of the cells.
For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.
In some embodiments, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agent includes a cytokine, such as IL-2, for example, to enhance persistence.
In some embodiments, the methods comprise administration of a chemotherapeutic agent, e.g., a conditioning chemotherapeutic agent, for example, to reduce tumor burden prior to the first or consecutive dose administrations.
Preconditioning subjects with immunodepleting (e.g., lymphodepleting) therapies can improve the effects of adoptive cell therapy (ACT). Preconditioning with lymphodepleting agents, including combinations of cyclosporine and fludarabine, have been effective in improving the efficacy of transferred tumor infiltrating lymphocyte (TIL) cells in cell therapy, including to improve response and/or persistence of the transferred cells. See, e.g., Dudley et al., 2002 Science, 298, 850-54; Rosenberg et al., Clin Cancer Res 2011, 17(13):4550-4557. Likewise, in the context of CAR+ T cells, several studies have incorporated lymphodepleting agents, most commonly cyclophosphamide, fludarabine, bendamustine, or combinations thereof, sometimes accompanied by low-dose irradiation. See Han et al. Journal of Hematology & Oncology 2013, 6:47; Kochenderfer et al., Blood 2012; 119: 2709-2720; Kalos et al., Sci Transl Med 2011, 3(95):95ra73; Clinical Trial Study Record Nos.: NCT02315612; NCT01822652. Such preconditioning can be carried out with the goal of reducing the risk of one or more of various outcomes that could dampen efficacy of the therapy. These include the phenomenon known as “cytokine sink,” by which T cells, B cells, NK cells compete with TILs for homeostatic and activating cytokines, such as IL-2, IL-7, and/or IL-15; suppression of TILs by regulatory T cells, NK cells, or other cells of the immune system; impact of negative regulators in the tumor microenvironment. Muranski et al., Nat Clin Pract Oncol. 2006 December; 3(12): 668-681.
Thus, in some embodiments, the methods include administering a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, to a subject prior to the first or subsequent dose. For example, the subject may be administered a preconditioning agent at least 2 days prior, such as at least 3, 4, 5, 6, or 7 days prior, to the first or subsequent dose. In some embodiments, the subject is administered a preconditioning agent no more than 7 days prior, such as no more than 6, 5, 4, 3, or 2 days prior, to the first or subsequent dose
In some embodiments, where the lymphodepleting agent comprises cyclophosphamide, the subject is administered between or between about 0.5 g/m2 and 5 g/m2, such as between or between about 1 g/m2 and 4 g/m2, 1 g/m2 and 3 g/m2, or 2 g/m2 and 4 g/m2 of cyclophosphamide. In some aspects, the subject is administered 2 g/m2 cyclophosphamide or about 2 g/m2 cyclophosphamide. In some embodiments, the subject is preconditioned with cyclophosphamide at a dose between or between about 20 mg/kg and 100 mg/kg, such as between or between about 40 mg/kg and 80 mg/kg. In some aspects, the subject is preconditioned with or with about 60 mg/kg of cyclophosphamide.
In some embodiments, where the lymphodepleting agent comprises fludarabine, the subject is administered fludarabine at a dose between or between about 1 g/m2 and 100 g/m2, such as between or between about 10 g/m2 and 75 g/m2, 15 g/m2 and 50 g/m2, 20 g/m2 and 30 g/m2, or 24 g/m2 and 26 g/m2. In some instances, the subject is administered 25 g/m2 of fludarabine. In some embodiments, the fludarabine can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. For example, in some instances, the agent, e.g., fludarabine, is administered between or between about 1 and 5 times, such as between or between about 3 and 5 times. In some embodiments, such plurality of doses is administered in the same day, such as 1 to 5 times or 3 to 5 times daily.
In some embodiments, the lymphodepleting agent comprises a combination of agents, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of agents may include cyclophosphamide at any dose or administration schedule, such as those described above, and fludarabine at any dose or administration schedule, such as those described above. For example, in some aspects, the subject is administered 60 mg/kg (˜2 g/m2) of cyclophosphamide and 3 to 5 doses of 25 mg/m2 fludarabine prior to the first or subsequent dose.
In some embodiments, the administration of the preconditioning agent prior to infusion of the first or subsequent dose improves an outcome of the treatment. For example, in some aspects, preconditioning improves the efficacy of treatment with the first or subsequent dose or increases the persistence of the recombinant receptor-expressing cells (e.g., CAR-expressing cells, such as CAR-expressing T cells) in the subject. In some embodiments, preconditioning treatment increases disease-free survival, such as the percent of subjects that are alive and exhibit no minimal residual or molecularly detectable disease after a given period of time following the first or subsequent dose. In some embodiments, the time to median disease-free survival is increased.
Once the cells are administered to the subject (e.g., human), the biological activity of the engineered cell populations in some aspects is measured by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells also can be measured by assaying expression and/or secretion of certain cytokines, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load. In some aspects, toxic outcomes, persistence and/or expansion of the cells, and/or presence or absence of a host immune response, are assessed.
In certain embodiments, engineered cells are modified in any number of ways, such that their therapeutic or prophylactic efficacy is increased. For example, the engineered CAR or TCR expressed by the population can be conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating compounds, e.g., the CAR or TCR, to targeting moieties is known in the art. See, for instance, Wadwa et al., J. Drug Targeting 3: 1 1 1 (1995), and U.S. Pat. No. 5,087,616.
III. Dosing
The timing and size of the multiple doses of cells generally are designed to reduce risk of or minimize toxic outcomes and/or to improve efficacy, such as by providing increased exposure of the subject to the cells, e.g., over time. The methods involve administering a first dose, generally followed by one or more consecutive doses, with particular time frames between the different doses.
In the context of adoptive cell therapy, administration of a given “dose” encompasses administration of the given amount or number of cells as a single composition and/or single uninterrupted administration, e.g., as a single injection or continuous infusion, and also encompasses administration of the given amount or number of cells as a split dose, provided in multiple individual compositions or infusions, over a specified period of time, which is no more than 3 days. Thus, in some contexts, the first or consecutive dose is a single or continuous administration of the specified number of cells, given or initiated at a single point in time. In some contexts, however, the first or consecutive dose is administered in multiple injections or infusions over a period of no more than three days, such as once a day for three days or for two days or by multiple infusions over a single day period.
Thus, in some aspects, the cells of the first dose are administered in a single pharmaceutical composition. In some embodiments, the cells of the consecutive dose are administered in a single pharmaceutical composition.
In some embodiments, the cells of the first dose are administered in a plurality of compositions, collectively containing the cells of the first dose. In some embodiments, the cells of the consecutive dose are administered in a plurality of compositions, collectively containing the cells of the consecutive dose. In some aspects, additional consecutive doses may be administered in a plurality of compositions over a period of no more than 3 days.
The term “split dose” refers to a dose that is split so that it is administered over more than one day. This type of dosing is encompassed by the present methods and is considered to be a single dose.
Thus, the first dose and/or consecutive dose(s) in some aspects may be administered as a split dose. For example, in some embodiments, the dose may be administered to the subject over 2 days or over 3 days. Exemplary methods for split dosing include administering 25% of the dose on the first day and administering the remaining 75% of the dose on the second day. In other embodiments, 33% of the first dose may be administered on the first day and the remaining 67% administered on the second day. In some aspects, 10% of the dose is administered on the first day, 30% of the dose is administered on the second day, and 60% of the dose is administered on the third day. In some embodiments, the split dose is not spread over more than 3 days.
With reference to a prior dose, such as a first dose, the term “consecutive dose” refers to a dose that is administered to the same subject after the prior, e.g., first, dose without any intervening doses having been administered to the subject in the interim. Nonetheless, the term does not encompass the second, third, and/or so forth, injection or infusion in a series of infusions or injections comprised within a single split dose. Thus, unless otherwise specified, a second infusion within a one, two or three-day period is not considered to be a “consecutive” dose as used herein. Likewise, a second, third, and so-forth in the series of multiple doses within a split dose also is not considered to be an “intervening” dose in the context of the meaning of “consecutive” dose. Thus, unless otherwise specified, a dose administered a certain period of time, greater than three days, after the initiation of a first or prior dose, is considered to be a “consecutive” dose even if the subject received a second or subsequent injection or infusion of the cells following the initiation of the first dose, so long as the second or subsequent injection or infusion occurred within the three-day period following the initiation of the first or prior dose.
Thus, unless otherwise specified, multiple administrations of the same cells over a period of up to 3 days is considered to be a single dose, and administration of cells within 3 days of an initial administration is not considered a consecutive dose and is not considered to be an intervening dose for purposes of determining whether a second dose is “consecutive” to the first.
In some embodiments, multiple consecutive doses are given, in some aspects using the same timing guidelines as those with respect to the timing between the first dose and first consecutive dose, e.g., by administering a first and multiple consecutive doses, with each consecutive dose given within a period of time that is greater than about 14 and less than about 28 days, e.g., about 21 days, after the administration of the first or immediately prior dose. The additional multiple additional consecutive dose or doses also are referred to as subsequent dose or subsequent consecutive dose.
As used herein, “first dose” is used to describe the timing of a given dose being prior to the administration of a consecutive or subsequent dose. The term does not necessarily imply that the subject has never before received a dose of cell therapy or even that the subject has not before received a dose of the same cells or cells expressing the same recombinant receptor or targeting the same antigen.
Dosage Amount or Size
The size of the first and/or one or more consecutive doses of cells are generally designed to provide improved efficacy and/or reduced risk of toxicity. In some embodiments, the number of cells in the first dose is between about 0.5×106 cells/kg body weight of the subject and 3×106 cells/kg, between about 0.75×106 cells/kg and 2.5×106 cells/kg or between about 1×106 cells/kg and 2×106 cells/kg, each inclusive.
In some embodiments, the first dose is a low dose. In particular embodiments, the first dose contains a number of cells, number of recombinant receptor (e.g., CAR)-expressing cells, number of T cells, or number of peripheral blood mononuclear cells (PBMCs) in the range from about 105 to about 106 of such cells per kilogram body weight of the subject, inclusive, and/or a number of such cells that is no more than about 105 or about 106 such cells per kilogram body weight of the subject, inclusive. For example, in some embodiments, the first dose includes less than or no more than at or about 1×105, at or about 2×105, at or about 5×105, or at or about 1×106 of such cells per kilogram body weight of the subject. In some embodiments, the first dose includes at or about 1×105, at or about 2×105, at or about 5×105, or at or about 1×106 of such cells per kilogram body weight of the subject, or a value within the range between any two of the foregoing values. In particular embodiments, the numbers and/or concentrations of cells refer to the number of recombinant receptor (e.g., CAR)-expressing cells. In other embodiments, the numbers and/or concentrations of cells refer to the number or concentration of all cells, T cells, or peripheral blood mononuclear cells (PBMCs) administered.
In some embodiments, for example, where the subject is a human, the first dose includes fewer than or equal to about 1×108 total recombinant receptor (e.g., CAR)-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), e.g., in the range of about 1×106 to 1×108 such cells, inclusive, such as no more than 2×106, 5×106, 1×107, 5×107, or 1×108 or total such cells, or the range between any two of the foregoing values.
In some embodiments, the first dose contains fewer than or equal to about 1×108 total recombinant receptor (e.g., CAR)-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs) cells per m2 of the subject, e.g., in the range of about 1×106 to 1×108 such cells per m2 of the subject, inclusive, such as no more than 2×106, 5×106, 1×107, 5×107, or 1×108 such cells per m2 of the subject, or the range between any two of the foregoing values.
In certain embodiments, for example, where risk of toxicity and/or disease burden in the subject is determined to be low, the first dose can be a relatively high dose, such as a dose that is greater than 1×106 cells/kg or that includes greater than 1×108 cells, such as T cells or PBMCs, or greater than 1×108 such cells per m2 of the subject. In some embodiments, disease burden is low if the subject does not exhibit substantial morphologic disease or does not exhibit morphologic disease, or that exhibits less than 20% of blast cells in bone marrow, less than 15% of blast cells in bone marrow, less than 10% blast cells in bone marrow or less than 5% blast cells in bone marrow. In some embodiments, disease burden is low if the subject exhibits non-morphologic disease, such as exhibits minimal residual disease or molecularly detectable disease but does not exhibit the features associated with morphological disease as known in the art of described elsewhere, such as does not exhibit greater than 5% of blast cells in bone marrow. In some embodiments, the number of cells, recombinant receptor (e.g., CAR)-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs) in the first dose is greater than about 1×106 such cells per kilogram body weight of the subject, e.g., 2×106, 3×106, 5×106, 1×107, 5×107, 1×108, 1×109, or 1×1010 such cells per kilogram of body weight and/or 1×108, 1×109, 1×1010 such cells per m2 of the subject or total, or the range between any two of the foregoing values.
In some embodiments, the number of cells administered in the consecutive dose is the same as or similar to the number of cells administered in the first dose in any of the embodiments herein, such as less than or no more than at or about 1×105, at or about 2×105, at or about 5×105, or at or about 1×106 of such cells per kilogram body weight of the subject. In some embodiments, the consecutive dose(s) contains at or about 1×105, at or about 2×105, at or about 5×105, or at or about 1×106 of such cells per kilogram body weight of the subject, or a value within the range between any two of the foregoing values.
In reference to cell numbers, in some embodiments, such values refer to numbers of recombinant receptor-expressing (e.g. CAR-expressing) cells; in other embodiments, they refer to number of T cells or PBMCs or total cells administered.
In some aspects, the consecutive dose is larger than the first dose. For example, in some embodiments, the consecutive dose contains more than about 1×106 cells, recombinant receptor (e.g. CAR)-expressing cells, T cells, and/or PBMCs per kilogram body weight of the subject, such as about or at least about 2×106, 3×106, 5×106, 1×107, 1×108, or 1×109 such cells per kilogram body weight of the subject. In some embodiments, the number of cells in the consecutive dose is between about 2×106 cells/kg body weight of the subject and 6×106 cells/kg, between about 2.5×106 cells/kg and 5.0×106 cells/kg, or between about 3.0×106 cells/kg and about 4.0×106 cells/kg, each inclusive. In some embodiments, the amount or size of the consecutive dose is sufficient to reduce disease burden or an indicator thereof, and/or one or more symptoms of the disease or condition. In some embodiments, the dose is of a size effective to improve survival of the subject, for example, to induce survival, relapse-free survival, or event-free survival of the subject for at least 6 months, or at least 1, 2, 3, 4, or 5 years. In some embodiments, the number of cells, recombinant receptor (e.g. CAR)-expressing cells, T cells, and/or PBMCs administered and/or number of such cells administered per body weight of the subject in the consecutive dose is at least 2-fold, 5-fold, 10-fold, 50-fold, or 100-fold or more greater than the number administered in the first dose. In some embodiments, disease burden, tumor size, tumor volume, tumor mass, and/or tumor load or bulk is reduced following the consecutive dose by at least at or about 50, 60, 70, 80, 90% or more compared to that immediately prior to the administration of the first dose or of the consecutive dose.
In other embodiments, the number of cells administered in the consecutive dose is lower than the number of cells administered in the first dose.
In some embodiments, multiple consecutive doses are administered following the first dose, such that an additional dose or doses are administered following administration of the consecutive dose. In some aspects, the number of cells administered to the subject in the additional dose or doses (i.e., the third, fourth, fifth, and so forth) is the same as or similar to the first dose and/or consecutive dose. In some embodiments, the additional dose or doses are larger than prior doses.
In some aspects, the size of the first and/or consecutive dose is determined based on one or more criteria such as response of the subject to prior treatment, e.g. chemotherapy, disease burden in the subject, such as tumor load, bulk, size, or degree, extent, or type of metastasis, stage, and/or likelihood or incidence of the subject developing toxic outcomes, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or a host immune response against the cells and/or recombinant receptors being administered.
In some aspects, the size of the first and/or consecutive dose is determined by the burden of the disease or condition in the subject. For example, in some aspects, the number of cells administered in the first dose is determined based on the tumor burden that is present in the subject immediately prior to administration of the first dose. In some embodiments, the size of the first and/or consecutive dose is inversely correlated with disease burden. In some aspects, as in the context of a large disease burden, the subject is administered a low number of cells, for example less than about 1×106 cells per kilogram of body weight of the subject. In other embodiments, as in the context of a lower disease burden, the subject is administered a larger number of cells, such as more than about 1×106 cells per kilogram body weight of the subject, for example more than about 2×106, 2.5×106 or 3×106 cells/kg.
In some aspects, the number of cells administered in the consecutive dose is determined based on the tumor burden that is present in the subject following administration of the first dose. In some embodiments, e.g. where the first dose results in reduced or decreased disease burden or has done so below a particular threshold amount or level, e.g., one above which there is an increased risk of toxic outcome, the consecutive dose is high or large, e.g. more than 1×106 cells (e.g., total cells, receptor-expressing cells, T cells, or PBMCs) per kilogram body weight, such as more than 2.0×106, 2.5×106 or 3.0×106 cells/kg, and/or is larger than the first dose. In some embodiments, a subject exhibits reduced or decreased disease burden if they exhibited morphological disease prior to treatment and exhibit complete remission (e.g., fewer than 5% blasts in bone marrow) with or without molecular disease (e.g., minimum residual disease (MRD) that is molecularly detectable, e.g., as detected by flow cytometry or quantitative PCR) after treatment. In some embodiments, a subject exhibits reduced or decreased disease burden if they exhibited molecular disease prior to treatment and do not exhibit molecular disease after treatment.
In other aspects, the number of cells administered in the consecutive dose is low, e.g. less than about 1×106, e.g. the same as or lower than the first dose, where the first dose has reduced tumor burden to a small extent or where the first dose has not led to a detectable reduction in tumor burden. In some cases, even if tumor burden is not reduced in a subject after receiving a first dose, a consecutive dose can be high or large, e.g. more than 1×106 cells (e.g., total cells, receptor-expressing cells, T cells, or PBMCs) per kilogram body weight, such as more than 2.0×106, 2.5×106 or 3.0×106 cells/kg, and/or is larger than the first dose.
In some embodiments, the first dose includes the cells in an amount that does not cause or reduces the likelihood of toxicity or toxic outcomes, such as cytokine release syndrome (CRS), severe CRS (sCRS), macrophage activation syndrome, tumor lysis syndrome, fever of at least at or about 38 degrees Celsius for three or more days and a plasma level of CRP of at least at or about 20 mg/dL, neurotoxicity and/or neurotoxicity. In some aspects, the number of cells administered in the first dose is determined based on the likelihood that the subject will exhibit toxicity or toxic outcomes, such as CRS, sCRS, and/or CRS-related outcomes following administration of the cells. For example, in some embodiments, the likelihood for the development of toxic outcomes in a subject is predicted based on tumor burden. In some embodiments, the methods include detecting or assessing the toxic outcome and/or disease burden prior to the administration of the dose.
In some aspects, the number of cells administered in the consecutive dose is determined based on the level of toxicity or toxic outcomes, e.g. CRS-related outcomes, following administration of the first dose. For example, in some embodiments, the number of cells administered in the consecutive dose is low, e.g. less than 1×106 cells per kilogram body weight, e.g. the same as or lower than the first dose, if the subject exhibits a detectable level of toxicity or toxic outcomes, e.g. CRS-related outcomes, following administration of the first dose.
In some embodiments, the subject is not administered the consecutive dose at a time following the first dose at which they exhibit toxicity or toxic outcomes, such as CRS-related outcomes, e.g. if a serum level of an indicator of CRS or other biochemical indicator of the toxicity is more than at or about 10 times, more than at or about 15 times, more than at or about 20 times, more than at or about 25 times, more than at or about 50 times, more than at or about 75 times, more than at or about 100 times, more than at or about 125 times, more than at or about 150 times, more than at or about 200 times, or more than at or about 250 times the baseline or pre-treatment level, such as the serum level of the indicator immediately prior to administration of the first dose.
In some embodiments, the subject is administered the consecutive dose, if and when, following the first dose, a biochemical indicator of or other indicator of a toxic outcome, e.g. a serum level of an indicator of CRS has not increased to above a given level, e.g., an acceptable level, such as more than at or about 10, 15, 20, 25, 50, 75, or 100 times the serum level of the indicator immediately prior to administration of the first dose, or has increased above an acceptable level but has declined to at or below the acceptable level. In some aspects, the consecutive dose is administered if and when the indicator declines below the acceptable level within 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days, e.g., does so within 21 days, and/or within 15-27 days, within 21 days, within 28 days, or within 35 days, of the administration of the first dose, but is not administered if the level of the indicator does not decline below the acceptable level within that time period.
In some embodiments, the consecutive dose is administered if and when a clinical risk for developing cytokine-release syndrome (CRS), macrophage activation syndrome, or tumor lysis syndrome, or neurotoxicity is not present or has passed or has subsided following the first administration, such as after a critical window after which such events generally have subsided and/or are less likely to occur, e.g., in 60, 70, 80, 90, or 95% of subjects with a particular disease or condition.
In some embodiments, whether a consecutive dose is administered, when the consecutive dose is administered, and/or the number of the cells administered in the consecutive dose is or are determined based on the presence, absence, or degree of an immune response or detectable immune response in the subject to the cells of the first dose or recombinant receptor expressed thereby. In some aspects, a consecutive dose containing cells expressing the receptor of the cells of the first dose will not be administered to a subject with a detectable host adaptive immune response, or an immune response that has become established or reached a certain level, stage, or degree.
Timing of Doses
In some aspects, the timing of the consecutive dose is measured from the initiation of the first dose to the initiation of the consecutive dose. In other embodiments, the timing of the consecutive dose is measured from the completion of the first dose, or from the median day of administration of the first dose, e.g. in the context of split dosing, described herein, where a dose is administered over more than one day, e.g. over 2 days or over 3 days.
In some embodiments, the consecutive dose is administered at a time at which a serum level of a factor indicative of CRS in the subject is no more than 10 times, 25 times, 50 times, or 100 times the serum level of said indicator in the subject immediately prior to the administration of the first dose.
In some embodiments, the consecutive dose is administered at a time after a CRS-related outcome, such as a serum factor associated with or indicative of CRS, or a clinical sign or symptom thereof such as fever, hypoxia, hypotension, or neurological disturbance, in the subject has reached a peak level and begun to decline following administration of the first dose. In some embodiments, the consecutive dose is administered at a time after which the outcome is observed to be on the decline compared with the highest level of such outcome measured following the administration, or at a time at which or after which the level is on the decline following the maximum value or level of the outcome reached after the administration.
In some embodiments, the consecutive dose is administered when the level of an indicator of a toxic outcome, such as a serum indicator of CRS, declines below 25 times the level of the indicator immediately prior to the first dose. In some aspects, the consecutive dose is administered at a time at which the subject does not exhibit CRS or does not exhibit severe CRS.
In some aspects, the consecutive dose is administered at a point in time at which the disease burden in the patient has decreased as compared to the disease burden immediately prior to administration of the first dose. In some embodiments, a subject exhibits reduced or decreased disease burden if they exhibited morphological disease prior to treatment and exhibit complete remission (e.g., fewer than 5% blasts in bone marrow) with or without molecular disease (e.g., minimum residual disease (MRD) that is molecularly detectable, e.g., as detected by flow cytometry or quantitative PCR) after treatment. In some embodiments, a subject exhibits reduced or decreased disease burden if they exhibited molecular disease prior to treatment and do not exhibit molecular disease after treatment. In some embodiments, the consecutive dose is administered at a time at which the disease burden or indicator thereof such as bulk or number or percentage of disease (e.g., tumor) cells in the blood, other fluid, organ or tissue of the subject, or size of the tumor, has decreased by 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more following administration of the first dose.
In some embodiments, the consecutive dose is administered at a point in time at which the disease or condition in the subject has not relapsed following the reduction in response to the first or prior dose. In some embodiments, the disease burden reduction is indicated by a reduction in one or more factors, such as load or number of disease cells in the subject or fluid or organ or tissue thereof, the mass or volume of a tumor, or the degree or extent of metastases. Such a factor is deemed to have relapsed if after reduction in the factor in response to an initial treatment or administration, the factor subsequently increases. In some embodiments, the relapse is in one or one or more factors, or in the disease burden generally. In some aspects, the consecutive dose is administered at a point in time at which the subject, disease burden, or factor thereof has relapsed as compared to the lowest point measured or reached following the first or prior administration, but still is lower compared to the time immediately prior to the first dose. In some embodiments, the subject is administered the consecutive dose at a point in time at which disease burden or factor indicative thereof has not changed, e.g. at a time when an increase in disease burden has been prevented.
In some embodiments, the consecutive dose is administered at a time when a host adaptive immune response is not detected, has not become established, or has not reached a certain level, degree, or stage. In some aspects, the consecutive dose is administered prior to the development of a memory immune response in the subject.
In some aspects, the time between the administration of the first dose and the administration of the consecutive dose is about 9 to about 35 days, about 14 to about 28 days, or 15 to 27 days. In some embodiments, the administration of the consecutive dose is at a time point more than about 14 days after and less than about 28 days after the administration of the first dose. In some embodiments, the administration of the consecutive dose is no more than about 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days or 28 days after the administration of the first dose. In some aspects, the time between the first and consecutive dose is about 14 days about 17 days or about 21 days.
In some embodiments, an additional or subsequent dose or doses, e.g. further consecutive doses, are administered following administration of the consecutive dose. In some aspects, the additional consecutive dose or doses are administered at least about 14 and less than about 28 days following administration of a prior dose, such as a prior consecutive dose. In some embodiments, an exemplary dosage regime includes a schedule of administration of recombinant receptor-expressing cells (e.g., CAR-expressing cells, such as CAR-expressing T cells) at or about In some embodiments, the additional dose is administered less than about 14 days following the prior dose, for example, less than 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days after the prior dose. In some embodiments, no dose is administered less than about 14 days following the prior dose and/or no dose is administered more than about 28 days after the prior dose.
In any of the embodiments, the methods in some cases include the administration of the first or prior dose and the consecutive dose(s), and in other cases include the administration of the consecutive dose(s) to a subject who has previously received the first or prior dose but do not include the administration of the first or prior dose itself. Thus, the methods in some cases involve the administration of consolidating treatment, such as by administering a consolidating consecutive dose to a subject that has previously received a dose, e.g., a debulking dose, of recombinant receptor-expressing, e.g., CAR-expressing, cells. In some aspects, the previous dose of receptor-expressing, e.g., CAR,-expressing, cells has been sufficient to reduce the burden of the disease or condition in the subject such that the efficacy and/or safety of the administration of the cells in the consecutive dose is improved relative to administering the dose without the subject having received the first dose.
In some embodiments, disease burden, tumor size, tumor volume, tumor mass, and/or tumor load or bulk is reduced following the consecutive dose by at least at or about 50, 60, 70, 80, 90% or more compared to that immediately prior to the administration of the first dose or of the consecutive dose.
IV. Toxicity and Toxic Outcomes
In some embodiments, the timing or amount of the doses reduces or prevents toxicity or an outcome or symptom thereof, for example, compared to administration of the same or similar total number of cells as a single-dose, administration of the consecutive dose without the subject having previously received the first dose, administration of the consecutive dose at a different time relative to the first dose, and/or administration of different amount(s) in one or more of the doses.
Administration of adoptive T cell therapy, such as treatment with T cells expressing chimeric antigen receptors, can induce toxic effects or outcomes such as cytokine release syndrome and neurotoxicity. In some examples, such effects or outcomes parallel high levels of circulating cytokines, which may underlie the observed toxicity.
In some embodiments, the provided methods are designed to or include features that result in a lower degree of toxicity, toxic outcome or symptom, toxicity-promoting profile, factor, or property, such as a symptom or outcome associated with or indicative of cytokine release syndrome (CRS), for example, compared to administration of the same or similar total number of cells as a single-dose, administration of the consecutive dose without the subject having previously received the first dose, administration of the consecutive dose at a different time relative to the first dose, and/or administration of different amount(s) in one or more of the doses.
In some aspects, the toxic outcome is or is associated with or indicative of cytokine release syndrome (CRS) or severe CRS (sCRS). CRS, e.g., sCRS, can occur in some cases following adoptive T cell therapy and administration to subjects of other biological products. See Davila et al., Sci Transl Med 6, 224ra25 (2014); Brentjens et al., Sci. Transl. Med. 5, 177ra38 (2013); Grupp et al., N. Engl. J. Med. 368, 1509-1518 (2013); and Kochenderfer et al., Blood 119, 2709-2720 (2012); Xu et al., Cancer Letters 343 (2014) 172-78.
Typically, CRS is caused by an exaggerated systemic immune response mediated by, for example, T cells, B cells, NK cells, monocytes, and/or macrophages. Such cells may release a large amount of inflammatory mediators such as cytokines and chemokines. Cytokines may trigger an acute inflammatory response and/or induce endothelial organ damage, which may result in microvascular leakage, heart failure, or death. Severe, life-threatening CRS can lead to pulmonary infiltration and lung injury, renal failure, or disseminated intravascular coagulation. Other severe, life-threatening toxicities can include cardiac toxicity, respiratory distress, neurologic toxicity and/or hepatic failure.
CRS may be treated using anti-inflammatory therapy such as an anti-IL-6 therapy, e.g., anti-IL-6 antibody, e.g., tocilizumab, or antibiotics. In some embodiments, the subject is treated with such a therapy following the first administration and the consecutive dose is administered only if and when the CRS-associated symptom(s) are reduced or declining or declined below an acceptable level following such treatment.
Outcomes, signs and symptoms of CRS are known and include those described herein. In some embodiments, where a particular dosage regimen or administration effects or does not effect a given CRS-associated outcome, sign, or symptom, particular outcomes, signs, and symptoms and/or quantities or degrees thereof may be specified.
In the context of administering CAR-expressing cells, CRS typically occurs 6-20 days after infusion of cells that express a CAR. See Xu et al., Cancer Letters 343 (2014) 172-78. In some cases, CRS occurs less than 6 days or more than 20 days after CAR T cell infusion. The incidence and timing of CRS may be related to baseline cytokine levels or tumor burden at the time of infusion. Commonly, CRS involves elevated serum levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and/or interleukin (IL)-2. Other cytokines that may be rapidly induced in CRS are IL-1β, IL-6, IL-8, and IL-10.
In some aspects, a lower degree of toxicity, outcome, symptom, profile, factor, or property is observed in the subjects to which the cells are administered by the dosing regimen of the present methods, for example, as compared to administration of the same or similar total number of cells as a single-dose, administration of the consecutive dose without the subject having previously received the first dose, administration of the consecutive dose at a different time relative to the first dose, and/or administration of different amount(s) in one or more of the doses. For example, in some embodiments, following administration of the first dose to the subject, the subject exhibits a lower degree of a CRS-related outcome, and/or exhibits a lower serum level of an inflammatory cytokine or factor indicative of CRS, as compared to administration of a larger number of cells to the subject. In some embodiments, following administration to the subject of the consecutive dose, the subject exhibits a lower degree of a CRS-related outcome, and/or exhibits a lower serum level of an inflammatory cytokine or outcome indicative of CRS, as compared to administration of the consecutive dose to the subject without the subject having received the first dose, or as compared to administering the first and consecutive dose(s) as a single dose, or as compared to administering the consecutive dose at a time that is earlier than or after the specified time period. In some embodiments, the subject does not exhibit CRS or severe CRS following administration of the first dose and/or following administration of the consecutive dose.
Exemplary outcomes associated with CRS include fever, rigors, chills, hypotension, dyspnea, acute respiratory distress syndrome (ARDS), encephalopathy, ALT/AST elevation, renal failure, cardiac disorders, hypoxia, neurologic disturbances, and death. Neurological complications include delirium, seizure-like activity, confusion, word-finding difficulty, aphasia, and/or becoming obtunded. Other CRS-related outcomes include fatigue, nausea, headache, seizure, tachycardia, myalgias, rash, acute vascular leak syndrome, liver function impairment, and renal failure. In some aspects, CRS is associated with an increase in one or more factors such as serum-ferritin, d-dimer, aminotransferases, lactate dehydrogenase and triglycerides, or with hypofibrinogenemia or hepatosplenomegaly.
In some embodiments, outcomes associated with CRS include one or more of: persistent fever, e.g., fever of a specified temperature, e.g., greater than at or about 38 degrees Celsius, for two or more, e.g., three or more, e.g., four or more days or for at least three consecutive days; fever greater than at or about 38 degrees Celsius; elevation of cytokines, such as a max fold change, e.g., of at least at or about 75, compared to pre-treatment levels of at least two cytokines (e.g., at least two of the group consisting of interferon gamma (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L, fracktalkine, and IL-5, and/or tumor necrosis factor alpha (TNFα)), or a max fold change, e.g., of at least at or about 250 of at least one of such cytokines; and/or at least one clinical sign of toxicity, such as hypotension (e.g., as measured by at least one intravenous vasoactive pressor); hypoxia (e.g., plasma oxygen (PO2) levels of less than at or about 90%); and/or one or more neurologic disorders (including mental status changes, obtundation, and seizures).
Exemplary CRS-related outcomes include increased or high serum levels of one or more factors, including cytokines and chemokines and other factors associated with CRS. Exemplary outcomes further include increases in synthesis or secretion of one or more of such factors. Such synthesis or secretion can be by the T cell or a cell that interacts with the T cell, such as an innate immune cell or B cell.
In some embodiments, the CRS-associated serum factors or CRS-related outcomes include inflammatory cytokines and/or chemokines, including interferon gamma (IFN-γ), TNF-α, IL-1β, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, sIL-2Ra, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage inflammatory protein (MIP)-1, tumor necrosis factor alpha (TNFα), IL-6, and IL-10, IL-1β, IL-8, IL-2, MIP-1, Flt-3L, fracktalkine, and/or IL-5. In some embodiments, the factor or outcome includes C reactive protein (CRP). In addition to being an early and easily measurable risk factor for CRS, CRP also is a marker for cell expansion. In some embodiments, subjects that are measured to have high levels of CRP, such as ≥15 mg/dL, have CRS. In some embodiments, subjects that are measured to have high levels of CRP do not have CRS. In some embodiments, a measure of CRS includes a measure of CRP and another factor indicative of CRS.
In some embodiments, one or more inflammatory cytokines or chemokines are monitored before, during, or after CAR treatment. In some aspects, the one or more cytokines or chemokines include IFN-γ, TNF-α, IL-2, IL-1β, IL-6, IL-7, IL-8, IL-10, IL-12, sIL-2Rα, granulocyte macrophage colony stimulating factor (GM-CSF), or macrophage inflammatory protein (MIP). In some embodiments, IFN-γ, TNF-α, and IL-6 are monitored.
CRS criteria that appear to correlate with the onset of CRS to predict which patients are more likely to be at risk for developing sCRS have been developed (see Davilla et al. Science translational medicine. 2014; 6(224):224ra25). Factors include fevers, hypoxia, hypotension, neurologic changes, elevated serum levels of inflammatory cytokines, such as a set of seven cytokines (IFNγ, IL-5, IL-6, IL-10, Flt-3L, fractalkine, and GM-CSF) whose treatment-induced elevation can correlate well with both pretreatment tumor burden and sCRS symptoms. Other guidelines on the diagnosis and management of CRS are known (see e.g., Lee et al, Blood. 2014; 124(2):188-95). In some embodiments, the criteria reflective of CRS grade are those detailed in Table 1 below.
TABLE 1
Exemplary Grading Criteria for CRS
Grade
Description of Symptoms
1
Not life-threatening, require only symptomatic
Mild
treatment such as antipyretics and anti-emetics (e.g.,
fever, nausea, fatigue, headache, myalgias, malaise)
2
Require and respond to moderate intervention:
Moderate
Oxygen requirement < 40%, or
Hypotension responsive to fluids or low dose of a
single vasopressor, or
Grade 2 organ toxicity (by CTCAE v4.0)
3
Require and respond to aggressive intervention:
Severe
Oxygen requirement ≥ 40%, or
Hypotension requiring high dose of a single
vasopressor (e.g., norepinephrine ≥ 20 μg/kg/min,
dopamine ≥ 10 μg/kg/min, phenylephrine ≥ 200
μg/kg/min, or epinephrine ≥ 10 μg/kg/min),
or
Hypotension requiring multiple vasopressors
(e.g., vasopressin + one of the above agents, or
combination vasopressors equivalent to ≥20
μg/kg/min norepinephrine), or
Grade 3 organ toxicity or Grade 4 transaminitis
(by CTCAE v4.0)
4
Life-threatening:
Life-threatening
Requirement for ventilator support, or
Grade 4 organ toxicity (excluding transaminitis)
5
Death
Fatal
As used herein, a subject is deemed to develop “severe CRS” (“sCRS”) in response to or secondary to administration of a cell therapy or dose of cells thereof, if, following administration, the subject displays: (1) fever of at least 38 degrees Celsius for at least three days; (2) cytokine elevation that includes either (a) a max fold change of at least 75 for at least two of the following group of seven cytokines compared to the level immediately following the administration: interferon gamma (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L, fracktalkine, and IL-5 and/or (b) a max fold change of at least 250 for at least one of the following group of seven cytokines compared to the level immediately following the administration: interferon gamma (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L, fracktalkine, and IL-5; and (c) at least one clinical sign of toxicity such as hypotension (requiring at least one intravenous vasoactive pressor) or hypoxia (PO2<90%) or one or more neurologic disorder(s) (including mental status changes, obtundation, and/or seizures). In some embodiments, severe CRS includes CRS with a grade of 3 or greater, such as set forth in Table 1.
In some embodiments, the CRS encompasses a combination of (1) persistent fever (fever of at least 38 degrees Celsius for at least three days) and (2) a serum level of CRP of at least at or about 20 mg/dL.
In some embodiments, the CRS encompasses hypotension requiring the use of two or more vasopressors or respiratory failure requiring mechanical ventilation.
The method of measuring or detecting the various outcomes may be specified.
In some aspects, prior to the administration of the first dose, subsequent to the administration of the first dose and before administration of the consecutive dose, or subsequent to the administration of the consecutive dose, a CRS-associated outcome is assessed in the subject. In some embodiments, the level of the toxic outcome, e.g. the CRS-related outcome, e.g. the serum level of an indicator of CRS, is measured by ELISA. In some embodiments, fever and/or levels of CRP can be measured. In some embodiments, subjects with a fever and a CRP≥15 mg/dL may be considered high-risk for developing severe CRS.
In some embodiments, the toxic outcome, toxicity, or symptom is measured at a specified time point following administration. In some embodiments, the toxic outcome, e.g. CRS, severe CRS, and/or the CRS-associated outcome or serum level, or the lower degree of the outcome or serum level, is measured at 24 hours, at day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 following the administration, or over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, CRP is measured at day 3, 4, 5, 6, 7, 8, or 9. In some embodiments, the fold change in a factor or multiple factors, such as a cytokine(s), is measured as fold change between the level prior to treatment and at day 2, 3, 4, 5, 6, 7, 14, or 20 or 21, following treatment.
In some embodiments, at the time of the administration of the consecutive dose, the level of the CRS-related outcome is no more than 50% of the peak level, is no more than 40% of the peak level, is no more than 30% of the peak level, is no more than 20% of the peak level, is no more than 15% of the peak level, is no more than 10% of the peak level, is no more than 5% of the peak level, or is at or about or below the level immediately prior to the administration of the first dose and/or is at or about or below baseline.
In some aspects, at the time of the administration of the consecutive dose, the level of the CRS-related outcome is no more than ten times the level immediately prior to the administration of the first dose.
In some embodiments, the CRS-related outcome in the subject at day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 following the administration of the consecutive dose is not detectable or is reduced as compared to a method where the subject is administered the consecutive dose without having been administered the first dose and/or a method in which the cells of the first and second doses are administered in a single dose, and/or a method in which the consecutive dose is given at a time which is earlier than the time between the first and consecutive doses specified by the method.
In some embodiments, the CRS-related outcome in the subject at day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 following the administration of the first dose is not detectable or is reduced as compared to that following administration to the subject a dose of 2-fold, 5-fold, 10-fold, or 100-fold the number of cells, T cells, cells expressing the recombinant receptor, or PBMCs.
In some embodiments, the area under the curve (AUC) for a CRS-related outcome or serum level of a factor indicative of CRS over time in the subject following administration of the consecutive dose is lower as compared to that of a method where the subject is administered the consecutive dose without having been administered the first dose, and/or a method in which the cells of the first and consecutive doses are administered in a single dose, and/or a method in which the consecutive dose is given at a time which is earlier than the time between the first and consecutive doses specified by the method.
In some embodiments, the area under the curve (AUC) for a CRS-related outcome or serum level of a factor indicative of CRS over time in the subject following administration of the first dose is lower as compared to that following administration to the subject dose of 2-fold, 5-fold, 10-fold, or 100-fold the number of cells, T cells, cells expressing the recombinant receptor, or PBMCs.
In some aspects, the toxic outcome is or is associated with neurotoxicity. In some embodiments, symptoms associated with a clinical risk of neurotoxicity include confusion, delirium, expressive aphasia, obtundation, myoclonus, lethargy, altered mental status, convulsions, seizure-like activity, seizures (optionally as confirmed by electroencephalogram [EEG]), elevated levels of beta amyloid (Aβ), elevated levels of glutamate, and elevated levels of oxygen radicals. In some embodiments, neurotoxicity is graded based on severity (e.g., using a Grade 1-5 scale (see, e.g., Guido Cavaletti & Paola Marmiroli Nature Reviews Neurology 6, 657-666 (December 2010); National Cancer Institute—Common Toxicity Criteria version 4.03 (NCI-CTCAE v4.03).
In some instances, neurologic symptoms may be the earliest symptoms of sCRS. In some embodiments, neurologic symptoms are seen to begin 5 to 7 days after cell therapy infusion. In some embodiments, duration of neurologic changes may range from 3 to 19 days. In some cases, recovery of neurologic changes occurs after other symptoms of sCRS have resolved. In some embodiments, time or degree of resolution of neurologic changes is not hastened by treatment with anti-IL-6 and/or steroid(s).
As used herein, a subject is deemed to develop “severe neurotoxicity” in response to or secondary to administration of a cell therapy or dose of cells thereof, if, following administration, the subject displays symptoms that limit self-care (e.g. bathing, dressing and undressing, feeding, using the toilet, taking medications) from among: 1) symptoms of peripheral motor neuropathy, including inflammation or degeneration of the peripheral motor nerves; 2) symptoms of peripheral sensory neuropathy, including inflammation or degeneration of the peripheral sensory nerves, dysesthesia, such as distortion of sensory perception, resulting in an abnormal and unpleasant sensation, neuralgia, such as intense painful sensation along a nerve or a group of nerves, and/or paresthesia, such as functional disturbances of sensory neurons resulting in abnormal cutaneous sensations of tingling, numbness, pressure, cold and warmth in the absence of stimulus. In some embodiments, severe neurotoxicity includes neurotoxicity with a grade of 3 or greater, such as set forth in Table 2.
TABLE 2
Exemplary Grading Criteria for neurotoxicity
Grade
Description of Symptoms
1
Mild or asymptomatic symptoms
Asymptomatic or Mild
2
Presence of symptoms that limit instrumental
Moderate
activities of daily living (ADL), such as
preparing meals, shopping for groceries or
clothes, using the telephone, managing money
3
Presence of symptoms that limit self-care ADL,
Severe
such as bathing, dressing and undressing, feeding
self, using the toilet, taking medications
4
Symptoms that are life-threatening, requiring
Life-threatening
urgent intervention
5
Death
Fatal
In some embodiments, the methods reduce symptoms associated with neurotoxicity compared to other methods. For example, subjects treated according to the present methods may have reduced symptoms of neurotoxicity, such as limb weakness or numbness, loss of memory, vision, and/or intellect, uncontrollable obsessive and/or compulsive behaviors, delusions, headache, cognitive and behavioral problems including loss of motor control, cognitive deterioration, and autonomic nervous system dysfunction, and sexual dysfunction, compared to subjects treated by other methods. In some embodiments, subjects treated according to the present methods may have reduced symptoms associated with peripheral motor neuropathy, peripheral sensory neuropathy, dysethesia, neuralgia or paresthesia.
In some embodiments, the methods reduce outcomes associated with neurotoxicity including damages to the nervous system and/or brain, such as the death of neurons. In some aspects, the methods reduce the level of factors associated with neurotoxicity such as beta amyloid (Aβ), glutamate, and oxygen radicals.
In some embodiments, subjects administered the consecutive dose following the first dose have reduced symptoms, outcomes, or factors associated with neurotoxicity compared to administration of the consecutive dose in the absence of the first dose, administration of the cells of the first and second doses in a single dose, and/or administration of the consecutive dose at a time which is earlier than the time between the first and consecutive doses specified by the method.
V. Host Immune Responses to Transferred Cells
In some embodiments, one or more of the doses, e.g., the consecutive dose(s), is administered at a time at which an immune response, e.g., an adaptive or specific immune response to the transgenic receptor or cells, in the subject is not present, not detectable, or not detectable above a certain level. The presence or degree of a specific immune response to the transgene generally is related to the immunogenicity of the receptor, e.g., the CAR or transgenic TCR, expressed by the cells, and/or the time during which the subject has been exposed to the cells. For example, in some embodiments, an immune response, e.g., a specific humoral and/or cell-mediated immune response against the receptor, is not detected before 28 days, 35 days, or 42 days following the first exposure of the subject to the cells expressing the receptor. Thus, in some embodiments, the consecutive dose is administered before an immune response, an adaptive or specific immune response, a detectable immune response, and/or a memory response against the recombinant receptor or cells has developed in the subject. In this regard, the ability of cells of the consecutive dose to expand and/or persist in the subject is improved in comparison to other methods in which a consecutive dose is given at a later time point in comparison with the prior or first dose.
The methods may involve the detection of the presence or absence or level of such an immune response or indicator thereof, for example, following the administration of a first or consecutive dose and before the administration of the consecutive or next consecutive dose.
In some embodiments, the decision of when and/or whether to administer the consecutive dose depends on whether the subject exhibits such an immune response or detectable readout thereof, e.g., a detectable specific or adaptive host immune response specific for the cells or recombinant receptor, e.g., CAR, expressed by the cells of the first dose, and/or whether such a response is detected above a certain level. In some embodiments, where such a response is detected, the subject is not administered the consecutive dose.
In general, the consecutive dose is administered at a time at which the subject does not exhibit a specific or adaptive, e.g., humoral or cell-mediated, immune response against the receptor, e.g., CAR, expressed by the cells of the first dose, or does not exhibit such a response or indicator thereof at a detectable level or above an acceptable level. In some aspects, at the time of administration of the consecutive dose, the subject exhibits a reduced humoral or cell-mediated immune response against the CAR expressed by the cells of the first dose as compared to when an initial dose is larger.
In some embodiments, the host immune response is or comprises a humoral immune response. The humoral immune response may be indicated by the presence of antibodies specific for the cells or receptors expressed thereby in the serum, other bodily fluid, and/or organ or tissue of the subject. In some embodiments, such antibodies of a particular isotype are present, such as IgM or IgG, e.g., IgG1, IgG2, IgG3, and/or IgG4; in some embodiments they include IgE.
In some embodiments, the immune response is or comprises a cell-mediated component. A cell-mediated response may be indicated by the presence of cells, e.g., T cells, e.g., helper or cytotoxic T cells, that specifically recognize one or more epitopes of the recombinant receptor or cells via a T cell receptor.
In some embodiments the immune response is a primary immune response; in some aspects, the immune response is a memory response.
In some of any of the above embodiments, a detectable immune response refers to an amount detectable by any of a number of known methods for assessing specific immune responses to particular antigens and cells. For example, in some embodiments, the immune response of the specified type is detectable by performing ELISpot, ELISAs, or cell-based antibody detection methods, for example, by flow cytometry, on serum from the subject to detect the presence of antibodies that specifically bind to and/or neutralize antigens present on the cells, e.g., binding to epitopes of the recombinant receptor, e.g., CAR. In some such assays, isotype of the detected antibody is determined and may indicate the type of response and/or whether the response is a memory response.
In some embodiments, the specified immune response is detectable by cytotoxic T-lymphocyte (CTL) assays for detection of CD8+ T cells that specifically bind to and induce cytotoxicity in response to epitopes in the recombinant receptor, and/or a mixed lymphocyte reaction, using cells, e.g., irradiated cells, expressing the recombinant receptor, as stimulator cells.
In some aspects, the detectable immune response is one that is detected by such a method above or significantly above the level of a control sample, such as a non-coated well or well coated with a control peptide or cells not expressing the recombinant receptor and/or levels detected based on pre-treatment serum or blood sample from the subject prior to treatment with the cells expressing the recombinant receptors.
In some aspects, the presence or absence of such a host immune response and/or quantity, degree, or extent thereof, is detected or measured, for example, following the administration of the first dose or consecutive dose.
Humoral immune responses may be detected by any of a number of well-known assays for detection of antibodies specific for particular antigens or cells, including binding assays, immunoassays, and cell-based assays. The assays may include those designed to assess the presence or absence of particular functions of the antibodies, such as their ability to carry out a particular effector function upon binding to the antigen, such as neutralizing antibody assays. In some embodiments, outcomes of humoral immune responses, such as antigen-specific antibodies, e.g., neutralizing antibodies, are detected using cell-based assays, e.g., by incubating pre- and post-treatment cells from the subject with cells expressing the recombinant receptor (and control cells) and detecting antigen-specific binding and/or other outcomes, such as neutralizing outcomes, e.g., by flow cytometry or enzymatic assays. In some embodiments, ELISA, and/or ELISpot assays are used to detect and quantify antibodies specific for the recombinant receptors, such as CARs, and epitopes mapped using known techniques, such as those using individual peptides representing portions of the receptor. See, e.g., Jensen et al. Biol Blood Marrow Transplant. 2010 September; 16(9): 1245-1256. In some embodiments, isotype of the detected antibodies are assessed, for example by using detection antibodies specific for particular isotypes, e.g., human isotypes.
Cellular or cell-based immune response to the cells and/or receptors may be detected and/or measured using any of a number of well-known techniques. Such techniques may include cytotoxic T-lymphocyte (CTL) assays for detection of CD8+ T cells that specifically bind to and induce cytotoxicity in response to epitopes in the recombinant receptor, e.g., CAR, and/or cells administered. In some embodiments, the assay is a mixed lymphocyte reaction, such as those using PBMCs or other host-derived cells from blood or other organ or tissue as responder cells, and cells induced to express the recombinant receptor, e.g., irradiated T cells expressing the CAR, as stimulator cells. The stimulator cells generally are autologous and may be the same cells administered to the subject, and may be irradiated. Non-transduced cells or cells not expressing the transgene of interest may be used as negative controls in place of the stimulator cells in control samples. Likewise, responder cell samples from pre-treatment time points or other subjects may be used in control samples. In some aspects, such assays assess the ability of host cells to carry out one or more effector functions, e.g., antigen-specific cell lysis, e.g., using a chromium release assay to detect cytotoxic T cells present in the subject which specifically recognize antigens present on or in the administered cells and induce a cytotoxic response. In some embodiments, peripheral blood cells, e.g., PBMCs, are obtained from a subject before and after administration of the cells, and each used in an assay, such as a cell lysis assay, using autologous T cells modified to express the recombinant receptor, which generally are irradiated. Specific lysis indicates the presence of receptor-specific cell-mediated immune response. Epitope mapping may be carried out using panels of peptides representing portions of the recombinant receptor. See, e.g., Riddell et al., Nature Medicine 2, 216-223 (1996); Lamers, Blood 2011 117: 72-82. HLA tetramer binding assays may be used for the enumeration of antigen-specific T cells. In some aspects, lymphoproliferative assays (LPAs) and/or assays to assess for secreted cytokines, such as ELISAs and/or intracellular staining and assessment by flow cytometry, are used for detection of transgene-specific CD4+ T cells.
In some embodiments, the method prevents the induction of or reduces the level of antibodies against the receptor, e.g. anti-CAR antibodies. For example, antibody titers of anti-receptor, e.g. anti-CAR, antibodies, for example, as measured in the serum of the subject by ELISA, are decreased following administration of the consecutive dose, as compared to methods in which a consecutive dose is administered at a different time relative to the administration of the first dose, such as at a later time, e.g., following relapse. Thus, in some embodiments, the methods improve efficacy by increasing exposure of the subject to the administered cells by preventing or reducing host immune responses that would otherwise clear or prevent expansion of the administered cells.
VI. Disease Burden
The administration, e.g., of one or more of the doses, generally reduces or prevents the expansion or burden of the disease or condition in the subject. For example, where the disease or condition is a tumor, the methods generally reduce tumor size, bulk, metastasis, percentage of blasts in the bone marrow or molecularly detectable cancer and/or improve prognosis or survival or other symptom associated with tumor burden. In some embodiments, administration of the consecutive dose is timed with respect to a decrease in burden and/or relapse following the first or prior dose.
Disease burden can encompass a total number of cells of the disease in the subject or in an organ, tissue, or bodily fluid of the subject, such as the organ or tissue of the tumor or another location, e.g., which would indicate metastasis. For example, tumor cells may be detected and/or quantified in the blood or bone marrow in the context of certain hematological malignancies. Disease burden can include, in some embodiments, the mass of a tumor, the number or extent of metastases and/or the percentage of blast cells present in the bone marrow.
In some embodiments, a subject has leukemia. The extent of disease burden can be determined by assessment of residual leukemia in blood or bone marrow. In some embodiments, a subject exhibits morphologic disease if there are greater than or equal to 5% blasts in the bone marrow, for example, as detected by light microscopy. In some embodiments, a subject exhibits complete or clinical remission if there are less than 5% blasts in the bone marrow.
In some embodiments, a subject may exhibit complete remission, but a small proportion of morphologically undetectable (by light microscopy techniques) residual leukemic cells are present. A subject is said to exhibit minimum residual disease (MRD) if the subject exhibits less than 5% blasts in the bone marrow and exhibits molecularly detectable cancer. In some embodiments, molecularly detectable cancer can be assessed using any of a variety of molecular techniques that permit sensitive detection of a small number of cells. In some aspects, such techniques include PCR assays, which can determine unique Ig/T-cell receptor gene rearrangements or fusion transcripts produced by chromosome translocations. In some embodiments, flow cytometry can be used to identify cancer cell based on leukemia-specific immunophenotypes. In some embodiments, molecular detection of cancer can detect as few as 1 leukemia cell in 100,000 normal cells. In some embodiments, a subject exhibits MRD that is molecularly detectable if at least or greater than 1 leukemia cell in 100,000 cells is detected, such as by PCR or flow cytometry. In some embodiments, the disease burden of a subject is molecularly undetectable or MRD−, such that, in some cases, no leukemia cells are able to be detected in the subject using PCR or flow cytometry techniques.
In some embodiments, the methods and/or administration of the first dose decrease(s) disease burden as compared with disease burden at a time immediately prior to the administration of the first dose. In some aspects, administration of the first dose reduces disease burden, e.g. tumor burden. In some embodiments, the consecutive dose effects a reduction, e.g., a further reduction, in disease burden.
In some embodiments, the first dose contains the cells in an amount that is effective to reduce burden of a disease or condition in the subject, e.g., tumor burden. In some embodiments, e.g. where the disease or condition is a tumor, administration of the first dose is one that debulks the tumor. As used herein, a “debulking dose” refers to a dose that is effective to at least partially reduce burden of the disease or condition, e.g. tumor burden, in the subject. In some aspects, the debulking dose may not completely eradicate the disease or condition.
In some aspects, administration of the first dose and/or consecutive dose may prevent an increase in disease burden, and this may be evidenced by no change in disease burden.
In some aspects, the disease or condition persists following administration of the first dose and/or administration of the first dose is not sufficient to eradicate the disease or condition in the subject.
In some aspects, administration of the consecutive dose reduces disease burden as compared to disease burden at a time immediately prior to the first dose, or at a time immediately prior to the consecutive dose. In some aspects, for example in the context of relapse, administration of the consecutive dose effects a reduction in disease burden as compared to the peak level of disease burden following administration of the first dose.
In some embodiments, the method reduces the burden of the disease or condition, e.g., number of tumor cells, size of tumor, duration of patient survival or event-free survival, to a greater degree and/or for a greater period of time as compared to the reduction that would be observed with a comparable method using an alternative dosing regimen, such as one in which the subject receives a single dose, e.g., a single large dose, of cells, e.g. administration of the total number of cells administered in the first dose and the consecutive dose, collectively, in the provided methods, instead as a single dose, or administration of multiple large doses or multiple doses spaced from one another by less than about 14 or more than about 28 days. In some embodiments, disease burden is reduced to a greater extent or for a greater duration following the consecutive dose compared to the reduction that would be effected by administering the consecutive dose to a subject having not received the first dose.
In some embodiments, the burden of a disease or condition in the subject is detected, assessed, or measured. Disease burden may be detected in some aspects by detecting the total number of disease or disease-associated cells, e.g., tumor cells, in the subject, or in an organ, tissue, or bodily fluid of the subject, such as blood or serum. In some embodiments, disease burden, e.g. tumor burden, is assessed by measuring the mass of a solid tumor and/or the number or extent of metastases. In some aspects, survival of the subject, survival within a certain time period, extent of survival, presence or duration of event-free or symptom-free survival, or relapse-free survival, is assessed. In some embodiments, any symptom of the disease or condition is assessed. In some embodiments, the measure of disease or condition burden is specified.
In some aspects, disease burden is measured or detected prior to administration of the first dose, following the administration of the first dose but prior to administration of the consecutive dose, and/or following administration of the consecutive dose. In the context of multiple consecutive doses, disease burden in some embodiments may be measured prior to or following any of the consecutive doses, or at a time between administration of consecutive doses.
In some embodiments, the burden is decreased by or by at least at or about 10, 20, 30, 40, 50, 60, 70, 90, or 100 percent after administration of the first dose. In some aspects, administration of the consecutive dose effects a further reduction in disease burden, e.g. tumor burden, such as a at or about 10, 20, 30, 40, 50, 60, 70, 90, or 100 percent decrease in burden compared to immediately prior to the administration of the consecutive dose or overall compared to immediately prior to the first dose. In some embodiments, disease burden, tumor size, tumor volume, tumor mass, and/or tumor load or bulk is reduced following the consecutive dose by at least at or about 50, 60, 70, 80, 90% or more compared to that immediately prior to the administration of the first dose or of the consecutive dose.
In some embodiments, reduction of disease burden by the method comprises an induction in morphologic complete remission, for example, as assessed at 1 month, 2 months, 3 months, or more than 3 months, after administration of, e.g., initiation of, the first or consecutive dose. In some aspects, an assay for minimal residual disease, for example, as measured by multiparametric flow cytometry, is negative, or the level of minimal residual disease is less than about 0.3%, less than about 0.2%, less than about 0.1%, or less than about 0.05%.
In some embodiments, the event-free survival rate or overall survival rate of the subject is improved by the methods, as compared with other methods. For example, in some embodiments, event-free survival rate or probability for subjects treated by the methods at 6 months following the first dose is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some aspects, overall survival rate is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some embodiments, the subject treated with the methods exhibits event-free survival, relapse-free survival, or survival to at least 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some embodiments, the time to progression is improved, such as a time to progression of greater than at or about 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
In some embodiments, following treatment by the method, the probability of relapse is reduced as compared to other methods. For example, in some embodiments, the probability of relapse at 6 months following the first dose is less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10%.
In some aspects, reduction of disease burden, e.g. debulking of the tumor, following the first dose reduces toxicity or toxic outcomes following the consecutive dose. Toxic outcomes following a reduction in tumor burden can be assessed as described herein.
In some aspects, reduction of disease burden, e.g. debulking of the tumor, resulting from the present methods improves the persistence of the cells in the subject. For example, in some aspects, administration of the first dose reduces disease burden, e.g. tumor burden, such that the cells administered in the consecutive dose persist for longer than cells administered by other dosing regimens, such as administering the consecutive dose to a subject that has not been administered the cells of the first dose.
VII. Cell Exposure and Persistence
In some embodiments, the dose amount(s) and/or timing thereof are designed to promote exposure of the subject to the cells, such as by promoting their expansion and/or persistence over time.
In some embodiments, the provided methods increase exposure of the subject to the administered cells (e.g., increased number of cells or duration over time) and/or improve efficacy and therapeutic outcomes in adoptive cell therapy. In some aspects, the methods are advantageous in that a greater and/or longer degree of exposure to the cells expressing the recombinant receptors, e.g., CAR-expressing cells, improves treatment outcomes as compared with other methods. Such outcomes may include patient survival and remission, even in individuals with severe tumor burden.
In some embodiments, the administration of the first dose, e.g., first low dose, increases maximum, total, and/or duration of exposure to the cells in the subject as compared to administration of a high initial dose of the cells. In some aspects, administration of the first dose in the context of high disease burden (and thus higher amounts of antigen) enhances efficacy as compared with administration of a larger dose in the same context, which may result in exhaustion which may prevent expansion and/or persistence of the cells. In some embodiments, administering the first dose in the context of high disease burden reduces exhaustion of the transferred cells, thereby increasing clinical efficacy as compared to other methods, such as those where a higher initial dose is administered.
In some embodiments, the presence and/or amount of cells expressing the recombinant receptor (e.g., CAR-expressing cells) in the subject following the first dose and/or following the consecutive dose is detected. In some aspects, quantitative PCR (qPCR) is used to assess the quantity of cells expressing the recombinant receptor (e.g., CAR-expressing cells) in the blood or serum or organ or tissue (e.g., disease site) of the subject. In some aspects, persistence is quantified as copies of DNA or plasmid encoding the receptor, e.g., CAR, per microgram of DNA, or as the number of receptor-expressing, e.g., CAR-expressing, cells per microliter of the sample, e.g., of blood or serum, or per total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or T cells per microliter of the sample.
In some embodiments, the cells are detected in the subject at or at least at 4, 14, 15, 27, or 28 days following the administration of the first dose. In some aspects, the cells are detected at or at least at 2, 4, or 6 weeks following, or 3, 6, or 12, 18, or 24, or 30 or 36 months, or 1, 2, 3, 4, 5, or more years, following administration of the first or consecutive dose.
In some embodiments, the persistence of receptor, e.g., CAR,-expressing cells in the subject by the methods, following the consecutive dose, and/or following administration of the first dose, is greater as compared to that which would be achieved by alternative methods such as those involving the administration of a single dose, e.g., containing a larger number of cells than the first dose, administration of the cells of the collective doses as a single dose, administration of the cells of the consecutive dose without the subject having received the first dose, and/or administration of the consecutive dose at a time that is outside the specified time window such as later than the time specified or following the mounting of an immune response by the subject against the receptor, e.g., the CAR.
In some embodiments, the persistence and/or expansion and/or presence of recombinant receptor-expressing, e.g., CAR-expressing, cells in the subject following administration of the consecutive dose is greater as compared to that achieved via a method using an alternative dosing regimen, such as one involving the administration of the cells of the consecutive dose without the subject having been administered the cells of the first dose or where the subject is administered the cells collectively administered in the first and consecutive doses as a single dose.
The exposure, e.g., number of cells, indicative of expansion and/or persistence, may be stated in terms of maximum numbers of the cells to which the subject is exposed, duration of detectable cells or cells above a certain number or percentage, area under the curve for number of cells over time, and/or combinations thereof and indicators thereof. Such outcomes may be assessed using known methods, such as qPCR to detect copy number of nucleic acid encoding the recombinant receptor compared to total amount of nucleic acid or DNA in the particular sample, e.g., blood or serum, and/or flow cytometric assays detecting cells expressing the receptor generally using antibodies specific for the receptors. Cell-based assays may also be used to detect the number or percentage of functional cells, such as cells capable of binding to and/or neutralizing and/or inducing responses, e.g., cytotoxic responses, against cells of the disease or condition or expressing the antigen recognized by the receptor.
In some aspects, increased exposure of the subject to the cells includes increased expansion of the cells. In some embodiments, the receptor- (e.g., CAR-) expressing cells expand in the subject following administration of the first dose and/or following administration of the consecutive dose. In some aspects, the methods result in greater expansion of the cells compared with other methods, such as those involving the administration of the cells as a single dose, administration of larger first doses, administration of the consecutive dose without administering the first dose, and/or methods in which a consecutive dose is administered before or after the specified window of time or time point, such that, for example, an immune response develops prior to the administration of the consecutive dose.
In some aspects, the method results in high in vivo proliferation of the administered cells, for example, as measured by flow cytometry. In some aspects, high peak proportions of the cells are detected. For example, in some embodiments, at a peak or maximum level following the first or consecutive administration, in the blood or disease-site of the subject or white blood cell fraction thereof, e.g., PBMC fraction or T cell fraction, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells express the recombinant receptor, e.g., the CAR.
In some embodiments, the method results in a maximum concentration, in the blood or serum or other bodily fluid or organ or tissue of the subject, of at least 100, 500, 1000, 1500, 2000, 5000, 10,000 or 15,000 copies of or nucleic acid encoding the receptor, e.g., the CAR per microgram of DNA, or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 receptor-expressing, e.g., CAR,-expressing cells per total number of peripheral blood mononuclear cells (PBMCs), total number of mononuclear cells, total number of T cells, or total number of microliters. In some embodiments, the cells expressing the receptor are detected as at least 10, 20, 30, 40, 50, or 60% of total PBMCs in the blood of the subject, and/or at such a level for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, or 52 weeks following the first or consecutive administration or for 1, 2, 3, 4, or 5, or more years following such administration.
In some aspects, the method results in at least a 2-fold, at least a 4-fold, at least a 10-fold, or at least a 20-fold increase in copies of nucleic acid encoding the recombinant receptor, e.g., CAR, per microgram of DNA, e.g., in the serum of the subject.
In some embodiments, cells expressing the receptor are detectable in the blood or serum of the subject, e.g., by a specified method, such as qPCR or flow cytometry-based detection method, at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 or more days following administration of the first dose or after administration of the consecutive dose, for at least at or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 or more weeks following the administration of the first dose or the consecutive dose.
In some aspects, at least about 1×102, at least about 1×103, at least about 1×104, at least about 1×105, or at least about 1×106 or at least about 5×106 or at least about 1×107 or at least about 5×107 or at least about 1×108 recombinant receptor-expressing, e.g., CAR-expressing cells, and/or at least 10, 25, 50, 100, 200, 300, 400, or 500, or 1000 receptor-expressing cells per microliter, e.g., at least 10 per microliter, are detectable or are present in the subject or fluid, tissue, or compartment thereof, such as in the blood, e.g., peripheral blood, or disease site thereof. In some embodiments, such a number or concentration of cells is detectable in the subject for at least about 20 days, at least about 40 days, or at least about 60 days, or at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 2 or 3 years, following administration of the first dose or following the administration of the consecutive dose(s). Such cell numbers may be as detected by flow cytometry-based or quantitative PCR-based methods and extrapolation to total cell numbers using known methods. See, e.g., Brentjens et al., Sci Transl Med. 2013 5(177), Park et al, Molecular Therapy 15(4):825-833 (2007), Savoldo et al., JCI 121(5):1822-1826 (2011), Davila et al. (2013) PLoS ONE 8(4):e61338, Davila et al., Oncoimmunology 1(9):1577-1583 (2012), Lamers, Blood 2011 117:72-82, Jensen et al. Biol Blood Marrow Transplant 2010 September; 16(9): 1245-1256, Brentjens et al., Blood 2011 118(18):4817-4828.
In some aspects, the copy number of nucleic acid encoding the recombinant receptor, e.g., vector copy number, per 100 cells, for example in the peripheral blood or bone marrow or other compartment, as measured by immunohistochemistry, PCR, and/or flow cytometry, is at least 0.01, at least 0.1, at least 1, or at least 10, at about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or at least about 6 weeks, or at least about 2, 3, 4, 5, 6, 7, 8. 9, 10, 11, or 12 months or at least 2 or 3 years following administration of the cells, e.g., the first or consecutive dose(s). In some embodiments, the copy number of the vector expressing the receptor, e.g. CAR, per microgram of genomic DNA is at least 100, at least 1000, at least 5000, or at least 10,000, or at least 15,000 or at least 20,000 at a time about 1 week, about 2 weeks, about 3 weeks, or at least about 4 weeks following administration of the first dose or consecutive dose(s) of receptor-expressing, e.g. CAR-expressing, cells, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or at least 2 or 3 years following such administration.
In some aspects, the receptor, e.g. CAR, expressed by the cells, is detectable by quantitative PCR (qPCR) or by flow cytometry in the subject, blood thereof, and/or disease site thereof, at a time that is at least about 3 months, at least about 6 months, at least about 12 months, at least about 1 year, at least about 2 years, at least about 3 years, or more than 3 years, following the administration of the cells, e.g., following the initiation of the administration of the first dose or the consecutive dose or subsequent consecutive dose.
In some embodiments, the area under the curve (AUC) for concentration of receptor- (e.g., CAR-) expressing cells in a fluid, tissue, or organ, e.g., blood, of the subject over time following the administration of the first dose is greater as compared to that achieved via an alternative dosing regimen where the subject is administered the cells of the first dose and the consecutive dose as a single dose.
In some aspects, the area under the curve (AUC) for concentration of receptor- (e.g., CAR-) expressing cells in a fluid, tissue, or organ, e.g., blood, of the subject over time following the administration of the consecutive dose is greater as compared to that achieved via an alternative dosing regimen where the subject is administered the consecutive dose without having been administered the first dose or in which the cells of the first and second doses are administered in a single dose.
VIII. Recombinant Receptors Expressed by the Cells
The cells generally express recombinant receptors, including antigen receptors such as functional non-TCR antigen receptors, e.g., chimeric antigen receptors (CARs), and other antigen-binding receptors such as transgenic T cell receptors (TCRs). Also among the receptors are other chimeric receptors.
Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in international patent application publication numbers WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the antigen receptors include a CAR as described in U.S. Pat. No. 7,446,190, and those described in International Patent Application Publication No.: WO/2014055668 A1. Examples of the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190, 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190, and 8,389,282.
Among the chimeric receptors are chimeric antigen receptors (CARs). The chimeric receptors, such as CARs, generally include an extracellular antigen binding domain, such as a portion of an antibody molecule, generally a variable heavy (VH) chain region and/or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment.
In some embodiments, the antibody portion of the recombinant receptor, e.g., CAR, further includes a spacer, which may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region, e.g., an IgG4 hinge region, and/or a CH1/CL and/or Fc region. In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgG1. In some aspects, the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. In some examples, the spacer is or is about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, international patent application publication number WO2014031687, U.S. Pat. No. 8,822,647 or published app. No. US2014/0271635.
In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgG1. In some embodiments, the spacer has the sequence ESKYGPPCPPCP (set forth in SEQ ID NO: 1), and is encoded by the sequence set forth in SEQ ID NO: 2. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 3. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 4. In some embodiments, the constant region or portion is of IgD. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 5. In some embodiments, the spacer has a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 1, 3, 4 or 5.
This antigen recognition domain generally is linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and/or signal via another cell surface receptor. Thus, in some embodiments, the antigen-binding component (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154 and/or transmembrane regions containing functional variants thereof such as those retaining a substantial portion of the structural, e.g., transmembrane, properties thereof. In some embodiments, the transmembrane domain is a transmembrane domain derived from CD4, CD28, or CD8, e.g., CD8alpha, or functional variant thereof. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and/or transmembrane domain(s).
Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
The receptor, e.g., the CAR, generally includes at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the antigen-binding portion is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR or other chimeric receptor includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25 or CD16.
In some embodiments, upon ligation of the CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptors to initiate signal transduction following antigen receptor engagement.
In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.
T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.
In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.
In some embodiments, the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR includes both the activating and costimulatory components.
In some embodiments, the activating domain is included within one CAR, whereas the costimulatory component is provided by another CAR recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see WO2014/055668). In some aspects, the cells include one or more stimulatory or activating CAR and/or a costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than the one associated with and/or specific for the disease or condition whereby an activating signal delivered through the disease-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.
In some embodiments, the intracellular signaling component of the recombinant receptor, such as CAR, comprises a CD3 zeta intracellular domain and a costimulatory signaling region. In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and/or CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.
In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.
In some embodiments, the CAR or other antigen receptor further includes a marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor, such as a truncated version of a cell surface receptor, such as truncated EGFR (tEGFR). In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. For example, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence. An exemplary polypeptide for a truncated EGFR (e.g. tEGFR) comprises the sequence of amino acids set forth in SEQ ID NO: 15 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 15. An exemplary T2A linker sequence comprises the sequence of amino acids set forth in SEQ ID NO: 14 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14.
In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.
In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self” by the immune system of the host into which the cells will be adoptively transferred.
In some embodiments, the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive transfer and encounter with ligand.
In some cases, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR is one that includes multiple costimulatory domains of different costimulatory receptors.
In some embodiments, the chimeric antigen receptor includes an extracellular portion containing an antibody or antibody fragment. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment includes an scFv and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain includes a signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some aspects, the transmembrane domain contains a transmembrane portion of CD28. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. The extracellular domain and transmembrane domain can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein. In some embodiments, the receptor contains extracellular portion of the molecule from which the transmembrane domain is derived, such as a CD28 extracellular portion. In some embodiments, the chimeric antigen receptor contains an intracellular domain derived from a T cell costimulatory molecule or a functional variant thereof, such as between the transmembrane domain and intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.
For example, in some embodiments, the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some embodiments, the CAR contains an antibody, e.g., antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4-1BB or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some such embodiments, the receptor further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4 hinge, such as a hinge-only spacer.
In some embodiments, the transmembrane domain of the recombinant receptor, e.g., the CAR, is or includes a transmembrane domain of human CD28 (e.g. Accession No. P01747.1) or variant thereof, such as a transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 6 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 6; in some embodiments, the transmembrane-domain containing portion of the recombinant receptor comprises the sequence of amino acids set forth in SEQ ID NO: 7 or a sequence of amino acids having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
In some embodiments, the intracellular signaling component(s) of the recombinant receptor, e.g. the CAR, contains an intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as a domain with an LL to GG substitution at positions 186-187 of a native CD28 protein. For example, the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 8 or 9 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8 or 9. In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of 4-1BB (e.g. (Accession No. Q07011.1) or functional variant or portion thereof, such as the sequence of amino acids set forth in SEQ ID NO: 10 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10.
In some embodiments, the intracellular signaling domain of the recombinant receptor, e.g. the CAR, comprises a human CD3 zeta stimulatory signaling domain or functional variant thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human CD3ζ (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. No. 7,446,190 or 8,911,993. For example, in some embodiments, the intracellular signaling domain comprises the sequence of amino acids as set forth in SEQ ID NO: 11, 12 or 13 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11, 12 or 13.
In some aspects, the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgG1, such as the hinge only spacer set forth in SEQ ID NO: 1. In other embodiments, the spacer is or contains an Ig hinge, e.g., an IgG4-derived hinge, optionally linked to a CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains, such as set forth in SEQ ID NO: 4. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a CH3 domain only, such as set forth in SEQ ID NO: 3. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers.
For example, in some embodiments, the CAR includes an antibody such as an antibody fragment, including scFvs, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR includes an antibody or fragment, such as scFv, a spacer such as any of the Ig-hinge containing spacers, a CD28-derived transmembrane domain, a 4-1BB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.
In some embodiments, such CAR constructs further includes a T2A ribosomal skip element and/or a tEGFR sequence, e.g., downstream of the CAR, such as set forth in SEQ ID NO: 14 and/or 15, respectively, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14 or 15.
The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided receptors and other polypeptides, e.g., linkers or peptides, may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
In some embodiments, the receptor, e.g., the CAR, expressed by the cells in the consecutive dose contains at least one immunoreactive epitope as the receptor, e.g., the CAR, expressed by the cells of the first dose. In some aspects, the receptor, e.g., the CAR, expressed by the cells administered in the consecutive dose is identical to the receptor, e.g., the CAR, expressed by the first dose or is substantially identical to the receptor, e.g., the CAR, expressed by the cells of administered in the first dose.
The recombinant receptors, such as CARs, expressed by the cells administered to the subject in the various doses generally recognize or specifically bind to a molecule that is expressed in, associated with, and/or specific for the disease or condition or cells thereof being treated. Upon specific binding to the molecule, e.g., antigen, the receptor generally delivers an immunostimulatory signal, such as an ITAM-transduced signal, into the cell, thereby promoting an immune response targeted to the disease or condition. For example, in some embodiments, the cells in the first dose express a CAR that specifically binds to an antigen expressed by a cell or tissue of the disease or condition or associated with the disease or condition.
The receptor, e.g., the CAR, expressed by the cells in the consecutive dose(s) generally specifically binds to the same antigen as the CAR of the first dose and is often the same receptor or extremely similar to the receptor in the cells of the first dose. In some embodiments, the receptor on the cells in the consecutive dose(s) is the same as or shares a large degree of identity with the receptor in the cells of the first dose.
In some embodiments, the CAR expressed by the cells of the consecutive dose contains the same scFv, the same signaling domains, and/or the same junctions as the CAR expressed by the cells of the first dose. In some embodiments, it further contains the same costimulatory, stimulatory, transmembrane, and/or other domains as that of the first dose. In some embodiments, one or more component of the CAR of the consecutive dose is distinct from the CAR of the first dose.
IX. Engineered Cells
Among the cells expressing the receptors and administered by the provided methods are engineered cells.
The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, and re-introducing them into the same subject, before or after cryopreservation.
Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naïve T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
In some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.
Vectors and Methods for Genetic Engineering
Also provided are methods, compositions, and kits, for producing the genetically engineered cells expressing recombinant receptors. The genetic engineering generally involves introduction of a nucleic acid encoding the recombinant or engineered component into the cell, such as by retroviral transduction, transfection, or transformation.
In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.
In some contexts, overexpression of a stimulatory factor (for example, a lymphokine or a cytokine) may be toxic to a subject. Thus, in some contexts, the engineered cells include gene segments that cause the cells to be susceptible to negative selection in vivo, such as upon administration in adoptive immunotherapy. For example in some aspects, the cells are engineered so that they can be eliminated as a result of a change in the in vivo condition of the subject to which they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell II:223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).
In some aspects, the cells further are engineered to promote expression of cytokines or other factors. Various methods for the introduction of genetically engineered components, e.g., antigen receptors, e.g., CARs, are well known and may be used with the provided methods and compositions. Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.
In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557.
In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
Methods of lentiviral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.
In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).
Other approaches and vectors for transfer of the nucleic acids encoding the recombinant products are those described, e.g., in international patent application, Publication No.: WO2014055668, and U.S. Pat. No. 7,446,190.
Among additional nucleic acids, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.
Preparation of Cells for Engineering
In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for introduction of the nucleic acid encoding the transgenic receptor such as the CAR, may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig.
In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.
In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++/Mg++ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.
In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques.
For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (markerhigh) on the positively or negatively selected cells, respectively.
In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining Tcm-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.
In embodiments, memory T cells are present in both CD62L+ and CD62L− subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L−CD8+ and/or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.
In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.
CD4+ T helper cells are sorted into naïve, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L− and CD45RO−.
In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, N.J.).
In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.
In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.
The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.
In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.
In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.
In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.
In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Number WO2009/072003, or US 20110003380 A1.
In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.
In some aspects, the separation and/or other steps is carried out using CliniMACS system (Miltenyi Biotic), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.
The CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.
In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.
In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.
In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are generally then frozen to −80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.
In some embodiments, the provided methods include cultivation, incubation, culture, and/or genetic engineering steps. For example, in some embodiments, provided are methods for incubating and/or engineering the depleted cell populations and culture-initiating compositions.
Thus, in some embodiments, the cell populations are incubated in a culture-initiating composition. The incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.
In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agents include IL-2 and/or IL-15, for example, an IL-2 concentration of at least about 10 units/mL.
In some aspects, incubation is carried out in accordance with techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, the T cells are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.
In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.
In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigen-specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.
X. Compositions and Formulations
Also provided are compositions including the cells for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.
The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
In some aspects, the choice of carrier is determined in part by the particular cell and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).
Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.
In some embodiments, the composition includes the cells in an amount effective to reduce burden of the disease or condition, and/or in an amount that does not result in CRS or severe CRS in the subject and/or to effect any of the other outcomes of the methods as described herein.
The cells and compositions may be administered using standard administration techniques, formulations, and/or devices. Administration of the cells can be autologous or heterologous. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyoi (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.
Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
XII. Articles of Manufacture
Also provided are articles of manufacture, such as kits and devices, for the administration of the cells to subjects in according to the provided methods for adoptive cell therapy, and for storage and administration of the cells and compositions.
The articles of manufacture include one or more containers, typically a plurality of containers, packaging material, and a label or package insert on or associated with the container or containers and/or packaging, generally including instructions for administration of the cells to a subject.
The containers generally contain the cells to be administered, e.g., one or more unit doses thereof. The article of manufacture typically includes a plurality of containers, each containing a single unit dose of the cells. The unit dose may be an amount or number of the cells to be administered to the subject in the first dose or twice the number (or more) the cells to be administered in the first or consecutive dose(s). It may be the lowest dose or lowest possible dose of the cells that would be administered to the subject in connection with the administration method. In some embodiments, the unit dose is the minimum number of cells or number of cells that would be administered in a single dose to any subject having a particular disease or condition or any subject, according to the methods herein. For example, the unit dose in some aspects may include a minimum number of cells that would be administered to a patient of a relatively lower body weight and/or with relatively low disease burden, such that one and in some cases more than one unit dose is administered to a given subject as a first dose and one or more than one unit dose is administered to a given subject in one or more consecutive dose, e.g., according to the provided methods. In some embodiments, the number of cells in the unit dose is the number of cells or number of recombinant receptor-expressing or CAR-expressing cells that it is desired to administer to a particular subject in a first dose, such as a subject from which the cells have been derived. In some embodiments, the cells have been derived from the subject to be treated by methods as provided herein or in need thereof.
In some embodiments, each of the containers individually comprises a unit dose of the cells, e.g., including the same or substantially the same number of cells. Thus in some embodiments, each of the containers comprises the same or approximately or substantially the same number of cells or number of recombinant receptor-expressing cells. In some embodiments, the unit dose includes less than about 1×108, less than about 5×107, less than about 1×106 or less than about 5×105 of the engineered cells, of total cells, of T cells, or PBMCs, per kg of the subject to be treated and/or from which the cells have been derived. In some embodiments, each unit dose contains at or about 2×106, 5×106, 1×107, 5×107, or 1×108 engineered cells, total cells, T cells, or PBMCs.
Suitable containers include, for example, bottles, vials, syringes, and flexible bags, such as infusion bags. In particular embodiments, the containers are bags, e.g., flexible bags, such as those suitable for infusion of cells to subjects, e.g., flexible plastic or PVC bags, and/or IV solution bags. The bags in some embodiments are sealable and/or able to be sterilized, so as to provide sterile solution and delivery of the cells and compositions. In some embodiments, the containers, e.g., bags, have a capacity of at or about or at least at or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000 ml capacity, such as between at or about 10 and at or about 100 or between at or about 10 and at or about 500 mL capacity. In some embodiments, the containers, e.g., bags, are and/or are made from material which is stable and/or provide stable storage and/or maintenance of cells at one or more of various temperatures, such as in cold temperatures, e.g. below at or about or at or about −20° C., −80° C., −120° C., 135° C. and/or temperatures suitable for cryopreservation, and/or other temperatures, such as temperatures suitable for thawing the cells and body temperature such as at or about 37° C., for example, to permit thawing, e.g., at the subject's location or location of treatment, e.g., at bedside, immediately prior to treatment.
The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container has one or more port, e.g., sterile access ports, for example, for connection of tubing or cannulation to one or more tubes, e.g., for intravenous or other infusion and/or for connection for purposes of transfer to and from other containers, such as cell culture and/or storage bags or other containers. Exemplary containers include infusion bags, intravenous solution bags, vials, including those with stoppers pierceable by a needle for injection.
The article of manufacture may further include a package insert or label with one or more pieces of identifying information and/or instructions for use. In some embodiments, the information or instructions indicates that the contents can or should be used to treat a particular condition or disease, and/or providing instructions therefor. The label or package insert may indicate that the contents of the article of manufacture are to be used for treating the disease or condition. In some embodiments, the label or package insert provides instructions to treat a subject, e.g., the subject from which the cells have been derived, via a method involving the administration of a first and one or more consecutive doses of the cells, e.g., according to any of the embodiments of the provided methods. In some embodiments, the instructions specify administration, in a first dose, of one unit dose, e.g., the contents of a single individual container in the article of manufacture, followed by one or more consecutive doses at a specified time point or within a specified time window and/or after the detection of the presence or absence or amount or degree of one or more factors or outcomes in the subject.
In some embodiments, the instructions specify administering a plurality of the unit doses to the subject by carrying out a first administration and a consecutive administration. In some embodiments, the first administration comprises delivering one of said unit doses to the subject and the consecutive administration comprises administering one or a plurality of said unit doses to the subject.
In some embodiments, the instructions specify that the consecutive administration is to be carried out at a time between about 15 and about 27 days or between about 9 and about 35 days, e.g., at or about 21 days, following the first administration, e.g., following the initiation of the first administration or the prior administration. In some embodiments, the instructions specify that the consecutive dose is to be administered at a time after which it has been determined that a serum level of a factor indicative of cytokine-release syndrome (CRS) in the subject is less than about 10 times, less than about 25 times, and/or less than about 50 times the serum level of the indicator in the subject immediately prior to said first administration, and/or that an indicator of CRS has peaked and is declining, and/or that the subject does not exhibit a detectable adaptive host immune response specific for the receptor, e.g., CAR, expressed by the cells.
In some embodiments, the label or package insert or packaging comprises an identifier to indicate the specific identity of the subject from which the cells are derived and/or are to be administered. In the case of autologous transfer, the identity of the subject from which the cells are derived is the same as the identity of the subject to which the cells are to be administered. Thus, the identifying information may specify that the cells are to be administered to a particular patient, such as the one from which the cells were originally derived. Such information may be present in the packaging material and/or label in the form of a bar code or other coded identifier, or may indication the name and/or other identifying characteristics of the subject.
The article of manufacture in some embodiments includes one or more, typically a plurality, of containers containing compositions comprising the cells, e.g., individual unit dose forms thereof, and further include one or more additional containers with a composition contained therein which includes a further agent, such as a cytotoxic or otherwise therapeutic agent, for example, which is to be administered in combination, e.g., simultaneously or sequentially in any order, with the cells. Alternatively, or additionally, the article of manufacture may further include another or the same container comprising a pharmaceutically-acceptable buffer. It may further include other materials such as other buffers, diluents, filters, tubing, needles, and/or syringes.
The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include “consisting” and/or “consisting essentially of” aspects and variations.
Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.
The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.
As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
As used herein, a statement that a cell or population of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.
As used herein, a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.
The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
XIII. Exemplary Embodiments
Among the embodiments provided herein are:
1. A method of treatment, comprising:
(a) administering to a subject having a disease or condition a first dose of cells expressing a chimeric antigen receptor (CAR), said first dose comprising no more than about 1×106 of the cells per kilogram body weight of the subject, no more than about 1×108 of the cells, and/or no more than about 1×108 of the cells/m2 of the subject; and
(b) administering to the subject a consecutive dose of cells expressing a CAR at a time point that is at least or more than about 14 days after and less than about 28 days after initiation of said administration in (a).
2. The method of embodiment 1, wherein, at the time of the administration in (b):
(i) the serum level in the subject of a factor indicative of cytokine release syndrome (CRS) is less than about 10 times, less than about 25 times, and/or less than about 50 times that in the subject immediately prior to said administration in (a); and/or
(ii) the subject does not exhibit grade 3 or higher neurotoxicity; and/or
(iii) a CRS-related outcome or symptom of neurotoxicity in the subject following said administration of said first dose has reached a peak level and begun to decline following the administration in (a); and/or
(iv) the subject does not exhibit a detectable humoral or cell-mediated immune response against the CAR expressed by the cells of said first dose.
3. A method of treatment, comprising:
(a) administering to a subject a first dose of cells expressing a chimeric antigen receptor (CAR), said first dose comprising the cells in an amount sufficient to reduce burden of a disease or condition in the subject; and
(b) administering to the subject a consecutive dose of CAR-expressing cells at a time at which:
(i) a clinical risk for neurotoxicity, cytokine-release syndrome (CRS), macrophage activation syndrome, or tumor lysis syndrome, is not present or has passed or has subsided following said administration in (a), (ii) a biochemical readout evidencing (CRS), neurotoxicity, macrophage activation syndrome, or tumor lysis syndrome, is not present or has passed or has subsided following said administration in (a), and/or (iii) a serum level of a factor indicative of cytokine-release syndrome (CRS) or neurotoxicity in the subject is less than about 10 times, less than about 25 times, and/or less than about 50 times the serum level of said indicator in the subject immediately prior to said administration in (a); and
the subject does not exhibit a detectable adaptive host immune response specific for the CAR expressed by the cells of said first dose.
4. A method of treatment, comprising:
(a) administering to a subject a first dose of cells expressing a chimeric antigen receptor (CAR), said first dose comprising the cells in an amount sufficient to reduce burden of a disease or condition in the subject; and
(b) administering to the subject a consecutive dose of CAR-expressing cells, at a time after a neurotoxicity and/or CRS-related outcome in the subject has reached a peak level and begun to decline following said administration in (a) and at which the subject does not exhibit a detectable humoral or cell-mediated immune response against the CAR expressed by the cells of said first dose.
5. The method of embodiment 3 or embodiment 4, wherein:
(i) the administration in (a) does not induce severe CRS in the subject or does not induce CRS in the subject;
(ii) the administration in (a) does not induce grade 3 or higher neurotoxicity in the subject;
(iii) based on clinical data, the administration of the dose of cells in (a) does not induce severe CRS in a majority of subjects so-treated; and/or
(iv) based on clinical data, the administration of the dose of cells in (a) does not induce grade 3 or higher neurotoxicity in a majority of subjects so-treated.
6. The method of any of embodiments 3-5, wherein:
the time between the initiation of the administration in (a) and the initiation of the administration in (b) is between about 9 and about 35 days, between about 14 and about 28 days, between 15 and 27 days or is between 17 days and about 21 days, each inclusive; and/or
said first dose comprises no more than about 1×106 of the cells per kilogram body weight of the subject, no more than about 1×108 of the cells, or no more than about 1×108 of the cells/m2 of the subject.
7. The method of any of embodiments 2-6, wherein the CRS-related outcome is selected from the group consisting of fever, hypotension, hypoxia, neurologic disturbances, or a serum level of an inflammatory cytokine or C reactive protein (CRP).
8. The method of any of embodiments 2-6, wherein the factor indicative of CRS is an inflammatory cytokine selected from the group consisting of interferon gamma (IFNγ), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFα), IL-6, IL-10, IL-1β, IL-8, IL-2, MIP-1, Flt-3L, fracktalkine, and IL-5 or is CRP.
9. The method of any of embodiments 2-8, wherein, at the time of the administration in (b):
said level of said CRS-related outcome is no more than 50% of the peak level, is no more than 20% of the peak level, or is no more than 5% of the peak level, or is at or about the level immediately prior to the administration in (a); or said serum level of said factor indicative of CRS is no more than ten times the level immediately prior to the administration in (a).
10. The method of any of embodiments 2-9, wherein symptoms associated with a clinical risk of neurotoxicity and/or grade 3 or higher neurotoxicity are selected from among confusion, delirium, expressive aphasia, obtundation, myoclonus, lethargy, altered mental status, convulsions, seizure-like activity, seizures (optionally as confirmed by electroencephalogram [EEG]), elevated levels of beta amyloid (Aβ), elevated levels of glutamate, and elevated levels of oxygen radicals.
11. The method of any of embodiments 1-10, wherein said subject has not received a dose of cells expressing the CAR expressed by the cells in the first dose prior to the administration in (a).
12. The method of any of embodiments 1-11, wherein the CAR expressed by the cells in the consecutive dose contains at least one immunoreactive epitope present in the CAR expressed by the cells in the first dose.
13. The method of embodiment 12, wherein the CAR expressed by the cells in the consecutive dose is identical to the CAR expressed by the cells in the first dose or is substantially identical to the CAR expressed by the cells in the first dose.
14. The method of any of embodiments 1-13, wherein the CAR expressed by the cells in the first dose specifically binds to an antigen expressed by a cell or tissue of the disease or condition or associated with the disease or condition.
15. The method of embodiment 14, wherein the disease or condition is a tumor or a cancer.
16. The method of embodiment 15, wherein the administration in (a) leads to a reduction in burden of the disease or condition in the subject, as indicated by a reduction in one or more factors indicative of disease burden following said administration in (a).
17. The method of embodiment 16, wherein at the time of the administration in (b), the subject has not relapsed and/or the one or more factors indicative of disease burden have not increased following said reduction.
18. The method of any of embodiments 1-17, wherein the consecutive dose of cells comprises cells in an amount sufficient for reduction in burden of a disease or condition in the subject.
19. The method of any of embodiments 16-18, wherein the administration in (b) leads to a further reduction in burden of the disease or condition in the subject.
20. The method of any of embodiments 1-19, wherein said administration of said consecutive dose leads to a reduction in burden of the disease or condition in the subject as compared with immediately prior to initiation of the administration of the consecutive dose.
21. The method of any of embodiments 1-20, wherein the method reduces burden of the disease or condition to a greater degree and/or for a greater period of time as compared to a method comprising an alternative dosing regimen wherein the subject is administered the cells in (a) and the cells in (b) in a single dose.
22. The method of any of embodiments 16-22, wherein:
said reduction in burden and/or further reduction in burden comprises a reduction in total number of cells of the disease in the subject, in an organ of the subject, in a tissue of the subject, or in a bodily fluid of the subject, a reduction in mass or volume of a tumor, and/or a reduction in number and/or extent of metastases.
23. The method of any of embodiments 1-22, wherein:
the disease is a cancer and the subject does not exhibit morphologic disease at the time of initiation of the administration in (b); and/or
the disease is a leukemia or lymphoma and the subject does not exhibit greater than 5% blast cells in the bone marrow at the time of the administration in (b).
24. The method of any of embodiments 1-23, wherein the disease or condition persists following the administration of said first dose and/or the administration of the first dose is not sufficient to eradicate the disease or condition in the subject.
25. The method of any of embodiments 1-24, wherein the subject exhibits detectable molecular disease and/or minimum residual disease at the time of the administration in (b).
26. The method of any of embodiments 1-25, wherein (i) the maximum number of CAR-expressing cells, (ii) the area under the curve (AUC) of CAR-expressing cells over time, and/or (iii) the duration of detectable CAR-expressing cells in the subject following said administration in (b) is greater, as compared to that achieved via a method comprising an alternative dosing regimen wherein the subject is administered the cells in (a) and the cells in (b) as a single dose.
27. The method of any of embodiments 1-26, wherein:
the method results in a maximum concentration or number of CAR-expressing cells in the blood of the subject of at least at or about 10 CAR-expressing cells per microliter, at least 50% of the total number of peripheral blood mononuclear cells (PBMCs), at least at least about 1×105 CAR-expressing cells, or at least 5,000 copies of CAR-encoding DNA per micrograms DNA; and/or
at day 90 following the initiation of the administration in (a), CAR-expressing cells are detectable in the blood or serum of the subject; and/or
at day 90 following the initiation of the administration in (a), the blood of the subject contains at least 20% CAR-expressing cells, at least 10 CAR-expressing cells per microliter or at least 1×104 CAR-expressing cells.
28. The method of any of embodiments 1-27, wherein the area under the curve (AUC) for blood concentration of CAR-expressing cells over time following the administration in (a) is greater as compared to that achieved via a method comprising an alternative dosing regimen wherein the subject is administered the cells in (a) and the cells in (b) as a single dose.
29. The method of any of embodiments 1-28, wherein a CRS-related outcome in the subject at day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 following the administration in (b) is not detectable or is reduced as compared to a method comprising an alternative dosing regimen wherein the subject is administered the cells in (b) without having been administered the first dose.
30. The method of any of embodiments 1-29, wherein the area under the curve (AUC) for a serum level of a factor indicative of CRS over time in the subject following the administration in (b) is lower as compared to that of a method comprising an alternative dosing regimen wherein the subject is administered the cells in (b) without having been administered the first dose.
31. The method of any of embodiments 15-30, wherein the subject has been treated with a therapeutic agent targeting the tumor or cancer prior to said administration in (a) and is refractory or non-responsive to said therapeutic agent at the time of the administration in (a).
32. The method of any of embodiments 1-31, further comprising, subsequent to administration in (a) and before said administration in (b), or prior to administration in (a), assessing a serum level of a factor indicative of CRS, a factor indicative of neurotoxicity, a factor indicative of disease burden, and/or an indicator of a host anti-CAR immune response in said subject.
33. The method of embodiment 32, wherein the factor indicative of disease burden is measured and comprises a total number of cells of the disease in the subject, in an organ of the subject, in a tissue of the subject, or in a bodily fluid of the subject, molecular detection by flow cytometry or quantitative PCR, mass or volume of a solid tumor, or number or extent of metastases.
34. The method of embodiment 32 or embodiment 33, comprising:
i) assessing a factor indicative of disease burden prior to administration in (b); and
ii) based on the result of the assessment, determining the consecutive dose of cells to be administered to the subject, and:
iii) if the assessment determines that the subject has morphologic disease, administering to the subject a consecutive dose comprising less than or about the same number of CAR-expressing cells as the number of CAR-expressing cells in the first dose; and/or if the assessment determines that the subject has minimal residual disease, administering to the subject a consecutive dose comprising an increased number of CAR-expressing cells as compared to the first dose.
35. The method of any of embodiments 1-33, wherein the consecutive dose comprises about the same number of CAR-expressing cells as the number of CAR-expressing cells in the first dose.
36. The method of any of embodiments 1-33, wherein the consecutive dose comprises an increased number of CAR-expressing cells as compared to the first dose.
37. A method of treatment, comprising administering a consecutive dose of cells expressing a chimeric antigen receptor (CAR) to a subject previously administered a first dose of cells expressing a CAR, wherein:
the consecutive dose of cells is administered at a time point that is at least or more than about 14 days after and less than about 28 days after initiation of the first dose; and/or
the number of CAR-expressing cells administered in the consecutive dose is increased as compared to the first dose.
38. The method of any of embodiments 1-37, wherein the number of cells administered in the first dose is between about 0.5×106 cells/kg body weight of the subject and 3×106 cells/kg, between about 0.75×106 cells/kg and 2.5×106 cells/kg or between about 1×106 cells/kg and 2×106 cells/kg, each inclusive.
39. The method of embodiment 34, 36 or 37, wherein the increased number is at least 2-fold, 5-fold, or 10-fold greater than the number in the first dose.
40. The method of any of embodiments 1-39, wherein the number of cells administered in the consecutive dose of CAR-expressing cells comprises between about 2×106 cells per kilogram (cells/kg) body weight and about 6×106 cells/kg, between about 2.5×106 cells/kg and about 5.0×106 cells/kg, or between about 3.0×106 cells/kg and about 4.0×106 cells/kg, each inclusive.
41. The method of any of embodiments 1-40, wherein the subject does not exhibit cytokine release syndrome (CRS), does not exhibit severe CRS, does not exhibit neurotoxicity, does not exhibit severe neurotoxicity, or does not exhibit neurotoxicity above grade 3 following administration of the first dose and/or following the administration in of the consecutive dose.
42. The method of any of embodiments 1-41, wherein the CAR-expressing cells in the first dose expand in the subject following administration of the first dose and/or following the administration of the consecutive dose.
43. The method of embodiment 42, wherein the expansion is evidenced by (i) an increase in serum CRP level following the administration of the first dose and/or consecutive dose as compared to just prior to the administration, and/or (ii) an increase in level of CAR-encoding nucleic acid in the serum, as measured by qPCR, following the administration of the first dose and/or consecutive dose as compared to just prior to the administration, wherein said increase is optionally at least 1, 2, or 3-fold.
44. The method of any of embodiments 1-43, wherein the time between the first and consecutive dose is between 15 and 27 days.
45. The method of embodiment 44, wherein the time between the first and consecutive dose is about 21 days.
46. The method of embodiment 44, wherein the time between the first and consecutive dose is about 17 days.
47. The method of any of embodiments 1-46, wherein the cells of the first dose are administered in a single pharmaceutical composition comprising the cells of the first dose and/or wherein the cells of the consecutive dose are administered in a single pharmaceutical composition comprising the cells of the consecutive dose.
48. The method of any of embodiments 1-47, wherein:
the first dose is a split dose, wherein the cells of the first dose are administered in a plurality of compositions, collectively comprising the cells of the first dose, over a period of no more than three days; and/or
the consecutive dose is a split dose, wherein the cells of the consecutive dose are administered in a plurality of compositions, collectively comprising the cells of the consecutive dose, over a period of no more than three days.
49. The method of any of embodiments 1-48, wherein the method further comprises administering a chemotherapeutic agent prior to the administration in (a) and/or the administration in (b) or wherein the subject has been previously treated with a chemotherapeutic agent prior to the administration of the first dose.
50. The method of embodiment 49, wherein the chemotherapeutic agent comprises an agent selected from the group consisting of cyclophosphamide, fludarabine, and/or a combination thereof.
51. The method of embodiment 49 or 50, wherein the administration of the chemotherapeutic agent comprises administration of a chemotherapeutic agent prior to the administration in (a) and optionally not prior to the administration in (b).
52. The method of any of embodiments 49-51, wherein the chemotherapeutic agent is administered between 2 and 5 days prior to the administration in (a) and/or is administered between 2 and 5 days prior to the administration in (b).
53. The method of any of embodiments 49-52, wherein the chemotherapeutic agent is administered at a dose of between at or about 1 g/m2 of the subject and at or about 100 g/m2, 15 g/m2 of the subject and 50 g/m2 of the subject, 0.5 g/m2 of the subject and 5 g/m2 of the subject, on g/m2 of the subject and at or about 3 g/m2 of the subject.
54. The method of any of embodiments 1-53, wherein the subject has received cryoreductive chemotherapy prior to the administration or the first dose or the method further comprises the administration of cryoreductive chemotherapy prior to the administration of the first dose.
55. The method of any of embodiments 49-54, wherein the chemotherapeutic agent comprises conditioning chemotherapy, which reduces burden of the disease or condition in the subject.
56. A method of providing consolidating treatment, comprising administering to a subject a consecutive dose of cells expressing a chimeric antigen receptor (CAR), wherein:
prior to said administration, the subject has received a previous dose of CAR-expressing in an amount sufficient to reduce burden of a disease or condition in the subject; and
at the time of administration, a serum level of a factor indicative of cytokine-release syndrome (CRS) in the subject is less than about 10 times, less than about 25 times, and/or less than about 50 times the serum level of said indicator in the subject immediately prior to said previous dose and the subject does not exhibit a detectable adaptive host immune response specific for the CAR expressed by the cells of said previous dose; and/or the time between said previous and consecutive doses is greater than about 14 days and less than about 28 days.
57. The method of embodiment 56, wherein the number of cells in the consecutive dose of CAR-expressing cells comprises between about 2×106 cells per kilogram (cells/kg) body weight and about 6×106 cells/kg, between about 2.5×106 cells/kg and about 5.0×106 cells/kg, or between about 3.0×106 cells/kg and about 4.0×106 cells/kg, each inclusive.
58. The method of any of embodiments 1-57, wherein the disease or condition is a leukemia or lymphoma.
59. The method of any of embodiments 1-58, wherein the disease or condition is acute lymphoblastic leukemia.
60. The method of embodiment 59, wherein the number of CAR+ cells per kilogram administered in the consecutive dose is greater than the number of CAR+ cells per kilogram administered in the first dose.
61. The method of embodiment 60, wherein the number of CAR+ cells per kilogram administered in the consecutive dose is at least at or about 2 times or at or about 3 times greater than the number of CAR+ cells per kilogram administered in the first dose.
62. The method of any of embodiments 1-58, wherein the disease or condition is a non-Hodgkin lymphoma (NHL).
63. The method of any of embodiments 1-62, wherein the number of CAR+ cells administered in the first dose is at or about or no more than at or about 1×106 per kilogram of the subject and/or the number of CAR+ cells administered in the consecutive dose is at or about 3×106 per kilogram of the subject.
64. The method of embodiment 62, wherein the number of CAR+ cells per kilogram administered in the consecutive dose is less than or about less than or is the same or about the same as the number of CAR+ cells per kilogram administered in the first dose.
65. The method of any of embodiments 1-64, further comprising administering to the subject one or more additional subsequent doses, wherein the first of said one or more additional subsequent doses is administered at a time that is at least or greater than 14 days after the initiation of the administration of the consecutive dose.
66. The method of embodiment 65, wherein the administration of the first, consecutive and subsequent doses comprises administering at least three of the doses within at or about 28 days.
67. The method of embodiment 65 or 66, wherein the consecutive dose is administered at about day 14 following the initiation of administration of the first dose, one of the at least one additional subsequent doses is administered at day 28 following the initiation of administration of the first dose, and optionally wherein additional subsequent doses are administered at day 42 and/or day 56 following the initiation of administration of the first dose.
68. The method of any of embodiments 1-67, wherein the cells are T cells.
69. The method of any of embodiments 1-68, wherein the T cells are autologous to the subject.
70. Use of a composition comprising cells expressing a chimeric antigen receptor (CAR) for manufacture of a medicament for treatment of a disease or condition in a subject previously treated with CAR-expressing cells, wherein:
the composition is for use 14 to 28 days after the previous treatment; and/or
the composition is formulated for administration of a consecutive dose in an amount sufficient for reduction in burden of a disease or condition in the subject having been previously treated with the CAR-expressing cells.
71. Cells expressing a chimeric antigen receptor (CAR) for use in treating a disease in a subject previously treated with CAR-expressing cells, wherein:
the cells are for use between about 14 and 28 days after the previous treatment; and
the cells are formulated for administration of a consecutive dose in an amount sufficient for reduction in burden of a disease or condition in the subject having been previously treated with the CAR-expressing cells.
72. The use of embodiment 70 or cells of embodiment 71, wherein the subject does not exhibit morphologic disease and/or the subject does not exhibit greater than 5% blast cells in the bone marrow.
73. Use of cells expressing a chimeric antigen receptor (CAR) in the manufacture of a medicament for use in a method for treating a disease or condition, said method comprising:
(a) administering to a subject having the disease or condition a first dose of cells expressing the CAR, said first dose comprising no more than about 1×106 of the cells per kilogram body weight of the subject, no more than about 1×108 of the cells, and/or no more than about 1×108 of the cells/m2 of the subject; and
(b) administering to the subject a consecutive dose of cells expressing a CAR at a time point that is at least or more than about 14 days after and less than about 28 days after initiation of said administration in (a).
74. Cells expressing a chimeric antigen receptor (CAR) for use in a method for treating a disease or condition, said method comprising:
(a) administering to a subject having the disease or condition a first dose of cells expressing the CAR, said first dose comprising no more than about 1×106 of the cells per kilogram body weight of the subject, no more than about 1×108 of the cells, and/or no more than about 1×108 of the cells/m2 of the subject; and
(b) administering to the subject a consecutive dose of cells expressing a CAR at a time point that is at least or more than about 14 days after and less than about 28 days after initiation of said administration in (a).
75. The use or cells of any of embodiments embodiment 70-74, wherein the cells are formulated for administration in an amount that (i) does not induce severe CRS in the subject or does not induce CRS in the subject; (ii) does not induce grade 3 or higher neurotoxicity in the subject; (iii) based on clinical data, does not induce severe CRS in a majority of subjects so-treated; and/or (iv) based on clinical data, does not induce grade 3 or higher neurotoxicity in a majority of subjects so-treated.
76. Use of cells expressing a chimeric antigen receptor (CAR) for manufacture of a medicament for the treatment of a disease or condition in a subject,
wherein the cells are formulated and/or packaged for administration to the subject in a first and a consecutive dose and/or the treatment comprises administering the cells to the subject in a first and a consecutive dose, wherein:
the first dose comprises no more than about 1×106 of the cells per kilogram body weight of the subject, no more than about 1×108 of the cells, and/or no more than about 1×108 of the cells/m2 of the subject, and
the consecutive dose is for administration at a time point (a) that is at least or more than about 14 days after and less than about 28 days after initiation of the first administration, and/or (b) at which (i) the serum level in the subject of a factor indicative of cytokine release syndrome (CRS) is less than about 10 times, less than about 25 times, and/or less than about 50 times that in the subject immediately prior to said first administration; (ii) the subject does not exhibit grade 3 or higher neurotoxicity; (iii) a CRS-related outcome or symptom of neurotoxicity in the subject following said administration of said first dose has reached a peak level and begun to decline following the first administration, and/or (iv) the subject does not exhibit a detectable humoral or cell-mediated immune response against the CAR expressed by the cells of said first dose.
77. Cells expressing a chimeric antigen receptor (CAR) for use in treatment of a disease or condition in a subject,
wherein the cells are formulated and/or packaged for administration to the subject in a first and a consecutive dose and/or the treatment comprises administering the cells to the subject in a first and a consecutive dose, wherein:
the first dose comprises no more than about 1×106 of the cells per kilogram body weight of the subject, no more than about 1×108 of the cells, and/or no more than about 1×108 of the cells/m2 of the subject, and
the consecutive dose is for administration at a time point (a) that is at least or more than about 14 days after and less than about 28 days after initiation of the first administration, and/or (b) at which (i) the serum level in the subject of a factor indicative of cytokine release syndrome (CRS) is less than about 10 times, less than about 25 times, and/or less than about 50 times that in the subject immediately prior to said first administration; (ii) the subject does not exhibit grade 3 or higher neurotoxicity; (iii) a CRS-related outcome or symptom of neurotoxicity in the subject following said administration of said first dose has reached a peak level and begun to decline following the first administration, and/or (iv) the subject does not exhibit a detectable humoral or cell-mediated immune response against the CAR expressed by the cells of said first dose.
78. The use or cells of any of embodiments 73-77, wherein the first and consecutive administrations comprise administering the cells in one or more unit dose, each unit dose comprising about between 5×107 of the cells and about 5×108 cells, about between 5×107 of the cells and about 2.5×108 cells or about between 2.5×108 cells and 4×108 cells; or the cells are formulated in a unit dose comprising no more than about 5×107 cell, no more than about 1×108 cells, no more than about 2×108 of the cells, no more than about 2.5×108 of the cells, no more than about 3.0×108 of the cells or no more than about 4×108 of the cells.
79. The use or cells of embodiment 78, wherein the first administration comprises administering a single unit dose.
80. The use or cells of embodiment 78 or embodiment 79, wherein the consecutive administration comprises administration of two or more unit doses.
81. The use or cells of embodiment 78 or embodiment 79, wherein the consecutive administration comprises administration a single unit dose.
82. The use, composition or cells of any of embodiments 68-81, wherein the disease or condition is a tumor or a cancer.
83. The use, composition or cells of embodiment 82, wherein the tumor or cancer is leukemia or lymphoma.
84. The use, composition or cells of any of embodiments 68-83, wherein the consecutive dose is formulated for administration of an increased number of CAR-expressing cells as compared to the first dose or previous dose.
85. The use, composition or cells of embodiment 84, wherein the composition is for use in treating acute lymphoblastic leukemia.
86. The use, composition or cells of any of embodiments 68-83, wherein the consecutive dose is formulated for administration of less than or about the same number of CAR-expressing cells as the number of CAR-expressing cells in the previous dose.
87. The use, composition or cells of embodiment 84 or embodiment 86, wherein the composition is for use in treating non-Hodgkin lymphoma (NHL).
88. An article of manufacture, comprising:
a plurality of sealable containers, each individually comprising a unit dose of cells expressing a chimeric antigen receptor (CAR) for administration to a subject, said unit dose comprising about 1×108 of the cells, no more than about 1×108 of the cells, about 5×107 of the cells, no more than about 5×107 of the cells, about 1×106 cells per kg of the subject, or no more than about 1×106 of the cells per kg of the subject;
packaging material; and
a label or package insert comprising instructions for administering a plurality of said unit doses to the subject by carrying out a first administration and a consecutive administration, said first administration comprising delivering one of said unit doses to the subject and said consecutive administration comprising administering one or a plurality of said unit doses to the subject.
89. The article of manufacture of embodiment 88, wherein the instructions specify that said consecutive administration is to be carried out at a time between about 14 or 15 and 27 days, optionally at about day 17 or about day 21, following said first administration.
90. The article of manufacture of embodiment 88 or embodiment 89, wherein the instructions specify that said consecutive administration is to be carried out at a time after which it has been determined that a serum level of a factor indicative of cytokine-release syndrome (CRS) in the subject is less than about 10 times, less than about 25 times, and/or less than about 50 times the serum level of said indicator in the subject immediately prior to said first administration.
91. The article of manufacture of any of embodiments 88-90, wherein the instructions specify that said consecutive administration is to be carried out at a time after which it has been determined that an indicator of CRS has peaked and is declining, and/or that the subject does not exhibit a detectable adaptive host immune response specific for the CAR expressed by the cells of said first dose.
92. The article of manufacture of any of embodiments 88-91, wherein the cells have been derived from the subject.
93. The article of manufacture of any of embodiments 88-92, wherein said label and/or said packaging material further comprises an identifier specific to the subject, indicating that the cells were derived from the subject and/or should be administered to the subject specifically.
94. The article of manufacture of any of embodiments 88-93, wherein the containers are or comprise flexible cell infusion bags.
95. A method of treatment, comprising:
a) assessing a factor indicative of disease burden in a subject having or suspected of having a tumor; and
b) based on the result of the assessment, determining the dose of cells expressing a chimeric antigen receptor (CAR) to be administered to the subject, wherein:
i) if the assessment determines that the subject does not have morphologic disease or does not have substantial morphologic disease or does not have a disease burden at or above a threshold level, administering to the subject a dose of CAR-expressing cells greater than 1×106 cells/kg; and
ii) if the assessment determines that the subject has morphologic disease or substantial morphologic disease or a disease burden at or above a threshold level, administering to the subject a dose of CAR-expressing cells that is relatively lower as compared to in ii).
96. The method of embodiment 95, wherein:
the subject does not exhibit morphologic disease or substantial morphologic disease or a disease burden at or above a threshold level and is administered a dose of cells that is greater than or about 2×106 cells/kg, 3×106 cells/kg, 4×106 cells/kg, 5×106 cells/kg, 6×106 cells/kg, 7×106 cells/kg, 8×106 cells/kg, 9×106 cells/kg, 1×107 cells/kg or 2×107 cells/kg; and/or
the subject exhibits morphologic disease or substantial morphologic disease or has a disease burden above a threshold level and is administered a dose of cells that is less than or less than about 1×106 cells/kg or 5×106 cells/kg.
97. The method of embodiment 95 or embodiment 96, wherein the subject exhibits morphologic disease and/or substantial morphologic disease and/or the disease burden is at or above a threshold level if there are greater than or equal to or about 5% blasts in the bone marrow, greater than or equal to or about 10% blasts in the bone marrow, greater than or equal to or about 15% blasts in the bone marrow or greater than or equal to or about 20% blasts in the bone marrow.
98. The method of any of embodiments 95 to 97, wherein:
if the subject exhibits less than or less than about 5% blasts the subject is administered a dose of cells of greater than or about 1×106 cells/kg 2×106 cells/kg, 3×106 cells/kg, 4×106 cells/kg, 5×106 cells/kg, 6×106 cells/kg, 7×106 cells/kg, 8×106 cells/kg, 9×106 cells/kg, 1×107 cells/kg or 2×107 cells/kg and/or if the subject exhibits greater than or equal to or about 5% blasts the subject is administered a dose of cells that is less than about 1×106 cells/kg or 5×106 cells/kg;
if the subject exhibits less than or less than about 10% blasts the subject is administered a dose of cells of greater than or about 1×106 cells/kg 2×106 cells/kg, 3×106 cells/kg, 4×106 cells/kg, 5×106 cells/kg, 6×106 cells/kg, 7×106 cells/kg, 8×106 cells/kg, 9×106 cells/kg, 1×107 cells/kg or 2×107 cells/kg and/or if the subject exhibits greater than or equal to or about 10% blasts the subject is administered a dose of cells that is less than about 1×106 cells/kg or 5×106 cells/kg;
if the subject exhibits less than or less than about 15% blasts the subject is administered a dose of cells of greater than or about 1×106 cells/kg 2×106 cells/kg, 3×106 cells/kg, 4×106 cells/kg, 5×106 cells/kg, 6×106 cells/kg, 7×106 cells/kg, 8×106 cells/kg, 9×106 cells/kg, 1×107 cells/kg or 2×107 cells/kg and/or if the subject exhibits greater than or equal to or about 15% blasts the subject is administered a dose of cells that is less than about 1×106 cells/kg or 5×106 cells/kg; if the subject exhibits less than or less than about 20% blasts the subject is administered a dose of cells of greater than or about 1×106 cells/kg 2×106 cells/kg, 3×106 cells/kg, 4×106 cells/kg, 5×106 cells/kg, 6×106 cells/kg, 7×106 cells/kg, 8×106 cells/kg, 9×106 cells/kg, 1×107 cells/kg or 2×107 cells/kg and/or if the subject exhibits greater than or equal to or about 20% blasts the subject is administered a dose of cells that is less than about 1×106 cells/kg or 5×106 cells/kg.
99. The method of any of embodiments 95 to 98, wherein the tumor is a leukemia or lymphoma, optionally a B cell-derived leukemia or lymphoma.
100. The method of any of embodiments 95 to 99, wherein the subject does not exhibit cytokine release syndrome (CRS), does not exhibit severe CRS, does not exhibit neurotoxicity, does not exhibit severe neurotoxicity, or does not exhibit neurotoxicity above grade 3 following administration of the dose of cells.
101. The method of any of embodiments 95 to 100, wherein the administration in (a) leads to a reduction in burden of the disease or condition in the subject, as indicated by a reduction in one or more factors indicative of disease burden following said administration in (a).
102. The method of any of embodiments 95 to 101, further comprising administering one or more consecutive doses to the subject.
EXAMPLES
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1
Treatment of Cancer Patients with First and Consecutive Doses of CAR-Expressing Autologous T Cells
T cells are isolated from peripheral blood of human subjects with cancer by immunoaffinity-based enrichment and the cells cultured and transduced with viral vectors encoding a chimeric antigen receptor (CAR) that specifically binds to an antigen expressed by the cancer in the subject, which is a tumor-associated or tumor-specific antigen. The cells are cryopreserved in infusion medium in individual flexible infusion bags, each containing a single unit dose of the cells, which is about 1×106 cells per kilogram body weight of the subject or about 5×105 cells per kilogram body weight of the subject. The cells are maintained at a temperature below −130° C. prior to infusion.
Prior to initiation of cell therapy, blood is obtained from the subjects and, optionally, the levels of one or more serum factors indicative of cytokine release syndrome (CRS) such as tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and IL-6, are assessed in the serum by ELISA. Tumor burden is optionally assessed by measurement of the size or mass of a solid tumor, such as by PET or CT scan, or by assessing the number of cells of the patient associated with the cancer, such as in the bone marrow or peripheral blood, before treatment begins.
The cells are thawed at bedside by warming to approximately 37° C. and subjects administered a first dose of the cells by single infusion. The amount of the first dose is a single unit dose. For subjects deemed to have low tumor burden, two unit doses may be administered in the first dose. The first dose is administered intravenously (IV) by continuous infusion, over a period of approximately 15 to 30 minutes.
Following administration of the first dose, the subjects receive physical examinations and are monitored for any symptoms of toxicity or toxic outcomes, such as fever, hypotension, hypoxia, neurologic disturbances, or an increased serum level of an inflammatory cytokine or C reactive protein (CRP). Optionally, following administration of the first dose, on one or more occasions, blood is obtained from the patients and the levels of serum factors indicative of CRS are assessed by ELISA. The levels of the serum factors are compared to those obtained immediately prior to administration of the first dose. If necessary, anti-IL6 or other CRS therapy is administered to reduce signs of CRS.
The presence or absence of an anti-CAR immune response in the subject is optionally detected following the administration of the first dose, for example, at 1, 2, 3, and/or 4 weeks following the initiation of the administration, for example, by ELISA, ELISPOT, cell-based antibody assay, and/or mixed-lymphocyte reaction.
The percent reduction in tumor burden achieved by the first dose is optionally measured on one or more occasions following administration of the first dose by scans, such as PET and CT scans, in patients with solid tumors, and/or by quantifying disease-positive cells in blood or tumor sites, e.g., in subjects with hematological cancers, and comparing the values to those observed immediately prior to the first dose.
A consecutive dose is administered. In some subjects, the consecutive dose is administered in 21 days following the initiation of the administration of the first dose. In some cases, the consecutive dose is only administered if the level of a tested CRS-related outcome or serum factor is below an acceptable level and if no anti-CAR immune response is detected in the subject at 21 days following the first dose administration. In other subjects, the consecutive does is administered at a time that is greater than 3 days following the administration of the first dose, and at which the subject is deemed to not have CRS or severe CRS, or at which levels of all tested serum factors indicative of CRS are below 20% of that observed at a peak following the first dose administration, and the subject is deemed not to have a detectable anti-CAR immune response.
The size of the consecutive dose is patient-specific, and is based on tumor burden, presence of an anti-CAR immune response, and level of CRS-related outcomes. Some patients are administered a consecutive dose containing 1, 2, 3, or even more unit doses of the cells. The consecutive dose is administered by continuous infusion, IV, over approximately 15 to 30 minutes.
Beginning after the first dose and continuing for up to several years, the subjects are monitored on a regular basis. Development of an anti-CAR immune response is assessed and tumor burden is measured. Optionally, during follow-up visits, the CAR-expressing cells are detected by flow cytometry and quantitative polymerase chain reaction (qPCR) to measure in vivo proliferation and persistence of the administered cells.
Example 2
Assessment of Neurotoxicity of CAR-T Cell Treatment in Subjects Having Morphological Disease Prior to Treatment
Subjects with CD19+ B cell acute lymphoblastic leukemia (ALL) were administered autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR). The CAR included a truncated EGFR (EGFRt) portion as a marker. Prior to administration of the cells, patients underwent leukapheresis and were treated with chemotherapy. To generate the autologous CAR-expressing T cells, T cells were isolated by immunoaffinity-based enrichment from leukapheresis samples from individual subjects, activated and transduced with a viral vector encoding an anti-CD19 CAR. The cells were expanded, frozen, and thawed at bedside prior to administration.
Tumor burden was assessed prior to treatment by evaluating bone marrow and the percent of bone marrow blasts was determined. Subjects having at least 5% blasts in bone marrow were deemed to have morphological disease (MD). Subjects exhibiting complete remission (CR) as defined below, including having less than 5% blasts in the bone marrow, but showing molecularly detectable disease in the bone marrow (by flow cytometry) were deemed to have minimal residual disease (MRD). CAR-expressing T cells were administered to subjects by single intravenous (IV) continuous infusion, over approximately 15-30 minutes, at varying doses ranging from about or approximately 0.9×106 CAR+ cells/kg to about or approximately 5.6×106 CAR+ cells/kg (see FIG. 1A-C). Cyclophosphamide was administered to subjects as a preconditioning chemotherapeutic treatment 2 to 7 days before cell infusion.
Disease status of subjects (MRD or MD) was assessed subsequent to administration of CAR-expressing T cells to assess response to treatment. Complete remission (CR) was determined in subjects if there was a restoration of normal hematopoiesis with neutrophil count >1,000×106/L, platelet count of >100,000×106/L and hemoglobin >10 g/dL; <5% blasts present in a post-treatment bone marrow differential; and no clinical evidence of leukemia for a minimum of four weeks. Following treatment, subjects also were assessed and monitored for neurotoxicity (neurological complications including symptoms of confusion, aphasia, seizures, convulsions, lethargy, and/or altered mental status), graded based on severity (using a Grade 1-5 scale (see, e.g., Guido Cavaletti & Paola Marmiroli Nature Reviews Neurology 6, 657-666 (December 2010), with grade 3 (severe symptoms), 4 (life-threatening symptoms) or 5 (death) being deemed severe neurotoxicity. Cytokine release syndrome (CRS) also was determined and monitored, graded based on severity.
Response, presence of severe CRS, and presence of severe neurotoxicity following treatment with a single infusion of varying doses of CAR-expressing T cells, compared to dosage, were assessed in groups of subjects separated based on disease burden prior to treatment. Results are shown in FIGS. 1A-C.
As shown in FIG. 1A, complete remission (CR) was observed in the majority of subjects treated with CAR-expressing T cells at all doses tested, regardless of disease burden (morphological or molecularly detectable disease) prior to treatment. CRS was observed primarily in subjects classified as having morphological disease prior to treatment. The results depicted in FIG. 1B, however, show that within this group, the presence of CRS was observed for subjects having received various dosage levels of CAR-expressing T cells.
As shown in FIG. 1C, severe neurotoxicity also was most frequently observed in subjects with morphological disease as composed to minimal residual disease prior to treatment in contrast to the results for CRS, however, neurotoxicity was observed only in those subjects receiving higher doses. Subjects analyzed in this study who had received a dose of CAR-expressing T cells lower than about 3.5×106 cells/kg, did not exhibit severe neurotoxicity after treatment. With one exception, severe neurotoxicity was generally not observed in subjects classified as having molecularly detectable disease prior to treatment. Thus, subjects receiving a lower dose of administered T cells and/or who had a lower disease burden prior to treatment did not generally exhibit severe neurotoxicity.
These results support, in order to minimize toxicity and maximize efficacy, using a dosage regime that includes administering to subjects, including those with morphologic disease, a first low dose of CAR-expressing T cells (which may not necessarily be sufficient to eradicate disease in all or most subjects) to reduce disease burden, followed by a consecutive or subsequent dose of cells after tumor burden has been reduced. The results also support a conclusion that if desired or needed in a particular disease or context (for example, to promote an increased response or efficacy), the consecutive or subsequent administration of cells (given after reduction in tumor burden, at which point all or most subjects should exhibit minimal disease), the consecutive dose can be carried out at a higher dose, without or with minimal risk of severe neurotoxicity.
Example 3
Assessment of Efficacy and Toxicity Following First and Consecutive Doses of CAR+ Autologous T Cells
Treatment efficacy and toxic effects were assessed in a subset of subjects in the study described in Example 2 who were administered multiple doses of the CAR+ T cells. Table 3 below sets forth the particular dosages of cells administered in the various multiple doses, and in each case, the time that elapsed between the first and consecutive dose. Table 3 also sets forth each subject's tumor burden (MRD or MD with % of blast cells in bone marrow; listed under the column labeled “Disease burden”) as assessed prior to administration of each dose, based on criteria as described in Example 2. Table 3 also lists results for each patient for response to each administration (under the column “Response,” listing the tumor burden following administration, which as compared to the pre-administration disease burden, indicates response to treatment), presence or absence (Y/N) of severe CRS and severe neurotoxicity as described in Example 2.
TABLE 3
Safety and Efficacy of First and Consecutive CAR+ T Cell Doses
First Dose
Consecutive Dose
Severe
Days
Severe
Disease
Dose ×
Severe
Neuro-
between
Disease
Dose ×
Severe
Neuro-
ID
burden
106/kg
CRS
toxicity
Response
doses
burden
106/kg
CRS
toxicity
Response
1
MRD+
1.74
N
N
MRD−
133
MRD+
1.37
N
N
MRD−
[2%]
CR
[2%]
CR
2
6%
3.08
N
N
MRD−
86
MRD+
1.50
N
N
MRD−
CR
[3%]
CR
2*
90%
4.27
N
N
48%
23
56%
3.27
N
N
NR
4
MRD+
0.47
N
N
MRD+
203
MRD+
3.84
N
N
MRD+
[4%]
CR
[3%]
5
97%
2.99
N
N
NR
15
75%
3.80
N
N
NR
6
60%
3.48
N
Y
MRD−
414
MRD+
N/A
N
N
MRD−
CR
[2%]
CR
7
MRD+
4.15
N
Y
MRD+
103
MRD+
4.53
N
N
MRD−
CRi
8
85%
4.10
Y
Y
MRD−
104
15%
4.27
N
Y
MRD−
CR
CR
9
25%
1.79
N
N
MRD+
79
87%
1.98
N
N
CR
[2%]
*3rd dose; subject in CR following 1st and 2nd doses
ID: Subject Number
MRD: Minimal Residual Disease
CR: complete remission
Cri: complete remission with incomplete blood count recovery
NR: No response
N/A: not available
N: No
Y: Yes
The results in Table 3 show that a majority of treated subjects were responsive to the first dose of cells as evidenced by a reduction in tumor burden in the subject, for example, reduction in tumor burden from presence of >5% of blast cells to MRD+ or in some cases MRD−, or from MRD+ to MRD−. Complete remission (CR) of some subjects also was observed. As shown in Table 3, tumor reduction also was observed for subjects following administration of a consecutive dose of cells as evidenced by complete remissions or disease reduction as assessed from flow cytometric analysis for presence of MRD (see patient ID No. 1, 2, 7, 8 and 9). Thus, this result demonstrates that administration of the consecutive dose of cells also was efficacious. Most subjects did not exhibit severe CRS at any of the doses. Further, consistent with Example 2 severe neurotoxicity was observed to occur following administration only in subjects who had received higher doses of CAR-expressing cells and generally only in subjects with morphologic disease.
In some cases, Table 3 demonstrates that some subjects were not responsive (NR) to the first dose (see patient ID No. 5 and ID No. 2*) so that a reduction in disease or tumor burden levels was not achieved. Further, the results show that such failure to achieve clinical remission after infusion of the first dose did not increase the risk or frequency of neurotoxicity in the subject when administered a consecutive higher dose, since severe neurotoxicity was not detected after receiving a consecutive higher dose in either of these subjects.
In some embodiments, lack of (or relatively lower) degrees of responsiveness or toxicity in a subject indicates that the subject may not have responded well to CAR+ T cell therapy, but is also not at risk for certain toxic adverse events upon another infusion, which may otherwise indicate a need to avoid higher doses for subsequent administration. Therefore, these results support a dosage regime (e.g., if desired or necessary for maximizing or improving efficacy) in which a first low dose administration is followed (e.g., in a relatively short time) by a consecutive administration, carried out using a higher dose as compared to the first dose. The results presented here show that even if certain subjects do not respond well to the first dose (and thus do not have low tumor burden at the time of the consecutive dose) the risk of severe neurotoxicity is low upon administration of the consecutive dose, even for these non-responding subjects.
To further assess efficacy of cells administered in the first and consecutive dose, biological activity of administered cells was compared based on peak levels of measurements that were taken of serum levels of C reactive protein (CRP). Elevated peak levels of CRP in serum were taken as evidence of T cell expansion following infusion. The results are set forth in FIG. 2. The results show evidence of cell expansion after infusion of both a first and consecutive dose of T cells. Thus, these results show that, while subjects exhibited reduced CRS and neurotoxicity following a consecutive as opposed to a first administration of CAR+ T cells, the biological activity of the cells administered in and their ability to expand was comparable in each administration.
Subjects in the study also received physical examinations and were monitored for symptoms of other adverse events (AE), including those set forth in Table 4. To assess differences between first and consecutive administrations of CAR+ T cells in subjects, these data were compared. A treatment emergent AE (TEAE) after infusion of the first dose was defined as any AE occurring within 30 days after receiving the first dose but prior to receiving the consecutive dose. A TEAE after infusion of the consecutive dose was defined as any AE occurring within 30 days after receiving the consecutive dose but prior to any subsequent dose. Table 4 sets forth the number of subjects that exhibited a TEAE and the percentage (in parenthesis) of subjects exhibiting such TEAE compared to the number of total subjects assessed for the same TEAE. The results show that the percentage of adverse events was comparable following infusion of the first dose and consecutive dose.
TABLE 4
Adverse Events
TEAE post-
TEAE post-
infusion dose
infusion dose
Adverse Event
#1 (n = 33)
#2 (n = 11)
All
30
(90.9)
8
(72.7)
Febrile neutropenia
15
(45.5)
2
(18.2)
Abdominal pain
3
(9.1)
1
(9.1)
Nausea
8
(24.2)
1
(9.1)
Chills
10
(30.3)
1
(9.1)
Pyrexia
12
(36.4)
3
(27.3)
Cytokine release syndrome
5
(15.2)
1
(9.1)
(CRS)
Alanine aminotransferase
3
(9.1)
1
(9.1)
increased
Aspartate aminotransferase
2
(6.1)
1
(9.1)
increased
Hyperglycaemia
5
(15.2)
1
(9.1)
Hypophosphataemia
14
(42.5)
1
(9.1)
Hypophosphataemia
14
(42.5)
1
(9.1)
Muscular weakness
3
(9.1)
1
(9.1)
Convulsion
6
(18.2)
1
(9.1)
Dizziness
2
(6.1)
1
(9.1)
Encephalopathy
9
(27.3)
1
(9.1)
Dermatitis acneiform
0
(0.0)
1
(9.1)
Hypotension
12
(36.4)
1
(9.1)
Example 4
Impact of CAR+ T Cell Dose on Overall Survival of Different Subject Populations
To further assess the impact of CAR+ T cell dose on overall treatment of subjects, overall survival of subjects in the study described in Example 2 was compared between small groups of subjects, separated based upon number of cells administered and disease state at the time of administration. Product-limit survival was determined by calculating the Kaplan-Meier estimate with censored observations, such as occurred, for example, if a patient withdrew from the study.
Comparing all subjects in a group of subjects who had been administered a dose of fewer than 2.5×106 CAR+ cells/kg and all subjects in another group of subjects who had been administered a dose with greater than 2.5×106 CAR+ T cells/kg, the results indicated an overall survival advantage for the group of subjects administered the higher dose. Looking just at subjects having morphologic disease at the time of administration, the observed effect on overall survival with a higher dose was even greater. Collectively, the results presented herein support a dosage regime in which a higher dose (as compared to a first dose) is used in a consecutive administration, when the consecutive dose is given at a time at which disease burden remains reduced in patients on average, but at which the risk of CRS and/or neurotoxicity remains low. The results in this example support a conclusion that use of a higher consecutive dose will promote increased efficacy. Moreover, as noted, the results presented in Example 2 support a conclusion that use of a higher dose upon subsequent administration (at which subjects should have low disease burden or be otherwise not at risk for toxicity) will not lead to an increased toxicity risk.
Example 5
Multiple Dose Regimen of CAR+ T Cells for Treating Acute Lymphoblastic Leukemia (ALL)
In an exemplary dose regimen, subjects with CD19+ B cell acute lymphoblastic leukemia (ALL) were treated with two doses of CAR-expressing T cells, which included administering a first low dose of cells and a consecutive higher dose of cells. Before treatment, autologous CAR-expressing T cells were generated substantially as described in Example 2. Subjects received preconditioning chemotherapy including a single intravenous lymphodepleting dose of 1.0-3.0 g/m2 cyclophosphamide at 2-5 days prior to the first dose of CAR-expressing T cells. The first dose included approximately 1×106 cells/kg patient weight. A consecutive dose of cells expressing the CAR (approximately 3×106 such cells/kg patient weight) was administered 14-28 days after the first dose at a dose.
Subjects were monitored for efficacy of treatment, including by bone marrow, peripheral blood, and cerebrospinal fluid (CSF) examination, evaluation of central nervous system (CNS) symptoms, in order to assess and monitor disease burden (including levels and presence or absence of morphologic and degree of molecularly-detectable disease), evidence of adverse events, including CRS and neurotoxicity, and survival.
Example 6
Repeated Dosing Schedule of CAR+ T Cells for Treating Acute Lymphoblastic Leukemia (ALL)
In an exemplary dose regimen, subjects with relapsed or refractory B cell acute lymphoblastic leukemia (ALL) are treated with repeated doses of CAR-expressing T cells, which includes administering at least three doses of cells within the first 28 days. Before treatment, autologous CAR-expressing T cells are generated substantially as described in Example 2. Optionally, subjects receive preconditioning immunosuppressive chemotherapy of cyclophosphamide and/or fludarabine (CY/FLU), which is administered at least two days before the first dose of CAR-expressing cells and generally no more than 5 or no more than 7 days before administration of cells.
Subjects receive a first dose of CAR-expressing cells that is less than or equal to about 1×106 cells/kg patient weight, such as ranging from about 0.4×106 cells/kg to about 1×106 cells/kg, inclusive. Within 14-28 days after administration of the first dose, and/or prior to development of an immune response to the CAR, subjects are infused with two additional higher doses of cells. In some embodiments, a consecutive dose of CAR-expressing cells is administered about 14 days after the first dose at a dose that is higher than the first dose, such as a dose ranging from about 2.5×106 cells/kg to about 4.5×106 cells/kg, such as approximately 3×106 cells/kg patient weight, followed by a third dose of CAR-expressing cells that is administered about 28 days after the first dose at a dose that is higher than the first dose, such as a dose ranging from about 2.5×106 cells/kg to about 4.5×106 cells/kg, such as approximately 3×106 cells/kg patient weight. In some embodiments, one or more subsequent doses of cells are administered. In some embodiments, a fourth dose of CAR-expressing cells is administered within about 42 to 56 days after the first dose at a dose that is higher than the first dose, such as a dose ranging from about 2.5×106 cells/kg to about 4.5×106 cells/kg, such as approximately 3×106 cells/kg patient weight.
Subjects are monitored for efficacy of treatment by measuring the overall remission rate (ORR) after the final dose of cells in subjects. In some embodiments, efficacy is monitored in subjects with morphologic evidence of disease prior to treatment (greater than or equal to 5% of cells in bone marrow were blasts). ORR is determined as the proportion of subjects with CR with incomplete blood count recovery (CRi), as determined by examination of the bone marrow, peripheral blood, and cerebrospinal fluid (CSF), as well as physical examination and evaluation of central nervous system (CNS) symptoms.
Example 7
Multiple Dose Regimen of CAR+ T Cells with Lymphodepleting Chemotherapy Pre-Conditioning for Treating Non-Hodgkin Lymphoma (NHL)
In exemplary dose regimens for treating CD19+ B cell Non-Hodgkin Lymphoma (NHL), a multiple dose regimen of CAR-expressing T cells is used to treat subjects. Before treatment, autologous CAR-expressing T cells are generated substantially as described in Example 2. Subjects with NHL are treated with at least two doses of CAR-expressing T cells. Subjects receive a first dose of CAR-expressing cells that is less than or equal to about 1×106 cells/kg patient weight, such as ranging from about 0.4×106 cells/kg to about 1×106 cells/kg, inclusive. In exemplary dosage regimes, a consecutive dose of CAR-expressing T cells is administered 14 to 28 days after the first dose.
In one exemplary dosage regime, subjects with NHL receive a consecutive dose of CAR-expressing at a dose that is the same or lower than the first dose of CAR-expressing T cells, such as a dose that is less than or equal to about 1×106 cells/kg patient weight, such as ranging from about 0.4×106 cells/kg to about 1×106 cells/kg, inclusive. Optionally, further repeat doses are administered 14 to 28 days after a prior administration at a dose that is less than or equal to the dose administered in the prior administration.
In one exemplary dosage regime, prior to receiving a consecutive dose, subjects are optionally monitored for tumor burden. If molecular remission is detected, as evidenced by tumor reduction from morphological disease to MRD, subjects are administered a consecutive dose of CAR-expressing T cells at a dose that is higher than the first dose, such as a dose ranging from about 2.5×106 cells/kg to about 4.5×106 cells/kg, such as approximately 3×106 cells/kg patient weight. If molecular remission has not occurred, subjects are administered a consecutive dose of CAR-expressing T cells at a dose that is the same or lower than the first dose of CAR-expressing T cells, such as a dose that is less than or equal to about 1×106 cells/kg patient weight, such as ranging from about 0.4×106 cells/kg to about 1×106 cells/kg, inclusive. Optionally, further repeat doses are administered 14 to 28 days after a prior administration, with higher doses being administered if molecular remission has occurred and lower doses being administered if molecular remission has not occurred.
In one exemplary dosage regime, prior to receiving the first dose, subjects receive an immunodepleting preconditioning chemotherapy of cyclophosphamide and fludarabine (CY/FLU), which is administered at least two days before the first dose of CAR-expressing cells and generally no more than 7 days before administration of cells. After preconditioning treatment, subjects are administered the first dose as described above at a dose of CAR-expressing T cells that is less than or equal to about 1×106 cells/kg patient weight, such as ranging from about 0.4×106 cells/kg to about 1×106 cells/kg, inclusive. Subjects are administered a consecutive dose of CAR-expressing T cells at a dose that is higher than the first dose, such as a dose ranging from about 2.5×106 cells/kg to about 4.5×106 cells/kg, such as approximately 3×106 cells/kg patient weight. Optionally, further repeat doses are administered 14 to 28 days after a prior administration at a dose ranging from about 2.5×106 cells/kg to about 4.5×106 cells/kg, such as approximately 3×106 cells/kg patient weight.
Example 8
Assessment of PD1/PD-L1 Expression In T-Cells Stimulated Through a Chimeric Antigen Receptor (CAR)
T cells were isolated by immunoaffinity-based enrichment from leukapheresis samples from human subjects, and cells were activated and transduced with a viral vector encoding an anti-CD19 chimeric antigen receptor (CAR) containing a human CD28-derived intracellular signaling domain and a human CD3 zeta-derived signaling domain. Surface expression on the resulting isolated compositions (of the CAR and of certain T cell markers) was assessed by flow cytometry, to determine, in the composition, the percentage of CAR+ cells among all T cells in the and among T cell subsets, as well as ratio of CD4+ to CD8+ T cells (see Table 5).
TABLE 5
Anti-CD19 CAR Expression on Transduced T cells
CD3+CAR+
CD4+CAR+
CD8+CAR+
CD3+CD4+
CD3+CD8+
percent
49.91
23.60
28.73
40.03
53.66
(average)
Standard
2.97
1.18
2.38
1.10
1.22
Deviation
The composition then was subdivided into different samples by incubation with: 1) K562 cells expressing the antigen for which the CAR was specific (K562-tCD19 cells) (antigen-specific coculture); 2) K562 cells expressing an unrelated antigen (K562-ROR1 cells) (non-specific coculture control); or 3) plate-bound anti-CD3 antibody and soluble anti-CD28 antibody (for stimulation via the TCR complex), initially using plate-bound anti-CD3 and soluble anti-CD28, and at day 3, where applicable, incubation. For (1) and (2), K562 (immortalized myelogenous leukemia line) cells, were engineered to express CD19 and ROR1, respectively, and incubated with the CAR-expressing T cells at a 1:1 ratio. For each of the conditions, CAR-expressing T cells were stimulated for 24 hours. An unstimulated sample (“media,” no K562 cells or stimulating antibodies) was used as an additional negative control.
After 24 hours in culture, flow cytometry was performed to assess surface expression of PD1, PD-L1, PD-L2, T cell markers, and CAR (based on goat-anti-mouse (“GAM”) staining to detect the murine variable region portion of the CAR) on the cells in each sample. Live, single cells with forward scatter and side scatter profiles matching lymphocytes were gated for analysis. Expression of PD1, PD-L1 and PD-L2 was assessed on various gated populations of T cells (CD4+/CAR+, CD4+/CAR−, CD8+/CAR+, and CD8+/CAR−), with gates set based on the surface expression of various markers, and using values for the negative control (“media”) sample to determine appropriate gating.
As shown in FIGS. 3A and 4A, PD1 and PD-L1 expression increased within twenty-four (24) hours in both CD4+/CAR+ and CD8+/CAR+ T cells when cultured with cells expressing the antigen to which the CAR was specific (K562-tCD19). This increase in expression of PD1 and PD-L1 was not observed within this timeframe in CAR+ cells incubated with cells of the same type expressing an irrelevant antigen cells (K562-ROR1) or in any of the CD4+ or CD8+ cell populations incubated under conditions designed to effect stimulation through the TCR complex (anti-CD3 and anti-CD28 antibodies). Expression of PD-L2 was not upregulated within this timeframe under any of the stimulated conditions tested.
As shown in FIGS. 3B and 4B, the increase in expression of PD-1 and PD-L1 in cells incubated with CD19-expressing cells was observed to be primarily due to expression of the anti-CD19 CAR. Neither the CD4+-gated nor the CD8+-gated T cells that did not express the CAR (“CAR-”) exhibited substantial increases in PD-1 or PD-L1 surface expression following incubation with the CD19-expressing cells.
Similar results were obtained in the presence of T cells genetically engineered with an anti-CD19 chimeric antigen receptor (CAR) containing a human 4-1BB-derived intracellular signaling domain and a human CD3 zeta-derived signaling domain. Thus, the results showed that the upregulation of PD-1 and PD-L1 occurred on T cells transduced with CAR constructs containing either a CD28 or 4-1BB costimulatory signaling domain. These data demonstrate upregulation of surface expression of PD1 and PD-L1 within twenty-four hours following stimulation through the chimeric antigen receptor, but not following stimulation under conditions designed to mimic signal through the canonical T cell antigen receptor complex (CD3/CD28 antibodies).
These data further support a conclusion that exposure, in vivo, upon administration to subjects, to antigen to which the CAR was specific may in some contexts result in upregulation of PD-L1 and/or PD-1, which may occur to a greater degree and/or more rapidly than (or in distinction from) what would occur following interaction with cognate antigen through an endogenous TCR.
Upregulation of such molecules and other inhibitory markers may contribute to loss of function and/or exhaustion of the T cells and for example may impair long-term exposure to the cells. A repeat or consecutive dose(s) of cells may be used to deliver cells not yet expressing the inhibitory molecules, such as PD-1 and/or PD-L1, or expressing them at lower levels compared to the cells present in the subject. Thus, these data provide further support for a multiple dosing schedule, in which a consecutive dose of T cells is administered to a subject following an initial dose, such as at a time at which PD-L1 or PD-1 has been or is upregulated on cells of the initial dose, following exposure to the target antigen.
In some embodiments, in the consecutive dose, the inhibitory molecule(s) are not expressed or substantially expressed (or expressed to the same degree as a reference cell population) on the cells therein (or on greater than 50, 40, 30, 20, 10, or 5% of the cells therein). In some embodiments, repeated doses of cells that do not express or do not substantially express inhibitory molecules, such as PD-1 and PD-L1, can extend the time during which functional CAR-expressing T cells or CAR-expressing T cells having robust function are present in the subject. In some embodiments, replenishing the army of genetically engineered T cells by administering one or more consecutive doses can lead to a greater and/or longer degree of exposure to the antigen receptor (e.g. CAR)-expressing cells and improve treatment outcomes. In some embodiments, the consecutive dose is administered at a time at which PD-L1 or PD-1 is upregulated compared to a reference level or population, such as compared to the cells in the composition of the first dose immediately prior to administration to the subject, for example, to a degree that is at least 10, 20, 30, 40, 50, 60, 70, or 80% higher surface expression as compared to the reference population.
Example 9
Assessment of Neurotoxicity in Subjects Based on Tumor Burden
Subjects with CD19+ B cell acute lymphoblastic leukemia (ALL) were administered autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR). Before treatment, autologous CAR-expressing T cells were generated substantially as described in Example 2
Neurotoxicity was observed in a cohort of patients after receiving a single IV continuous infusion of CAR-expressing T cells, at a dose of either 2×105 cells/kg (N=13), 2×106 cells/kg (N=15) or 2×107 cells/kg (N=2) (see FIGS. 5A and 5B). In some cases, a preconditioning chemotherapeutic treatment of cyclophosphamide (about 2 g/m2), cyclophosphamide (2 g/m2) and etoposide (100 mg/m2, administered three times daily) or cyclophosphamide (60 mg/kg) and fludarabine (25 mg/m2, administered three to five times daily) was administered to subjects prior to infusion.
The number of CAR-T cells present in peripheral blood of treated subjects was determined post-treatment by performing flow cytometry for surface expression of EGFRt (CAR-specific marker) and CD4 or CD8. Neurotoxicity also was determined and monitored, and graded based on severity as described above.
As shown in FIG. 5A, the degree of neurotoxicity observed in this study in treated subjects correlated to the presence of relatively higher tumor burden prior to treatment. As shown in FIG. 5A, subjects exhibiting severe symptoms of neurotoxicity with a grade of 3 or higher also exhibited a greater number of CD8+ or CD4+ CAR-T cells in peripheral blood following treatment at the time assessed, indicating a correlation between tumor burden, the degree of expansion of CAR+ T cells and neurotoxicity. FIG. 5B shows that subjects determined to require intensive care unit (ICU) care following treatment were among those having relatively higher percentages of bone marrow blasts prior to treatment.
Example 10
Risk-Adapted Dosing of CAR+ T Cells
Five subjects, among those treated as described in Example 9, were administered the anti-CD19 CAR-T cells in doses adapted based on tumor burden.
Prior to treatment, tumor burden was assessed by evaluating the percent of marrow blasts present in the bone marrow. Subjects having greater than 20% blasts (>20%) in bone marrow were selected for administration of a dose of CAR-T cells (2×105 T cells/kg) that was relatively lower as compared to the dose administered to subjects having less than or equal to 20% blasts. Subjects having less than or equal to 20% blasts (≤20%) were selected for administration of a relatively higher dose (2×106 CAR-T cells/kg). CAR-expressing T cells were administered to subjects by single intravenous (IV) continuous infusion.
At days 0, 1, 3, 7, 10, 14, 21 and 28 post-treatment, the number of CAR-T cells present in peripheral blood of treated subjects was determined by performing flow cytometry of a cell sample for surface expression of EGFRt (CAR-specific marker) and CD4 or CD8. Subjects also were monitored for toxicity, including severe toxic outcomes requiring intensive care unit (ICU) care.
CAR-T cells were observed to expand in all subjects. As compared to a group of subjects receiving similar treatment, but for which the administered dosage was not based on tumor burden, the risk-adapted dosing resulted in a reduced incidence of serious toxicity. For example, in subjects for which the administered dosage was not based on tumor burden, seven of eleven subjects were required to have ICU care. In contrast, none (0%) of the five subjects for which risk-adapted dosing was used required ICU care.
Example 11
Pre-Conditioning with Fludarabine Prior to CAR-T Cell Administration in Subjects with B Cell Acute Lymphoblastic Leukemia (ALL)
Subjects with CD19+ B cell acute lymphoblastic leukemia (ALL) were administered 2×106 cells/kg of autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR). Before treatment, autologous CAR-expressing T cells were generated substantially as described in Example 2.
Prior to administration of the CAR-expressing T cells, subjects were treated either with 1) 2 g/m2 cyclophosphamide (with or without administration of 100 mg/m2 etoposide three times daily) (N-=5, no Flu treated group), or were treated with 60 mg/kg (˜2 g/m2) cyclophosphamide and 3 to 5 doses of 25 mg/m2 fludarabine (N=10, designated Flu treated group).
At days 0, 1, 3, 7, 10, 14, 21 and 28 post-treatment, the number of CAR-T cells present in peripheral blood of treated subjects was determined by performing flow cytometry of a cell sample for surface expression of EGFRt (CAR-specific marker) and CD4 or CD8.
As shown in FIGS. 6A and 6B, a greater degree of CAR-T cell expansion and/or persistence, as measured by the presence of CD8+ (FIG. 6A) or CD4+ (FIG. 6B) CAR-T cells in peripheral blood, was observed from days 7 to 28 post-treatment in subjects who were pre-conditioned with cyclophosphamide/fludarabine as compared with those who did not receive fludarabine prior to the administration of CAR-T cells. This result demonstrates that pre-conditioning with cyclophosphamide/fludarabine can impact in vivo CAR-T cell expansion.
As shown in FIG. 6C, a greater percentage of patients exhibiting disease-free survival over the time period shown was observed among subjects having been pre-treated with both cyclophosphamide and fludarabine.
Example 12
Pre-Conditioning with Fludarabine Prior to CAR-T Cell Administration in Subjects with Non-Hodgkin Lymphoma (NHL)
Subjects with Non-Hodgkin Lymphoma (NHL) were administered 2×107 cells/kg of autologous T cells expressing an anti-CD19 chimeric antigen receptor (CAR). Before treatment, autologous CAR-expressing T cells were generated substantially as described in Example 2.
Prior to administration of the CAR-expressing T cells, subjects were treated either with 1) 2-4 g/m2 cyclophosphamide (with or without administration of 100-200 mg/m2 etoposide three times daily) (N=3, designated no Flu treated group), or 2) with 30-60 mg/kg (˜1-2 g/m2) cyclophosphamide and 3 to 5 doses of 25 mg/m2 fludarabine (N=6, designated Cy/Flu treated group).
At days 0, 1, 3, 7, 10, 14, 21 and 28 post-treatment, the number of CAR-T cells present in peripheral blood of treated subjects was determined by performing flow cytometry of a cell sample for surface expression of EGFRt (CAR-specific marker) and CD4 or CD8.
The results are depicted in FIGS. 7A and 7B, which set forth the CAR-expressing CD8+ or CD4+ T cells, respectively, as total cells per μL or as the total percentage of CD4+ or CD8+ cells, respectively, in the sample. As shown in FIGS. 7A and 7B, a greater degree of CAR-T cell expansion and/or persistence, as measured by the presence of CD8+ (FIG. 7A) or CD4+ (FIG. 7B) CAR-T cells in peripheral blood following treatment, was observed from days 3 to 28 post-treatment in subjects who were pre-conditioned with cyclophosphamide/fludarabine as compared to those who did not receive fludarabine prior to the administration of CAR-T cells. This result demonstrated that pre-conditioning with cyclophosphamide/fludarabine can impact in vivo CAR-T cell expansion in subjects with NHL.
Subjects were monitored for efficacy of treatment by measuring the overall remission rate (ORR). ORR was determined as the proportion of subjects with complete remission (CR) or partial remission (PR), as determined by examination of the bone marrow, peripheral blood, and cerebrospinal fluid (CSF), as well as physical examination and evaluation of central nervous system (CNS) symptoms.
The overall remission rate for subjects pre-conditioned with cyclophosphamide/fludarabine was 62% (8/13), including a 38% CR rate and 23% PR rate (CR=5/13, PR=3/13), while subjects who did not receive fludarabine pre-conditioning had an overall remission rate of 50% (6/12), including an 8% CR rate and a 42% PR rate (CR=1/12, PR=5/12).
The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.
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14918451
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juno therapeutics, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Mar 30th, 2022 06:04PM
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Mar 30th, 2022 06:04PM
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Bristol-Myers Squibb
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:bmy
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Juno Therapeutics
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Sep 29th, 2020 12:00AM
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Jul 15th, 2016 12:00AM
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https://www.uspto.gov?id=US10786533-20200929
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Engineered cells for adoptive cell therapy
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Provided are engineered cells for adoptive therapy, including NK cells and T cells. Also provided are compositions for engineering and producing the cells, compositions containing the cells, and methods for their administration to subjects. In some embodiments, the cells contain genetically engineered antigen receptors that specifically bind to antigens, such as chimeric antigen receptors (CARs) and costimulatory receptors. In some embodiments, the cells include receptors targeting multiple antigens. In some embodiments, the cells include repression of one or more gene product, for example, by disruption of a gene encoding the gene product. In some embodiments, a gene encoding an antigen recognized by the engineered antigen receptor is disrupted, reducing the likelihood of targeting of the engineered cells. In some embodiments, the antigen recognized by the engineered antigen receptor is related to a tumor antigen recognized by the engineered antigen receptor.
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10786533
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1. An engineered immune cell comprising:
a recombinant receptor comprising an anti-B cell maturation antigen (BCMA) antibody or an antigen-binding fragment thereof that specifically binds to a BCMA, wherein the recombinant receptor also binds or is suspected of binding to a related antigen that is a transmembrane activator and CAML Interactor (TACI) or a B-cell activating factor receptor (BAFF-R); and
a genetic disruption in the gene encoding the related antigen in the engineered immune cell.
2. The engineered immune cell of claim 1, wherein the genetic disruption has been induced by a targeted nuclease.
3. The engineered immune cell of claim 2, wherein the targeted nuclease is an RNA-guided nuclease.
4. The engineered immune cell of claim 3, wherein the RNA-guided nuclease is a CRISPR-Cas9 combination comprising a Cas9 enzyme and a guide RNA (gRNA) that specifically binds to, recognizes, or hybridizes to the gene encoding the related antigen.
5. A pharmaceutical composition comprising the engineered immune cell of claim 4 and a pharmaceutically acceptable carrier.
6. The engineered immune cell of claim 4, wherein the immune cell is a T cell.
7. The engineered immune cell of claim 6, wherein the T cell is a CD4+ or CD8+ T cell.
8. The engineered immune cell of claim 4, wherein the immune cell is a natural killer (NK) cell.
9. A pharmaceutical composition comprising the engineered immune cell of claim 2 and a pharmaceutically acceptable carrier.
10. The engineered immune cell of claim 2, wherein the targeted nuclease is a fusion protein comprising a DNA-targeting protein and a nuclease.
11. The engineered cell of claim 10, wherein the fusion protein is a zinc finger nuclease (ZFN) or a TAL-effector nuclease (TALEN) that specifically binds to or recognizes the gene encoding the related antigen.
12. The engineered immune cell of claim 2, wherein the immune cell is a T cell.
13. The engineered immune cell of claim 12, wherein the T cell is a CD4+ or CD8+ T cell.
14. The engineered immune cell of claim 2, wherein the immune cell is a natural killer (NK) cell.
15. The engineered immune cell of claim 1, wherein the recombinant receptor is a chimeric antigen receptor (CAR).
16. The engineered immune cell of claim 15, wherein the immune cell is a T cell.
17. The engineered immune cell of claim 16, wherein the T cell is a CD4+ or CD8+ T cell.
18. The engineered immune cell of claim 15, wherein the immune cell is a natural killer (NK) cell.
19. A pharmaceutical composition comprising the engineered immune cell of claim 15 and a pharmaceutically acceptable carrier.
20. The engineered immune cell of claim 1, wherein:
the disruption comprises disrupting the gene at the DNA level,
the disruption is not reversible; or
the disruption is not transient.
21. A pharmaceutical composition comprising the engineered immune cell of claim 1 and a pharmaceutically acceptable carrier.
22. The engineered immune cell of claim 1, wherein the immune cell is a T cell.
23. The engineered immune cell of claim 22, wherein the T cell is a CD4+ or CD8+ T cell.
24. The engineered immune cell of claim 1, wherein the immune cell is a natural killer (NK) cell.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Phase Application of International Application No. PCT/US2016/042647, filed Jul. 15, 2016, which claims priority from U.S. provisional application No. 62/281,722 filed Jan. 21, 2016, entitled “Engineered Cells for Adoptive Cell Therapy” and is a continuation-in-part of International PCT Appl. No. PCT/US15/40660, filed Jul. 15, 2015, entitled “Engineered Cells for Adoptive Cell Therapy,” the contents of which are incorporated by reference in their entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042000530SeqList.txt, created Jul. 12, 2018, which is 72,762 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.
FIELD
The present disclosure relates in some aspects to engineered cells for adoptive therapy, including NK cells and T cells. In some aspects, the disclosure further relates to methods and compositions for engineering and producing the cells, compositions containing the cells, and methods for their administration to subjects. In some aspects, features of the cells and methods provide specificity and/or efficacy. In some embodiments, the cells contain genetically engineered antigen receptors that specifically bind to antigens, such as chimeric antigen receptors (CARs) and costimulatory receptors. In some embodiments, the cells include receptors targeting multiple antigens. In some embodiments, the cells include repression of one or more gene product, for example, by disruption of a gene encoding the gene product. In some embodiments, a gene encoding an antigen recognized by the engineered antigen receptor is disrupted, reducing the likelihood of targeting of the engineered cells. In some embodiments, the engineered antigen receptor may recognize, such as specifically bind to, one antigen, such as a target antigen, e.g., a disease-specific (e.g., tumor-specific or tumor-associated) antigen, and additionally may also specifically bind to or recognize another antigen, which in some cases is not expressed or is not associated with or specifically expressed or overexpressed or upregulated on or in the disease or condition or target cell or tissue or pathogen being treated. In some such aspects, however, the another antigen may be a family member of, share structural and/or sequence similarity with, and/or an epitope with, and/or be related to, the first antigen, such as the disease-specific, e.g., tumor-specific antigen. Thus, in some embodiments, the receptor is one that specifically binds to a first antigen that is a disease-specific antigen and also to a second antigen that is not a disease-specific antigen for the disease or condition or cell or tissue or organism being treated.
BACKGROUND
Various strategies are available for producing and administering engineered cells for adoptive therapy. For example, strategies are available for engineering immune cells expressing genetically engineered antigen receptors, such as CARs, and for suppression or repression of gene expression in the cells. Improved strategies are needed, for example, to provide a wider range of target antigens and diseases that may be treated using such cells, to improve specificity or selectivity of the cells, e.g., to avoid off-target effects, and to improve efficacy of the cells, for example, by avoiding suppression of effector functions and improving the activity and/or survival of the cells upon administration to subjects. Provided are methods, cells, compositions, kits, and systems that meet such needs.
SUMMARY
Provided are cells, including engineered cells, such as engineered immune or immunostimulatory cells, as well as methods for producing and using the cells, such as in adoptive therapy, and compositions, such as pharmaceutical compositions, containing the cells. Among the cells are those having one or more features, such as dual-antigen targeting features and/or gene disruptions, which on their own or collectively provide improved safety, specificity, selectivity, and/or efficacy, and/or allow for the targeting of a broader range of antigens or diseases by adoptive cell therapy.
In some embodiments, the cells are engineered cells, such as engineered immune cells including: a genetically engineered antigen receptor that specifically binds to a target antigen (such as a target antigen associated with or expressed, e.g., specifically expressed, on or in a disease or condition to be treated). In some embodiments, a target antigen that is expressed on or in, specifically expressed on or in, or associated with, the disease or condition being treated may be referred to herein as a “disease-specific target” “disease-specific target antigen” or “disease-specific antigen”. In some aspects, the cells further include a disruption in a gene. Thus, such terms are not meant to refer only those antigens expressed only on or in the disease, but encompass antigens that are associated with or expressed in such disease or condition even if also expressed in certain other cell types or tissue types or in the context of non-disease tissue or cells. In some aspects, the gene disrupted is a gene encoding an antigen, such as the target antigen (for example, the antigen expressed on or in or associated with the disease or condition, i.e., the disease-specific antigen), or such as an antigen related to such target antigen. The disruption generally results in reduced expression, in the engineered cell, of the target antigen, an epitope thereof, or another antigen related to or sharing similarity or an epitope with such disease-specific target antigen, in the engineered immune cell. In some aspects, the target antigen such as the disease-specific target antigen (and/or the another antigen) is an antigen expressed (or exhibiting heightened expression as compared to other or normal or control cells) in, or on the surface of, resting T cells, activated T cells (and in some such aspects not resting T cells), or both. In some aspects, the target antigen such as the disease-specific target antigen is expressed on or in or is upregulated on or in the cell used to generate the engineered cell, or such cell in an activated form, such as on or in T cells, NK cells, CD4+ T cells, CD8+ T cells, and/or stem cells, such as induced pluripotent stem cell (iPS cells). In some aspects, the target antigen is expressed on the cell surface in a cancer, such as a hematologic cancer, an immune cancer, a leukemia, a lymphoma, and/or a myeloma, such as multiple myeloma. Thus, in some aspects, the disease or condition being treated is a cancer or tumor such as hematologic cancer, an immune cancer, a leukemia, a lymphoma, and/or a myeloma, such as multiple myeloma, and/or a solid tumor and/or a cell or tissue associated with such cancer or tumor. In some embodiments, the disease or condition is an autoimmune or inflammatory disease or condition or is an infectious disease or condition or cell or pathogen associated therewith. In some aspects, the another antigen is one that is not expressed or is not specifically expressed or associated with the disease or condition, and in some aspects is not expressed or specifically expressed or associated with any cancer.
In some aspects, the target antigen (e.g., the disease-specific target antigen) is not overexpressed or is not expressed on the surface of the cell type used to produce the engineered cell, or such cell in its resting or activated form, such as in resting T cells or activated T cells and/or in resting or activated NK cells, CD4+ T cells, CD8+ T cells, stem cells, and/or induced pluripotent stem cell (iPS cell). In some such aspects, however, one or more antigens that are related to such target antigen, such as share sequence or structural similarity with and/or a common epitope with such target antigen (e.g., the antigen associated and/or expressed with or by the disease or condition) are expressed or overexpressed on the surface of or within such cell type, e.g., resting and/or activated T cells (and in some cases in activated but not resting T cells). In some aspects, the antigen receptor is cross-reactive to (for example, specifically binds to) both the target antigen (such as the disease-specific target antigen) and the antigen(s) related to the target antigen or sharing similarity or epitope(s) therewith, which for example may be not associated with the disease or condition.
In some embodiments where the target antigen, or related antigen thereof, is expressed on or in activated T cells, such antigen is not expressed on or in such cells, e.g., on or in resting or non-activated T cells, or is not expressed on 50, 60, 70, 80, 90, or 95% of resting or non-activated T cells. In some aspects, the antigen is expressed in activated T cells at a higher level compared to resting or non-activated T cells. In specific embodiments, the antigen is expressed on or in activated T cells at a level (e.g., as measured by flow cytometry for example, mean fluorescence intensity, and/or as measured by quantitative PCR, e.g., quantitative RT-PCR such as qPCR) that is at least 50%, 60%, 70%, 80%, 90%, or 95% more and/or 1-fold, 1.5-fold, 2-fold, 3-fold more as compared to resting or non-activated T cells. In some cases, when expression levels, such as surface expression levels are compared in different T cell populations, the comparison is between two different such populations derived from the same individual, such as the subject being treated and in some aspects from the same sample or tissue or fluid.
In some aspects, such as where the antigen receptor induces an activating signal or one that causes an immune response directed at cells expressing the target antigen, the target antigen is one that is expressed on a disease or condition to be treated, such as cancer but that also is ordinarily expressed on the cell being engineered or used for adoptive cell therapy. In some aspects, the cell being engineered or used for adoptive cell therapy expresses or ordinarily expresses one or more antigens related to or sharing similarity with the target antigen. In some aspects, that target antigen, such as the disease-specific target, is a universal tumor antigen, in some aspects one that is expressed naturally on or in the engineered cells and/or expressed on or in or the expression of which is upregulated on or in, activated T cells. In some aspects, the universal tumor antigen, is a human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2/neu, Wilms' tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53 or cyclin (D1). For example, the target antigen is hTERT or survivin. In some aspects, the target antigen is CD38. In some aspects, the target antigen is B-cell maturation antigen (BCMA, BCM). In some aspects, the target antigen is BCMA, B-cell activating factor receptor (BAFFR, BR3), and/or transmembrane activator and CAML interactor (TACI), or a related protein thereof. For example, the target antigen in some embodiments is or is related to BAFFR or TACI. In some aspects, the target antigen is CD33 or TIM-3. In some aspects, it is CD26, CD30, CD53, CD92, CD100, CD148, CD150, CD200, CD261, CD262, or CD362.
In some embodiments, the gene or antigen the expression of which is disrupted antigen is another antigen, which is related to the target antigen (e.g., related to or sharing similarity or epitope with, the disease-specific target antigen). In some such embodiments, whereas the target antigen (e.g., the disease-specific antigen) may not be expressed naturally on or in the engineered cells, on or in the engineered cells in an activated form, and/or on or in activated T cells, and/or may not be upregulated or overexpressed in T cells compared to other cells or on or in or activated T cells compared to non-activated or normal or control T cells, In some such embodiments, however, the related antigen is one that is expressed naturally on or in the engineered cells and/or upregulated on or in activated T cells whereas the target antigen may not expressed naturally on or in the engineered cells and/or upregulated on or in activated T cells. In some aspects, the gene or antigen disrupted (or the expression of which is disrupted) is or related to any of the target antigens disclosed herein. In some aspects, the target antigen and/or the antigen disrupted or the expression of which is disrupted is BCMA, TACI, BAFF-R, and/or Fc Receptor-like 5 (FCRLS, FcRH5). In some aspects, both the target antigen and one or more antigens disrupted, such as antigens related to the target antigen, are expressed on or in activated T cells.
In some aspects, such as where the receptor induces a suppressive or inhibitory, e.g., immunosuppressive, signal, the antigen is one that is not expressed in the disease or condition. In some aspects, wherein the genetically engineered antigen receptor is capable of inducing an inhibitory or immunosuppressive or repressive signal to the cell upon recognition of the target antigen. In some aspects, the antigen is an antigen that is not expressed on the surface of a cancer cell or infected cell or the expression of which is downregulated on a cancer cell or an infected cell. Exemplary of such antigens are MHC-class I molecules.
In some embodiments, the antigen is a gene product that is naturally expressed in the cell type of the engineered cell. In some embodiments, expression of the target antigen in the engineered immune cell is reduced by at least 50, 60, 70, 80, 90, or 95% as compared to the expression in the immune cell in the absence of said gene disruption. In some embodiments, the disruption comprises a deletion of at least a portion of at least one exon of the gene; comprises a deletion, mutation, and/or insertion in the gene resulting in the presence of a premature stop codon in the gene; and/or comprises a deletion, mutation, and/or insertion within a first or second exon of the gene.
Among the cell types are T cells, NK cells, CD4+ T cells, CD8+ T cells, and stem cells, such as an induced pluripotent stem cell (iPS cell).
In some embodiments, the genetically engineered antigen receptor is capable of inducing an activating signal to the cell. In some aspects, the genetically engineered antigen receptor comprises an intracellular domain with an ITAM-containing motif. In some aspects, the genetically engineered antigen receptor is a T cell receptor (TCR) or a functional non-TCR antigen recognition receptor. In some aspects, it is a chimeric antigen receptor (CAR), such as an activating or stimulatory CAR, an inhibitory CAR and/or a costimulatory CAR. Among the CARs are those with an extracellular antigen-recognition domain that specifically binds to the target antigen and an intracellular signaling domain comprising an ITAM, such as an intracellular domain of a CD3-zeta (CD3) chain those that further comprise a costimulatory signaling region, such as a signaling domain of CD28 or 41BB.
In some aspects, engineered cells include an engineered receptor such as a CAR with an extracellular antigen-recognition domain that specifically binds to a first target antigen such as a disease-specific antigen, and further include a second receptor, such as second CAR that also binds to one or more other, e.g., second, antigen that is not expressed on or in the disease or condition (e.g., on or in tumor cells) but that is expressed on normal or control cells or tissues that also express the first target antigen. In some aspects, the, and an intracellular signaling domain that comprises a signaling portion that induces a negative or immunoinhibitory signal and/or otherwise dampens a signal induced by a receptor targeting the first antigen, such as a signaling domain of an immune checkpoint molecule, such as PD-1 or CTLA4. In some aspects, such cells further comprise disruption of such molecule or gene encoding it.
In some aspects, the cell comprises another genetically engineered antigen receptor, such as a costimulatory receptor, such as a chimeric costimulatory receptor, that specifically binds to another antigen and is capable of inducing a costimulatory signal to the cell. In some aspects, such another target antigen and the first target antigen recognized by the first receptor are distinct. In some embodiments, at least one of such antigens is selected from among a human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2/neu, Wilms' tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, cyclin (D1), BCMA, BAFFR, FcRH5, or TACI, and the other is another antigen expressed on a tumor or cancer, and in some cases is expressed on a particular type of tumor or cancer, and not on one or more certain other types of cancers. In some aspects, such other antigen is a multiple myeloma-associated or multiple myeloma-specific antigen such as CD38 or CD138 or BCMA or CS-1; in some aspects, such other antigen is expressed on one or more blood cancers or one or more solid tumor types. In some aspects, the target antigen and said another antigen are distinct and are selected, individually, from the group consisting of CD38 and CD138.
In some such aspects, the cell contains a first genetically engineered antigen receptor and further comprises an additional genetically engineered antigen receptor which recognizes an antigen expressed on a disease or condition to be treated and induces a stimulatory or activating signal, which is dampened by the first genetically engineered antigen receptor.
Also provided are methods for producing the cells and cells produced by such methods. In some embodiments, the methods are carried out by (a) introducing into an immune cell a genetically engineered antigen receptor that specifically binds to a target antigen; and (b) effecting repression of expression of the target antigen, e.g., the disease-specific antigen (and/or the another antigen, such as the related antigen that is related to or shares similarity or an epitope with the disease-specific antigen not a disease-specific antigen for the disease or condition being treated) in the immune cell, thereby producing a genetically engineered immune cell in which expression of the target antigen is repressed. In some aspects, steps (a) and (b) are carried out simultaneously or sequentially in any order.
In some embodiments, the effecting in (b) comprises disrupting a gene encoding the target antigen or other antigen to be disrupted, the disruption comprises disrupting the gene at the DNA level and/or the disruption is not reversible; and/or the disruption is not transient. In some aspects, the disruption comprises introducing into the immune cell a DNA binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to the gene.
In some embodiments, the disruption comprises introducing: (a) a fusion protein comprising a DNA-targeting protein and a nuclease or (b) an RNA-guided nuclease. For example, in some embodiments, the DNA-targeting protein or RNA-guided nuclease comprises a zinc finger protein (ZFP), a TAL protein, or a clustered regularly interspaced short palindromic nucleic acid (CRISPR) specific for the gene. In some embodiments, the disruption comprises introducing a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or and a CRISPR-Cas9 combination that specifically binds to, recognizes, or hybridizes to the gene. In some embodiments, the introducing is carried out by introducing into the cell a nucleic acid comprising a sequence encoding the DNA-binding protein, DNA-binding nucleotide, and/or complex comprising the DNA-binding protein or DNA-binding nucleotide. In some embodiments, the nucleic acid is a viral vector.
In some embodiments, the specific binding to the gene is within an exon of the gene and/or is within a portion of the gene encoding an N-terminus of the target antigen or other antigen. In some embodiments, the introduction thereby effects a frameshift mutation in the gene and/or an insertion of an early stop codon within the coding region of the gene.
In some of any of the embodiments, the methods further include (c) introducing another genetically engineered antigen receptor, which in some aspects is a chimeric costimulatory receptor that specifically binds to another antigen, which in some aspects is a disease-specific antigen with respect to the same disease or condition being treated, and is capable of inducing a costimulatory signal to the cell, wherein steps (a), (b) and (c) are carried out simultaneously or sequentially in any order. Also provided are cells produced by the methods.
In some embodiments, the engineered cells comprise: (a) a first genetically engineered antigen receptor, which specifically binds to a first antigen (generally a disease-specific antigen) and is capable of inducing an activating signal to the cell; and (b) a second genetically engineered antigen receptor, which is a costimulatory receptor such as a chimeric costimulatory receptor that specifically binds to a second antigen (generally a disease-specific antigen, e.g., one that is specific to or associated with the same disease or condition) and is capable of inducing a costimulatory signal to the cell (such as one that is necessary for full activation of the cell or a particular effector function thereof following binding of the first receptor to its antigen).
In some embodiments, the provided methods include administration of two or more different engineered cells, e.g., in the same composition and/or in separate compositions, respectively containing the two or more engineered cells, each of which specifically recognizes or binds to the first and second, and optionally, third, and so forth, antigens. In some embodiments, the first and second different cells express the first and second engineered receptors, respectively.
In some embodiments, the disease-specific antigen may be of a type that is not itself necessarily expressed on or in (or specifically expressed on or in or associated with) the disease or condition, but an epitope of which is disease-specific and/or is expressed or overexpressed only (or comparatively more) on cells or tissues of the disease or condition.
In some such embodiments, the first and second antigens are distinct and, at least one is selected from the group consisting of human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2/neu, Wilms' tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, cyclin (D1), BCMA, BAFF-R, or TACI. The other of the first second antigen can be a different antigen from any of hTERT, survivin, MDM2, CYP1B, HER2/neu, WT1, livin, AFP, CEA, MUC16, MUC1, PSMA, p53, cyclin (D1), BCMA, BAFFR, or TACI, or can be another tumor antigen.
In some embodiments, the engineered cells comprise: (a) a first genetically engineered antigen receptor, which specifically binds to a first antigen and is capable of inducing an activating signal to the cell; and (b) a second genetically engineered antigen receptor, which is a costimulatory receptor such as a chimeric costimulatory receptor that specifically binds to a second antigen and is capable of inducing a costimulatory signal to the cell (such as one that is necessary for full activation of the cell or a particular effector function thereof following binding of the first receptor to its antigen). In some such embodiments, the first and second antigens are distinct and, individually, are selected from the group consisting of CD38, CS-1, and CD138. In some embodiments, they are selected from the group consisting of BCMA and any one or more of CD38, CS-1, and CD138 or any one or more of CD38, CS-1, and CD138, TACI, and BAFFR, and/or any one or more of TACI and BAFFR.
In some embodiments, the engineered cell comprises (a) a first genetically engineered antigen receptor, which specifically binds to a first antigen and is capable of inducing an activating signal to the cell; and (b) a second genetically engineered antigen receptor, which is a chimeric costimulatory receptor that specifically binds to a second antigen and is capable of inducing a costimulatory signal to the cell, wherein the first and second antigens are distinct and, individually, are selected from the group consisting of CD38 and CD138.
In some embodiments, the engineered immune cell comprises (a) a first genetically engineered antigen receptor that specifically binds to a first antigen and is capable of inducing an activating signal to the cell; and (b) a second genetically engineered antigen receptor which is a chimeric costimulatory receptor that specifically binds to a second antigen and is capable of inducing a costimulatory signal to the cell, wherein the first and second antigens are distinct and the first or the second antigen is CS-1.
In some embodiments, the second antigen is an antigen expressed in multiple myeloma. In some embodiments, the first antigen is an antigen expressed in multiple myeloma.
In some embodiments, the first genetically engineered antigen receptor comprises an ITAM-containing sequence, the first genetically engineered antigen receptor comprises an intracellular signaling domain of a CD3-zeta (CD3) chain, and/or the first genetically engineered antigen receptor does not comprise a signaling domain from a T cell costimulatory molecule, such as one having an intracellular signaling domain of a T cell costimulatory molecule, such one or more molecules selected from the group consisting of CD28 and 41BB.
In some aspects, (a) the first antigen is CD38 and the second antigen is CD138; (b)) the first antigen is CD38 and the second antigen is CS-1; (c) the first antigen is CD138 and the second antigen is CD38; (d) the first antigen is CD138 and the second antigen is CS-1; (e) the first antigen is CS-1 and the second antigen is CD38; or (f) the first antigen is CS-1 and the second antigen is CD138, or (g) the first antigen is CD38 and the second antigen is BCMA, (h) the first antigen is BCMA and the second antigen is CS-1; (c) the first antigen is CD138 and the second antigen is BCMA; (d) the first antigen is BCMA and the second antigen is TACI; (e) the first antigen is BCMA and the second antigen is BAFF-R or (f) the first antigen is BCMA and the second antigen is BAFF-R. In some cases, the cell further comprises a third genetically engineered antigen receptor recognizing a third antigen, such as an additional antigen of any of the above combinations.
In some aspects, the first genetically engineered antigen receptor contains an extracellular antigen recognition domain which specifically binds to the first target antigen at a dissociation constant (KD) of at least 10−8M, at least 10−7M, at least 10−6M, at least 10−5 M, 10−5 M, or 10−4 M. In some aspects, ligation of the first genetically engineered antigen receptor and ligation of the second genetically engineered antigen receptor induces a response in the cell, which response is not induced by ligation of either of the genetically engineered antigen receptors alone.
In some embodiments, the response is selected from the group consisting of proliferation, secretion of a cytokine, and cytotoxic activity.
In some embodiments, such as any of the embodiments in which a gene is disrupted, the gene that is disrupted or the antigen the expression of which is disrupted in the provided cells includes a gene encoding the first antigen or includes the first antigen, and/or includes a gene encoding the second antigen or includes the second antigen. In some aspects the disruption results in reduced expression of the first and/or second antigen in the engineered immune cell, such as by disruption as described herein. In some examples the disrupted gene encodes CD38, TACI, BAFF-R, or BCMA, and/or hTERT, survivin, MDM2, CYP1B, HER2/neu, WT1, livin, AFP, CEA, MUC16, MUC1, PSMA, p53, cyclin (D1) In some embodiments, the antigen is a tumor-associated form of any of such an antigen, such as one including a tumor-specific or associated epitope. The antigen may be a neoantigen. In some such aspects, the gene disrupted may be the corresponding non-tumor-associated antigen that shares identity with the neoantigen, e.g., is otherwise the same or similar but does not contain the tumor-specific epitope, variation or modification.
Also provided are compositions, including pharmaceutical compositions comprising the cells, and in some aspects, a carrier, such as a pharmaceutically acceptable carrier. The disease sand conditions include cancers and infectious diseases, such as hematological cancers, leukemias, lymphomas, and multiple myeloma, as well as solid tumors and other non-blood or non-hematological cancers. In some embodiments, the provided cells and compositions are useful to treat multiple types of cancers.
DETAILED DESCRIPTION
I. Compositions and Methods Providing Specificity and/or Efficacy in Adoptive Cell Therapy
Provided are cells for adoptive cell therapy, e.g., adoptive immunotherapy. The cells include immune cells such as T cells and NK cells, and generally express genetically engineered antigen receptors such as engineered TCRs and/or chimeric antigen receptors (CARs). Also provided are methods and uses of the cells, such as in adoptive therapy in the treatment of cancers including multiple myeloma (MM). Also provided are methods for engineering, preparing, and producing the cells, compositions containing the cells, and kits and devices containing and for using, producing and administering the cells. In some aspects, the provided embodiments provide improved selectivity, specificity, and/or efficacy of antigen-specific adoptive cell therapy, and/or expand the scope of diseases, conditions, and/or target antigens which may be targeted via adoptive cell therapy. In some aspects, they improve the exposure to the cells being administered.
The cells generally are engineered by introducing one or more genetically engineered nucleic acid or product thereof. Among such products are genetically engineered antigen receptors, including engineered T cell receptors (TCRs) and functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs), including activating, stimulatory, and costimulatory CARs, and combinations thereof.
Also among the cells are those in which certain genes and/or gene products have been disrupted, modified and/or repressed, such as via disruption that impairs or reduces expression. Such genes and gene products may include, or include those encoding, one or more antigen recognized or specifically bound by a chimeric antigen receptor (CAR) or other engineered receptor such as antigen receptor expressed by the cells, such as a disease-specific target antigen, disease-specific epitope thereof and/or one that is related thereto. In some embodiments, the gene or gene product disrupted or repressed is or is a gene encoding an antigen targeted by the genetically engineered antigen receptor, such as an activating, inhibitory, or costimulatory CAR, and generally an activating or costimulatory CAR. Thus, also provided are nucleic acids effecting repression of expression and/or disruption of endogenous genes, such as by gene editing. Also provided are cells with reduced expression of a gene or genes and methods for effecting the repression.
In some embodiments, the cells express an engineered receptor targeting (e.g., specifically binding to or recognizing) an antigen, such as a disease-specific target antigen corresponding to the disease or condition to be treated. In some such embodiments, the target antigen is not expressed, or may not be overexpressed, on or in the engineered cells. In some aspects, such a receptor, e.g., TCR or CAR, may further specifically bind to or recognize with, e.g., may cross-react with, one or more other antigens, which may be expressed or overexpressed on or in the engineered cells or activated cells of the same type. In some aspects, the other antigens are related to, e.g., contain sequence or structural similarity to or an epitope of, the disease-specific target antigen. Such related antigen may be a disease-specific antigen or one that is expressed on the disease or condition to be treated, or may be one that is not disease specific or is not expressed on or in such disease or condition.. Such cross-reactivity can in some aspects produce unwanted effects and/or reduce efficacy. For example, if an engineered cell expresses an antigen that is structurally similar to a target tumor antigen specifically bound by the receptor, the tumor antigen-specific CAR or TCR may recognize and bind to, e.g., specifically bind to, the similar antigen and induce unwanted killing or inhibition of the engineered cell itself. In some embodiments, such related antigen or expression thereof is disrupted in the engineered cell, addressing or reducing risk of such effects. In some embodiments, the engineered cell expresses the same antigen that is targeted by the CAR or TCR and/or may express at least the target antigen and one or more antigens related to the target antigen.
In some embodiments, for example, in the context of an activating or costimulatory antigen receptor, e.g., activating CAR or costimulatory CAR, the repression of a gene that encodes an antigen (such as a disease-specific target antigen and/or antigen related thereto, e.g., sharing sequence or structural similarity or epitope therewith) expressed in the engineered cells avoids or reduces the likelihood of the engineered cells themselves being targeted for killing or inhibition, thereby improving efficacy of, and/or persistence or expansion of cells in, the adoptive cell therapy. Thus, in some aspects, such disruption avoids or reduces the likelihood of targeting, by the engineered cells, of the engineered cells themselves.
For example, antigen-specific cells, such as antigen-specific T cells, targeting antigens also expressed by such cells, can induce fratricide killing of the cells. In some cases, fratricide killing of T cells is observed in cultures of antigen-specific T cells against survivin, hTERT, p53 and others, such as CS1, which are protein antigens expressed in activated T cells (Turksma et al. (2013) Journal of Translational Medicine, 11:152; Leisegang et al. (2010) J Clin. Invest., 120:3869-3877; Chen et al. (2007) Cancer Biol Ther., 6:1991-1996; Theoret et al. (2008) Hum Gene Ther., 19:1219-1232). Methods of detecting fratricide, such as self-killing, of the immune cells can include, for example, methods in which T cells genetically engineered with an antigen receptor (e.g. TCR or CAR or other antigen receptor) are cultured in vitro, e.g., for up to 2 weeks, and monitored over time using any of a variety of assays for cell proliferation, viability and/or cytotoxicity. In some cases, the cultured cells can be stained with 7-AAD or other staining or other reagent used to discriminate viability and cell death or apoptosis or activation of apoptotic pathways, such as by detection of caspase or activated caspase molecules. In some examples, cytotoxicity induced by fratricide killing of antigen-specific immune cells can be assessed using a cytotoxicity assay, such as an assay to assess chromium release. In some cases, the self-killing of the immune cells themselves expressing the genetically engineered antigen receptor can limit methods of using certain antigen-specific immune cells in adoptive cell therapy, since such cells can be eliminated by the fratricide killing ex vivo prior to administration and/or are upon administration in vivo. Self-killing of engineered cells within a population may be by killing of a single cell within such population of engineered cells by the same cell or by cells within the population killing one another.
Provided are cells and methods that overcome such problems. For example, in some embodiments, the expression of a gene encoding an antigen specifically bound by one or more of the genetically engineered antigen receptors is repressed in the cell. In some embodiments, the expression of a gene encoding an antigen that is related to an antigen targeted by one or more of the genetically engineered antigen receptors is repressed in the cell. By repressing an antigen as described herein in the immune cell, in some embodiments, the fratricide killing of the immune cells themselves is prevented or reduced. In some embodiments, the fratricide killing (e.g. as assessed by a proliferation, viability and/or cytotoxicity assay) is reduced by at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, such as compared to a cell or cell population in which the cells express the engineered antigen receptor but in which the gene or antigen targeted thereby or expression thereof has not been disrupted. Such features in some aspects confer improved specificity and/or efficacy in the context of adoptive cell therapy.
Among such antigens are those expressed in cancers of the immune system, such as leukemias, lymphomas, and/or myelomas, such as multiple myeloma, and/or expressed on T cells and/or NK cells, including activated T cells. Exemplary antigens are those expressed on activated or stimulated T cells or NK cells, and subsets thereof, particularly activated cells produced by stimulatory conditions used to promote the introduction of the nucleic acid encoding the CAR or other engineered receptor. For example, among such antigens are those not generally expressed on resting T cells but expression of which is induced upon T cell activation and/or that are expressed on activated T cells. In some embodiments, the gene encodes a universal tumor antigen, or a family member thereof, such as hTERT, survivin, MDM2, CYP1B, HER2/neu, WT1, livin, AFP, CEA, MUC16, MUC1, PSMA, p53 or cyclin (D1). For example, the gene encodes hTERT or survivin. In some aspects, the gene encodes BCMA, BAFFR, or TACI (Kimberley et al., (2008) J Cellular Physiology, 218:1; Pelekanou, et al., (2013) PloS One, 8:12; Wang et al., (2001) Nat Immunol, 2:7; Mariette, X, (2011) Sjögren's Syndrome; Salzer et al., (2005) Curr Opin Allergy Clin Immunol, 5:6) and/or encodes FcRH5. In some embodiments, the gene encodes the antigen CD33 or TIM-3; in some embodiments, it encodes the antigen CD26, CD30, CD53, CD92, CD100, CD148, CD150, CD200, CD261, CD262, or CD362.
In some embodiments, the repression is effected via disruption of the gene encoding the antigen, such as by deletion, e.g., deletion of an entire gene, exon, or region, and/or replacement with an exogenous sequence, and/or by mutation, e.g., frameshift or missense mutation, within the gene, typically within an exon of the gene. In some embodiments, the disruption results in a premature stop codon being incorporated into the gene, such that the antigen is not expressed or is not expressed in a form that is recognized by the antigen receptor. The disruption is generally carried out at the DNA level. The disruption generally is permanent, irreversible, or not transient. In other embodiments, transient or reversible repression strategies are used, such as gene knockdown using RNAi. In some embodiments, by disrupting or otherwise repressing expression of the antigen on the engineered cells, the methods and compositions provided here avoid or reduce the likelihood of killing of the engineered cells by the engineered cells themselves, thereby promoting efficacy.
In some embodiments, the cells include features for increased efficacy of the cells and methods are provided by the disruption of gene expression in the engineered cells. In some aspects, the gene disrupted encodes a checkpoint molecule, immunosuppressive molecule, such as a receptor that delivers an immunosuppressive signal to the cell, and/or any molecule which could reduce the robustness of the response of the engineered cell, for example, following administration in connection with immunotherapy.
In some embodiments, for example, in the context of an antigen that is recognized by an inhibitory CAR, the gene disruption prevents or reduces the likelihood of the inhibitory CAR expressed by the engineered cell of itself binding to a molecule also expressed by the engineered cells, thereby inducing a dampening effect on the signaling or targeted immune response by the engineered cells. Exemplary of such antigens are those expressed on normal or non-targeted or off-target cells (such that an inhibitory CAR molecule is included to prevent off-target effects), but is also expressed on the cell type used for genetic engineering, such as the T cell or NK cell. Exemplary antigens are MHC molecules, such as MHC-class I molecules, which can in some cases be downregulated in the context of immune evasion, cancer, or infection, but are generally expressed on nucleated cells. Other examples are any inhibitory CAR target that is also expressed on a T cell, NK cell, or other cell engineered for cell therapy.
In other embodiments, the gene or genes disrupted is a gene other than that encoding the antigen, such as a gene encoding an immunosuppressive molecule, e.g., checkpoint molecule or adenosine receptor, e.g., A2AR.
In some aspects, the disruption is carried out by gene editing, such as using a DNA binding protein or DNA-binding nucleic acid, which specifically binds to or hybridizes to the gene at a region targeted for disruption. In some aspects, the protein or nucleic acid is coupled to or complexed with a nuclease, such as in a chimeric or fusion protein. For example, in some embodiments, the disruption is effected using a fusion comprising a DNA-targeting protein and a nuclease, such as a Zinc Finger Nuclease (ZFN) or TAL-effector nuclease (TALEN), or an RNA-guided nuclease such as a clustered regularly interspersed short palindromic nucleic acid (CRISPR)-Cas system, such as CRISPR-Cas9 system, specific for the gene being disrupted.
In some embodiments, improved selectivity and specificity is achieved through strategies targeting multiple antigens. Such strategies generally involve multiple antigen-binding domains, which typically are present on distinct genetically engineered antigen receptors and specifically bind to distinct antigens. Thus, in some embodiments, the cells are engineered with the ability to bind more than one antigen. In some aspects, a plurality of genetically engineered antigen receptors are introduced into the cell, which specifically bind to different antigens, each expressed in or on the disease or condition to be targeted with the cells or tissues or cells thereof. Such features can in some aspects address or reduce the likelihood of off-target effects. For example, where a single antigen expressed in a disease or condition is also expressed on or in non-diseased or normal cells, such multi-targeting approaches can provide selectivity for desired cell types by requiring binding via multiple antigen receptors in order to activate the cell or induce a particular effector function.
In some embodiments, the cell expresses a first genetically engineered antigen receptor recognizing a first antigen and a second genetically engineered antigen receptor recognizing a second antigen. In some aspects, the second receptor is a costimulatory receptor recognizing the second antigen. In some aspects, both receptors are activating receptors. In some embodiments, additional receptors recognize third, or more antigens. In some aspects, each of the antigens is expressed in the context of a target cell, disease, or condition, such as in a neoplasia, cancer, malignancy, or infectious disease, for example, is a disease-specific target. In some aspects, one or more of the antigens is also expressed on cells or in tissues or environments which it is not desired to target with the cell therapy, such as in normal or non-diseased tissue. In some embodiments, the presence of multiple receptors and/or multiple antigen-recognition components avoids or reduces the likelihood of off-target effects on such tissues, cells, or environments. Thus, in some aspects, features of the provided cells and methods avoid or reduce the likelihood of inappropriate activation or response of cells directed at cells, tissues, or environments other than those being targeted by the cell therapy. For example, in some embodiments, they avoid or reduce the likelihood of off-target effects. In some embodiments, the ability of the cells or the compositions or methods provided herein to target two or more disease-specific targets avoids or reduces the risk of unwanted effects due to antigen or epitope loss, for example, by allowing activation or stimulation in the presence of either of two or more disease-specific antigens. In some such aspects, the two or more receptors targeting the two or more disease-specific antigen receptors may be expressed on the same cell, e.g., from the same or different construct and/or control of the same promoter, and/or may be expressed by different cells in the same composition, and/or may be present in two different compositions both administered to the same subject.
In some embodiments, at least one of the two or more antigens is a tumor-specific or associated target, a universal tumor antigen and/or a multiple myeloma-specific or associated target. In some aspects, one or more of the two or more antigens is selected from among a human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2/neu, Wilms' tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, cyclin (D1), BCMA, BAFFR, FcRH5, or TACI, and the other is another antigen expressed on a tumor or cancer, and in some cases is expressed on a particular type of tumor or cancer, and not on one or more certain other types of cancers. In some aspects, one or more of the antigens is a multiple myeloma-associated or multiple myeloma-specific antigen such as CD38 or CD138 or BCMA or CS-1 and/or BAFF-R, FcRH5, or TACI; in some aspects, such other antigen is expressed on one or more blood cancers or one or more solid tumor types. In some aspects, the target antigen and said another antigen are distinct and are selected, individually, from the group consisting of CD38 and CD138, CS-1, BCMA, BAFF-R, FcRH5 and/or TACI. In some such aspects, the cell further comprises an additional genetically engineered antigen receptor which recognizes an antigen expressed on a disease or condition to be treated and induces a stimulatory or activating signal, which is dampened by the first genetically engineered antigen receptor.
In some aspects, at least one of the genetically engineered antigen receptor is specific for a target antigen that is a universal tumor antigen, or a family member thereof, and/or is expressed on one or on more than one type of tumor. For example, among the cells are those targeting multiple antigens where at least one of the targeted antigens is hTERT, survivin, MDM2, CYP1B, HER2/neu, WT1, livin, AFP, CEA, MUC16, MUC1, PSMA, p53, cyclin (D1), and/or in some embodiments at least one of the target antigens is or comprises BCMA, BAFFR, FcRH5 and/or TACI. In some embodiments, the one or other antigens targeted by one or more other genetically engineered antigen receptors on the cells is one that is expressed on a tumor or cancer.
In some aspects, the disease or condition is multiple myeloma, the target cell is a multiple myeloma cell, and/or the target antigens are antigens expressed in multiple myeloma. Among the cells are those targeting multiple antigens, such multiple antigens expressed in a disease or condition such as multiple myeloma, such as combinations of CD38, CD138 CS-1, BCMA, BAFF-R, TACI, and/or FcRH5. Exemplary combinations of antigens to be targeted include two or more of CD38, CD138, and/or CS-1; two or more of BCMA, BAFF-R, TACI, and/or two or more of BCMA, FcRH5 and/or CS-1, a combination of BCMA and any one or more of CD38, CD138 CS-1, BAFF-R, TACI, and/or FcRH5; and/or a combination of BCMA and any one or more of CD38, CD138, CS-1 and/or FcRH5. In some aspects, the cells recognize two or more antigens expressed in multiple myeloma, one or more of which may also be expressed on normal or non-cancerous cells. In some embodiments, the antigens include two or more of CD38, CD138, and CS-1; and/or two or more of BCMA, CS-1, CD38, CD138 and/or FcRH5.
In some embodiments, the multiple antigen-recognition domains or multiple antigen receptors are designed or engineered such that a response is induced in the cell only upon specific binding or ligation of the plurality of receptors or antigen-recognition domains, and not upon ligation or binding to antigen by a single receptor or domain alone. In some aspects, the first receptor includes an activation domain, such as an ITAM-containing motif, such as an immunostimulatory domain of a CD3-zeta chain. In some such aspects, the first receptor exhibits low affinity and/or specific binding by the receptor does not induce a particular activity in the cell, such as cytotoxic activity and/or proliferation and/or cytokine production. In some aspects, the second receptor is a costimulatory receptor, which does not include such an activation domain but includes a costimulatory domain, e.g., which enhances the signal induced by ligation of the first receptor. In some aspects, the normal cells also express or can express the antigen recognized by the activating receptor.
In some embodiments, the cell includes multiple genetically engineered antigen receptors recognizing different antigens and also includes a gene disruption, for example, disruption in a gene encoding the antigen targeted by one or more of the antigen receptors. In some embodiments, the gene disruption is carried out prior to the introduction of the antigen receptor(s); in some embodiments, it is carried out simultaneously to introduction of the antigen receptor(s). In some aspects, the introduction of the antigen receptor(s) effects the gene disruption, such as by insertion into the disrupted gene locus by homologous recombination.
Also provided are methods, compounds, and compositions for producing the engineered cells. Provided are methods for cell isolation, genetic engineering and gene disruption. Provided are nucleic acids, such as constructs, e.g., viral vectors encoding the genetically engineered antigen receptors and/or encoding nucleic acids and/or proteins for targeted gene disruption, and methods for introducing such nucleic acids into the cells, such as by transduction. Also provided are compositions containing the engineered cells, and methods, kits, and devices for administering the cells and compositions to subjects, such as for adoptive cell therapy. In some aspects, the cells are isolated from a subject, engineered, and administered to the same subject. In other aspects, they are isolated from one subject, engineered, and administered to another subject.
A. Cells, Cell Preparation and Culture
In some embodiments, the cells, e.g., engineered cells, are eukaryotic cells, such as mammalian cells, e.g., human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). In some aspects, the cells are human cells. The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, as described herein, and re-introducing them into the same patient, before or after cryopreservation.
Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naïve T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
In some embodiments, one or more of the T cell populations is enriched for or depleted of cells that are positive for (marker+) or express high levels (markerhigh) of one or more particular markers, such as surface markers, or that are negative for (marker−) or express relatively low levels (markerlow) of one or more markers. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (such as non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (such as memory cells). In one embodiment, the cells (such as the CD8+ cells or the T cells, e.g., CD3+ cells) are enriched for (i.e., positively selected for) cells that are positive or expressing high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and/or CD62L and/or depleted of (e.g., negatively selected for) cells that are positive for or express high surface levels of CD45RA. In some embodiments, cells are enriched for or depleted of cells positive or expressing high surface levels of CD122, CD95, CD25, CD27, and/or IL7-Rα (CD127). In some examples, CD8+ T cells are enriched for cells positive for CD45RO (or negative for CD45RA) and for CD62L.
In some embodiments, a CD4+ T cell population and a CD8+ T cell sub-population, e.g., a sub-population enriched for central memory (TCM) cells.
In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
Cell Preparation
The cells and compositions containing the cells for engineering typically are isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated as one having a particular disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a mammal, such as a human, such as a subject in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig.
Cell Processing, Preparation, and Non-Affinity-Based Separation
In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.
In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++/Mg++ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.
In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
Separation Based on Affinity and/or Marker Profile
In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques.
For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (markerhigh) on the positively or negatively selected cells, respectively.
In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood.1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.
In embodiments, memory T cells are present in both CD62L+ and CD62L-subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+ and/or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.
In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.
CD4+ T helper cells are sorted into naïve, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L− and CD45RO−.
In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher© Humana Press Inc., Totowa, N.J.).
In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.
In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.
The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.
In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.
In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.
In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.
In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Number WO2009/072003, or US 20110003380 A1.
In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.
In some aspects, the separation and/or other steps is carried out using CliniMACS system (Miltenyi Biotic), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.
The CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.
In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al.(2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.
In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.
Cryopreservation
In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to −80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.
In some embodiments, the provided methods include cultivation, incubation, culture, and/or genetic engineering steps. For example, in some embodiments, provided are methods for incubating and/or engineering the depleted cell populations and culture-initiating compositions.
Thus, in some embodiments, the cell populations are incubated in a culture-initiating composition. The incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.
Incubation and Culture
In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a genetically engineered antigen receptor.
The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agents include 1L-2 and/or IL-15, for example, an IL-2 concentration of at least about 10 units/mL.
In some aspects, incubation is carried out in accordance with techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al.(2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, the T cells are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.
In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.
In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigen-specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.
In some aspects, the methods include assessing expression of one or more markers on the surface of the engineered cells or cells being engineered. In one embodiment, the methods include assessing surface expression of one or more target antigen (e.g., antigen recognized by the genetically engineered antigen receptor) sought to be targeted by the adoptive cell therapy, for example, by affinity-based detection methods such as by flow cytometry. In some aspects, where the method reveals surface expression of the antigen or other marker, the gene encoding the antigen or other marker is disrupted or expression otherwise repressed for example, using the methods described herein.
B. Genetically Engineered Antigen Receptors
In some embodiments, the cells comprise one or more nucleic acids introduced via genetic engineering that encode one or more antigen receptors, and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.
Among the genetically engineered products are genetically engineered antigen receptors. Among such antigen receptors are genetically engineered T cell receptors (TCRs) and components thereof, and functional non-TCR antigen receptors, such as chimeric antigen receptors (CAR), including chimeric activating receptors and chimeric costimulatory receptors. In some embodiments, the CAR contains an extracellular antigen-recognition domain that specifically binds to an antigen. In some embodiments, the antigen is a protein expressed on the surface of cells. In some embodiments, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which, like a TCR, is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule.
Exemplary antigen receptors, including CARs and recombinant TCRs, as well as methods for engineering and introducing the receptors into cells, include those described, for example, in international patent application publication numbers WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the genetically engineered antigen receptors include a CAR as described in U.S. Pat. No. 7,446,190, and those described in International Patent Application Publication No.: WO/2014055668 A1.
Chimeric Antigen Receptors (CARs)
In some embodiments, the engineered antigen receptors include chimeric antigen receptors (CARs), including activating or stimulatory CARs, costimulatory CARs (see WO2014/055668), and/or inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013). The CARs generally include an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some aspects via linkers and/or transmembrane domain(s). Such molecules typically mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone.
In some embodiments, CAR is constructed with a specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type to be targeted by adoptive therapy, e.g., a cancer marker, and/or an antigen intended to induce a dampening response, such as an antigen expressed on a normal or non-diseased cell type. Thus, the CAR typically includes in its extracellular portion one or more antigen binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable domains, and/or antibody molecules. In some embodiments, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).
The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab′)2 fragments, Fab′ fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
In some embodiments, the antigen-binding proteins, antibodies and antigen binding fragments thereof specifically recognize an antigen of a full-length antibody. In some embodiments, the heavy and light chains of an antibody can be full-length or can be an antigen-binding portion (a Fab, F(ab′)2, Fv or a single chain Fv fragment (scFv)). In other embodiments, the antibody heavy chain constant region is chosen from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly chosen from, e.g., IgG1, IgG2, IgG3, and IgG4, more particularly, IgG1 (e.g., human IgG1). In another embodiment, the antibody light chain constant region is chosen from, e.g., kappa or lambda, particularly kappa.
Among the provided antibodies are antibody fragments. An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; variable heavy chain (VH) regions, single-chain antibody molecules such as scFvs and single-domain VH single antibodies; and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody. In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds the antigen, such as a cancer marker or cell surface antigen of a cell or disease to be targeted, such as a tumor cell or a cancer cell, such as any of the target antigens described herein or known in the art.
Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some aspects, the antibody fragments are scFvs.
A “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
In some embodiments, the CAR contains an antibody or an antigen-binding fragment (e.g. scFv) that specifically recognizes an antigen, such as an intact antigen, expressed on the surface of a cell, such as a disease-specific antigen. In some embodiments, the CAR further recognizes or binds to, e.g., is cross-reactive with, one or more antigens that are related to, such as by way of sharing similarity or identity in sequence, epitope, or structure, the target antigen that the CAR was designed to recognize, such as the disease-specific antigen.
In some embodiments, the CAR contains a TCR-like antibody, such as an antibody or an antigen-binding fragment (e.g. scFv) that specifically recognizes an intracellular antigen, such as a tumor-associated antigen, presented on the cell surface as a MHC-peptide complex. In some embodiments, an antibody or antigen-binding portion thereof that recognizes an MHC-peptide complex can be expressed on cells as part of a recombinant receptor, such as an antigen receptor. Among the antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Generally, a CAR containing an antibody or antigen-binding fragment that exhibits TCR-like specificity directed against peptide-MHC complexes also may be referred to as a TCR-like CAR.
Reference to “Major histocompatibility complex” (MHC) refers to a protein, generally a glycoprotein, that contains a polymorphic peptide binding site or binding groove that can, in some cases, complex with peptide antigens of polypeptides, including peptide antigens processed by the cell machinery. In some cases, MHC molecules can be displayed or expressed on the cell surface, including as a complex with peptide, i.e. MHC-peptide complex, for presentation of an antigen in a conformation recognizable by an antigen receptor on T cells, such as a TCRs or TCR-like antibody. Generally, MHC class I molecules are heterodimers having a membrane spanning α chain, in some cases with three a domains, and a non-covalently associated (32 microglobulin. Generally, MHC class II molecules are composed of two transmembrane glycoproteins, α and β, both of which typically span the membrane. An MHC molecule can include an effective portion of an MHC that contains an antigen binding site or sites for binding a peptide and the sequences necessary for recognition by the appropriate antigen receptor. In some embodiments, MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where a MHC-peptide complex is recognized by T cells, such as generally CD8+ T cells, but in some cases CD4+ T cells. In some embodiments, MHC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are typically recognized by CD4+ T cells. Generally, MHC molecules are encoded by a group of linked loci, which are collectively termed H-2 in the mouse and human leukocyte antigen (HLA) in humans. Hence, typically human MHC can also be referred to as human leukocyte antigen (HLA).
The term “MHC-peptide complex” or “peptide-MHC complex” or variations thereof, refers to a complex or association of a peptide antigen and an MHC molecule, such as, generally, by non-covalent interactions of the peptide in the binding groove or cleft of the MHC molecule. In some embodiments, the MHC-peptide complex is present or displayed on the surface of cells. In some embodiments, the MHC-peptide complex can be specifically recognized by an antigen receptor, such as a TCR, TCR-like CAR or antigen-binding portions thereof.
The term “peptide antigen” or “peptide epitope” refers to a peptide of a polypeptide that can associate with an MHC molecule, such as for recognition by an antigen receptor. Generally, the peptide is derived from or based on a fragment of a longer biological molecule, such as a polypeptide or protein. In some embodiments, the peptide typically is about 8 to about 24 amino acids in length. In some embodiments, a peptide has a length of from or from about 9 to 22 amino acids for recognition in the MHC Class II complex. In some embodiments, a peptide has a length of from or from about 8 to 13 amino acids for recognition in the MHC Class I complex. In some embodiments, upon recognition of the peptide in the context of an MHC molecule, such as MHC-peptide complex, the antigen receptor, such as TCR or TCR-like CAR, produces or triggers an activation signal to the T cell that induces a T cell response, such as T cell proliferation, cytokine production, a cytotoxic T cell response or other response.
In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to a MHC-peptide complex, can be produced by immunizing a host with an effective amount of an immunogen containing a specific MHC-peptide complex. In some cases, the peptide of the MHC-peptide complex is an epitope of antigen capable of binding to the MHC, such as a tumor antigen, for example a universal tumor antigen, or a family member thereof, myeloma antigen or other antigen as described below. In some embodiments, an effective amount of the immunogen is then administered to a host for eliciting an immune response, wherein the immunogen retains a three-dimensional form thereof for a period of time sufficient to elicit an immune response against the three-dimensional presentation of the peptide in the binding groove of the MHC molecule. Serum collected from the host is then assayed to determine if desired antibodies that recognize a three-dimensional presentation of the peptide in the binding groove of the MHC molecule is being produced. In some embodiments, the produced antibodies can be assessed to confirm that the antibody can differentiate the MHC-peptide complex from the MHC molecule alone, the peptide of interest alone, and a complex of MHC and irrelevant peptide. The desired antibodies can then be isolated.
In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to an MHC-peptide complex can be produced by employing antibody library display methods, such as phage antibody libraries. In some embodiments, phage display libraries of mutant Fab, scFV or other antibody forms can be generated, for example, in which members of the library are mutated at one or more residues of a CDR or CDRs. Exemplary of such methods are known in the art (see e.g. US published application No. US20020150914, US2014/0294841; and Cohen C J. et al. (2003) J Mol. Recogn. 16:324-332).
In some aspects, the antigen-specific binding, or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR includes a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, e.g., CD8alpha, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, and CD 154. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
The CAR generally includes at least one intracellular signaling component or components. In some embodiments, the CAR includes an intracellular component of the TCR complex, such as a TCR CD3+ chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the antigen binding molecule is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the CAR further includes a portion of one or more additional molecules such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR includes a chimeric molecule containing signaling domains of CD3-zeta (CD3-ζ) or Fc receptor γ and signaling domains or transmembrane domains of CD8, CD4, CD25 and/or CD16.
In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling domain of the CAR activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the cell. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule. Such truncated portion in some aspects is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functional capability.
In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal. In some aspects, the cell comprises a first CAR which contains signaling domains to induce the primary signal and a second CAR which binds to a second antigen and contains the component for generating a costimulatory signal. For example, a first CAR can be an activating CAR and the second CAR can be a costimulatory CAR. In some aspects, both CARs must be ligated in order to induce a particular effector function in the cell, which can provide specificity and selectivity for the cell type being targeted.
T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.
In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD79a, and CD79b. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.
In some embodiments, the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR includes both the activating and costimulatory components; in other aspects, the activating domain is provided by one CAR whereas the costimulatory component is provided by another CAR recognizing another antigen.
In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta or other primary or activating intracellular domain.
In some embodiments, the CAR encompasses two or more costimulatory domain combined with an activation domain, e.g., primary activation domain, in the cytoplasmic portion. One example is a receptor including intracellular components of CD3-zeta, CD28, and 4-1BB.
In some embodiments, the CAR or other antigen receptor further includes a marker to confirm transduction or engineering of the cell to express the receptor, such as a truncated version of a cell surface receptor, such as truncated EGFR, EGFRt). In some aspects, the marker is under the control of the same promoter or regulatory element such as enhancer as the CAR or other receptor. In some aspects, where multi-targeting approaches are used, multiple markers are used for each different receptor.
In some cases, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR in some aspects is one that include multiple costimulatory domains of different costimulatory receptors.
In some aspects, the CAR or other antigen receptor is an inhibitory CAR (e.g. iCAR) and includes intracellular components that dampen or suppress a response, such as an immune response, such as an ITAM- and/or costimulatory-promoted response in the cell. Exemplary of such intracellular signaling components are those found on immune checkpoint molecules, including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptors, EP2/4 Adenosine receptors including A2AR. In some aspects, the engineered cell includes an inhibitory CAR including a signaling domain of or derived from such an inhibitory molecule, such that it serves to dampen the response of the cell, for example, that induced by an activating and/or costimulatory CAR. Such CARs are used, for example, to reduce the likelihood of off-target effects in the context in which the antigen recognized by the activating receptor, e.g, CAR, is also expressed or may also be expressed on the surface of normal cells. In some aspects, an inhibitory receptor, e.g., iCAR is introduced which recognizes a marker specific to the normal cell. In some aspects, where such an antigen or antigen related thereto is also expressed on the engineered cell, the antigen is the target of gene editing or disruption approaches provided herein. In some aspects, such disruption avoids dampening of the engineered cells' response induced by the engineered cells themselves.
TCRs
In some embodiments, the genetically engineered antigen receptors include recombinant T cell receptors (TCRs) and/or TCRs cloned from naturally occurring T cells and/or pairs of chains of TCRs cloned from naturally occurring T cells.
In general, TCRs contain a variable α and β chains (also known as TCRα and TCRβ, respectively) or a variable γ and δ chain (also known as TCRγ and TCRδ, respectively) or antigen-binding portion(s) thereof, and in general are capable of specifically binding to an antigen peptide bound to a MHC receptor. In some embodiments, the TCR is in the αβ form. Typically, TCRs that exist in αβ and γδ forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions. A TCR can be found on the surface of a cell or in soluble form. Generally, a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, a TCR also can contain a constant domain, a transmembrane domain and/or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997). For example, in some aspects, each chain of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end. In some embodiments, a TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. Unless otherwise stated, the term “TCR” should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full-length TCRs, including TCRs in the αβ form or γδ form.
Thus, for purposes herein, reference to a TCR includes any TCR or functional fragment, such as an antigen-binding portion of a TCR that binds to a specific antigenic peptide bound in an MHC molecule, i.e. MHC-peptide complex. An “antigen-binding portion” or antigen-binding fragment” of a TCR, which can be used interchangeably, refers to a molecule that contains a portion of the structural domains of a TCR, but that binds the antigen (e.g. MHC-peptide complex) to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable α chain and variable β chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex, such as generally where each chain contains three complementarity determining regions.
In some embodiments, the variable domains of the TCR chains associate to form loops, or complementarity determining regions (CDRs) analogous to immunoglobulins, which confer antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule and determine peptide specificity. Typically, like immunoglobulins, the CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the main CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the beta chain interacts with the C-terminal part of the peptide. CDR2 is thought to recognize the MHC molecule. In some embodiments, the variable region of the β-chain can contain a further hypervariability (HV4) region.
In some embodiments, the TCR chains contain a constant domain. For example, like immunoglobulins, the extracellular portion of TCR chains (e.g., α-chain, β-chain) can contain two immunoglobulin domains, a variable domain (e.g., Vα or Vβ; typically amino acids 1 to 116 based on Kabat numbering Kabat et al., “Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.) at the N-terminus, and one constant domain (e.g., α-chain constant domain or Cα, typically amino acids 117 to 259 based on Kabat, β-chain constant domain or Cβ, typically amino acids 117 to 295 based on Kabat) adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains, and two membrane-distal variable domains containing CDRs. The constant domain of the TCR domain contains short connecting sequences in which a cysteine residue forms a disulfide bond, making a link between the two chains. In some embodiments, a TCR may have an additional cysteine residue in each of the α and β chains such that the TCR contains two disulfide bonds in the constant domains.
In some embodiments, the TCR chains can contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chains contains a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules like CD3. For example, a TCR containing constant domains with a transmembrane region can anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex.
Generally, CD3 is a multi-protein complex that can possess three distinct chains (γ, δ, and ε) in mammals and the ζ-chain. For example, in mammals the complex can contain a CD3γ chain, a CD3δ chain, two CD3ε chains, and a homodimer of CD3ζ chains. The CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T cell receptor chains. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, whereas each CD3ζ chain has three. Generally, ITAMs are involved in the signaling capacity of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in propagating the signal from the TCR into the cell. The CD3- and ζ-chains, together with the TCR, form what is known as the T cell receptor complex.
In some embodiments, the TCR may be a heterodimer of two chains α and β (or optionally γ and δ) or it may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (α and β chains or γ and δ chains) that are linked, such as by a disulfide bond or disulfide bonds.
In some embodiments, a TCR for a target antigen (e.g., a cancer antigen) is identified and introduced into the cells. In some embodiments, a TCR for a target antigen also specifically binds to, e.g., is cross-reactive with, one or more peptide epitopes of one or more other antigens, such as those that are related to (e.g., by way of sharing sequence or structural similarity with) the target antigen. The cross-reactive antigen may have an epitope that is the same as or has one or more amino acid differences as compared to the target antigen, such as one, two, or three differences. In some embodiments, nucleic acid encoding the TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In some embodiments, the TCR is obtained from a biological source, such as from cells such as from a T cell (e.g. cytotoxic T cell), T-cell hybridomas or other publicly available source. In some embodiments, the T-cells can be obtained from in vivo isolated cells. In some embodiments, a such as a high-affinity T cell clone can be isolated from a patient, and the TCR isolated. In some embodiments, the T-cells can be a cultured T-cell hybridoma or clone. In some embodiments, the TCR clone for a target antigen has been generated in transgenic mice engineered with human immune system genes (e.g., the human leukocyte antigen system, or HLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al. (2009) Clin Cancer Res. 15:169-180 and Cohen et al. (2005) J Immunol. 175:5799-5808. In some embodiments, phage display is used to isolate TCRs against a target antigen (see, e.g., Varela-Rohena et al. (2008) Nat Med. 14:1390-1395 and Li (2005) Nat Biotechnol. 23:349-354. In some embodiments, the TCR or antigen-binding portion thereof can be synthetically generated from knowledge of the sequence of the TCR.
In some embodiments, after the T-cell clone is obtained, the TCR alpha and beta chains are isolated and cloned into a gene expression vector. In some embodiments, the TCR alpha and beta genes are linked via a picornavirus 2A ribosomal skip peptide so that both chains are coexpression. In some embodiments, genetic transfer of the TCR is accomplished via retroviral or lentiviral vectors, or via transposons (see, e.g., Baum et al. (2006) Molecular Therapy: The Journal of the American Society of Gene Therapy. 13:1050-1063; Frecha et al. (2010) Molecular Therapy: The Journal of the American Society of Gene Therapy. 18:1748-1757; an Hackett et al. (2010) Molecular Therapy: The Journal of the American Society of Gene Therapy. 18:674-683.
Antigens
Among the antigens targeted by the genetically engineered antigen receptors are those expressed in the context of (e.g., that are specifically expressed on or in, overexpressed on or in, or associated with) a disease, condition, or cell type to be targeted via the adoptive cell therapy. In some embodiments, such antigens are referred to as disease-specific antigens, disease-specific targets and/or disease-specific target antigens. In some embodiments, the disease-specific target antigen differs from a non-disease-specific target antigen by only an epitope or relatively small change, e.g., an epitope that is exposed or present only in the disease or condition. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and/or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas.
In some embodiments, the antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on the engineered cells. In some such embodiments, a multi-targeting and/or gene disruption approach as provided herein is used to improve specificity and/or efficacy.
In some embodiments, the antigen is a universal tumor antigen, or a family member thereof. The term “universal tumor antigen” refers to an immunogenic molecule, such as a protein, that is, generally, expressed at a higher level in tumor cells than in non-tumor cells and also is expressed in tumors of different origins. In some embodiments, the universal tumor antigen is expressed in more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or more of human cancers. In some embodiments, the universal tumor antigen is expressed in at least three, at least four, at least five, at least six, at least seven, at least eight or more different types of tumors. In some cases, the universal tumor antigen may be expressed in non-tumor cells, such as normal cells, but at lower levels than it is expressed in tumor cells. In some cases, the universal tumor antigen is not expressed at all in non-tumor cells, such as not expressed in normal cells. Exemplary universal tumor antigens include, for example, human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2/neu, Wilms' tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, cyclin (D1). Peptide epitopes of tumor antigens, including universal tumor antigens, are known in the art and, in some aspects, can be used to generate MHC-restricted antigen receptors, such as TCRs or TCR-like CARs (see e.g. published PCT application No. WO2011009173 or WO2012135854 and published U.S. application No. US20140065708).
In some aspects, the target antigen is or includes a disease-specific antigen such as a tumor antigen, which is not expressed naturally on or in the engineered cells and/or is not upregulated on or in activated T cells or activated cells of a type used to generate the engineered cells. In some such aspects, however, the receptor further binds to or recognizes, or has the potential to bind to or recognize (such as by cross-reactivity) another antigen, such as an antigen that is related to the target antigen, e.g., by way of sequence or structural similarity, and in some aspects that is expressed naturally on or in the engineered cells or activated types or forms thereof thereof and/or is upregulated on or in activated T cells or activated cells of the type being used. For example, in some such cases, if a T cell or activated T cell expresses or is suspected of expressing or upregulating an antigen that is related to a tumor antigen, an antibody or binding protein that binds to the tumor antigen but may also bind to and be cross-reactive to the antigen expressed in the T cell. In some aspects, the target antigen is or is a family member of BCMA, TACI, or BAFFR. For example, in some aspects, the target antigen is BCMA and the cells may express TACI and/or BAFFR and in some examples the receptor may cross-react therewith.
An exemplary description of one or more antigens that are related to or a family member of a target antigen includes antigens that exhibit structural and/or sequence similarity to a target antigen, e.g., by having the same or a similar epitope(s), for example in some cases, such that antibodies, receptors, engineered receptors, or other binding agents or ligands that bind to the target antigen also bind to (e.g. are cross-reactive to) the related antigen. For example, an agent, such as an engineered receptor such as an engineered antigen receptor, that specifically binds to and/or recognizes BCMA may in some aspects be cross-reactive to TACI, for example, such as an agent as described in WO 2002/066516, and/or BAFF-R. Likewise, agents that bind to and/or recognize TACI may be cross-reactive to BCMA or BAFF-R. Antigens that are related to or a family member of a target antigen may be evolutionarily related and/or related by structure and/or sequence. Antigens that are related to or a family member of a target antigen may exhibit a percent sequence identity to the target antigen by nucleic acid and/or amino acid sequence. Antigens that are related to or a family member of a target antigen may in some aspects exhibit at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the target antigen either by nucleic acid or amino acid sequence. In some aspects, they may exhibit 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity over a portion comprising an epitope, such as an epitope recognized by the receptor. In some aspects, such a portion may include at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, such as contiguous amino acids. In some embodiments, the related antigens may not exhibit a high degree of similarity in sequence over a contiguous stretch of amino acids but may share a similar or the same epitope within the respective folded proteins, such as an epitope comprising multiple contiguous portions. In some embodiments, the related antigens bind to, e.g., specifically bind to, the same ligand or receptor. In some embodiments, an antigen that is related to or a family member of a target antigen may exhibit a certain percentage sequence identity or structural similarity over only a portion of the full-length sequence or structure of the antigen, such as over a series of predicted epitopes. For example, a target antigen may comprise a number of peptide epitopes that are recognized by the engineered binding proteins described herein. In some embodiments, an antigen that is related to a target antigen may comprise epitopes that are structurally and/or sequentially similar to a relevant epitope of a target antigen. In some embodiments, these epitopes are peptides of about 8 to about 24 amino acids in length.
In some embodiments, the antigen, e.g., the target antigen, such as the disease-specific antigen and/or related antigen, is hTERT. hTERT is a tumor antigen that is widely expressed in various cancers. In some aspects, hTERT has or contains a sequence of amino acids set forth in SEQ ID NO:3 (GenBank Accession No. NP_937983.2) and encoded by a sequence of nucleotides set forth in SEQ ID NO:4 (GenBank Accession No. NM_198253.2). Generally, hTERT is a reverse transcriptase that is part of the human telomerase complex, which is a complex that can maintain the telomeric ends of linear chromosomes and, in some cases, protects chromosomes from degradation and end-to-end fusion. In some aspects, hTERT expression results in teleomere synthesis from an RNA template, and its expression can correlate with telomerase activity because it is the rate-limiting component of the complex. Generally, hTERT is not expressed in most human cells, including normal cells, but is expressed or upregulated in tumors and cancer cells. For example, more than 85% of human tumors express hTERT. Thus, in some aspects, hTERT can be a universal tumor antigen. HLA-restricted peptide epitopes of hTERT are known (see e.g. U.S. Pat. No. 7,718,777, and published PCT application Nos. WO2000025813 and WO2013135553), and, in some aspects, such can be used to generate or identify antigen receptors against hTERT. Non-limiting examples of hTERT peptide epitopes include any set forth in any of SEQ ID NOS: 7-19. Antigen receptors against hTERT, such as TCR or TCR-like antibodies, have been generated and are known in the art (see e.g., U.S. Pat. No. 7,718,777 and published U.S. patent applications US20090226474 and US20030223994; see also Ugel et al. (2010) Blood, 115:1374-1384).
In some embodiments, the antigen, e.g., the target antigen, such as the disease-specific antigen and/or related antigen, is survivin (also called baculoviral inhibitor of apoptosis repeat-containing 5, BRCS). In some aspects, survivin has or contains a sequence of amino acids set forth in SEQ ID NO:5 (GenBank Accession No. NP_001159) and encoded by a sequence of nucleotides set forth in SEQ ID NO:6 (GenBank Accession No. NM_001168.2). In some cases, other transcript isoforms can exist, such as that have or contain an amino acid or nucleotide sequence referenced at GenBank Accession No. NP_001012270 or NM_001012270, respectively, or NP_001012271.1 or NM_001012271.1, respectively. Generally, survivin is a member of the family of inhibitors of apoptosis proteins (IAPs). In some cases, survivin is upregulated in many types of cancers, such as lung, colon, breast, pancreas, prostate, melanoma and others, but it is not generally expressed or upregulated in normal cells or tissues. In some aspects, the expression of survivin in cancer may be related to a general role of apoptosis inhibition in tumor progression. Thus, in some aspects, survivin can be a universal tumor antigen. HLA-restricted peptide epitopes of survivn are known (see e.g. U.S. Pat. No. 7,892,559 and published U.S. patent application No. US20090004214), and, in some cases, such peptide epitopes can be used to generate or identify antigen receptors against survivin, including TCRs or TCR-like CARs. Non-limiting examples of survivin peptide epitopes include any set forth in any of SEQ ID NOS: 20-30. Antigen receptors against survivin, such as TCR or TCR-like antibodies, have been generated and are known in the art (see e.g., published PCT application No. WO2010075417).
In some aspects, the antigen, e.g., the target antigen, such as the disease-specific antigen and/or related antigen, is expressed on multiple myeloma, such as CD38, CD138, and/or CS-1 and/or such as BCMA, BAFF-R, TACI, and/or FcRH5. Other exemplary multiple myeloma antigens include CD56, TIM-3, CD33, CD123, and/or CD44. Antibodies or antigen-binding fragments directed against such antigens are known and include, for example, those described in U.S. Pat. Nos. 8,153,765; 8,603,477, 8,008,450; U.S. published application No. US20120189622 or US20100260748A1; and/or published international PCT application Nos. WO2006099875, WO2009080829 or WO2012092612 or WO2014210064A1. In some embodiments, such antibodies or antigen-binding fragments thereof (e.g. scFv) can be used to generate a CAR.
In some embodiments, the antigen, e.g., the target antigen, such as the disease-specific antigen and/or related antigen, is a CD38 antigen that is a human CD38. In some aspects, the CD38 has or contains an amino acid sequence referenced at GenBank Accession No.: BAA18966, e.g., at BAA18966.1, or that is referenced at GI:1911103. In some aspects, it has or contains the following sequence: mancefspvs gdkpccrlsr raq1clgvsi lvlilvvvla vvvprwrqqw sgpgttkrfp etvlarcvky teihpemrhv dcqsvwdafk gafiskhpcn iteedyqplm klgtqtvpcn killwsrikd lahqftqvqr dmftledtll gyladdltwc gefntskiny qscpdwrkdc snnpvsvfwk tvsrrfaeaa cdvvhvmlng srskifdkns tfgsvevhnl qpekvqtlea wvihggreds rdlcqdptik elesiiskrn iqfsckniyr pdkflqcvkn pedssctsei (SEQ ID NO:1). In some aspects, the CD38 antigen can be encoded by the sequence of nucleotides set forth in SEQ ID NO:2. In some embodiments, the portion of the antigen to which the antigen-binding domain specifically binds typically is within the extracellular region of the antigen. For example, with reference to SEQ ID NO:1, the extracellular region corresponds to amino acid residues 43-300 of SEQ ID NO:1, In some embodiments, the antigen may be one that is expressed or upregulated on cancer or tumor cells, but that also may be expressed in an immune cell, such as a resting or activated T cell. For example, in some cases, expression of hTERT, survivin and other universal tumor antigens are reported to be present in lymphocytes, including activated T lymphocytes (see e.g., Weng et al. (1996) J Exp. Med., 183:2471-2479; Hathcock et al. (1998) J Immunol., 160:5702-5706; Liu et al. (1999) Proc. Natl Acad Sci., 96:5147-5152; Turksma et al. (2013) Journal of Translational Medicine, 11:152). Likewise, in some cases, CD38 and other tumor antigens also can be expressed in immune cells, such as T cells, such as upregulated in activated T cells.
In some aspects, the antigen may be expressed (or upregulated) only in activated T cells and not in resting T cells or not in a certain percentage of resting cells. For example, in some aspects, the target antigen or the related antigen, such as the tumor-specific antigen, is a BCMA and/or a BCMA family member, such as TACI or BAFF-R, which in some aspects is (or a family member thereof is) or is at risk of being expressed not only in B cells or in other tumor cells but also in activated T cells. See, e.g., Wang et al., (2001) Nat Immunol, 2:7; Mariette, X, (2011) Sjögren's Syndrome; Salzer et al., (2005) Curr Opin Allergy Clin Immunol, 5:6. In some aspects of such embodiments and/or of other embodiments herein, by further providing for the disruption of a gene encoding the target antigen(s) and/or one or more antigen related thereto, the provided embodiments permit a multi-targeting strategy in which multiple receptors are targeted even if potentially expressed on the engineered cells, and/or permit the use of an engineered receptor that not only binds to the target antigen but also cross-reacts with the one or more related antigens such as the one or more related family members, which may also be expressed on the disease or condition.
For example, in some aspects, CD38 is a known T cell activation marker. In some embodiments as provided herein, an immune cell, such as a T cell, can be engineered to repress or disrupt the gene encoding the antigen in the immune cell so that the expressed genetically engineered antigen receptor does not specifically bind the antigen in the context of its expression on the immune cell itself. Thus, in some aspects, this may avoid off-target effects, such as binding of the engineered immune cells to themselves, which may reduce the efficacy of the engineered in the immune cells, for example, in connection with adoptive cell therapy.
In some embodiments, the antigen, e.g., the target antigen, such as the disease-specific antigen and/or related antigen, is a B cell maturation antigen (BCMA; also called tumor necrosis factor receptor superfamily 17, TNFRSF17) or antigen belonging to the same family thereof or binding to the same ligand. In some aspects, the BCMA has or contains the sequence of amino acids set forth in SEQ ID NO:31 and/or is encoded by the sequence of nucleotides set forth in SEQ ID NO:32, or is a splice variant or isoform, allelic variant or other variant thereof, such as any expressed by an immune cell (e.g. T cell, such as activated T cell). In some embodiments, an antibody or antigen-binding fragment thereof (e.g. scFv) against such antigen can be used to generate a CAR. In some aspects, the antigen such as the BCMA or BCMA family member is expressed in cancers other than multiple myeloma, such as glioma. See Kimberley et al., (2008) J Cellular Physiology, 218:1; Pelekanou, et al., (2013) PloS One, 8:12.
In some embodiments, the antigen, e.g., the target antigen, such as the disease-specific antigen and/or related antigen, is a Transmembrane activator and CAML interactor (TACI; also called tumor necrosis factor receptor superfamily member 13B, TNFRSF13B). In some aspects, the TACI has or contains the sequence of amino acids set forth in SEQ ID NO:33 and/or is encoded by the sequence of nucleotides set forth in SEQ ID NO:34, or is a splice variant or isoform, allelic variant or other variant thereof, such as any expressed by an immune cell (e.g. T cell, such as activated T cell). In some embodiments, an antibody or antigen-binding fragment thereof (e.g. scFv) against such antigen can be used to generate a CAR.
In some embodiments, the antigen, e.g., the target antigen, such as the disease-specific antigen and/or related antigen, is a B-cell-activating factor receptor (BAFF receptor, also called tumor necrosis factor receptor superfamily member 13C, TNFRSF13C). In some aspects, the BAFF receptor has or contains a sequence of amino acids set forth in SEQ ID NO:35 and/or is encoded by the sequence of nucleotides set forth in SEQ ID NO:36, or is a splice variant or isoform, allelic variant or other variant thereof, such as any expressed by an immune cell (e.g. T cell, such as activated T cell). In some embodiments, an antibody or antigen-binding fragment thereof (e.g. scFv) against such antigen can be used to generate a CAR.
In some embodiments, the antigen is Fc receptor homolog 5 (FcRH5, also called FcR-like protein 5, FcRL5). In some aspects, FcRH5 has or contains a sequence of amino acids set forth in SEQ ID NO:55 and/or is encoded by the sequence of nucleotides set forth in SEQ ID NO:56, or is a splice variant or isoform, allelic variant or other variant thereof, such as any expressed by an immune cell (e.g. T cell, such as activated T cell). In some embodiments, an antibody or antigen-binding fragment thereof (e.g. scFv) against such antigen can be used to generate a CAR. In some embodiments, FcRH5 and/or a related antigen such as a homolog or family member, such as one sharing one or more domains, similarity in sequence or structure and/or epitopes therewith, such as one that is expressed or may be expressed on the cells or activated forms thereof, such as FcRH3, e.g., which may be expressed in non-disease cells such as engineered cells or activated forms thereof such as T cells or NK Cells. In some embodiments, the target antigen or related antigen and/or the antigen to be deleted is FcRH3. See Polson A G et al., Int. Immunol. 2006 Sep. 18(9): 1368-73.
In some embodiments, such as in the case of an inhibitory CAR, the target is an off-target marker, such as an antigen not expressed on the diseased cell or cell to be targeted, but that is expressed on a normal or non-diseased cell which also expresses a disease-specific target being targeted by an activating or stimulatory receptor in the same engineered cell. Exemplary such antigens are MHC molecules, such as MHC class I molecules, for example, in connection with treating diseases or conditions in which such molecules become downregulated but remain expressed in non-targeted cells. In some embodiments, the engineered immune cells can contain an antigen that targets one or more other antigens. In some embodiments, the one or more other antigens is a tumor antigen or cancer marker. Other antigen targeted by antigen receptors on the provided immune cells can, in some embodiments, include orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, fetal acethycholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1), a cyclin, such as cyclin A1 (CCNA1), and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens.
In some embodiments, the engineered immune cells contain or are suspected of being at risk for containing and/or upregulating (such as upon activation or stimulation) an antigen that is related to any target antigen described herein such that the engineered receptor (CAR or TCR, for example) is cross-reactive with the engineered immune cells and the target antigen.
In some embodiments, the receptor, e.g., the CAR binds a pathogen-specific antigen. In some embodiments, the CAR is specific for viral antigens (such as HIV, HCV, HBV, etc.), bacterial antigens, and/or parasitic antigens. In some embodiments, the receptor, e.g., the CAR, is specific for an inflammatory marker.
Multi-Targeting
In some embodiments, the cells and methods include multi-targeting strategies, such as in some aspects, expression of two or more genetically engineered receptors on the cell, each recognizing a different antigen and in some aspects, each including a different intracellular signaling component. The receptors may be encoded by the same or a different vector, under the same or a different promoter, and be associated with the expression of one or more of the same or different expression markers. In some aspects, the multi-targeting approaches encompass the administration of multiple cells each expressing different such engineered receptors, such as different receptors recognizing different antigens. In some aspects, the cells are in the same composition; in other aspects, they are in different compositions, which may be administered at the same time or sequentially. In some embodiments, a composition administered comprises cells expressing one of the two or more receptors and other cells expressing another one of (but not the first one of) the two or more receptors, and other cells expressing both receptors.
Certain multi-targeting strategies for use with embodiments provided herein are described, for example, in International Patent Application, Publication No.: WO 2014055668 A1 (describing combinations of activating and costimulatory CARs, e.g., targeting two different antigens present individually on off-target, e.g., normal cells, but present together only on cells of the disease or condition to be treated) and Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013) (describing cells expressing an activating and an inhibitory CAR, such as those in which the activating CAR binds to one antigen expressed on both normal or non-diseased cells and cells of the disease or condition to be treated, and the inhibitory CAR binds to another antigen expressed only on the normal cells or cells which it is not desired to treat).
In some embodiments, the multi-targeting strategy is employed in a case where an antigen associated with a particular disease or condition is expressed on a non-diseased cell and/or is expressed on the engineered cell itself, either transiently (e.g., upon stimulation in association with genetic engineering) or permanently. In such cases, by requiring ligation of two separate and individually specific antigen receptors, specificity, selectivity, and/or efficacy may be improved.
For example, in some embodiments, the cells include a receptor expressing a first genetically engineered antigen receptor (e.g., CAR or TCR) which is capable of inducing an activating signal to the cell, generally upon specific binding to the antigen recognized by the first receptor, e.g., the first antigen. In some embodiments, the cell further includes a second genetically engineered antigen receptor (e.g., CAR or TCR), e.g., a chimeric costimulatory receptor, which is capable of inducing a costimulatory signal to the immune cell, generally upon specific binding to a second antigen recognized by the second receptor. In some aspects, the first receptor includes an intracellular signaling component containing ITAM or ITAM-like motifs. In some aspects, the second receptor includes intracellular signaling domains of costimulatory receptors such as CD28, CD137 (4-1 BB), OX40, and/or ICOS.
In some embodiments, the activation induced by the first receptor involves a signal transduction or change in protein expression in the cell resulting in initiation of an immune response, such as ITAM phosphorylation and/or initiation of ITAM-mediated signal transduction cascade, formation of an immunological synapse and/or clustering of molecules near the bound receptor (e.g. CD4 or CD8, etc.), activation of one or more transcription factors, such as NF-κB and/or AP-1, and/or induction of gene expression of factors such as cytokines, proliferation, and/or survival.
In some embodiments, the costimulatory signal induced by the second receptor, in combination with the signal induced by the first receptor, is one that results in an immune response, such as a robust and sustained immune response, such as increased gene expression, secretion of cytokines and other factors, and T cell mediated effector functions such as cell killing.
In some embodiments, neither ligation of the first receptor alone nor ligation of the second receptor alone induces a robust immune response. In some aspects, if only one receptor is ligated, the cell becomes tolerized or unresponsive to antigen, or inhibited, and/or is not induced to proliferate or secrete factors or carry out effector functions. In some such embodiments, however, when the plurality of receptors are ligated, such as upon encounter of a cell expressing the first and second antigens, a desired response is achieved, such as full immune activation or stimulation, e.g., as indicated by secretion of one or more cytokine, proliferation, persistence, and/or carrying out an immune effector function such as cytotoxic killing of a target cell.
In some embodiments, the plurality of antigens, e.g., the first and second antigens, are expressed on the cell, tissue, or disease or condition being targeted, such as on the cancer cell. In some aspects, the cell, tissue, disease or condition is multiple myeloma or a multiple myeloma cell. One or more of the plurality of antigens generally also is expressed on a cell which it is not desired to target with the cell therapy, such as a normal or non-diseased cell or tissue, and/or the engineered cells themselves. In such embodiments, by requiring ligation of multiple receptors to achieve a response of the cell, specificity and/or efficacy is achieved.
In some embodiments, among the combinations of antigens for multi-targeting strategies includes those in which at least one of the antigens is a universal tumor antigen, or a family member thereof, such as hTERT, survivin, MDM2, CYP1B, HER2/neu, WT1, livin, AFP, CEA, MUC16, MUC1, PSMA, p53, cyclin (D1), each individually in combination with a second antigen that also targets an antigen expressed on a tumor. In some embodiments, the universal tumor antigen and the second antigen target an antigen on the same tumor type. In some embodiments, the second antigen may also be another different universal tumor antigen or may be a tumor antigen specific to the tumor type. In some embodiments, the second antigen may be a tumor antigen, such as ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, fetal acethycholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1), a cyclin, such as cyclin A1 (CCNA1), and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens, and in some aspects, neoepitopes or neoantigens thereof.
In some embodiments, among the combinations of antigens for multi-targeting strategies include those expressed on multiple myeloma, such as CD38 (cyclic ADP ribose hydrolase), CD138 (syndecan-1, syndecan, SYN-1), and CS-1 (CS1, CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), each individually in combination with a second antigen that is also expressed in multiple myeloma. In some embodiments, the plurality of antigens, such as those recognized by activating and costimulatory engineered receptors, respectively (or those targeted by other multi-targeting approaches such as those using two different receptors each having a costimulatory and primary or activating signaling ability, on the same or different cells), are CD38 and CD138, CD38 and CS-1, CD138 and CD38, CD138 and CS-1, CS-1 and CD38, and/or CS-1 and CD138. In some embodiments, the plurality of antigens, such as those recognized by activating and costimulatory engineered receptors, respectively, are BCMA and TACI, BCMA and BAFFR, BCMA and FcRH5, BCMA and CS-1, BCMA and CD38, BCMA and CD138, and/or any combination thereof. Additional multiple-myeloma-specific antigens include CD20, CD40, CD56, CD74, CD200, EGFR, and β2-Microglobulin, HM1.24, IGF-1R, IL-6R, TRAIL-R1, and the activin receptor type IIA (ActRIIA). See Benson and Byrd, Journal of Clinical Oncology, Jun. 1, 2012, 30(16): 2013-15; Tao and Anderson, Bone Marrow Research, Volume 2011 (2011), Article ID 924058, 14 pages; Chu et al., Leukemia (26 September 2013) 2014 April; 28(4):917-27; Garfall et al., Discov Med. 2014 January; 17(91):37-46. In some embodiments, the antigens include those present on lymphoid tumors, myeloma, AIDS-associated lymphoma, and/or post-transplant lymphoproliferations, such as CD38.
In some embodiments, one or more of the multiple antigen receptors recognizes or binds to its antigen with relatively low affinity. In some aspects, the receptor binds to its antigen with a dissociation constant (KD) of at least at or about 10−8M, at least at or about 10−7 M, at least at or about 10−6M, at least at or about 10−5 M, and/or at least at or about 10−4 M. In some such embodiments, a relatively lower affinity of the antigen receptor for the antigen helps ensure that a full response is not achieved by ligation of only one of the multiple genetically engineered antigen receptor. In some embodiments, the CD38-targeting receptor includes an antigen binding domain as disclosed, for example, in U.S. Patent Application, Publication No.: US20120189622 A1, such as antigen binding portion(s) of the antibody designated as OKT10 and of other antibodies described in Table 1 of the application, and of antibodies binding to similar or the same epitopes.
In some embodiments, the two receptors induce, respectively, an activating and an inhibitory signal to the cell, such that binding by one of the receptor to its antigen activates the cell or induces a response, but binding by the second inhibitory receptor to its antigen induces a signal that suppresses or dampens that response. Examples are combinations of activating CARs and inhibitory CARs or iCARs. Such a strategy may be used, for example, in which the activating CAR binds an antigen expressed in a disease or condition but which is also expressed on normal cells, and the inhibitory receptor binds to a separate antigen which is expressed on the normal cells but not cells of the disease or condition.
Vectors and Methods for Genetic Engineering
Also provided are methods, nucleic acids, compositions, and kits, for producing the genetically engineered cells. In some aspects, the genetic engineering involves introduction of a nucleic acid encoding the genetically engineered component or other component for introduction into the cell, such as a component encoding a gene-disruption protein or nucleic acid.
In some embodiments, gene transfer is accomplished by first stimulating cell growth, e.g., T cell growth, proliferation, and/or activation, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.
In some contexts, overexpression of a stimulatory factor (for example, a lymphokine or a cytokine) may be toxic to a subject. Thus, in some contexts, the engineered cells include gene segments that cause the cells to be susceptible to negative selection in vivo, such as upon administration in adoptive immunotherapy. For example in some aspects, the cells are engineered so that they can be eliminated as a result of a change in the in vivo condition of the patient to which they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell II:223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT)gene, the cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).
In some aspects, the cells further are engineered to promote expression of cytokines or other factors. Various methods for the introduction of genetically engineered components, e.g., antigen receptors, e.g., CARs, are well known and may be used with the provided methods and compositions. Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.
In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 April 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November; 29(11): 550-557.
In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
Methods of lentiviral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.
In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).
In other embodiments, the cells, e.g., T cells, are not engineered to express recombinant receptors, but rather include naturally occurring antigen receptors specific for desired antigens, such as tumor-infiltrating lymphocytes and/or T cells cultured in vitro or ex vivo, e.g., during the incubation step(s), to promote expansion of cells having particular antigen specificity. For example, in some embodiments, the cells are produced for adoptive cell therapy by isolation of tumor-specific T cells, e.g. autologous tumor infiltrating lymphocytes (TIL). The direct targeting of human tumors using autologous tumor infiltrating lymphocytes can in some cases mediate tumor regression (see Rosenberg S A, et al.(1988) N Engl J Med. 319:1676-1680). In some embodiments, lymphocytes are extracted from resected tumors. In some embodiments, such lymphocytes are expanded in vitro. In some embodiments, such lymphocytes are cultured with lymphokines (e.g., IL-2). In some embodiments, such lymphocytes mediate specific lysis of autologous tumor cells but not allogeneic tumor or autologous normal cells.
Other approaches and vectors for transfer of the genetically engineered nucleic acids encoding the genetically engineered products are those described, e.g., in international patent application, Publication No.: WO2014055668, and U.S. Pat. No. 7,446,190.
Among additional nucleic acids, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.
C. Repression of Gene Expression, Activity, or Function
Among the provided immune cells are immune cells in which the expression, activity, and/or function of one or more genes encoding an antigen of a genetically engineered antigen receptor is repressed in the cell. In some cases, a particular antigen of interest may be one that is expressed in a disease or condition, such as a tumor or cancer, but that may also be expressed by the immune cell, such as a T cell or activated T cell. In some aspects, expression of the antigen by the immune cell may be undesirable, such as in the context of engineering the immune cell with a genetically engineered antigen receptor for the antigen, for example, in the context of adoptive immunotherapy. Thus, in some embodiments, provided are immune cells, such as T cells, that contains a genetically engineered antigen receptor specific for a target antigen in a disease or condition, for example a tumor or cancer, and that also contains a disruption or repression of an endogenous gene in the cell encoding the antigen.
Also provided are methods for effecting such gene repression. In some embodiments, the gene repression is carried out by effecting a disruption in the gene, such as a knock-out, insertion, mis sense or frameshift mutation, such as a biallelic frameshift mutation, deletion of all or part of the gene, e.g., one or more exon or portion thereof, and/or knock-in. Such disruptions in some embodiments are effected by sequence-specific or targeted nucleases, including DNA-binding targeted nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases such as a CRISPR-associated nuclease (Cas), specifically designed to be targeted to the sequence of a gene or a portion thereof.
In some embodiments, the methods of repressing or disrupting a gene in the cell can be performed prior, simultaneous or subsequent to the methods of introducing a nucleic acid molecule encoding a genetically engineered antigen receptor into the cells. For example, in some cases, the methods of repressing or disrupting a gene in the cell is performed prior to introducing a nucleic acid molecule encoding a genetically engineered antigen receptor into the cell.
Repressed/Disrupted Genes
In some embodiments, the gene repressed and/or disrupted encodes a product capable of having or contributing to an inhibitory effect on the engineered cell, such as inhibiting an immune response by the cell. For example, in some cases, the gene repressed and/or disrupted encodes an antigen recognized by a genetically engineered antigen receptor that is or is planned to be expressed on the immune cells, such as T cells.
In some embodiments, the gene encodes a target antigen specifically bound by an antigen receptor on the engineered cell, such as a genetically engineered antigen receptor, e.g., CAR. Thus, in some aspects, the gene being repressed encodes the target antigen of a CAR or other antigen receptor. Such aspects typically arise in the case of a target antigen expressed in a particular disease or condition but that is expressed on the cell type being engineered, or a subset thereof, such as upon activation of the engineered cell. Exemplary antigens are those expressed on activated or stimulated T cells or NK cells, and subsets thereof, particularly activated cells produced by stimulatory conditions used to promote the introduction of the nucleic acid encoding the CAR or other engineered receptor. In some embodiments, by disrupting or otherwise repressing expression of the antigen on the engineered cells, the methods and compositions provided here avoid or reduce the likelihood of killing of the engineered cells by the engineered cells themselves, thereby promoting efficacy.
In some embodiments, the gene encodes an antigen that is an activating and/or costimulatory CAR target. In some embodiments, the gene encodes an antigen that is a target in a multi-targeting strategy employing two or more genetically engineered antigen receptors, where at least one of the receptors is specific for an antigen that may be or is expressed on the immune cell, such as T cell or activated T cell.
In some embodiments, the gene disrupted or the expression or function of which is otherwise repressed is any that encodes a universal tumor antigen, or a family member thereof, and particularly a universal tumor antigen, or a family member thereof, that is expressed or upregulated on the immune cell, such as on a T cell or an activated T cells. In some embodiments, the gene that is disrupted or repressed is one that encodes hTERT or survivin. In some cases, other exemplary genes that can be disrupted or repressed include those encoding the antigens MDM2, CYP1B, HER2/neu, WT1, livin, AFP, CEA, MUC16, MUC1, PSMA, p53, cyclin (D1), BCMA, BAFFR, or TACI. In some embodiments, a targeted nuclease is targeted to a gene encoding hTERT, survivin, p53 or other universal tumor antigen, or a family member thereof. In some aspects, the targeting disrupts the gene, e.g., decreases or abolishes the expression of hTERT, survivin, p53 or other targeted universal tumor antigen, or a family member thereof, in the cell.
For example, in some embodiments, provided are immune cells, such as T cells, that contain a genetically engineered antigen receptor that specifically binds an antigen from among hTERT, survivin MDM2, CYP1B, HER2/neu, WT1, livin, AFP, CEA, MUC16, MUC1, PSMA, p53, cyclin (D1), BCMA, BAFFR, or TACI and that contains disruption or expression of the corresponding endogenous gene in the immune cells. In some aspects, provided are immune cells, such as T cells, that contain a genetically engineered antigen receptor that specifically binds to an hTERT antigen and that contains disruption or repression of the corresponding endogenous hTERT gene in the cell. In other aspects, provided are immune cells, such as T cells, that contain a genetically engineered antigen receptor that specifically binds to a survivin gene and that contains disruption or repression of the corresponding endogenous survivin gene in the cell.
In particular embodiments, the gene disrupted or the expression or function of which is otherwise repressed encodes CD38, such as in a cell expressing an anti-CD38 engineered receptor, such as an anti-CD38 CAR. Other exemplary genes include those encoding antigens CD33 and TIM-3. See Mardiros et al., Blood 122 (18) (Oct. 31, 2013). Other exemplary genes include those encoding the antigens CD26, CD30, CD53, CD92, CD100, CD148, CD150, CD200, CD261, CD262, and CD362. In some embodiments, a targeted nuclease is targeted to a CD38 gene on the engineered cell. In some aspects, the targeting disrupts the CD38 gene, e.g., decreases or abolishes the expression of CD38 on the engineered cell. In some aspects, provided are immune cells, such as T cells, that contain a genetically engineered antigen receptor that specifically binds to CD38 antigen and that contains disruption or repression of the corresponding endogenous CD38 gene in the cell.
In some embodiments, the gene is an inhibitory CAR target. For example, in the context of an antigen that binds to an inhibitory CAR, gene disruption in some contexts prevents or reduces the likelihood of a molecule that is expressed by the engineered cells themselves inducing a dampening effect on a response of the engineered cells. Exemplary of such antigens are those expressed on normal or non-targeted or off-target cells (such that an inhibitory CAR molecule is included to prevent off-target effects), but is also expressed on the cell type used for genetic engineering, such as the T cell or NK cell. Exemplary antigens are MHC molecules, such as MHC-class I molecules, which can in some cases be downregulated in the context of immune evasion, cancer, or infection, but are generally expressed on nucleated cells. Other examples are any inhibitory CAR target that is also expressed on a T cell, NK cell, or other cell engineered for cell therapy.
In some embodiments, the gene encodes an immunoinhibitory molecule or other product that dampens or prevents activation or stimulation or other effector function of the engineered cell, such as its ability to proliferate, survive, secrete one or more factors, or recruit or carry out cell killing, for example, in response to binding via the engineered antigen receptor.
In some embodiments, the gene repressed encodes a protein that suppresses the immune system. In some embodiments, the repression reduces the suppressive effects of the gene product on the immune system. In some aspects, this increases the immune response to tumor cells. In some embodiments, the gene encodes an adenosine receptor, such as A2AR. Thus, in some embodiments, the expression, activity, and/or function of A2AR is repressed.
Techniques for Gene Repression
In some embodiments, the repression of the expression, activity, and/or function of the gene is carried out by disrupting the gene. In some aspects, the gene is disrupted so that its expression is reduced by at least at or about 20, 30, or 40%, generally at least at or about 50, 60, 70, 80, 90, or 95% as compared to the expression in the absence of the gene disruption or in the absence of the components introduced to effect the disruption.
In some embodiments, gene disruption is carried out by induction of one or more double-stranded breaks and/or one or more single-stranded breaks in the gene, typically in a targeted manner. In some embodiments, the double-stranded or single-stranded breaks are made by a nuclease, e.g. an endonuclease, such as a gene-targeted nuclease. In some aspects, the breaks are induced in the coding region of the gene, e.g. in an exon. For example, in some embodiments, the induction occurs near the N-terminal portion of the coding region, e.g. in the first exon, in the second exon, or in a subsequent exon.
In some aspects, the double-stranded or single-stranded breaks undergo repair via a cellular repair process, such as by non-homologous end-joining (NHEJ) or homology-directed repair (HDR). In some aspects, the repair process is error-prone and results in disruption of the gene, such as a frameshift mutation, e.g., biallelic frameshift mutation, which can result in complete knockout of the gene. For example, in some aspects, the disruption comprises inducing a deletion, mutation, and/or insertion. In some embodiments, the disruption results in the presence of an early stop codon. In some aspects, the presence of an insertion, deletion, translocation, frameshift mutation, and/or a premature stop codon results in repression of the expression, activity, and/or function of the gene.
In some embodiments, the repression is transient or reversible, such that expression of the gene is restored at a later time. In other embodiments, the repression is not reversible or transient, e.g., is permanent.
In some embodiments, gene repression is achieved using antisense techniques, such as by RNA interference (RNAi), short interfering RNA (siRNA), short hairpin (shRNA), and/or ribozymes are used to selectively suppress or repress expression of the gene. siRNA technology includes that based on RNAi utilizing a double-stranded RNA molecule having a sequence homologous with the nucleotide sequence of mRNA which is transcribed from the gene, and a sequence complementary with the nucleotide sequence. siRNA generally is homologous/complementary with one region of mRNA which is transcribed from the gene, or may be siRNA including a plurality of RNA molecules which are homologous/complementary with different regions.
DNA-Targeting Molecules and Complexes; Targeted Endonucleases
In some embodiments, the repression is achieved using a DNA-targeting molecule, such as a DNA-binding protein or DNA-binding nucleic acid, or complex, compound, or composition, containing the same, which specifically binds to or hybridizes to the gene. In some embodiments, the DNA-targeting molecule comprises a DNA-binding domain, e.g., a zinc finger protein (ZFP) DNA-binding domain, a transcription activator-like protein (TAL) or TAL effector (TALE) DNA-binding domain, a clustered regularly interspaced short palindromic repeats (CRISPR) DNA-binding domain, or a DNA-binding domain from a meganuclease.
Zinc finger, TALE, and CRISPR system binding domains can be “engineered” to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger or TALE protein. Engineered DNA binding proteins (zinc fingers or TALEs) are proteins that are non-naturally occurring. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP and/or TALE designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Publication No. 20110301073.
In some embodiments, the DNA-targeting molecule, complex, or combination contains a DNA-binding molecule and one or more additional domain, such as an effector domain to facilitate the repression or disruption of the gene. For example, in some embodiments, the gene disruption is carried out by fusion proteins that comprise DNA-binding proteins and a heterologous regulatory domain or functional fragment thereof. In some aspects, domains include, e.g., transcription factor domains such as activators, repressors, co-activators, co-repressors, silencers, oncogenes, DNA repair enzymes and their associated factors and modifiers, DNA rearrangement enzymes and their associated factors and modifiers, chromatin associated proteins and their modifiers, e.g. kinases, acetylases and deacetylases, and DNA modifying enzymes, e.g. methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases, and their associated factors and modifiers. See, for example, U.S. Patent Application Publication Nos. 20050064474; 20060188987 and 2007/0218528, incorporated by reference in their entireties herein, for details regarding fusions of DNA-binding domains and nuclease cleavage domains. In some aspects, the additional domain is a nuclease domain. Thus, in some embodiments, gene disruption is facilitated by gene or genome editing, using engineered proteins, such as nucleases and nuclease-containing complexes or fusion proteins, composed of sequence-specific DNA-binding domains fused to or complexed with non-specific DNA-cleavage molecules such as nucleases.
In some aspects, these targeted chimeric nucleases or nuclease-containing complexes carry out precise genetic modifications by inducing targeted double-stranded breaks or single-stranded breaks, stimulating the cellular DNA-repair mechanisms, including error-prone non-homologous end joining (NHEJ) and homology-directed repair (HDR). In some embodiments the nuclease is an endonuclease, such as a zinc finger nuclease (ZFN), TALE nuclease (TALEN), an RNA-guided endonuclease (RGEN), such as a CRISPR-associated (Cas) protein, or a meganuclease.
In some embodiments, a donor nucleic acid, e.g., a donor plasmid or nucleic acid encoding the genetically engineered antigen receptor, is provided and is inserted by HDR at the site of gene editing following the introduction of the DSBs. Thus, in some embodiments, the disruption of the gene and the introduction of the antigen receptor, e.g., CAR, are carried out simultaneously, whereby the gene is disrupted in part by knock-in or insertion of the CAR-encoding nucleic acid.
In some embodiments, no donor nucleic acid is provided. In some aspects, NHEJ-mediated repair following introduction of DSBs results in insertion or deletion mutations that can cause gene disruption, e.g., by creating missense mutations or frameshifts.
ZFPs and ZFNs; TALs, TALEs, and TALENs
In some embodiments, the DNA-targeting molecule includes a DNA-binding protein such as one or more zinc finger protein (ZFP) or transcription activator-like protein (TAL), fused to an effector protein such as an endonuclease. Examples include ZFNs, TALEs, and TALENs. See Lloyd et al., Fronteirs in Immunology, 4(221), 1-7 (2013).
In some embodiments, the DNA-targeting molecule comprises one or more zinc-finger proteins (ZFPs) or domains thereof that bind to DNA in a sequence-specific manner. A ZFP or domain thereof is a protein or domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.
Among the ZFPs are artificial ZFP domains targeting specific DNA sequences, typically 9-18 nucleotides long, generated by assembly of individual fingers.
ZFPs include those in which a single finger domain is approximately 30 amino acids in length and contains an alpha helix containing two invariant histidine residues coordinated through zinc with two cysteines of a single beta turn, and having two, three, four, five, or six fingers. Generally, sequence-specificity of a ZFP may be altered by making amino acid substitutions at the four helix positions (−1, 2, 3 and 6) on a zinc finger recognition helix. Thus, in some embodiments, the ZFP or ZFP-containing molecule is non-naturally occurring, e.g., is engineered to bind to a target site of choice. See, for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528; 2005/0267061, all incorporated herein by reference in their entireties.
In some aspects, repression of the gene is carried out by contacting a first target site in the gene with a first ZFP, thereby repressing the gene. In some embodiments, the target site in the gene is contacted with a fusion ZFP comprising six fingers and the regulatory domain, thereby inhibiting expression of the gene.
In some embodiments, the step of contacting further comprises contacting a second target site in the gene with a second ZFP. In some aspects, the first and second target sites are adjacent. In some embodiments, the first and second ZFPs are covalently linked. In some aspects, the first ZFP is a fusion protein comprising a regulatory domain or at least two regulatory domains. In some embodiments, the first and second ZFPs are fusion proteins, each comprising a regulatory domain or each comprising at least two regulatory domains. In some embodiments, the regulatory domain is a transcriptional repressor, a transcriptional activator, an endonuclease, a methyl transferase, a histone acetyltransferase, or a histone deacetylase.
In some embodiments, the ZFP is encoded by a ZFP nucleic acid operably linked to a promoter. In some aspects, the method further comprises the step of first administering the nucleic acid to the cell in a lipid:nucleic acid complex or as naked nucleic acid. In some embodiments, the ZFP is encoded by an expression vector comprising a ZFP nucleic acid operably linked to a promoter. In some embodiments, the ZFP is encoded by a nucleic acid operably linked to an inducible promoter. In some aspects, the ZFP is encoded by a nucleic acid operably linked to a weak promoter.
In some embodiments, the target site is upstream of a transcription initiation site of the gene. In some aspects, the target site is adjacent to a transcription initiation site of the gene. In some aspects, the target site is adjacent to an RNA polymerase pause site downstream of a transcription initiation site of the gene.
In some embodiments, the DNA-targeting molecule is or comprises a zinc-finger DNA binding domain fused to a DNA cleavage domain to form a zinc-finger nuclease (ZFN). In some embodiments, fusion proteins comprise the cleavage domain (or cleavage half-domain) from at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. In some embodiments, the cleavage domain is from the Type IIS restriction endonuclease Fok I. Fok I generally catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31,978-31,982.]
In some embodiments, ZFNs target a gene present in the engineered cell. In some aspects, the ZFNs efficiently generate a double strand break (DSB), for example at a predetermined site in the coding region of the gene. Typical regions targeted include exons, regions encoding N-terminal regions, first exon, second exon, and promoter or enhancer regions. In some embodiments, transient expression of the ZFNs promotes highly efficient and permanent disruption of the target gene in the engineered cells. In particular, in some embodiments, delivery of the ZFNs results in the permanent disruption of the gene with efficiencies surpassing 50%.
Many gene-specific engineered zinc fingers are available commercially. For example, Sangamo Biosciences (Richmond, Calif., USA) has developed a platform (CompoZr) for zinc-finger construction in partnership with Sigma-Aldrich (St. Louis, Mo., USA), allowing investigators to bypass zinc-finger construction and validation altogether, and provides specifically targeted zinc fingers for thousands of proteins. Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405. In some embodiments, commercially available zinc fingers are used or are custom designed. (See, for example, Sigma-Aldrich catalog numbers CSTZFND, CSTZFN, CTI1-1KT, and PZD0020).
TALEs and TALENs
In some embodiments, the DNA-targeting molecule comprises a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, such as in a transcription activator-like protein effector (TALE) protein, See, e.g., U.S. Patent Publication No. 20110301073, incorporated by reference in its entirety herein.
A TALE DNA binding domain or TALE is a polypeptide comprising one or more TALE repeat domains/units. The repeat domains are involved in binding of the TALE to its cognate target DNA sequence. A single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. Each TALE repeat unit includes 1 or 2 DNA-binding residues making up the Repeat Variable Diresidue (RVD), typically at positions 12 and/or 13 of the repeat. The natural (canonical) code for DNA recognition of these TALEs has been determined such that an HD sequence at positions 12 and 13 leads to a binding to cytosine (C), NG binds to T, NI to A, NN binds to G or A, and NG binds to T and non-canonical (atypical) RVDs are also known. See, U.S. Patent Publication No. 20110301073. In some embodiments, TALEs may be targeted to any gene by design of TAL arrays with specificity to the target DNA sequence. The target sequence generally begins with a thymidine.
In some embodiments, the molecule is a DNA binding endonuclease, such as a TALE-nuclease (TALEN). In some aspects the TALEN is a fusion protein comprising a DNA-binding domain derived from a TALE and a nuclease catalytic domain to cleave a nucleic acid target sequence. In some embodiments, the TALE DNA-binding domain has been engineered to bind a target sequence within genes that encode the target antigen and/or the immunosuppressive molecule. For example, in some aspects, the TALE DNA-binding domain may target CD38 and/or an adenosine receptor, such as A2AR.
In some embodiments, the TALEN recognizes and cleaves the target sequence in the gene. In some aspects, cleavage of the DNA results in double-stranded breaks. In some aspects the breaks stimulate the rate of homologous recombination or non-homologous end joining (NHEJ). Generally, NHEJ is an imperfect repair process that often results in changes to the DNA sequence at the site of the cleavage. In some aspects, repair mechanisms involve rejoining of what remains of the two DNA ends through direct re-ligation (Critchlow and Jackson, Trends Biochem Sci. 1998 October; 23(10):394-8) or via the so-called microhomology-mediated end joining. In some embodiments, repair via NHEJ results in small insertions or deletions and can be used to disrupt and thereby repress the gene. In some embodiments, the modification may be a substitution, deletion, or addition of at least one nucleotide. In some aspects, cells in which a cleavage-induced mutagenesis event, i.e. a mutagenesis event consecutive to an NHEJ event, has occurred can be identified and/or selected by well-known methods in the art.
In some embodiments, TALE repeats are assembled to specifically target a gene. (Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405). A library of TALENs targeting 18,740 human protein-coding genes has been constructed (Kim et al., Nature Biotechnology. 31, 251-258 (2013)). Custom-designed TALE arrays are commercially available through Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, Ky., USA), and Life Technologies (Grand Island, N.Y., USA). Specifically, TALENs that target CD38 are commercially available (See Gencopoeia, catalog numbers HTN222870-1, HTN222870-2, and HTN222870-3, available on the World Wide Web at www.genecopoeia.com/product/search/detail.php?prt=26&cid=&key=HTN222870). Exemplary molecules are described, e.g., in U.S. Patent Publication Nos. US 2014/0120622, and 2013/0315884.
In some embodiments the TALENs are introduced as transgenes encoded by one or more plasmid vectors. In some aspects, the plasmid vector can contain a selection marker which provides for identification and/or selection of cells which received said vector.
RGENs (CRISPR/Cas Systems)
In some embodiments, the repression is carried out using one or more DNA-binding nucleic acids, such as disruption via an RNA-guided endonuclease (RGEN), or other form of repression by another RNA-guided effector molecule. For example, in some embodiments, the repression is carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. See Sander and Joung, Nature Biotechnology, 32(4): 347-355.
In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus.
In some embodiments, the CRISPR/Cas nuclease or CRISPR/Cas nuclease system includes a non-coding RNA molecule (guide) RNA, which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease domains).
In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes or Staphylococcus aureus.
In some embodiments, a Cas nuclease and gRNA (including a fusion of crRNA specific for the target sequence and fixed tracrRNA) are introduced into the cell. In general, target sites at the 5′ end of the gRNA target the Cas nuclease to the target site, e.g., the gene, using complementary base pairing. In some embodiments, the target site is selected based on its location immediately 5′ of a protospacer adjacent motif (PAM) sequence, such as typically NGG, or NAG. In this respect, the gRNA is targeted to the desired sequence by modifying the first 20 nucleotides of the guide RNA to correspond to the target DNA sequence.
In some embodiments, the CRISPR system induces DSBs at the target site, followed by disruptions as discussed herein. In other embodiments, Cas9 variants, deemed “nickases” are used to nick a single strand at the target site. In some aspects, paired nickases are used, e.g., to improve specificity, each directed by a pair of different gRNAs targeting sequences such that upon introduction of the nicks simultaneously, a 5′ overhang is introduced. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain such as a transcriptional repressor or activator, to affect gene expression.
In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence. Typically, the In the context of formation of a CRISPR complex, “target sequence” generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.
The target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell. In some embodiments, the target sequence may be within an organelle of the cell. Generally, a sequence or template that may be used for recombination into the targeted locus comprising the target sequences is referred to as an “editing template” or “editing polynucleotide” or “editing sequence”. In some aspects, an exogenous template polynucleotide may be referred to as an editing template. In some aspects, the recombination is homologous recombination.
Typically, in the context of an endogenous CRISPR system, formation of the CRISPR complex (comprising the guide sequence hybridized to the target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. Without wishing to be bound by theory, the tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of the CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. In some embodiments, the tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of the CRISPR complex.
As with the target sequence, in some embodiments, complete complementarity is not necessarily needed. In some embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, one or more vectors driving expression of one or more elements of the CRISPR system are introduced into the cell such that expression of the elements of the CRISPR system direct formation of the CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. In some embodiments, CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5′ with respect to (“upstream” of) or 3′ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.
In some embodiments, a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In some embodiments, one or more insertion sites (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and/or downstream of one or more sequence elements of one or more vectors. In some embodiments, a vector comprises an insertion site upstream of a tracr mate sequence, and optionally downstream of a regulatory element operably linked to the tracr mate sequence, such that following insertion of a guide sequence into the insertion site and upon expression the guide sequence directs sequence-specific binding of the CRISPR complex to a target sequence in a eukaryotic cell. In some embodiments, a vector comprises two or more insertion sites, each insertion site being located between two tracr mate sequences so as to allow insertion of a guide sequence at each site. In such an arrangement, the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations of these. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell. For example, a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences. In some embodiments, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containing vectors may be provided, and optionally delivered to the cell.
In some embodiments, a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding the CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas 1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of S. pyogenes Cas9 protein may be found in the SwissProt database under accession number Q99ZW2. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In some embodiments the CRISPR enzyme is Cas9, and may be Cas9 from S. pyogenes, S. aureus or S. pneumoniae. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.
In some embodiments, a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme. Non-limiting examples of mutations in a Cas9 protein are known in the art (see e.g. WO2015/161276), any of which can be included in a CRISPR/Cas9 system in accord with the provided methods. In some embodiments, the CRISPR enzyme is mutated such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). In some embodiments, a Cas9 nickase may be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ.
In some embodiments, an enzyme coding sequence encoding the CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding the CRISPR enzyme corresponds to the most frequently used codon for a particular amino acid.
In general, a guide sequence includes a targeting domain comprising a polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. In some examples, the targeting domain of the gRNA is complementary, e.g., at least 80, 85, 90, 95, 98 or 99% complementary, e.g., fully complementary, to the target sequence on the target nucleic acid.
Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of the CRISPR complex to a target sequence may be assessed by any suitable assay. For example, the components of the CRISPR system sufficient to form the CRISPR complex, including the guide sequence to be tested, may be provided to the cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of the CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
A guide sequence may be selected to target any target sequence. In some embodiments, the target sequence is a sequence within a genome of a cell. Exemplary target sequences include those that are unique in the target genome. In some embodiments, a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.
In general, a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized to the tracr sequence. In general, degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences.
Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the tracr sequence or tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the tracr sequence and tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. In some aspects, loop forming sequences for use in hairpin structures are four nucleotides in length, and have the sequence GAAA. However, longer or shorter loop sequences may be used, as may alternative sequences. In some embodiments, the sequences include a nucleotide triplet (for example, AAA), and an additional nucleotide (for example C or G). Examples of loop forming sequences include CAAA and AAAG. In some embodiments, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In some embodiments, the transcript has two, three, four or five hairpins. In a further embodiment, the transcript has at most five hairpins. In some embodiments, the single transcript further includes a transcription termination sequence, such as a polyT sequence, for example six T nucleotides.
In some embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme). A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a fusion protein comprising a CR ISPR enzyme are described in US20110059502, incorporated herein by reference. In some embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence.
In some embodiments, a CRISPR enzyme in combination with (and optionally complexed with) a guide sequence is delivered to the cell. In some embodiments, methods for introducing a protein component into a cell according to the present disclosure (e.g. Cas9/gRNA RNPs) may be via physical delivery methods (e.g. electroporation, particle gun, Calcium Phosphate transfection, cell compression or squeezing), liposomes or nanoparticles.
For example, CRISPR/Cas9 technology may be used to knock-down gene expression of the target antigen in the engineered cells. In an exemplary method, Cas9 nuclease (e.g., that encoded by mRNA from Staphylococcus aureus or from Stretpococcus pyogenes, e.g. pCW-Cas9, Addgene #50661, Wang et al. (2014) Science, 3:343-80-4; or nuclease or nickase lentiviral vectors available from Applied Biological Materials (ABM; Canada) as Cat. No. K002, K003, K005 or K006) and a guide RNA specific to the target antigen gene are introduced into cells, for example, using lentiviral delivery vectors or any of a number of known delivery method or vehicle for transfer to cells, such as any of a number of known methods or vehicles for delivering Cas9 molecules and guide RNAs. Non-specific or empty vector control T cells also are generated. Degree of Knockout of a gene (e.g., 24 to 72 hours after transfer) is assessed using any of a number of well-known assays for assessing gene disruption in cells.
It is within the level of a skilled artisan to design or identify a gRNA sequence that is or comprises a sequence targeting a target antigen of interest, such as any described herein, including the exon sequence and sequences of regulatory regions, including promoters and activators. A genome-wide gRNA database for CRISPR genome editing is publicly available, which contains exemplary single guide RNA (sgRNA) target sequences in constitutive exons of genes in the human genome or mouse genome (see e.g., genescript.com/gRNA-database.html; see also, Sanjana et al. (2014) Nat. Methods, 11:783-4; http://www.e-crisp.org/E-CRISP/; http://crispr.mit.edu/; https://www.dna20.com/eCommerce/cas9/input). In some embodiments, the gRNA sequence is or comprises a sequence with minimal off-target binding to a non-target gene.
Commercially available kits, gRNA vectors and donor vectors, for knockout of a universal tumor antigen or other target antigen, or a family member thereof, such any as described herein, for example one or more of MDM2, CYP1B, HER2/neu, WT1, livin, AFP, CEA, MUC16, MUC1, PSMA, p53, cyclin (D1), BCMA, BAFFR, or TACI are available, for example, from Origene (Rockville, Md.), GenScript (Atlanta, Ga.), Applied Biological Materials (ABM; Richmond, British Colombia), BioCat (Heidelberg, Germany) or others. For example, commercially available kits for knockout of hTERT via CRISPR include, for example, those available as catalog numbers K0009801, K0009802, K009803 and/or K0009804 each available from ABM. Commercially available kits for knockout of survivin via CRISPR include, for example, catalog numbers KN205935 available from Origene and catalog numbers K0184401, K0184402, K0184403, K0184404 each available from ABM. Commercially available kits for knockout of MDM2 via CRISPR include, for example, KN219518 from Origene and catalog number K1283521 from ABM. Commercially available kits for knockout of Her2/neu via CRISPR include, for example, KN212583 from Origene. Commercially available kits for knockout of Cyp1B1 via CRISPR include, for example, KN204074-OR available from BioCat. Commercially available kits for knockout of WT1 via CRISPR include, for example, KN220079 from Origine.
Commercially available kits, gRNA vectors and donor vectors, for knockout of CD38 via CRISPR are available, for example, from OriGene. See www.origene.com/CRISPR-CAS9/Product.aspx?SKU=KN203179; catalog numbers KN203179G1, KN203179G2, KN203179D.
Commercially available kits, gRNA vectors and donor vectors for knockout of BCMA, TACI or BAFF receptor, FcRH5 or other antigen as described are available, for example, from Santa Cruz Biotechnology (Dallas, Tex.), Origene (Rockville, Md.), GenScript (Piscataway, N.J.). In some embodiments, gRNA sequences for CRISPR knockout of BCMA include, for example, any set forth in any of SEQ ID NOS: 37-42, which, in some cases, can be used with a Cas9 nuclease to introduce a double stranded break by gene editing (see e.g. Sanjana et al. (2014) Nat. Methods, 11:783-4). Commercially available kits for knockout of BCMA via CRISPR include, for example, catalog number KN208851 from Origene. In some embodiments, gRNA sequences for CRISPR knockout of TACI include, for example, any set forth in any of SEQ ID NOS: 43-48, which, in some cases, can be used with a Cas9 nuclease to introduce a double stranded break by gene editing. Commercially available kits for knockout of TACI via CRISPR include, for example, catalog number KN204672 from Origene. In some embodiments, gRNA sequences for CRISPR knockout of BAFF receptor include, for example, any set forth in any of SEQ ID NOS: 49-54, which, in some cases, can be used with a Cas9 nuclease to introduce a double stranded break by gene editing. Commercially available kits for knockout of BAFF receptor via CRISPR include, for example, catalog number KN211270 from Origene.
In some embodiments, gRNA sequences for CRISPR knockout of FcRH5 include, for example, any set forth in any of SEQ ID NOS: 57-62, which, in some cases, can be used with a Cas9 nuclease to introduce a double stranded break by gene editing. Commercially available kits for knockout of FcRH5 via CRISPR include, for example, catalog number KN221468 from Origene.
In some embodiments, design gRNA guide sequences and/or vectors for any of the antigens as described herein are generated using any of a number of known methods, such as those for use in gene knockdown via CRISPR-mediated, TALEN-mediated and/or related methods.
In some aspects, target polynucleotides are modified in a eukaryotic cell. In some embodiments, the method comprises allowing the CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises the CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
In some aspects, the methods include modifying expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing the CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
In some embodiments, a CRISPR/Cas system can be used for knocking down, such as reducing or suppressing, the expression of a target sequence. Exemplary features of CRISPR/Cas systems are described below and can be adapted for use in reducing or suppressing expression of a molecule, rather than disrupting or deleting a gene encoding the molecule, by using an enzymatically inactive nuclease. In some embodiments, a guide RNA (gRNA) targeting a gene of interest, such as any described herein, or the promoter, enhancer or other cis- or trans-acting regulatory regions associated therewith, can be introduced in combination with a modified Cas9 protein or a fusion protein containing the modified Cas9 protein, to suppress the expression of, e.g., knock-down, of the gene(s). In some embodiments, the Cas9 molecule is an enzymatically inactive Cas9 (eiCas9) molecule, which comprises a mutation, e.g., a point mutation, that causes the Cas9 molecule to be inactive, e.g., a mutation that eliminates or substantially reduces the Cas9 molecule cleavage activity (see e.g. WO2015/161276). In some embodiments, the eiCas9 molecule is fused, directly or indirectly to, a transcription activator or repressor protein.
Delivery of Nucleic Acids Encoding the Gene Disrupting Molecules and Complexes
In some aspects, a nucleic acid encoding the DNA-targeting molecule, complex thereof (e.g. Cas9/gRNA RNPs), or combination, is administered or introduced to the cell. In some embodiments, such nucleic acid molecule or complex thereof can be introduced into cells, such as T cells, by methods well known in the art. Such methods include, but are not limited to, introduction in the form of recombinant viral vectors (e.g. retroviruses, lentiviruses, adenoviruses), liposomes or nanoparticles. In some embodiments, methods can include microinjection, electroporation, particle bombardment, Calcium Phosphate transfection, cell compression, or squeezing. In some embodiments, such nucleic acid molecule or complex thereof can be introduced in the form of an expression vector, such as a viral expression vector. In some aspects, the expression vector is a retroviral expression vector, an adenoviral expression vector, a DNA plasmid expression vector, or an AAV expression vector. In some aspects, one or more polynucleotides encoding the disruption molecule or complex, such as the DNA-targeting molecule, is delivered to the cell. In some aspects, the delivery is by delivery of one or more vectors, one or more transcripts thereof, and/or one or proteins transcribed therefrom, is delivered to the cell.
In some embodiments, the polypeptides are synthesized in situ in the cell as a result of the introduction of polynucleotides encoding the polypeptides into the cell. In some aspects, the polypeptides could be produced outside the cell and then introduced thereto. Methods for introducing a polynucleotide construct into animal cells are known and include, as non-limiting examples stable transformation methods wherein the polynucleotide construct is integrated into the genome of the cell, transient transformation methods wherein the polynucleotide construct is not integrated into the genome of the cell, and virus mediated methods. In some embodiments, the polynucleotides may be introduced into the cell by for example, recombinant viral vectors (e.g. retroviruses, adenoviruses), liposome and the like. For example, in some aspects, transient transformation methods include microinjection, electroporation, or particle bombardment. In some embodiments, the polynucleotides may be included in vectors, more particularly plasmids or virus, in view of being expressed in the cells.
In some embodiments, viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CRISPR, ZFP, ZFN, TALE, and/or TALEN system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon. TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bohm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).
Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
In some embodiments, delivery is via the use of RNA or DNA viral based systems for the delivery of nucleic acids. Viral vectors in some aspects may be administered directly to patients (in vivo) or they can be used to treat cells in vitro or ex vivo, and then administered to patients. Viral-based systems in some embodiments include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer.
In some aspects, a reporter gene which includes but is not limited to glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP), may be introduced into the cell to encode a gene product which serves as a marker by which to measure the alteration or modification of expression of the gene product. In a further embodiment, the DNA molecule encoding the gene product may be introduced into the cell via a vector. In some embodiments, the gene product is luciferase. In a further embodiment, the expression of the gene product is decreased.
In some embodiments, an agent capable of inducing a genetic disruption, such as a knockdown or a knockout of a target gene or genes, such as any encoding an antigen described herein, is introduced as a complex, such as a ribonucleoprotein (RNP) complex. RNP complexes include a sequence of ribonucleotides, such as an RNA or a gRNA molecule, and a polypeptide, such as a Cas9 protein or variant thereof. In some embodiments, the Cas9 protein is delivered as an RNP complex that comprises a Cas9 protein and a gRNA molecule, e.g., a gRNA targeted for a gene encoding the target antigen. In some embodiments, the RNP that includes one or more gRNA molecules targeted for a gene encoding the target antigen, and a Cas9 enzyme or variant thereof, is directly introduced into the cell via physical delivery (e.g., electroporation, particle gun, Calcium Phosphate transfection, cell compression or squeezing), liposomes or nanoparticles. In particular embodiments, the RNP includes one or more gRNA molecules targeted for a gene encoding the target antigen and a Cas9 enzyme or variant thereof is introduced via electroporation.
In some embodiments, the degree of knockout of a gene, such as any encoding an antigen described herein, at various time points, e.g., 24 to 72 hours after introduction of agent, can be assessed using any of a number of well-known assays for assessing gene disruption in cells. Degree of knockdown of a gene at various time points, e.g., 24 to 72 hours after introduction of agent, can be assessed using any of a number of well-known assays for assessing gene expression in cells, such as assays to determine the level of transcription or protein expression or cell surface expression.
II. COMPOSITIONS, FORMULATIONS, KITS, DEVICES, METHODS, AND USES
Also provided are cells, cell populations, and compositions containing the cells produced by the provided methods. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administrating the cells and compositions to subjects, e.g., patients.
Provided are methods and uses of the cells, including therapeutic methods and uses, such as in adoptive cell therapy. In some embodiments, the methods include administration of the cells or a composition containing the cells to a subject, tissue, or cell, such as one having, at risk for, or suspected of having a disease, condition or disorder. In some embodiments, the methods treat cancers and other diseases, conditions, and disorders. In some embodiments, the cells, populations, and compositions are administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells or compositions are administered to the subject, such as a subject having or at risk for the disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptom of, the disease or condition, such as by lessening tumor burden in a cancer expressing an antigen recognized by the engineered cell.
Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.
In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.
In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.
In some embodiments, the subject, e.g., patient, to whom the cells, cell populations, or compositions are administered is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent. In some examples, the patient or subject is a validated animal model for disease, adoptive cell therapy, and/or for assessing toxic outcomes such as cytokine release syndrome (CRS).
Also provided are pharmaceutical compositions for use in such methods.
Among the diseases, conditions, and disorders are tumors, including solid tumors, hematologic malignancies, and melanomas, and infectious diseases, such as infection with a virus or other pathogen, e.g., HIV, HCV, HBV, CMV, and parasitic disease. In some embodiments, the disease or condition is a tumor, cancer, malignancy, neoplasm, or other proliferative disease. Such diseases include but are not limited to cancers of the immune system, leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), ALL, non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B cell lymphoma, B cell malignancies, cancers of the colon, lung, liver, breast, prostate, ovarian, skin (including melanoma), bone, and brain cancer, ovarian cancer, epithelial cancers, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin lymphoma, cervical carcinoma, colorectal cancer, glioblastoma, glioma, neuroblastoma, Ewing sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and/or mesothelioma. In one embodiment, the disease or condition is or is associated with multiple myeloma.
In some embodiments, the disease or condition is an infectious disease or condition, such as, but not limited to, viral, retroviral, bacterial, and protozoal infections, immunodeficiency, Cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus. In some embodiments, the disease or condition is an autoimmune or inflammatory disease or condition, such as arthritis, e.g., rheumatoid arthritis (RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Grave's disease, Crohn's disease multiple sclerosis, asthma, and/or a disease or condition associated with transplant.
In some embodiments, the one or more genetically engineered antigen receptors specifically binds to a target antigen associated with the disease or disorder. In some embodiments, the one or more genetically engineered antigen receptors binds to a target antigen as well as one or more antigens related to the target antigen. In some cases, two more genetically engineered antigen receptors bind to two or more different antigens associated with the disease or disorder, or related antigens thereof. In some embodiments, at least one antigen associated with the disease or disorder is a universal tumor antigen, or a family member thereof. For example, in some cases the antigen is hTERT, survivin, MDM2, CYP1B, HER2/neu, WT1, livin, AFP, CEA, MUC16, MUC1, PSMA, p53, cyclin (D1), BCMA, BAFFR, or TACI. For example, the universal tumor antigen is hTERT or survivin. In some embodiments, at least one antigen associated with the disease or disorder is a myeloma antigen. For example, in some cases, the myeloma antigen is CD38, CD138, CS-1, CD56, TIM-3, CD33, CD123 or CD44. For example, the myeloma antigen is CD38.
In some embodiments, one or more other antigens associated with the disease or disorder is selected from the group consisting of orphan tyrosine kinase receptor ROR1, EGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, OEPHa2, ErbB2, 3, or 4, FBP, fetal acethycholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1), a cyclin, such as cyclin A1 (CCNA1), and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens. The antigens include proteins, carbohydrates, and other molecules.
In some embodiments, the cells and cell populations are administered to a subject in the form of a composition, such as a pharmaceutical composition. In some embodiments, the pharmaceutical composition further comprises other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the cell populations are administered in the form of a salt, e.g., a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p-toluenesulphonic acid.
In some aspects, the choice of carrier can in the pharmaceutical composition is determined in part by the particular engineered CAR or TCR, vector, or cells expressing the CAR or TCR, as well as by the particular method used to administer the vector or host cells expressing the CAR. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition.
In addition, buffering agents in some aspects are included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
In certain embodiments, a pharmaceutical composition comprising a cell population described herein can be formulated as an inclusion complex, such as cyclodextrin inclusion complex, or as a liposome. Liposomes can serve to target the host cells (e.g., T-cells or NK cells) to a particular tissue. Many methods are available for preparing liposomes, such as those described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
The pharmaceutical composition in some aspects can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Many types of release delivery systems are available and known to those of ordinary skill in the art. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.
The pharmaceutical composition in some embodiments comprises the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
In certain embodiments, a subject is administered the range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges.
The cells and compositions in some embodiments are administered using standard administration techniques, formulations, and/or devices. Provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. Administration can be autologous or heterologous. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells of the invention or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition of the present invention (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cell populations are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
Compositions of the cells in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyoi (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
Sterile injectable solutions can be prepared by incorporating the genetically engineered in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.
Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
The cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cell populations are administered prior to the one or more additional therapeutic agents. In some embodiments, the cell populations are administered after to the one or more additional therapeutic agents.
Once the cells are administered to a mammal (e.g., a human), the biological activity of the engineered cell populations in some aspects is measured by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells also can be measured by assaying expression and/or secretion of certain cytokines, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
In certain embodiments, engineered cells are modified in any number of ways, such that their therapeutic or prophylactic efficacy is increased. For example, the engineered CAR or TCR expressed by the population can be conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating compounds, e.g., the CAR or TCR, to targeting moieties is known in the art. See, for instance, Wadwa et al., J. Drug Targeting 3: 1 1 1 (1995), and U.S. Pat. No. 5,087,616.
III. DEFINITIONS
As used herein, “repression” of gene expression refers to the elimination or reduction of expression of one or more gene products encoded by the subject gene in a cell, compared to the level of expression of the gene product in the absence of the repression. Exemplary gene products include mRNA and protein products encoded by the gene. Repression in some cases is transient or reversible and in other cases is permanent. Repression in some cases is of a functional or full-length protein or mRNA, despite the fact that a truncated or non-functional product may be produced. In some embodiments herein, gene activity or function, as opposed to expression, is repressed. Gene repression is generally induced by artificial methods, i.e., by addition or introduction of a compound, molecule, complex, or composition, and/or by disruption of nucleic acid of or associated with the gene, such as at the DNA level. Exemplary methods for gene repression include gene silencing, knockdown, knockout, and/or gene disruption techniques, such as gene editing. Examples include antisense technology, such as RNAi, siRNA, shRNA, and/or ribozymes, which generally result in transient reduction of expression, as well as gene editing techniques which result in targeted gene inactivation or disruption, e.g., by induction of breaks and/or homologous recombination.
As used herein, a “disruption” of a gene refers to a change in the sequence of the gene, at the DNA level. Examples include insertions, mutations, and deletions. The disruptions typically result in the repression and/or complete absence of expression of a normal or “wild type” product encoded by the gene. Exemplary of such gene disruptions are insertions, frameshift and missense mutations, deletions, knock-in, and knock-out of the gene or part of the gene, including deletions of the entire gene. Such disruptions can occur in the coding region, e.g., in one or more exons, resulting in the inability to produce a full-length product, functional product, or any product, such as by insertion of a stop codon. Such disruptions may also occur by disruptions in the promoter or enhancer or other region affecting activation of transcription, so as to prevent transcription of the gene. Gene disruptions include gene targeting, including targeted gene inactivation by homologous recombination.
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.”
Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.
The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.
As used herein, a subject includes any living organism, such as humans and other mammals. Mammals include, but are not limited to, humans, and non-human animals, including farm animals, sport animals, rodents and pets.
As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
As used herein, the terms “treatment,” “treat,” and “treating,” refer to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. In certain embodiments, the effect is therapeutic, such that it partially or completely cures a disease or condition or adverse symptom attributable thereto.
As used herein, a “therapeutically effective amount” of a compound or composition or combination refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered.
As used herein, a statement that a cell or population of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.
As used herein, a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.
In some embodiments, a decrease in expression of one or markers refers to loss of 1 log10 in the mean fluorescence intensity and/or decrease of percentage of cells that exhibit the marker of at least about 20% of the cells, 25% of-the cells, 30% of the cells, 35% of the cells, 40% of the cells, 45% of the cells, 50% of the cells, 55% of the cells, 60% of the cells, 65% of the cells, 70% of the cells, 75% of the cells, 80% of the cells, 85% of the cells, 90% of the cell, 95% of the cells, and 100% of the cells and any % between 20 and 100% when compared to a reference cell population. In some embodiments, a cell population positive for one or markers refers to a percentage of cells that exhibit the marker of at least about 50% of the cells, 55% of the cells, 60% of the cells, 65% of the cells, 70% of the cells, 75% of the cells, 80% of the cells, 85% of the cells, 90% of the cell, 95% of the cells, and 100% of the cells and any % between 50 and 100% when compared to a reference cell population.
Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
IV. EXEMPLARY EMBODIMENTS
Among the embodiments provided herein are:
1. An engineered immune cell comprising:
a genetically engineered antigen receptor that specifically binds to a target antigen that is a universal tumor antigen; and
a genetic disruption resulting in reduced expression of the target antigen in the engineered immune cell.
2. The engineered cell of embodiment 1, wherein the universal tumor antigen is a human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2/neu, Wilms' tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53 or cyclin (D1).
3. The engineered cell of embodiment 1 or embodiment 2, wherein the universal tumor antigen is hTERT or survivin.
4. An engineered immune cell comprising:
a genetically engineered antigen receptor that specifically binds to a target antigen, wherein the target antigen is B cell maturation antigen (BCMA), transmembrane activator and CAML Interactor (TACI) or B-cell activating factor receptor (BAFF-R); and
a genetic disruption resulting in reduced expression of the target antigen in the engineered immune cell.
5. The engineered immune cell of any of embodiments 1-4, wherein the genetic disruption comprises a disruption in a gene encoding the target antigen.
6. The engineered immune cell of any of embodiments 1-5, wherein the target antigen is a gene product that is capable of being naturally expressed in the cell type of the engineered immune cell.
7. The engineered immune cell of any of embodiments 1-6, wherein the target antigen is an antigen expressed by resting T cells, activated T cells, or both.
8. The engineered immune cell of any of embodiments 1-7, wherein the target antigen is an antigen expressed by activated T cells and expressed at a reduced level by resting T cells.
9. The engineered immune cell of embodiment 8, wherein expression of the target antigen by activated T cells at a level that is more than 50%, 60%, 70%, 80%, 90%, 95% or more and/or 1-fold, 1.5-fold, 2-fold, 3-fold or more compared to resting or non-activated T cells.
10. The engineered immune cell of embodiment 8 or embodiment 9, wherein the target antigen is not expressed on resting T cells.
11. An engineered immune cell comprising:
a genetically engineered antigen receptor that specifically binds to a disease-specific target antigen, wherein the receptor also binds or is suspected of binding to a related antigen, which is related to the target antigen; and
a genetic disruption in a gene encoding the related antigen and/or a genetic disruption that results in or is capable of resulting in reduced or repressed expression of the related antigen in the engineered immune cell.
12. The engineered immune cell of embodiment 11, wherein the target antigen is a B cell maturation antigen (BCMA) and the related antigen is a different BCMA family member.
13. The engineered immune cell of embodiment 12, wherein the BCMA family member is TACI or BAFF-R.
14. The engineered immune cell of any of embodiments 11-13, wherein the target antigen is a gene product that is not naturally expressed in the cell type of the engineered immune cell and the related antigen is a gene product that is naturally expressed in the cell type of the engineered immune cell.
15. The engineered immune cell of any of embodiments 1-14, wherein the immune cell is a T cell or an NK cell.
16. The engineered immune cell of any of embodiments 1-15, wherein the immune cell is a T cell that is a CD4+ or CD8+ T cell.
17. The engineered immune cell of any of embodiments 11-12, wherein the related antigen is an antigen that is expressed by resting T cells, activated T cells, or both.
18. The engineered immune cell of any of embodiments 11-17, wherein the related antigen is an antigen expressed by activated T cells and expressed at a reduced level by resting T cells.
19. The engineered immune cell of embodiment 18, wherein expression of the related antigen by activated T cells at a level that is more than 50%, 60%, 70%, 80%, 90%, 95% or more and/or 1-fold, 1.5-fold, 2-fold, 3-fold or more compared to resting or non-activated T cells.
20. The engineered immune cell of embodiment 18 or embodiment 19, wherein the related antigen is not expressed on resting T cells.
21. The engineered immune cell of any of embodiments 8-10 or 17-19, wherein:
the activated T cells are surface positive for a T cell activation marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L (CD154) and 4-1BB (CD137) and/or contain intracellular expression of a cytokine selected from the group consisting of IL-2, IFNgamma and TNF-alpha; and/or
the resting T cells are surface negative for a T cell activation marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L (CD154) and 4-1BB (CD137) or lack intracellular expression of a cytokine selected from the group consisting of IL-2, IFNgamma and TNF-alpha.
22. The cell of any of embodiments 1-21, wherein expression of the target antigen or the related antigen in the engineered immune cell is reduced by at least 50, 60, 70, 80, 90, or 95% as compared to the expression in the immune cell in the absence of said disruption.
23. The engineered immune cell of any of embodiments 1-22, wherein:
the target antigen is expressed on the cell surface in a cancer; or
the target antigen is expressed on or in a cell or tissue of a cancer.
24. The engineered immune cell of embodiment 23, wherein the cancer is a hematologic cancer, an immune cancer, a leukemia, a lymphoma, and/or a myeloma, which optionally is multiple myeloma.
25. The engineered immune cell of any of embodiments 1-24, wherein the genetically engineered antigen receptor is a T cell receptor (TCR) or a functional non-TCR antigen recognition receptor.
26. The engineered immune cell of any of embodiments 1-25, wherein the genetically engineered antigen receptor is a chimeric antigen receptor (CAR) comprising an extracellular antigen-recognition domain that specifically binds to the target antigen.
27. The engineered immune cell of any of embodiments 1-26, wherein the genetically engineered antigen receptor is a chimeric antigen receptor (CAR) comprising an extracellular antigen-recognition domain that specifically binds to a peptide of the target antigen in the context of a major histocompatibility complex (MHC) molecule.
28. The engineered immune cell of any of embodiments 1-27, wherein the genetically engineered antigen receptor is capable of inducing an activating signal to the engineered immune cell.
29. The engineered immune cell of embodiment 28, wherein the genetically engineered antigen receptor comprises an intracellular domain with an ITAM-containing motif.
30. The engineered immune cell of embodiment 29, wherein the intracellular signaling domain comprises an intracellular domain of a CD3-zeta (CD3ζ) chain.
31. The engineered immune cell of any of embodiments 28-30, wherein the genetically engineered receptor is a CAR and further comprises a costimulatory signaling region.
32. The engineered immune cell of embodiment 31, wherein the costimulatory signaling region comprises a signaling domain of CD28.
33. The engineered immune cell of any of embodiments 1-32, further comprising another genetically engineered antigen receptor, which is a chimeric costimulatory receptor that specifically binds to another antigen and is capable of inducing a costimulatory signal to the cell.
34. The engineered immune cell of any of embodiments 1-32, further comprising another genetically engineered antigen receptor, which is a chimeric inhibitory receptor that specifically binds to a second target antigen and is capable of inducing an inhibitory or immunosuppressive or repressive signal to the cell upon recognition of the other target antigen.
35. The engineered immune cell of embodiment 34, wherein the second target antigen is an antigen that is not expressed on the surface of a cancer cell or infected cell or the expression of which is downregulated on a cancer cell or an infected cell.
36. The engineered immune cell of embodiment 34 or embodiment 35, wherein the second target antigen is an MHC-class I molecule.
37. The engineered immune cell of any of embodiments 34-36, wherein the genetically engineered antigen receptor is a chimeric antigen receptor (CAR).
38. The engineered immune cell of embodiment 37, wherein the CAR comprises an extracellular antigen-recognition domain that specifically binds to the target antigen and an intracellular signaling domain that comprises a signaling portion of an immune checkpoint molecule.
39. The engineered immune cell of embodiment 38, wherein the immune checkpoint molecule is PD-1 or CTLA4.
40. A method of producing a genetically engineered immune cell, comprising:
(a) introducing into an immune cell a genetically engineered antigen receptor that specifically binds to a target antigen that is a universal tumor antigen; and
(b) effecting repression of expression of the target antigen in the immune cell,
thereby producing a genetically engineered immune cell in which expression of the target antigen is repressed,
wherein steps (a) and (b) are carried out simultaneously or sequentially in any order.
41. The method of embodiment 40, wherein the universal tumor antigen is human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2/neu, Wilms' tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53 or cyclin (D1).
42. The method of embodiment 40 or embodiment 41, wherein the universal tumor antigen is hTERT or survivin.
43. A method of producing a genetically engineered immune cell, comprising:
(a) introducing into an immune cell a genetically engineered antigen receptor that specifically binds to a target antigen, wherein the target antigen is BCMA, TACI or BAFF receptor; and
(b) effecting repression of expression of the target antigen in the immune cell,
thereby producing a genetically engineered immune cell in which expression of the target antigen is repressed,
wherein steps (a) and (b) are carried out simultaneously or sequentially in any order.
44. The method of any of embodiments 40-43, wherein the target antigen is a gene product that is capable of being naturally expressed in the cell type of the engineered immune cell.
45. The method of any of embodiments 40-44, wherein the target antigen is an antigen expressed by resting T cells, activated T cells, or both.
46. The method of any of embodiments 40-45, wherein the target antigen is an antigen expressed by activated T cells and expressed at a reduced level by resting T cells.
47. The method of embodiment 46, wherein expression of the target antigen by activated T cells at a level that is more than 50%, 60%, 70%, 80%, 90%, 95% or more and/or 1-fold, 1.5-fold, 2-fold, 3-fold or more compared to resting or non-activated T cells.
48. The method of embodiment 46 or embodiment 47, wherein the target antigen is not expressed on resting T cells.
49. A method of producing a genetically engineered immune cell, comprising:
(a) introducing into an immune cell a genetically engineered antigen receptor that specifically binds to a target antigen, wherein the target antigen is BCMA, TACI or BAFF receptor; and
(b) effecting repression of expression of a related antigen in the immune cell, wherein the receptor also binds is suspected of binding to the related antigen, which is related to the target antigen,
thereby producing a genetically engineered immune cell in which expression of the related antigen is repressed,
wherein steps (a) and (b) are carried out simultaneously or sequentially in any order.
50. The method of embodiment 49, wherein the target antigen is a B cell maturation antigen (BCMA) and the related antigen is a different BCMA family member.
51. The method of embodiment 50, wherein the BCMA family member is TACI or BAFF-R.
52. The method of any of embodiments 49-51, wherein the target antigen is a gene product that is not naturally expressed in the cell type of the engineered immune cell and the related antigen is a gene product that is naturally expressed in the cell type of the engineered immune cell.
53. The method of any of embodiments 40-52, wherein the immune cell is a T cell or an NK cell.
54. The method of any of embodiments 40-53, wherein the immune cell is a T cell that is a CD4+ or CD8+ T cell.
55. The method of any of embodiments 49-54, wherein the related antigen is an antigen that is expressed by resting T cells, activated T cells, or both.
56. The method of any of embodiments 49-55, wherein the related antigen is an antigen expressed by activated T cells and expressed at a reduced level by resting T cells.
57. The method of embodiment 56, wherein expression of the related antigen by activated T cells is at a level that is more than 50%, 60%, 70%, 80%, 90%, 95% or more and/or 1-fold, 1.5-fold, 2-fold, 3-fold or more compared to resting or non-activated T cells.
58. The method of embodiment 56 or embodiment 57, wherein the related antigen is not expressed on resting T cells.
59. The method of any of embodiments 45-48 and 55-58, wherein:
the activated T cells are surface positive for a T cell activation marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L (CD154) and 4-1BB (CD137) and/or contain intracellular expression of a cytokine selected from the group consisting of IL-2, IFNgamma and TNF-alpha; and/or
the resting T cells are surface negative for a T cell activation marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L (CD154) and 4-1BB (CD137) or lack intracellular expression of a cytokine selected from the group consisting of IL-2, IFNgamma and TNF-alpha.
60. The method of any of embodiments 40-59, wherein the effecting in (b) comprises disrupting a gene encoding the target antigen or the related antigen.
61. The method of embodiment 60, wherein:
the disruption comprises disrupting the gene at the DNA level and/or
the disruption is not reversible; and/or
the disruption is not transient.
62. The method of embodiment 60 or 61, wherein the disruption comprises introducing into the immune cell a DNA binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to the gene.
63. The method of embodiment 62, wherein the disruption comprises introducing: (a) a fusion protein comprising a DNA-targeting protein and a nuclease or (b) an RNA-guided nuclease.
64. The method of embodiment 63, wherein the DNA-targeting protein or RNA-guided nuclease comprises a zinc finger protein (ZFP), a TAL protein, or a clustered regularly interspaced short palindromic nucleic acid (CRISPR) specific for the gene.
65. The method of any of embodiments 40-64, wherein the disruption comprises introducing a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or and a CRISPR-Cas9 combination that specifically binds to, recognizes, or hybridizes to the gene.
66. The method of any of embodiments 40-65, wherein the target antigen is a gene product that is naturally expressed in the immune cell and/or the expression of which is induced by said introduction of said genetically engineered antigen receptor.
67. The method of any of embodiments 49-65, wherein the target antigen is a gene product that is not naturally expressed in the cell type of the engineered immune cell and the related antigen is a gene product that is naturally expressed in the cell type of the engineered immune cell.
68. The method of any of embodiments 40-67, wherein said repression reduces expression of the target antigen or the related antigen in the engineered immune cell by at least 50, 60, 70, 80, 90, or 95% as compared to an engineered cell produced by the method in the absence of the repression.
69. The method of any of embodiments 40-68, wherein the target antigen is expressed in multiple myeloma.
70. The method of any of embodiments 40-69, further comprising:
(c) introducing into the immune cell another genetically engineered antigen receptor, which is a chimeric costimulatory receptor that specifically binds to another antigen and is capable of inducing a costimulatory signal to the cell, wherein steps (a), (b) and (c) are carried out simultaneously or sequentially in any order.
71. The method of any of embodiments 40-70, wherein the genetically engineered antigen receptor is a T cell receptor (TCR) or a functional non-TCR antigen recognition receptor.
72. The method of any of embodiments 40-71, wherein the genetically engineered antigen receptor is a chimeric antigen receptor (CAR).
73. A cell produced by the method of any of embodiments 40-72.
74. A method of producing a genetically engineered immune cell, the method comprising:
(a) introducing into an immune cell a first genetically engineered antigen receptor that specifically binds to a first antigen; and
(b) introducing into the immune cell a second genetically engineered antigen receptor, which is a chimeric costimulatory receptor and specifically binds to a second antigen,
thereby producing the engineered immune cell,
wherein the first and second antigens are distinct and at least the first or the second antigen is a universal tumor antigen and (a) and (b) are carried out simultaneously or sequentially in any order.
75. The method of embodiment 74, wherein the universal tumor antigen is human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2/neu, Wilms' tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53 or cyclin (D1).
76. The method of embodiment 74 or embodiment 75, wherein the universal tumor antigen is hTERT or survivin.
77. A method of producing a genetically engineered immune cell, the method comprising:
(a) introducing into an immune cell a first genetically engineered antigen receptor that specifically binds to a first antigen; and
(b) introducing into the immune cell a second genetically engineered antigen receptor, which is a chimeric costimulatory receptor and specifically binds to a second antigen, thereby producing the engineered immune cell,
wherein the first and second antigens are distinct and at least the first or the second antigen is B cell maturation antigen (BCMA), transmembrane activator and CAML Interactor (TACI) or B-cell activating factor receptor (BAFF-R) and (a) and (b) are carried out simultaneously or sequentially in any order.
78. A method of producing a genetically engineered immune cell, the method comprising:
(a) introducing into an immune cell a first genetically engineered antigen receptor that specifically binds to a first antigen; and
(b) introducing into the immune cell a second genetically engineered antigen receptor that specifically binds to a second antigen,
thereby producing the engineered immune cell,
wherein the first and second antigens are distinct and at least the first or the second antigen is B cell maturation antigen (BCMA), transmembrane activator and CAML Interactor (TACI) or B-cell activating factor receptor (BAFF-R) and (a) and (b) are carried out simultaneously or sequentially in any order.
79. The method of any of embodiments 74-78, wherein the other of the first or second antigen is an antigen expressed in a tumor.
80. The method of any of embodiments 77-79, wherein the first or second antigen is BCMA.
81. The method of embodiment 80, wherein the other of the first or second antigen is a BCMA-related family member.
82. The method of embodiment 81, wherein the BCMA-related family member is TACI or BAFF-R.
83. The method of any of embodiments 78-82, wherein the other of the first or the second antigen is FcRH5, CS1, CD38 or CD138.
84. The method of any of embodiments 74-83, further comprising (c) effecting repression of expression of the first and/or second antigen in the immune cell.
85. The method of embodiment 84, wherein effecting repression comprises introducing into the immune cell a DNA binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to the gene.
86. The method of embodiment 84 or embodiment 85, wherein effecting repression comprises introducing a D a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or and a CRISPR-Cas9 combination that specifically binds to, recognizes, or hybridizes to the gene.
87. The method of any of embodiments 84-86, wherein said repression reduces expression of the target antigen in the engineered immune cell by at least 50, 60, 70, 80, 90, or 95% as compared to an engineered cell produced by the method in the absence of the repression.
88. A cell produced by the method of any of embodiments 74-87.
89. An engineered immune cell, comprising:
(a) a first genetically engineered antigen receptor that specifically binds to a first antigen and is capable of inducing an activating signal to the cell; and
(b) a second genetically engineered antigen receptor which is a chimeric costimulatory receptor that specifically binds to a second antigen and is capable of inducing a costimulatory signal to the cell,
wherein the first and second antigens are distinct and at least one of the first or the second antigen is a universal tumor antigen.
90. The engineered immune cell of embodiment 89, wherein the universal tumor antigen is human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2/neu, Wilms' tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53 or cyclin (D1).
91. The engineered immune cell of embodiment 89 or embodiment 90, wherein the universal tumor antigen is hTERT or survivin.
92. An engineered immune cell, comprising:
(a) a first genetically engineered antigen receptor that specifically binds to a first antigen and is capable of inducing an activating signal to the cell; and
(b) a second genetically engineered antigen receptor which is a chimeric costimulatory receptor that specifically binds to a second antigen and is capable of inducing a costimulatory signal to the cell,
wherein the first and second antigens are distinct and at least one of the first or the second antigen is B cell maturation antigen (BCMA), transmembrane activator and CAML Interactor (TACI) or B-cell activating factor receptor (BAFF-R).
93. An engineered immune cell, comprising:
(a) a first genetically engineered antigen receptor that specifically binds to a first antigen; and
(b) a second genetically engineered antigen receptor that specifically binds to a second antigen;
wherein the first and second antigens are distinct and at least one of the first or the second antigen is a B cell maturation antigen (BCMA), transmembrane activator and CAML Interactor (TACI) or B-cell activating factor receptor (BAFF-R).
94. The engineered immune cell of any of embodiments 89-93, wherein the other of the first or second antigen is an antigen expressed in a tumor.
95. The engineered immune cell of any of embodiments 92-94, wherein the first or second antigen is BCMA.
96. The engineered immune cell of any of embodiments 92-95, wherein the other of the first or second antigen is a BCMA-related family member.
97. The engineered immune cell of embodiment 96, wherein the BCMA related family member is TACI or BAFF-R.
98. The engineered immune cell of any of embodiments 92-95, wherein the other of the first or the second antigen is FcRH5, CS1, CD38 or CD138.
99. The engineered immune cell of any of embodiments 89-98, wherein the first genetically engineered antigen receptor comprises an ITAM-containing sequence.
100. The engineered immune cell of embodiment 99, wherein the first genetically engineered antigen receptor comprises an intracellular signaling domain of a CD3-zeta (CD3ζ) chain.
101. The engineered immune cell of embodiment 99 or embodiment 100, wherein the first genetically engineered antigen receptor does not comprise a signaling domain from a T cell costimulatory molecule.
102. The engineered immune cell of any of embodiments 89-101, wherein the receptor comprises an intracellular signaling domain of a T cell costimulatory molecule.
103. The engineered immune cell of embodiment 102, wherein the T cell costimulatory molecule comprises one or more molecules selected from the group consisting of CD28 and 41BB.
104. The engineered immune cell of any of embodiments89-103, further comprising a disruption in a gene encoding the first antigen, and/or in a gene encoding the second antigen, said disruption resulting in reduced expression of the first and/or second antigen in the engineered immune cell.
105. The engineered immune cell of embodiment 104, wherein expression of the first and/or second antigen in the engineered immune cell is reduced by at least 50, 60, 70, 80, 90, or 95% as compared to the expression in the immune cell in the absence of said gene disruption.
106. The engineered immune cell of any of embodiments 89-105, wherein the immune cell is a T cell.
107. The engineered immune cell of embodiment 106, wherein the immune cell is a CD4+ or CD8+ T cell.
108. The engineered immune cell of any of embodiments 89-107, wherein the first genetically engineered antigen receptor is a T cell receptor (TCR) or a functional non-TCR antigen recognition receptor.
109. The engineered immune cell of embodiment 108, wherein the first genetically engineered antigen receptor is a chimeric antigen receptor (CAR).
110. A pharmaceutical composition comprising the engineered immune cell of any of embodiments 1-39 and any of embodiments 89-109 and a pharmaceutically acceptable carrier.
111. A composition or combination, comprising:
(a) a first engineered immune cell expressing a first genetically engineered antigen receptor that specifically binds to a first antigen and not expressing a second engineered antigen receptor that specifically binds to a second antigen; and
(b) a second engineered immune cell expressing the genetically engineered antigen receptor,
wherein the first and second antigens are distinct and at least one of the first or the second antigen is a B cell maturation antigen (BCMA), transmembrane activator and CAML Interactor (TACI) or B-cell activating factor receptor (BAFF-R).
112. The composition or combination of embodiment 111, wherein the first or second antigen is BCMA.
113. The composition or combination of embodiment 112, wherein the other of the first or second antigen is a BCMA-related family member.
114. The composition or combination of embodiment 113, wherein the BCMA related family member is TACI or BAFF-R.
115. The composition or combination of embodiment 111 or 112, wherein the other of the first or the second antigen is FcRH5, CS1, CD38 or CD138.
116. The composition or combination of any of embodiments 111-115 that is a pharmaceutical composition.
117. A method of treatment, comprising administering to a subject having a disease or condition the cell of any of embodiments 1-39 and 89-109 or the composition or combiantion of any of embodiments 111-116.
118. The method of embodiment 117, wherein the genetically engineered antigen receptor or receptors specifically binds to an antigen associated with the disease or condition.
119. The method of embodiment 117 or embodiment 118, wherein the disease or condition is a cancer.
120. Use of cells of any of embodiments 1-39 and 89-109, the composition of embodiment 110 or the composition or combination of any of embodiments 111-116 in the manufacture of a medicament for use in a method for treating a disease or condition.
121. A composition of embodiment 110 or the composition or combination of any of embodiments 111-116 for use in treating a disease or condition.
122. The use of embodiment 120 or composition or combination of embodiment 121, wherein the genetically engineered antigen receptor or receptors specifically bind to an antigen associated with the disease or condition.
123. The use, composition or combination of embodiment 121 or embodiment 122, wherein the disease or condition is a tumor or a cancer.
V. EXAMPLES
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1: Assessment of Cytotoxic Activity of T-Cells Engineered With a Chimeric Antigen Receptor (CAR) Specific for hTERT in the Presence or Absence of Endogenous hTERT Repression
Exemplary studies are carried out to assess of T cells expressing an engineered antigen receptor specific for a particular antigen, which is expressed in or on T cells, following repression of the expression of and/or genetic disruption or knockout of such antigen in the T cells. This exemplary study is carried out by disrupting expression of the universal tumor protein antigen, human telomerase reverse transcriptase (hTERT). In other studies, similar methods are carried out by disrupting other antigen targets of interest (in cells expressing a genetically engineered antigen receptor recognizing such antigen), such as other universal protein antigens (e.g. survivin) and/or other antigens of interest including those naturally expressed in T cells and/or activated T cells (e.g. CD38).
T cells are isolated by immunoaffinity-based selection from a human apheresis product sample from a subject expressing the HLA-A*0201 allele, such as, in some cases, a subject having a cancer. The resulting cells are activated using an anti-CD3/anti-CD28 reagent in the presence of IL-2 (100 IU/mL), for example, for 72 hours at 37° C.
The CRISPR/Cas9 technology is used to knock-down gene expression of hTERT in the activated T cells. In an exemplary method, Cas9 nuclease (e.g., encoded by mRNA from Staphylococcus aureus or from Stretpococcus pyogenes, e.g. pCW-Cas9, Addgene #50661, Wang et al. (2014) Science, 3:343-80-4; or nuclease or nickase lentiviral vectors available from Applied Biological Materials (ABM; Canada) as Cat. No. K002, K003, K005 or K006) and hTERT guide RNA (gRNA, e.g. exemplary gRNA vector available from ABM, Cat. No. K0009811) are introduced into cells, for example, using lentiviral delivery vectors or any of a number of known delivery method or vehicle for transfer to cells, such as any of a number of known methods or vehicles for delivering Cas9 molecules and guide RNAs. Non-specific or empty vector control T cells also are generated. Degree of Knockout of hTERT (e.g., 24 to 72 hours after transfer) is assessed using any of a number of well-known assays for assessing gene disruption in cells.
Within 24 to 96 hours after transduction of CRISPR/Cas9 system, cells (hTERT knock-down and control cells) are transduced with an empty vector or with a viral vector encoding an hTERT antigen receptor, such as a T cell receptor (TCR) or an anti-hTERT chimeric TCR-like antibody or binding fragment thereof (e.g. scFv), that specifically bind an hTERT-derived peptide epitope in the context of HLA-A*0201, e.g. as described in U.S. Pat. No. 7,718,777, including antibody binding molecules specific for any of the peptides set forth in SEQ ID NOS: 7, 8 or 10-14. Thus, in total, four groups of cells are generated as follows: 1) hTERT knockout (without genetically engineered receptor); 2) hTERT knockout/genetically engineered hTERT-antigen receptor; 3) hTERT wildtype T cells/genetically engineered hTERT-antigen receptor; and hTERT wildtype (without genetically engineered receptor) (control).
Following the introduction, cells are further cultured, generally at 37 degrees C., for example, to allow for cell expansion. Antigen-induced cytotoxicity and/or activation of cells in the culture of each group is assessed by performing a cell proliferation assay, a chromium release assay, ELISPOT assay for IFN-γ, granzyme B and/or perforin and/or an assay for cell viability, such as by using CellTiter-Glo® (CTG)-assay or other assay that measures proliferation, viability and/or cytotoxicity of cells. The cytotoxicity of cells is compared among the different groups of generated cells. As a positive control for cytotoxicity or activation or other function, cells known to express hTERT and/or to present a peptide derived therefrom recognize by the cell, are included to confirm the hTERT-specific function(s) of the engineered cells assessed.
In additional studies, cells in the various condition groups are administered to animal subjects and their persistence followed over time and compared, to assess impacts of the knockout on self-killing by such cells and persistence of such cells over time, and efficacy in targeting cells expressing the target antigen, such as tumor cells.
Optionally, autologous hTERT knockout/hTERT-antigen specific T cells engineered as above, which are found to avoid or exhibit reduced potential for self-killing in culture, are administered to the subject to treat the cancer at a dosage of, for example, 1×107 cells to 5×1010 cells.
Example 2: Assessment of Cytotoxic Activity of T-Cells Engineered With a Chimeric Antigen Receptor (CAR) Specific for a Disease-Specific Target Antigen in the Presence or Absence of Endogenous Repression of Another Related Antigen
Exemplary studies are carried out to assess activities and properties of T cells expressing an engineered antigen receptor specific for a particular disease-specific target antigen that is known to or that may be cross-reactive to a related antigen that shares similarity or epitopes with the target antigen. The T cells are also engineered to effect repression of the expression and/or genetic disruption or knockout of such related antigen in the T cells. Specifically, in one exemplary study, T cells are engineered to express a chimeric antigen receptor (CAR) that specifically binds BCMA and also are disrupted for expression of one or more other related antigens, such as BAFF-R or TACI.
T cells are isolated by immunoaffinity-based selection from a human apheresis product sample from a subject, such as, in some cases, a subject having a cancer. The resulting cells are activated using an anti-CD3/anti-CD28 reagent in the presence of IL-2 (100 IU/mL), for example, for 72 hours at 37° C.
The CRISPR/Cas9 technology is used to knock-down gene expression of BAFF-R or TACI in the activated T cells. In an exemplary method, Cas9 nuclease (e.g., encoded by mRNA from Staphylococcus aureus or from Stretpococcus pyogenes, e.g. pCW-Cas9, Addgene #50661, Wang et al. (2014) Science, 3:343-80-4; or nuclease or nickase lentiviral vectors available from Applied Biological Materials (ABM; Canada) as Cat. No. K002, K003, K005 or K006) and either TACI guide RNA (gRNA, e.g. exemplary reagents available from Origene, Cat. No. KN204672) or BAFF-R guide RNA (gRNA, e.g. exemplary reagents available from Origene, Cat. No. KN211270) are introduced into cells, for example, using lentiviral delivery vectors or any of a number of known delivery method or vehicle for transfer to cells, such as any of a number of known methods or vehicles for delivering Cas9 molecules and guide RNAs. Non-specific or empty vector control T cells also are generated. Degree of Knockout of TACI or BAFF-R (e.g., 24 to 72 hours after transfer) is assessed using any of a number of well-known assays for assessing gene disruption in cells.
Within 24 to 96 hours after transduction of CRISPR/Cas9 system, cells (TACI knockout, BAFF-R knockout and control cells) are transduced with an empty vector or with a viral vector encoding a BCMA-specific antigen receptor, such as chimeric antigen receptor containing an anti-BCMA antibody or binding fragment thereof (e.g. scFv) that specifically binds BCMA. Thus, in total, six groups of cells are generated as follows: 1) TACI knockout (without genetically engineered receptor); 2) TACI knockout/genetically engineered BCMA-specific antigen receptor; 3) BAFF-R knockout (without genetically engineered receptor); 4) BAFF-R knockout/genetically engineered BCMA-specific antigen receptor; 5) wildtype T cells/genetically engineered BCMA-specific antigen receptor; and 6) wildtype cells (without genetically engineered receptor) (control).
Following the introduction, cells are further cultured, generally at 37 degrees C., for example, to allow for cell expansion. Antigen-induced cytotoxicity and/or activation of cells in the culture of each group is assessed by performing a cell proliferation assay, a chromium release assay, ELISPOT assay for IFN-γ, granzyme B and/or perforin and/or an assay for cell viability, such as by using CellTiter-Glo® (CTG)-assay or other assay that measures proliferation, viability and/or cytotoxicity of cells. The cytotoxicity of cells is compared among the different groups of generated cells. As a positive control for cytotoxicity or activation or other function, cells known to express BCMA are included to confirm the BCMA-specific function(s) of the engineered cells assessed.
In additional studies, cells in the various condition groups are administered to animal subjects and their persistence followed over time and compared, to assess impacts of the knockout on self-killing by such cells and persistence of such cells over time, and efficacy in targeting cells expressing the target antigen, such as tumor cells.
Optionally, autologous TACI and/or BAFF-R knockout and BCMA-specific antigen specific T cells engineered as above, which are found to avoid or exhibit reduced potential for self-killing in culture, are administered to the subject to treat the cancer at a dosage of, for example, 1×107 cells to 5×1010 cells.
The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the compositions and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.
SEQUENCE TABLE
SEQ
ID
NO:
Type
Sequence
Description
1
Protein
mancefspvs gdkpccrlsr raqlclgvsi lvlilvvvla
CD38;
vvvprwrqqw sgpgttkrfp etvlarcvky teihpemrhv
GenBank:
dcqsvwdafk gafiskhpcn iteedyqplm klgtqtvpcn
BAA18966
killwsrikd lahqftqvqr dmftledtll gyladdltwc
gefntskiny qscpdwrkdc snnpvsvfwk tvsrrfaeaa
cdvvhvmlng srskifdkns tfgsvevhnl qpekvqtlea
wvihggreds rdlcqdptik elesiiskrn iqfsckniyr
pdkflqcvkn pedssctsei
2
Nucleotide
AGTGAAACAGAAGGGGAGGTGCAGTTTCAGAACCCAGCCAGCCTCTCTC
CD38;
TTGCTGCCTAGCCTCCTGCCG
GenBank
GCCTCATCTTCGCCCAGCCAACCCCGCCTGGAGCCCTATGGCCAACTGC
NM_001775.2
GAGTTCAGCCCGGTGTCCGGG
GACAAACCCTGCTGCCGGCTCTCTAGGAGAGCCCAACTCTGTCTTGGCG
TCAGTATCCTGGTCCTGATCC
TCGTCGTGGTGCTCGCGGTGGTCGTCCCGAGGTGGCGCCAGCAGTGGAG
CGGTCCGGGCACCACCAAGCG
CTTTCCCGAGACCGTCCTGGCGCGATGCGTCAAGTACACTGAAATTCAT
CCTGAGATGAGACATGTAGAC
TGCCAAAGTGTATGGGATGCTTTCAAGGGTGCATTTATTTCAAAACATC
CTTGCAACATTACTGAAGAAG
ACTATCAGCCACTAATGAAGTTGGGAACTCAGACCGTACCTTGCAACAA
GATTCTTCTTTGGAGCAGAAT
AAAAGATCTGGCCCATCAGTTCACACAGGTCCAGCGGGACATGTTCACC
CTGGAGGACACGCTGCTAGGC
TACCTTGCTGATGACCTCACATGGTGTGGTGAATTCAACACTTCCAAAA
TAAACTATCAATCTTGCCCAG
ACTGGAGAAAGGACTGCAGCAACAACCCTGTTTCAGTATTCTGGAAAAC
GGTTTCCCGCAGGTTTGCAGA
AGCTGCCTGTGATGTGGTCCATGTGATGCTCAATGGATCCCGCAGTAAA
ATCTTTGACAAAAACAGCACT
TTTGGGAGTGTGGAAGTCCATAATTTGCAACCAGAGAAGGTTCAGACAC
TAGAGGCCTGGGTGATACATG
GTGGAAGAGAAGATTCCAGAGACTTATGCCAGGATCCCACCATAAAAGA
GCTGGAATCGATTATAAGCAA
AAGGAATATTCAATTTTCCTGCAAGAATATCTACAGACCTGACAAGTTT
CTTCAGTGTGTGAAAAATCCT
GAGGATTCATCTTGCACATCTGAGATCTGAGCCAGTCGCTGTGGTTGTT
TTAGCTCCTTGACTCCTTGTG
GTTTATGTCATCATACATGACTCAGCATACCTGCTGGTGCAGAGCTGAA
GATTTTGGAGGGTCCTCCACA
ATAAGGTCAATGCCAGAGACGGAAGCCTTTTTCCCCAAAGTCTTAAAAT
AACTTATATCATCAGCATACC
TTTATTGTGATCTATCAATAGTCAAGAAAAATTATTGTATAAGATTAGA
ATGAAAATTGTATGTTAAGTT
ACTTCACTTTAATTCTCATGTGATCCTTTTATGTTATTTATATATTGGT
AACATCCTTTCTATTGAAAAA
TCACCACACCAAACCTCTCTTATTAGAACAGGCAAGTGAAGAAAAGTGA
ATGCTCAAGTTTTTCAGAAAG
CATTACATTTCCAAATGAATGACCTTGTTGCATGATGTATTTTTGTACC
CTTCCTACAGATAGTCAAACC
ATAAACTTCATGGTCATGGGTAAA
3
Protein
MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRA
Telomerase
LVAQCLVCVPWDARPPPAAPS
reverse
FRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSV
transcriptase
RSYLPNTVTDALRGSGAWGLL
(hTERT);
LRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPH
GenBank
ASGPRRRLGCERAWNHSVREA
NP_937983.2
GVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHP
GRTRGPSDRGFCVVSPARPAE
EATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETK
HFLYSSGDKEQLRPSFLLSSL
RPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGN
HAQCPYGVLLKTHCPLRAAVT
PAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACL
RRLVPPGLWGSRHNERRFLRN
TKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREE
ILAKFLHWLMSVYVVELLRSF
FYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQH
REARPALLTSRLRFIPKPDGL
RPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGAS
VLGLDDIHRAWRTFVLRVRAQ
DPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQK
AAHGHVRKAFKSHVSTLTDLQ
PYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVR
IRGKSYVQCQGIPQGSILSTL
LCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVR
GVPEYGCVVNLRKTVVNFPVE
DEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLT
FNRGFKAGRNMRRKLFGVLRL
KCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNP
TFFLRVISDTASLCYSILKAK
NAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTA
QTQLSRKLPGTTLTALEAAAN
PALPSDFKTILD
4
Nucleotide
CAGGCAGCGCTGCGTCCTGCTGCGCACGTGGGAAGCCCTGGCCCCGGCC
telomerase
ACCCCCGCGATGCCGCGCGCT
reverse
CCCCGCTGCCGAGCCGTGCGCTCCCTGCTGCGCAGCCACTACCGCGAGG
transcriptase
TGCTGCCGCTGGCCACGTTCG
(hTERT),
TGCGGCGCCTGGGGCCCCAGGGCTGGCGGCTGGTGCAGCGCGGGGACCC
GenBank
GGCGGCTTTCCGCGCGCTGGT
NM_198253.2
GGCCCAGTGCCTGGTGTGCGTGCCCTGGGACGCACGGCCGCCCCCCGCC
GCCCCCTCCTTCCGCCAGGTG
TCCTGCCTGAAGGAGCTGGTGGCCCGAGTGCTGCAGAGGCTGTGCGAGC
GCGGCGCGAAGAACGTGCTGG
CCTTCGGCTTCGCGCTGCTGGACGGGGCCCGCGGGGGCCCCCCCGAGGC
CTTCACCACCAGCGTGCGCAG
CTACCTGCCCAACACGGTGACCGACGCACTGCGGGGGAGCGGGGCGTGG
GGGCTGCTGCTGCGCCGCGTG
GGCGACGACGTGCTGGTTCACCTGCTGGCACGCTGCGCGCTCTTTGTGC
TGGTGGCTCCCAGCTGCGCCT
ACCAGGTGTGCGGGCCGCCGCTGTACCAGCTCGGCGCTGCCACTCAGGC
CCGGCCCCCGCCACACGCTAG
TGGACCCCGAAGGCGTCTGGGATGCGAACGGGCCTGGAACCATAGCGTC
AGGGAGGCCGGGGTCCCCCTG
GGCCTGCCAGCCCCGGGTGCGAGGAGGCGCGGGGGCAGTGCCAGCCGAA
GTCTGCCGTTGCCCAAGAGGC
CCAGGCGTGGCGCTGCCCCTGAGCCGGAGCGGACGCCCGTTGGGCAGGG
GTCCTGGGCCCACCCGGGCAG
GACGCGTGGACCGAGTGACCGTGGTTTCTGTGTGGTGTCACCTGCCAGA
CCCGCCGAAGAAGCCACCTCT
TTGGAGGGTGCGCTCTCTGGCACGCGCCACTCCCACCCATCCGTGGGCC
GCCAGCACCACGCGGGCCCCC
CATCCACATCGCGGCCACCACGTCCCTGGGACACGCCTTGTCCCCCGGT
GTACGCCGAGACCAAGCACTT
CCTCTACTCCTCAGGCGACAAGGAGCAGCTGCGGCCCTCCTTCCTACTC
AGCTCTCTGAGGCCCAGCCTG
ACTGGCGCTCGGAGGCTCGTGGAGACCATCTTTCTGGGTTCCAGGCCCT
GGATGCCAGGGACTCCCCGCA
GGTTGCCCCGCCTGCCCCAGCGCTACTGGCAAATGCGGCCCCTGTTTCT
GGAGCTGCTTGGGAACCACGC
GCAGTGCCCCTACGGGGTGCTCCTCAAGACGCACTGCCCGCTGCGAGCT
GCGGTCACCCCAGCAGCCGGT
GTCTGTGCCCGGGAGAAGCCCCAGGGCTCTGTGGCGGCCCCCGAGGAGG
AGGACACAGACCCCCGTCGCC
TGGTGCAGCTGCTCCGCCAGCACAGCAGCCCCTGGCAGGTGTACGGCTT
CGTGCGGGCCTGCCTGCGCCG
GCTGGTGCCCCCAGGCCTCTGGGGCTCCAGGCACAACGAACGCCGCTTC
CTCAGGAACACCAAGAAGTTC
ATCTCCCTGGGGAAGCATGCCAAGCTCTCGCTGCAGGAGCTGACGTGGA
AGATGAGCGTGCGGGACTGCG
CTTGGCTGCGCAGGAGCCCAGGGGTTGGCTGTGTTCCGGCCGCAGAGCA
CCGTCTGCGTGAGGAGATCCT
GGCCAAGTTCCTGCACTGGCTGATGAGTGTGTACGTCGTCGAGCTGCTC
AGGTCTTTCTTTTATGTCACG
GAGACCACGTTTCAAAAGAACAGGCTCTTTTTCTACCGGAAGAGTGTCT
GGAGCAAGTTGCAAAGCATTG
GAATCAGACAGCACTTGAAGAGGGTGCAGCTGCGGGAGCTGTCGGAAGC
AGAGGTCAGGCAGCATCGGGA
AGCCAGGCCCGCCCTGCTGACGTCCAGACTCCGCTTCATCCCCAAGCCT
GACGGGCTGCGGCCGATTGTG
AACATGGACTACGTCGTGGGAGCCAGAACGTTCCGCAGAGAAAAGAGGG
CCGAGCGTCTCACCTCGAGGG
TGAAGGCACTGTTCAGCGTGCTCAACTACGAGCGGGCGCGGCGCCCCGG
CCTCCTGGGCGCCTCTGTGCT
GGGCCTGGACGATATCCACAGGGCCTGGCGCACCTTCGTGCTGCGTGTG
CGGGCCCAGGACCCGCCGCCT
GAGCTGTACTTTGTCAAGGTGGATGTGACGGGCGCGTACGACACCATCC
CCCAGGACAGGCTCACGGAGG
TCATCGCCAGCATCATCAAACCCCAGAACACGTACTGCGTGCGTCGGTA
TGCCGTGGTCCAGAAGGCCGC
CCATGGGCACGTCCGCAAGGCCTTCAAGAGCCACGTCTCTACCTTGACA
GACCTCCAGCCGTACATGCGA
CAGTTCGTGGCTCACCTGCAGGAGACCAGCCCGCTGAGGGATGCCGTCG
TCATCGAGCAGAGCTCCTCCC
TGAATGAGGCCAGCAGTGGCCTCTTCGACGTCTTCCTACGCTTCATGTG
CCACCACGCCGTGCGCATCAG
GGGCAAGTCCTACGTCCAGTGCCAGGGGATCCCGCAGGGCTCCATCCTC
TCCACGCTGCTCTGCAGCCTG
TGCTACGGCGACATGGAGAACAAGCTGTTTGCGGGGATTCGGCGGGACG
GGCTGCTCCTGCGTTTGGTGG
ATGATTTCTTGTTGGTGACACCTCACCTCACCCACGCGAAAACCTTCCT
CAGGACCCTGGTCCGAGGTGT
CCCTGAGTATGGCTGCGTGGTGAACTTGCGGAAGACAGTGGTGAACTTC
CCTGTAGAAGACGAGGCCCTG
GGTGGCACGGCTTTTGTTCAGATGCCGGCCCACGGCCTATTCCCCTGGT
GCGGCCTGCTGCTGGATACCC
GGACCCTGGAGGTGCAGAGCGACTACTCCAGCTATGCCCGGACCTCCAT
CAGAGCCAGTCTCACCTTCAA
CCGCGGCTTCAAGGCTGGGAGGAACATGCGTCGCAAACTCTTTGGGGTC
TTGCGGCTGAAGTGTCACAGC
CTGTTTCTGGATTTGCAGGTGAACAGCCTCCAGACGGTGTGCACCAACA
TCTACAAGATCCTCCTGCTGC
AGGCGTACAGGTTTCACGCATGTGTGCTGCAGCTCCCATTTCATCAGCA
AGTTTGGAAGAACCCCACATT
TTTCCTGCGCGTCATCTCTGACACGGCCTCCCTCTGCTACTCCATCCTG
AAAGCCAAGAACGCAGGGATG
TCGCTGGGGGCCAAGGGCGCCGCCGGCCCTCTGCCCTCCGAGGCCGTGC
AGTGGCTGTGCCACCAAGCAT
TCCTGCTCAAGCTGACTCGACACCGTGTCACCTACGTGCCACTCCTGGG
GTCACTCAGGACAGCCCAGAC
GCAGCTGAGTCGGAAGCTCCCGGGGACGACGCTGACTGCCCTGGAGGCC
GCAGCCAACCCGGCACTGCCC
TCAGACTTCAAGACCATCCTGGACTGATGGCCACCCGCCCACAGCCAGG
CCGAGAGCAGACACCAGCAGC
CCTGTCACGCCGGGCTCTACGTCCCAGGGAGGGAGGGGCGGCCCACACC
CAGGCCCGCACCGCTGGGAGT
CTGAGGCCTGAGTGAGTGTTTGGCCGAGGCCTGCATGTCCGGCTGAAGG
CTGAGTGTCCGGCTGAGGCCT
GAGCGAGTGTCCAGCCAAGGGCTGAGTGTCCAGCACACCTGCCGTCTTC
ACTTCCCCACAGGCTGGCGCT
CGGCTCCACCCCAGGGCCAGCTTTTCCTCACCAGGAGCCCGGCTTCCAC
TCCCCACATAGGAATAGTCCA
TCCCCAGATTCGCCATTGTTCACCCCTCGCCCTGCCCTCCTTTGCCTTC
CACCCCCACCATCCAGGTGGA
GACCCTGAGAAGGACCCTGGGAGCTCTGGGAATTTGGAGTGACCAAAGG
TGTGCCCTGTACACAGGCGAG
GACCCTGCACCTGGATGGGGGTCCCTGTGGGTCAAATTGGGGGGAGGTG
CTGTGGGAGTAAAATACTGAA
TATATGAGTTTTTCAGTTTTGAAAAAAA
5
Protein
MGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTE
BIRC5
NEPDLAQCFFCFKELEGWEPD
(survivin)
DDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKIAKETNN
GenBank
KKKEFEETAEKVRRAIEQLAA
NP_001159
MD
6
Nucleotide
CCCAGAAGGCCGCGGGGGGTGGACCGCCTAAGAGGGCGTGCGCTCCCGA
BIRC5
CATGCCCCGCGGCGCGCCATT
(survivin)
AACCGCCAGATTTGAATCGCGGGACCCGTTGGCAGAGGTGGCGGCGGCG
GenBank
GCATGGGTGCCCCGACGTTGC
NM_001168.2
CCCCTGCCTGGCAGCCCTTTCTCAAGGACCACCGCATCTCTACATTCAA
GAACTGGCCCTTCTTGGAGGG
CTGCGCCTGCACCCCGGAGCGGATGGCCGAGGCTGGCTTCATCCACTGC
CCCACTGAGAACGAGCCAGAC
TTGGCCCAGTGTTTCTTCTGCTTCAAGGAGCTGGAAGGCTGGGAGCCAG
ATGACGACCCCATAGAGGAAC
ATAAAAAGCATTCGTCCGGTTGCGCTTTCCTTTCTGTCAAGAAGCAGTT
TGAAGAATTAACCCTTGGTGA
ATTTTTGAAACTGGACAGAGAAAGAGCCAAGAACAAAATTGCAAAGGAA
ACCAACAATAAGAAGAAAGAA
TTTGAGGAAACTGCGGAGAAAGTGCGCCGTGCCATCGAGCAGCTGGCTG
CCATGGATTGAGGCCTCTGGC
CGGAGCTGCCTGGTCCCAGAGTGGCTGCACCACTTCCAGGGTTTATTCC
CTGGTGCCACCAGCCTTCCTG
TGGGCCCCTTAGCAATGTCTTAGGAAAGGAGATCAACATTTTCAAATTA
GATGTTTCAACTGTGCTCTTG
TTTTGTCTTGAAAGTGGCACCAGAGGTGCTTCTGCCTGTGCAGCGGGTG
CTGCTGGTAACAGTGGCTGCT
TCTCTCTCTCTCTCTCTTTTTTGGGGGCTCATTTTTGCTGTTTTGATTC
CCGGGCTTACCAGGTGAGAAG
TGAGGGAGGAAGAAGGCAGTGTCCCTTTTGCTAGAGCTGACAGCTTTGT
TCGCGTGGGCAGAGCCTTCCA
CAGTGAATGTGTCTGGACCTCATGTTGTTGAGGCTGTCACAGTCCTGAG
TGTGGACTTGGCAGGTGCCTG
TTGAATCTGAGCTGCAGGTTCCTTATCTGTCACACCTGTGCCTCCTCAG
AGGACAGTTTTTTTGTTGTTG
TGTTTTTTTGTTTTTTTTTTTTTGGTAGATGCATGACTTGTGTGTGATG
AGAGAATGGAGACAGAGTCCC
TGGCTCCTCTACTGTTTAACAACATGGCTTTCTTATTTTGTTTGAATTG
TTAATTCACAGAATAGCACAA
ACTACAATTAAAACTAAGCACAAAGCCATTCTAAGTCATTGGGGAAACG
GGGTGAACTTCAGGTGGATGA
GGAGACAGAATAGAGTGATAGGAAGCGTCTGGCAGATACTCCTTTTGCC
ACTGCTGTGTGATTAGACAGG
CCCAGTGAGCCGCGGGGCACATGCTGGCCGCTCCTCCCTCAGAAAAAGG
CAGTGGCCTAAATCCTTTTTA
AATGACTTGGCTCGATGCTGTGGGGGACTGGCTGGGCTGCTGCAGGCCG
TGTGTCTGTCAGCCCAACCTT
CACATCTGTCACGTTCTCCACACGGGGGAGAGACGCAGTCCGCCCAGGT
CCCCGCTTTCTTTGGAGGCAG
CAGCTCCCGCAGGGCTGAAGTCTGGCGTAAGATGATGGATTTGATTCGC
CCTCCTCCCTGTCATAGAGCT
GCAGGGTGGATTGTTACAGCTTCGCTGGAAACCTCTGGAGGTCATCTCG
GCTGTTCCTGAGAAATAAAAA
GCCTGTCATTTCAAACACTGCTGTGGACCCTACTGGGTTTTTAAAATAT
TGTCAGTTTTTCATCGTCGTC
CCTAGCCTGCCAACAGCCATCTGCCCAGACAGCCGCAGTGAGGATGAGC
GTCCTGGCAGAGACGCAGTTG
TCTCTGGGCGCTTGCCAGAGCCACGAACCCCAGACCTGTTTGTATCATC
CGGGCTCCTTCCGGGCAGAAA
CAACTGAAAATGCACTTCAGACCCACTTATTTCTGCCACATCTGAGTCG
GCCTGAGATAGACTTTTCCCT
CTAAACTGGGAGAATATCACAGTGGTTTTTGTTAGCAGAAAATGCACTC
CAGCCTCTGTACTCATCTAAG
CTGCTTATTTTTGATATTTGTGTCAGTCTGTAAATGGATACTTCACTTT
AATAACTGTTGCTTAGTAATT
GGCTTTGTAGAGAAGCTGGAAAAAAATGGTTTTGTCTTCAACTCCTTTG
CATGCCAGGCGGTGATGTGGA
TCTCGGCTTCTGTGAGCCTGTGCTGTGGGCAGGGCTGAGCTGGAGCCGC
CCCTCTCAGCCCGCCTGCCAC
GGCCTTTCCTTAAAGGCCATCCTTAAAACCAGACCCTCATGGCTACCAG
CACCTGAAAGCTTCCTCGACA
TCTGTTAATAAAGCCGTAGGCCCTTGTCTAAGTGCAACCGCCTAGACTT
TCTTTCAGATACATGTCCACA
TGTCCATTTTTCAGGTTCTCTAAGTTGGAGTGGAGTCTGGGAAGGGTTG
TGAATGAGGCTTCTGGGCTAT
GGGTGAGGTTCCAATGGCAGGTTAGAGCCCCTCGGGCCAACTGCCATCC
TGGAAAGTAGAGACAGCAGTG
CCCGCTGCCCAGAAGAGACCAGCAAGCCAAACTGGAGCCCCCATTGCAG
GCTGTCGCCATGTGGAAAGAG
TAACTCACAATTGCCAATAAAGTCTCATGTGGTTTTATCTAAAAAAAAA
AAAAAAAAAAAAAAAA
7
ILAKFLHWL
hTERT
peptide
T540 HLA-
A*0201
8
RLVDDFLLV
hTERT
peptide
peptide
T865 HLA-
A*0201
9
YAETKHFLY
hTERT
325-333
HLA-A*0101
10
ALLTSRLRFI
hTERT
615-624
HLA-A*0201
11
GLLGASVLGL
hTERT
674-683
HLA-A*0201
12
ILAKFLHWL
hTERT
540-548
HLA-A*0201
13
RLTSRVKAL
hTERT
653-661
HLA-A*0201
14
YLQVNSLQTV
hTERT
988-997
HLA-A*0201
15
KLFGVLRLK
hTERT
973-981
HLA-A*0301
16
VYAETKHFL
hTERT
324-332
HLA-A*2402
17
VYGFVRACL
hTERT
461-469
HLA-A*2402
18
APRCRAVRSL
hTERT4-13
HLA-B*0702
19
APSFRQVSCL
hTERT68-77
HLA-B*0702
20
MAEAGFIHY
Survivin
38-46 (mod)
HLA-
A*01:01
21
PTENEPDLAY
Survivin
47-56
(mod)
HLA-A*01:01
22
ELTLGEFLKL
Survivin
95-104
HLA-A*02:01
23
LMLGEFLKL
Survivin
96-104
(mod)
HLA-A*02:01
24
TLPPAWQPFL
Survivin
5-14
HLA-A*02:01
25
RISTFKNWPK
Survivin
18-27
(modK)
HLA-A*03:01
26
DLAQCFFCFK
Survivin
56-62
HLA-A*11:01
27
AYACNTSTL
Survivin
80-88
HLA-A*24:02
28
STFKNWPFL
Survivin
20-28
HLA-A*24:02
29
LPPAWQPFL
Survivin
6-14
HLA-B*07:02
30
EPDLAQCFY
Survivin
51-59
HLA-B*35:01
31
Protein
MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSV
BCMA
KGTNAILWTCLGLSLIISLAV
(TNFRSF17)
FVLMFLLRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGL
GenBank
EYTVEECTCEDCIKSKPKVDS
NP_001183.2
DHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR
32
Nucleotide
AAGACTCAAACTTAGAAACTTGAATTAGATGTGGTATTCAAATCCTTAG
BCMA
CTGCCGCGAAGACACAGACAG
(TNFRSF17)
CCCCCGTAAGAACCCACGAAGCAGGCGAAGTTCATTGTTCTCAACATTC
GenBank
TAGCTGCTCTTGCTGCATTTG
NM_001192
CTCTGGAATTCTTGTAGAGATATTACTTGTCCTTCCAGGCTGTTCTTTC
TGTAGCTCCCTTGTTTTCTTT
TTGTGATCATGTTGCAGATGGCTGGGCAGTGCTCCCAAAATGAATATTT
TGACAGTTTGTTGCATGCTTG
CATACCTTGTCAACTTCGATGTTCTTCTAATACTCCTCCTCTAACATGT
CAGCGTTATTGTAATGCAAGT
GTGACCAATTCAGTGAAAGGAACGAATGCGATTCTCTGGACCTGTTTGG
GACTGAGCTTAATAATTTCTT
TGGCAGTTTTCGTGCTAATGTTTTTGCTAAGGAAGATAAACTCTGAACC
ATTAAAGGACGAGTTTAAAAA
CACAGGATCAGGTCTCCTGGGCATGGCTAACATTGACCTGGAAAAGAGC
AGGACTGGTGATGAAATTATT
CTTCCGAGAGGCCTCGAGTACACGGTGGAAGAATGCACCTGTGAAGACT
GCATCAAGAGCAAACCGAAGG
TCGACTCTGACCATTGCTTTCCACTCCCAGCTATGGAGGAAGGCGCAAC
CATTCTTGTCACCACGAAAAC
GAATGACTATTGCAAGAGCCTGCCAGCTGCTTTGAGTGCTACGGAGATA
GAGAAATCAATTTCTGCTAGG
TAATTAACCATTTCGACTCGAGCAGTGCCACTTTAAAAATCTTTTGTCA
GAATAGATGATGTGTCAGATC
TCTTTAGGATGACTGTATTTTTCAGTTGCCGATACAGCTTTTTGTCCTC
TAACTGTGGAAACTCTTTATG
TTAGATATATTTCTCTAGGTTACTGTTGGGAGCTTAATGGTAGAAACTT
CCTTGGTTTCATGATTAAACT
CTTTTTTTTCCTGA
33
Protein
MSGLGRSRRGGRSRVDQEERFPQGLWTGVAMRSCPEEQYWDPLLGTCMS
TACI
CKTICNHQSQRTCAAFCRSLS
(TNFRSF13B)
CRKEQGKEYDHLLRDCISCASICGQHPKQCAYFCENKLRSPVNLPPELR
GenBank
RQRSGEVENNSDNSGRYQGLE
NP_036584.1
HRGSEASPALPGLKLSADQVALVYSTLGLCLCAVLCCFLVAVACFLKKR
GDPCSCQPRSRPRQSPAKSSQ
DHAMEAGSPVSTSPEPVETCSFCFPECRAPTQESAVTPGTPDPTCAGRW
GCHTRTTVLQPCPHIPDSGLG
IVCVPAQEGGPGA
34
Nucleotide
AGCATCCTGAGTAATGAGTGGCCTGGGCCGGAGCAGGCGAGGTGGCCGG
TACI
AGCCGTGTGGACCAGGAGGAG
(TNFRSF13B)
CGCTTTCCACAGGGCCTGTGGACGGGGGTGGCTATGAGATCCTGCCCCG
GenBank
AAGAGCAGTACTGGGATCCTC
NM_012452.2
TGCTGGGTACCTGCATGTCCTGCAAAACCATTTGCAACCATCAGAGCCA
GCGCACCTGTGCAGCCTTCTG
CAGGTCACTCAGCTGCCGCAAGGAGCAAGGCAAGTTCTATGACCATCTC
CTGAGGGACTGCATCAGCTGT
GCCTCCATCTGTGGACAGCACCCTAAGCAATGTGCATACTTCTGTGAGA
ACAAGCTCAGGAGCCCAGTGA
ACCTTCCACCAGAGCTCAGGAGACAGCGGAGTGGAGAAGTTGAAAACAA
TTCAGACAACTCGGGAAGGTA
CCAAGGATTGGAGCACAGAGGCTCAGAAGCAAGTCCAGCTCTCCCGGGG
CTGAAGCTGAGTGCAGATCAG
GTGGCCCTGGTCTACAGCACGCTGGGGCTCTGCCTGTGTGCCGTCCTCT
GCTGCTTCCTGGTGGCGGTGG
CCTGCTTCCTCAAGAAGAGGGGGGATCCCTGCTCCTGCCAGCCCCGCTC
AAGGCCCCGTCAAAGTCCGGC
CAAGTCTTCCCAGGATCACGCGATGGAAGCCGGCAGCCCTGTGAGCACA
TCCCCCGAGCCAGTGGAGACC
TGCAGCTTCTGCTTCCCTGAGTGCAGGGCGCCCACGCAGGAGAGCGCAG
TCACGCCTGGGACCCCCGACC
CCACTTGTGCTGGAAGGTGGGGGTGCCACACCAGGACCACAGTCCTGCA
GCCTTGCCCACACATCCCAGA
CAGTGGCCTTGGCATTGTGTGTGTGCCTGCCCAGGAGGGGGGCCCAGGT
GCATAAATGGGGGTCAGGGAG
GGAAAGGAGGAGGGAGAGAGATGGAGAGGAGGGGAGAGAGAAAGAGAGG
TGGGGAGAGGGGAGAGAGATA
TGAGGAGAGAGAGACAGAGGAGGCAGAGAGGGAGAGAAACAGAGGAGAC
AGAGAGGGAGAGAGAGACAGA
GGGAGAGAGAGACAGAGGGGAAGAGAGGCAGAGAGGGAAAGAGGCAGAG
AAGGAAAGAGACAGGCAGAGA
AGGAGAGAGGCAGAGAGGGAGAGAGGCAGAGAGGGAGAGAGGCAGAGAG
ACAGAGAGGGAGAGAGGGACA
GAGAGAGATAGAGCAGGAGGTCGGGGCACTCTGAGTCCCAGTTCCCAGT
GCAGCTGTAGGTCGTCATCAC
CTAACCACACGTGCAATAAAGTCCTCGTGCCTGCTGCTCACAGCCCCCG
AGAGCCCCTCCTCCTGGAGAA
TAAAACCTTTGGCAGCTGCCCTTCCTCAAAAAAAAAAAAAAAAAAAA
35
MRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGAS
BAFF
SPAPRTALQPQESVGAGAGEA
Receptor
ALPLPGLLFGAPALLGLALVLALVLVGLVSWRRRQRRLRGASSAEAPDG
(TNFRSF13C)
DKDAPEPLDKVIILSPGISDA
GenBank
TAPAWPPPGEDPGTTPPGHSVPVPATELGSTELVTTKTAGPEQQ
NP_443177.1
36
AGCTCAGCCTCAGTCCCCGCAGCTTGTGCGGCGGCGTCGGCACCATGAG
BAFF
GCGAGGGCCCCGGAGCCTGCG
Receptor
GGGCAGGGACGCGCCAGCCCCCACGCCCTGCGTCCCGGCCGAGTGCTTC
(TNFRSF13C)
GACCTGCTGGTCCGCCACTGC
GenBank
GTGGCCTGCGGGCTCCTGCGCACGCCGCGGCCGAAACCGGCCGGGGCCA
NM_012452.2
GCAGCCCTGCGCCCAGGACGG
CGCTGCAGCCGCAGGAGTCGGTGGGCGCGGGGGCCGGCGAGGCGGCGCT
GCCCCTGCCCGGGCTGCTCTT
TGGCGCCCCCGCGCTGCTGGGCCTGGCACTGGTCCTGGCGCTGGTCCTG
GTGGGTCTGGTGAGCTGGAGG
CGGCGACAGCGGCGGCTTCGCGGCGCGTCCTCCGCAGAGGCCCCCGACG
GAGACAAGGACGCCCCAGAGC
CCCTGGACAAGGTCATCATTCTGTCTCCGGGAATCTCTGATGCCACAGC
TCCTGCCTGGCCTCCTCCTGG
GGAAGACCCAGGAACCACCCCACCTGGCCACAGTGTCCCTGTGCCAGCC
ACAGAGCTGGGCTCCACTGAA
CTGGTGACCACCAAGACGGCCGGCCCTGAGCAACAATAGCAGGGAGCCG
GCAGGAGGTGGCCCCTGCCCT
CCCTCTGGACCCCCAGCCAGGGGCTTGGAAATCAAATTCAGCTCTTCAC
TCCAGCATGCACATGCCCTCT
TTCTGGGACCAGGCTAACTCTGCAGAAGCACAGACACTACAGACCACAG
CATTCAGCCCCCATGGAGTTT
GGTGTGCTTGCCTTTGGCTTCAGACCTCACCATCTTTGACAGCCCTTGA
AGGTGGTAGCCCAGCTCCTGT
TCCTGTGCCTTCAAAAGGCTGGGGCACTATGAGTAAAAGACCGCTTTTA
AAATGGGGAAGGCACCATTAA
GCCAAAATGAATCTGAAAAAAGAC
37
CAATGGTCAGAGTCGACCTT
BCMA
CRISPR
guide RNA
38
ATTCTTCCACCGTGTACTCG
BCMA
CRISPR
guide RNA
39
CAATAACGCTGACATGTTAG
BCMA
CRISPR
guide RNA
40
TATTAAGCTCAGTCCCAAAC
BCMA
CRISPR
guide RNA
41
GGTGTGACCAATTCAGTGAA
BCMA
CRISPR
guide RNA
42
GAAGAACATCGAAGTTGACA
BCMA
CRISPR
guide RNA
43
TCATAGCCACCCCCGTCCAC
TACI
CRISPR
guide RNA
44
CCAGTACTGCTCTTCGGGGC
TACI
CRISPR
guide RNA
45
AGTTCTATGACCATCTCCTG
TACI
CRISPR
guide RNA
46
AGGGTCACTCAGCTGCCGCA
TACI
CRISPR
guide RNA
47
GCTACTCACAGCGCTCCTCC
TACI
CRISPR
guide RNA
48
TCCGGCCACCTCGCCTGCTC
TACI
CRISPR
guide RNA
49
CCCTTACCCGGTTTCGGCCG
BAFF
receptor
CRISPR
guide RNA
50
GCGTCGGCACCATGAGGCGA
BAFF
receptor
CRISPR
guide RNA
51
CTCACCGTCCTTGTCTCCGT
BAFF
receptor
CRISPR
guide RNA
52
TCGCGGCGCGTCCTCCGCAG
BAFF
receptor
CRISPR
guide RNA
53
TGAACTGGTGACCACCAAGA
BAFF
receptor
CRISPR
guide RNA
54
CTTGGTGGTCACCAGTTCAG
BAFF
receptor
CRISPR
guide RNA
55
Protein
MLLWVILLVLAPVSGQFARTPRPIIFLQPPWTTVFQGERVTLTCKGFRF
FcRH5
YSPQKTKWYHRYLGKEILRET
GenBank
PDNILEVQESGEYRCQAQGSPLSSPVHLDFSSASLILQAPLSVFEGDSV
NP_112571.2
VLRCRAKAEVTLNNTIYKNDN
VLAFLNKRTDFHIPHACLKDNGAYRCTGYKESCCPVSSNTVKIQVQEPF
TRPVLRASSFQPISGNPVTLT
CETQLSLERSDVPLRFRFFRDDQTLGLGWSLSPNFQITAMWSKDSGFYW
CKAATMPYSVISDSPRSWIQV
QIPASHPVLTLSPEKALNFEGTKVTLHCETQEDSLRTLYRFYHEGVPLR
HKSVRCERGASISFSLTTENS
GNYYCTADNGLGAKPSKAVSLSVTVPVSHPVLNLSSPEDLIFEGAKVTL
HCEAQRGSLPILYQFHHEGAA
LERRSANSAGGVAISFSLTAEHSGNYYCTADNGFGPQRSKAVSLSVTVP
VSHPVLTLSSAEALTFEGATV
TLHCEVQRGSPQILYQFYHEDMPLWSSSTPSVGRVSFSFSLTEGHSGNY
YCTADNGFGPQRSEVVSLFVT
VPVSRPILTLRVPRAQAVVGDLLELHCEAPRGSPPILYWFYHEDVTLGS
SSAPSGGEASFNLSLTAEHSG
NYSCEANNGLVAQHSDTISLSVIVPVSRPILTFRAPRAQAVVGDLLELH
CEALRGSSPILYWFYHEDVTL
GKISAPSGGGASFNLSLTTEHSGIYSCEADNGLEAQRSEMVTLKVAVPV
SRPVLTLRAPGTHAAVGDLLE
LHCEALRGSPLILYRFFHEDVTLGNRSSPSGGASLNLSLTAEHSGNYSC
EADNGLGAQRSETVTLYITGL
TANRSGPFATGVAGGLLSIAGLAAGALLLYCWLSRKAGRKPASDPARSP
SDSDSQEPTYHNVPAWEELQP
VYTNANPRGENVVYSEVRIIQEKKKHAVASDPRHLRNKGSPIIYSEVKV
ASTPVSGSLFLASSAPHR
56
Nucleotide
AATTCACTAATGCATTCTGCTCTTTTTGAGAGCACAGCTTCTCAGATGT
FcRH5
GCTCCTTGGAGCTGGTGTGCA
GenBank
GTGTCCTGACTGTAAGATCAAGTCCAAACCTGTTTTGGAATTGAGGAAA
NM_0031281.2
CTTCTCTTTTGATCTCAGCCC
TTGGTGGTCCAGGTCTTCATGCTGCTGTGGGTGATATTACTGGTCCTGG
CTCCTGTCAGTGGACAGTTTG
CAAGGACACCCAGGCCCATTATTTTCCTCCAGCCTCCATGGACCACAGT
CTTCCAAGGAGAGAGAGTGAC
CCTCACTTGCAAGGGATTTCGCTTCTACTCACCACAGAAAACAAAATGG
TACCATCGGTACCTTGGGAAA
GAAATACTAAGAGAAACCCCAGACAATATCCTTGAGGTTCAGGAATCTG
GAGAGTACAGATGCCAGGCCC
AGGGCTCCCCTCTCAGTAGCCCTGTGCACTTGGATTTTTCTTCAGCTTC
GCTGATCCTGCAAGCTCCACT
TTCTGTGTTTGAAGGAGACTCTGTGGTTCTGAGGTGCCGGGCAAAGGCG
GAAGTAACACTGAATAATACT
ATTTACAAGAATGATAATGTCCTGGCATTCCTTAATAAAAGAACTGACT
TCCATATTCCTCATGCATGTC
TCAAGGACAATGGTGCATATCGCTGTACTGGATATAAGGAAAGTTGTTG
CCCTGTTTCTTCCAATACAGT
CAAAATCCAAGTCCAAGAGCCATTTACACGTCCAGTGCTGAGAGCCAGC
TCCTTCCAGCCCATCAGCGGG
AACCCAGTGACCCTGACCTGTGAGACCCAGCTCTCTCTAGAGAGGTCAG
ATGTCCCGCTCCGGTTCCGCT
TCTTCAGAGATGACCAGACCCTGGGATTAGGCTGGAGTCTCTCCCCGAA
TTTCCAGATTACTGCCATGTG
GAGTAAAGATTCAGGGTTCTACTGGTGTAAGGCAGCAACAATGCCTTAC
AGCGTCATATCTGACAGCCCG
AGATCCTGGATACAGGTGCAGATCCCTGCATCTCATCCTGTCCTCACTC
TCAGCCCTGAAAAGGCTCTGA
ATTTTGAGGGAACCAAGGTGACACTTCACTGTGAAACCCAGGAAGATTC
TCTGCGCACTTTGTACAGGTT
TTATCATGAGGGTGTCCCCCTGAGGCACAAGTCAGTCCGCTGTGAAAGG
GGAGCATCCATCAGCTTCTCA
CTGACTACAGAGAATTCAGGGAACTACTACTGCACAGCTGACAATGGCC
TTGGCGCCAAGCCCAGTAAGG
CTGTGAGCCTCTCAGTCACTGTTCCCGTGTCTCATCCTGTCCTCAACCT
CAGCTCTCCTGAGGACCTGAT
TTTTGAGGGAGCCAAGGTGACACTTCACTGTGAAGCCCAGAGAGGTTCA
CTCCCCATCCTGTACCAGTTT
CATCATGAGGGTGCTGCCCTGGAGCGTAGGTCGGCCAACTCTGCAGGAG
GAGTGGCCATCAGCTTCTCTC
TGACTGCAGAGCATTCAGGGAACTACTACTGCACAGCTGACAATGGCTT
TGGCCCCCAGCGCAGTAAGGC
GGTGAGCCTCTCCGTCACTGTCCCTGTGTCTCATCCTGTCCTCACCCTC
AGCTCTGCTGAGGCCCTGACT
TTTGAAGGAGCCACTGTGACACTTCACTGTGAAGTCCAGAGAGGTTCCC
CACAAATCCTATACCAGTTTT
ATCATGAGGACATGCCCCTGTGGAGCAGCTCAACACCCTCTGTGGGAAG
AGTGTCCTTCAGCTTCTCTCT
GACTGAAGGACATTCAGGGAATTACTACTGCACAGCTGACAATGGCTTT
GGTCCCCAGCGCAGTGAAGTG
GTGAGCCTTTTTGTCACTGTTCCAGTGTCTCGCCCCATCCTCACCCTCA
GGGTTCCCAGGGCCCAGGCTG
TGGTGGGGGACCTGCTGGAGCTTCACTGTGAGGCCCCGAGAGGCTCTCC
CCCAATCCTGTACTGGTTTTA
TCATGAGGATGTCACCCTGGGGAGCAGCTCAGCCCCCTCTGGAGGAGAA
GCTTCTTTCAACCTCTCTCTG
ACTGCAGAACATTCTGGAAACTACTCATGTGAGGCCAACAATGGCCTAG
TGGCCCAGCACAGTGACACAA
TATCACTCAGTGTTATAGTTCCAGTATCTCGTCCCATCCTCACCTTCAG
GGCTCCCAGGGCCCAGGCTGT
GGTGGGGGACCTGCTGGAGCTTCACTGTGAGGCCCTGAGAGGCTCCTCC
CCAATCCTGTACTGGTTTTAT
CATGAAGATGTCACCCTGGGTAAGATCTCAGCCCCCTCTGGAGGAGGGG
CCTCCTTCAACCTCTCTCTGA
CTACAGAACATTCTGGAATCTACTCCTGTGAGGCAGACAATGGTCTGGA
GGCCCAGCGCAGTGAGATGGT
GACACTGAAAGTTGCAGTTCCGGTGTCTCGCCCGGTCCTCACCCTCAGG
GCTCCCGGGACCCATGCTGCG
GTGGGGGACCTGCTGGAGCTTCACTGTGAGGCCCTGAGAGGCTCTCCCC
TGATCCTGTACCGGTTTTTTC
ATGAGGATGTCACCCTAGGAAATAGGTCGTCCCCCTCTGGAGGAGCGTC
CTTAAACCTCTCTCTGACTGC
AGAGCACTCTGGAAACTACTCCTGTGAGGCCGACAATGGCCTCGGGGCC
CAGCGCAGTGAGACAGTGACA
CTTTATATCACAGGGCTGACCGCGAACAGAAGTGGCCCTTTTGCCACAG
GAGTCGCCGGGGGCCTGCTCA
GCATAGCAGGCCTTGCTGCGGGGGCACTGCTGCTCTACTGCTGGCTCTC
GAGAAAAGCAGGGAGAAAGCC
TGCCTCTGACCCCGCCAGGAGCCCTTCAGACTCGGACTCCCAAGAGCCC
ACCTATCACAATGTACCAGCC
TGGGAAGAGCTGCAACCAGTGTACACTAATGCAAATCCTAGAGGAGAAA
ATGTGGTTTACTCAGAAGTAC
GGATCATCCAAGAGAAAAAGAAACATGCAGTGGCCTCTGACCCCAGGCA
TCTCAGGAACAAGGGTTCCCC
TATCATCTACTCTGAAGTTAAGGTGGCGTCAACCCCGGTTTCCGGATCC
CTGTTCTTGGCTTCCTCAGCT
CCTCACAGATGAGTCCACACGTCTCTCCAACTGCTGTTTCAGCCTCTGC
ACCCCAAAGTTCCCCTTGGGG
GAGAAGCAGCATTGAAGTGGGAAGATTTAGGCTGCCCCAGACCATATCT
ACTGGCCTTTGTTTCACATGT
CCTCATTCTCAGTCTGACCAGAATGCAGGGCCCTGCTGGACTGTCACCT
GTTTCCCAGTTAAAGCCCTGA
CTGGCAGGTTTTTTAATCCAGTGGCAAGGTGCTCCCACTCCAGGGCCCA
GCACATCTCCTGGATTCCTTA
GTGGGCTTCAGCTGTGGTTGCTGTTCTGAGTACTGCTCTCATCACACCC
CCACAGAGGGGGTCTTACCAC
ACAAAGGGAGAGTGGGCCTTCAGGAGATGCCGGGCTGGCCTAACAGCTC
AGGTGCTCCTAAACTCCGACA
CAGAGTTCCTGCTTTGGGTGGATGCATTTCTCAATTGTCATCAGCCTGG
TGGGGCTACTGCAGTGTGCTG
CCAAATGGGACAGCACACAGCCTGTGCACATGGGACATGTGATGGGTCT
CCCCACGGGGGCTGCATTTCA
CACTCCTCCACCTGTCTCAAACTCTAAGGTCGGCACTTGACACCAAGGT
AACTTCTCTCCTGCTCATGTG
TCAGTGTCTACCTGCCCAAGTAAGTGGCTTTCATACACCAAGTCCCAAG
TTCTTCCCATCCTAACAGAAG
TAACCCAGCAAGTCAAGGCCAGGAGGACCAGGGGTGCAGACAGAACACA
TACTGGAACACAGGAGGTGCT
CAATTACTATTTGACTGACTGACTGAATGAATGAATGAATGAGGAAGAA
AACTGTGGGTAATCAAACTGG
CATAAAATCCAGTGCACTCCCTAGGAAATCCGGGAGGTATTCTGGCTTC
CCTAAGAAACAATGGAAGAGA
AGGAGCTTGGATGAGGAAACTGTTCAGCAAGAGGAAGGGCTTCTCACAC
TTTCATGTGCTTGTGGATCAC
CTGAGGATCCTGTGAAAATACAGATACTGATTCAGTGGGTCTGCGTAGA
GCCTGAGACTGCCATTCTAAC
ATGTTCCCAGGGGATGCTGATGCTGCTGGCCCTGGGACTGCACTGCATG
CATGTGAAGCCCTATAGGTCT
CAGCAGAGGCCCATGGAGAGGGAATGTGTGGCTCTGGCTGCCCAGGGCC
CAACTCGGTTCACACGGATCG
TGCTGCTCCCTGGCCAGCCTTTGGCCACAGCACCACCAGCTGCTGTTGC
TGAGAGAGCTTCTTCTCTGTG
ACATGTTGGCTTTCATCAGCCACCCTGGGAAGCGGAAAGTAGCTGCCAC
TATCTTTGTTTCCCCACCTCA
GGCCTCACACTTTCCCATGAAAAGGGTGAATGTATATAACCTGAGCCCT
CTCCATTCAGAGTTGTTCTCC
CATCTCTGAGCAATGGGATGTTCTGTTCCGCTTTTATGATATCCATCAC
ATCTTATCTTGATCTTTGCTC
CCAGTGGATTGTACAGTGATGACTTTTAAGCCCCACGGCCCTGAAATAA
AATCCTTCCAAGGGCATTGGA
AGCTCACTCCACCTGAACCATGGCTTTTCATGCTTCCAAGTGTCAGGGC
CTTGCCCAGATAGACAGGGCT
GGCTCTGCTGCCCCAACCTTTCAAGGAGGAAACCAGACACCTGAGACAG
GAGCCTGTATGCAGCCCAGTG
CAGCCTTGCAGAGGACAAGGCTGGAGGCATTTGTCATCACTACAGATAT
GCAACTAAAATAGACGTGGAG
CAAGAGAAATGCATTCCCACCGAGGCCGCTTTTTTAGGCCTAGTTGAAA
GTCAAGAAGGACAGCAGCAAG
CATAGGCTCAGGATTAAAGAAAAAAATCTGCTCACAGTCTGTTCTGGAG
GTCACATCACCAACAAAGCTC
ACGCCCTATGCAGTTCTGAGAAGGTGGAGGCACCAGGCTCAAAAGAGGA
AATTTAGAATTTCTCATTGGG
AGAGTAAGGTACCCCCATCCCAGAATGATAACTGCACAGTGGCAGAACA
AACTCCACCCTAATGTGGGTG
GACCCCGTCCAGTCTGTTGAAGGCCTGAATGTAACAAAAGGGCTTATTC
TTCCTCAAGTAAGGGGGAACT
CCTGCTTTGGGCTGGGACATAAGTTTTTCTGCTTTCAGACGCAAACTGA
AAAATGGCTCTTCTTGGGTCT
TGAGCTTGCTGGCATATGGACTGAAAGAAACTATGCTATTGGATCTCCT
GGATCTCCAGCTTGCTGACTG
CAGATCTTGAGATATGTCAGCCTCTACAGTCACAAGAGCTAATTCATTC
TAATAAACCAATCTTTCTGTA
AAAAA
57
GGAAATAGGTCGTCCCCCTC
FcRH5
CRISPR
guide RNA
58
AGTTGGCCGACCTACGCTCC
FcRH5
CRISPR
guide RNA
59
AGCGGACTGACTTGTGCCTC
FcRH5
CRISPR
guide RNA
60
ACAAGTCAGTCCGCTGTGAA
FcRH5
CRISPR
guide RNA
61
CTCACCGCCTTACTGCGCTG
FcRH5
CRISPR
guide RNA
62
TTCAGTTCCGGTGTCTCGCC
FcRH5
CRISPR
guide RNA
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15744744
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juno therapeutics, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Mar 30th, 2022 06:04PM
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Mar 30th, 2022 06:04PM
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Bristol-Myers Squibb
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:bmy
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Juno Therapeutics
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Jan 12th, 2021 12:00AM
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Jun 4th, 2018 12:00AM
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https://www.uspto.gov?id=US10889652-20210112
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Antibodies and chimeric antigen receptors specific for ROR1
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Provided are ROR1 binding molecules, including anti-ROR1 antibodies, including antibody fragments such as variable heavy chain (VH) regions, single-chain fragments, and chimeric receptors including the antibodies, such as chimeric antigen receptors (CARs). In some embodiments, the antibodies specifically bind to ROR1. Among the antibodies are human antibodies, including those that compete for binding to ROR1 with reference antibodies, such as a non-human reference antibody. Also provided are genetically engineered cells expressing the chimeric receptors, and uses of the binding molecules and cells adoptive cell therapy.
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10889652
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1. An anti-human receptor tyrosine kinase-like orphan receptor 1 (ROR1) antibody or an antigen-binding fragment thereof comprising:
a heavy chain variable (VH) region comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 21, and 22, respectively, and a light chain variable (VL) region comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 23, 24, and 25 respectively;
a heavy chain variable (VH) region comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 26, and 22, respectively, and a light chain variable (VL) region comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively;
a heavy chain variable (VH) region comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively, and a light chain variable (VL) region comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 218, 229, and 39, respectively;
a heavy chain variable (VH) region comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively, and a light chain variable (VL) region comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 30, 31, and 32, respectively;
a heavy chain variable (VH) region comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively, and a light chain variable (VL) region comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 216, 227, and 40, respectively;
a heavy chain variable (VH) region comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively, and a light chain variable (VL) region comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 219, 230, and 43, respectively;
a heavy chain variable (VH) region comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and a light chain variable (VL) region comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 217, 228, and 41, respectively;
a heavy chain variable (VH) region comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and a light chain variable (VL) region comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 212, 223, and 42, respectively;
a heavy chain variable (VH) region comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and a light chain variable (VL) region comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 36, 37, and 38, respectively;
a heavy chain variable (VH) region comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and a light chain variable (VL) region comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 215, 226, and 44, respectively;
a heavy chain variable (VH) region comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and a light chain variable (VL) region comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 211, 222, and 48, respectively;
a heavy chain variable (VH) region comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 164, and 45, respectively, and a light chain variable (VL) region comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 220, 231, and 46, respectively;
a heavy chain variable (VH) region comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 318, and 35, respectively, and a light chain variable (VL) region comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 210, 221, and 49, respectively;
a heavy chain variable (VH) region comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 318, and 35, respectively, and a light chain variable (VL) region comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 210, 221, and 233, respectively; or
a heavy chain variable (Vii) region comprising a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 155, 34 and 35, respectively, and a light chain variable (VL) region comprising a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 213, 224, and 47, respectively.
2. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 1, wherein:
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 19 and 18, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 17 and 18, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 15 and 16, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 13 and 14, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 182 and 242 respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 182 and 246, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 182 and 247, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 185 and 248, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 186 and 248, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 175 and 234, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 175 and 235, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 176 and 236, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 176 and 237, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 177 and 238, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 179 and 240 respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 180 and 241, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 181 and 241, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 183 and 243 respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 183 and 244 respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 184 and 243, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 184 and 244 respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 183 and 245, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 184 and 245, respectively; or
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 178 and 239, respectively.
3. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 1, wherein:
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 19 and 18, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 17 and 18, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 15 and 16, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 13 and 14, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 182 and 242 respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 182 and 246, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 182 and 247, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 185 and 248, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 186 and 248, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 175 and 234, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 175 and 235, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 176 and 236, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 176 and 237, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 177 and 238, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 179 and 240 respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 180 and 241, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 181 and 241, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 183 and 243 respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 183 and 244 respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 184 and 243, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 184 and 244 respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 183 and 245, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 184 and 245, respectively; or
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 178 and 239, respectively.
4. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is human.
5. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 1 that is a single chain fragment.
6. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 5, wherein the single chain fragment is a single chain variable fragment (scFv).
7. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 6, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 12, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, or 268, or a sequence of amino acids that has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 12, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, or 268.
8. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 6, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 12, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, or 268.
9. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 6, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO: 4, 6, or 12, or a sequence of amino acids that has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4, 6, or 12.
10. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 6, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO: 6, or a sequence of amino acids that has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6.
11. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 1, wherein:
the CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences set forth in SEQ ID NOs: 20, 26, and 22, respectively; and the CDR-L1, CDR-L2, and CDR-L3 comprise the amino acid sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively; and the CDR-L1, CDR-L2, and CDR-L3 comprise the amino acid sequences set forth in SEQ ID NOs: 30, 31, and 32, respectively; or
the CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively; and the CDR-L1, CDR-L2, and CDR-L3 comprise the amino acid sequences set forth in SEQ ID NOs: 36, 37, and 38, respectively.
12. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 1, wherein the CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively; and the CDR-L1, CDR-L2, and CDR-L3 comprise the amino acid sequences set forth in SEQ ID NOs: 36, 37, and 38, respectively.
13. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 1, wherein:
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 19 and 18, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 15 and 16, respectively; or
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 13 and 14, respectively.
14. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 1, wherein the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 13 and 14, respectively.
15. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 1, wherein:
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 21, and 22, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 23, 24, and 25 respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 26, and 22, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 218, 229, and 39, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 30, 31, and 32, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 216, 227, and 40, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 219, 230, and 43, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 217, 228, and 41, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 212, 223, and 42, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 36, 37, and 38, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 215, 226, and 44, respectively; or
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 164, and 45, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 220, 231, and 46, respectively.
16. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 1, wherein:
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 19 and 18, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 17 and 18, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 15 and 16, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 13 and 14, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 182 and 242 respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 182 and 246, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 182 and 247, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 185 and 248, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 186 and 248, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 177 and 238, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 180 and 241, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 181 and 241, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 183 and 243 respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 183 and 244 respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 184 and 243, respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 184 and 244 respectively;
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 183 and 245, respectively; or
the VH and VL regions comprise amino acid sequences having at least 90% identity to SEQ ID NOs: 184 and 245, respectively.
17. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 1, wherein:
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 19 and 18, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 17 and 18, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 15 and 16, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 13 and 14, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 182 and 242 respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 182 and 246, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 182 and 247, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 185 and 248, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 177 and 238, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 180 and 241, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 181 and 241, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 183 and 243 respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 183 and 244 respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 184 and 243, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 184 and 244 respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 183 and 245, respectively; or
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 184 and 245, respectively.
18. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 12, 253, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, or 268, or a sequence of amino acids that has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 12, 253, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, or 268.
19. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 1, wherein:
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 21, and 22, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 23, 24, and 25 respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 20, 26, and 22, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 218, 229, and 39, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 30, 31, and 32, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 216, 227, and 40, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 219, 230, and 43, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 217, 228, and 41, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 212, 223, and 42, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 36, 37, and 38, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 215, 226, and 44, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 211, 222, and 48, respectively;
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 164, and 45, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 220, 231, and 46, respectively; or
the VH region comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 155, 34 and 35, respectively, and the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 213, 224, and 47, respectively.
20. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 1, wherein:
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 19 and 18, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 17 and 18, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 15 and 16, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 13 and 14, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 182 and 242 respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 182 and 246, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 182 and 247, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 185 and 248, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 176 and 236, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 176 and 237, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 177 and 238, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 180 and 241, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 181 and 241, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 183 and 243 respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 183 and 244 respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 184 and 243, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 184 and 244 respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 183 and 245, respectively;
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 184 and 245, respectively; or
the VH and VL regions comprise the amino acid sequences set forth in SEQ ID NOs: 178 and 239, respectively.
21. A conjugate, comprising the anti-human ROR1 antibody or antigen-binding fragment thereof of claim 1 and a heterologous molecule or moiety.
22. A composition comprising the anti-human ROR1 antibody or antigen-binding fragment thereof of claim 1.
23. A composition comprising the anti-human ROR1 antibody or antigen-binding fragment thereof of claim 3.
24. A composition comprising the anti-human ROR1 scFv of claim 7.
25. An anti-human receptor tyrosine kinase-like orphan receptor 1 (ROR1) antibody or an antigen-binding fragment thereof comprising:
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:19; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:18;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:17; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:18;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:15; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:16;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:13; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:14;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:182; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:242;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:182; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:246;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:182; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:247;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:185; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:248;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:186; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:248;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:175; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:234;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:175; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:235;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:176; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:236;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:176; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:237;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:177; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:238;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:180; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:241;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:181; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:241;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:183; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:243;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:183; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:244;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:184; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:243;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:184; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:244;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:183; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:245;
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:184; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:245; or
a heavy chain variable (VH) region comprising the CDR-H1, the CDR-H2, and the CDR-H3 of the VH region sequence set forth in SEQ ID NO:178; and a light chain variable (VL) region comprising the CDR-L1, the CDR-L2, and the CDR-L3 of the VL region sequence set forth in SEQ ID NO:239.
26. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 25, wherein the antibody or antigen-binding fragment thereof is human.
27. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 25 that is a single chain fragment.
28. The anti-human ROR1 antibody or antigen-binding fragment thereof of claim 27, wherein the single chain fragment is a single chain variable fragment (scFv).
29. A composition comprising the anti-human ROR1 antibody or antigen-binding fragment thereof of claim 25.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 14/997,991, filed Jan. 18, 2016, entitled “ANTIBODIES AND CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR ROR1,” which claims priority from U.S. provisional application No. 62/104,664 filed Jan. 16, 2015, entitled “ANTIBODIES AND CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR ROR1,” the contents of which are incorporated by reference in their entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042001701SeqList.txt, created Jun. 4, 2018, which is 215,680 bytes in size. The information in electronic format of the Sequence Listing is incorporated by reference in its entirety.
FIELD
The present disclosure relates in some aspects to ROR1 binding molecules, in particular, to anti-ROR1 antibodies, including antibody fragments. The present disclosure further relates to recombinant receptors containing such antibodies, including chimeric antigen receptors (CARs), which contain such antibodies. The disclosure further relates to genetically engineered cells expressing such receptors and antibodies, and use thereof in adoptive cell therapy.
BACKGROUND
Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a transmembrane receptor that is expressed during embryogenesis, but generally is not expressed on normal adult cells. ROR1, however, is expressed in the context of a variety of different cancers, where it is involved in cell signaling to promote tumor cell survival. Because ROR1 is generally not expressed on normal cells, ROR1 is a tumor-specific and/or tumor-associated target for therapy. Various ROR1-binding molecules, including anti-ROR1 antibodies are available. Chimeric antigen receptors containing anti-ROR1 antibody portions, and cells expressing such chimeric receptors, also are available. Improved ROR1-binding molecules and engineered ROR1-targeting cells are needed. For example, there is a need for molecules and cells with reduced immunogenicity and fully human antibodies, including antibody fragments, that specifically bind to ROR1, and chimeric receptors expressing such human antibodies for use in adoptive cell therapy. Provided are embodiments that meet such needs.
SUMMARY
Provided are ROR1-binding molecules, including polypeptides, such as anti-ROR1 antibodies, including antigen-binding antibody fragments such as single domain antibodies (e.g. VH alone), single-chain fragments including scFv fragments, and polypeptides containing such antibodies, including fusion proteins, receptors, e.g., recombinant receptors, including chimeric receptors such as chimeric antigen receptors (CARs) containing the antibody as an antigen-recognition component. In particular embodiments, the antibodies are human antibodies, such as human single-chain fragments including scFvs.
Provided are antibodies or antigen-binding fragments thereof, including those that specifically bind to ROR1, such as specifically bind to human ROR1. In some embodiments, the antibodies contain particular complementarity determining regions (CDRs), including heavy chain CDRs (CDR-Hs) and light chain CDRs (CDR-Ls), such as any described. In some embodiments, the antibody or antigen-binding fragment thereof includes a heavy chain variable (VH) region. In some embodiments, the antibody or antigen-binding fragment thereof includes a VH region and a light chain variable (VL) region.
In some embodiments, provided are antibodies or antigen-binding fragments thereof that include a heavy chain variable (VH) region containing a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence set forth in SEQ ID NO: 22, 29, 35, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 174 and/or a CDR-H3 contained within the heavy chain variable (VH) sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209.
Provided are antibody or antigen-binding fragments thereof that include a heavy chain variable (VH) region comprising at least 90% sequence identity to the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209. In some embodiments, provided are antibodies or antigen-binding fragments thereof that include a heavy chain variable (VH) region having at least 90% sequence identity to the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
In some of any such embodiments, the VH region comprises a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence set forth in SEQ ID NO: 22, 29, 35, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 174; or a CDR-H3 contained within the VH sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209. In some of any such embodiments, the VH region includes a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence set forth in SEQ ID NO: 22, 29, 35, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 174 and/or a CDR-H3 contained within the VH sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19. In some embodiments, the VH region includes a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence set forth in SEQ ID NO: 72 and/or a CDR-H3 contained within the VH sequence set forth in SEQ ID NO: 85.
In some of any such embodiments, the VH region includes a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO: 20, 27, 33, 155, 156, 157, 158, 159, 160, 161, 162 or 163, and/or comprising the amino acid sequence set forth in SEQ ID NO: 269, 270, 271, 272, 273, 274, 275, 276, 278 or 279 and/or comprising the amino acid sequence set forth in SEQ ID NO: 75, 77, 79, 280, 281, 282, 283, 284, 285, 286, 287, 288 or 289 and/or a CDR-H1 contained within the VH sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209; and/or a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence set forth in SEQ ID NO: 21, 26, 28, 34, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173 or 318 and/or comprising the amino acid sequence set forth in SEQ ID NO: 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302 or 303 and/or comprising the amino acid sequence set forth in SEQ ID NO: 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316 or 317 and/or a CDR-H2 contained within the VH sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209. In some of any such embodiments, the VH region includes a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence set forth in SEQ ID NO: 20, 27, or 33 and/or a CDR-H1 contained within the VH sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19; and/or a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence set forth in SEQ ID NO: 21, 26, 28, or 34 and/or a CDR-H2 contained within the VH sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19.
Provided are antibodies or antigen-binding fragments thereof that include a heavy chain variable (VH) region comprising a heavy chain complementarity determining region 1 (CDR-H1), CDR-H2, and CDR-H3, wherein the CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 20, 27, 33, 74, 75, 76, 77, 78, 79, 155, 156, 157, 158, 159, 160, 161, 162 or 163; the CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 21, 26, 28, 34, 80, 81, 82, 83, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, or 318; and/or the CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 22, 29, 35, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 174. In some embodiments, provided are antibodies or antigen-binding fragments thereof that include a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 20, 27, or 33; a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 21, 26, 28, or 34; and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 22, 29, 35, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 174.
In some of any of such embodiments, the antibody includes a CDR-H3 that has the amino acid sequence VSNYEYYFDY (SEQ ID NO: 29), DFGRWGYYFDY (SEQ ID NO: 52), DFGRWSYYFDY (SEQ ID NO: 35) or DSSYDAFDI (SEQ ID NO: 22).
In some of any of such embodiments, the antibody includes a VH region that includes the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 20, 21, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 20, 26, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 27, 28, and 29, respectively; the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 33, 34, and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 33, 34, and 52, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 27, 164 and 45, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 27, 164 and 68, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 27, 164 and 64, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 27, 164 and 66, respectively; the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 33, 318, and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 34 and 70, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 34 and 55, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 34 and 53, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 34 and 56, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 34 and 61, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 34 and 59, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 171 and 60, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 155, 34 and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 156, 34 and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 162, 170 and 50, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 162, 170 and 51, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 161, 169 and 54, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs:159, 167 and 57, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 160, 168 and 58, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 158, 166 and 62, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 158, 166 and 63, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 158, 166 and 65, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 157, 165 and 67, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 163, 173 and 69, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 160, 172, 71, respectively; or the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 160, 172, 174, respectively. In some embodiments, any of the above provided antibodies or fragments can contain the corresponding CDR (e.g. CDR-H1 or CDR-H2) in accord with an alternative numbering scheme as is described and known to a skilled artisan.
In some of any of such embodiments, the antibody includes a VH region that includes the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 20, 21, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 20, 26, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 27, 28, and 29, respectively; the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 33, 34, and 35, respectively; or the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 33, 34, and 52, respectively. In some embodiments, the antibody includes a VH region that includes the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 27, 28, and 29, respectively; or the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 34, and 52, respectively. In some embodiments, the antibody includes a VH region that includes the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 20, 21, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 20, 26, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 27, 28, and 29, respectively; the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 33, 34, and 35, respectively.
Provided are antibodies or antigen-binding fragments thereof that includes a heavy chain complementarity determining region 1 (CDR-H1), a CDR-H2, and a CDR-H3, respectively, comprising the amino acid sequences of a CDR-H1, a CDR-H2, and a CDR-H3 contained within the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209. In some embodiments, provided are antibodies or antigen-binding fragments thereof includes a VH region that includes a heavy chain CDR-H1, a CDR-H2, and a CDR-H3, respectively having the amino acid sequences of a CDR-H1, a CDR-H2, and a CDR-H3 contained within the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19. In some embodiments, the antibody includes a VH region that includes a heavy chain CDR-H1, a CDR-H2, and a CDR-H3, respectively having the amino acid sequences of a CDR-H1, a CDR-H2, and a CDR-H3 contained within the VH region amino acid sequence set forth in SEQ ID NO: 8 or 10. In some embodiments, the antibody includes a VH region that includes a heavy chain CDR-H1, a CDR-H2, and a CDR-H3, respectively having the amino acid sequences of a CDR-H1, a CDR-H2, and a CDR-H3 contained within the VH region amino acid sequence set forth in SEQ ID NO: 13, 15, 17, or 19.
In some of any such embodiments, the VH region contains a framework region 1 (FR1), a FR2, a FR3, and/or a FR4 comprising at least 90% sequence identity, respectively, to a FR1, FR2, FR3, and/or FR4 of the amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209. In some of any such embodiments, the VH region contains a framework region 1 (FR1), a FR2, a FR3, and/or a FR4 sequence having at least 90% sequence identity, respectively, to a FR1, FR2, FR3, and/or FR4 of the amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17 or 19. In some embodiments, the VH region contains a framework region 1 (FR1), a FR2, a FR3, and/or a FR4 sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% sequence identity, respectively, to a FR1, FR2, FR3, and/or FR4 of the amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17 or 19. In some embodiments, the VH region contains a framework region 1 (FR1), a FR2, a FR3, and a FR4 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% sequence identity, respectively, to a FR1, FR2, FR3, and FR4 of the amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17 or 19.
In some of any of such embodiments, the VH region has the sequence of amino acids set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209. In some of any such embodiments, the VH region has the sequence of amino acids set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19. In some embodiments, the VH region has the sequence of amino acids set forth in SEQ ID NO: 8 or 10. In some embodiments, the VH region has the sequence of amino acids set forth in SEQ ID NO: 13, 15, 17, or 19.
In some of any of such embodiments, the VH region comprises the CDR-H1, CDR-H2, and CDR-H3 set forth in SEQ ID NOS: 20, 26, and 22, respectively, and/or the VH region comprises the sequence of amino acids set forth in SEQ ID NO: 19. In some of any of such embodiments, the VH region comprises the CDR-H1, CDR-H2, and CDR-H3 set forth in SEQ ID NOS: 33, 34, and 52, respectively, and/or the VH region comprises the sequence of amino acids set forth in SEQ ID NO: 10. In some of any of such embodiments, the VH region comprises the CDR-H1, CDR-H2, and CDR-H3 set forth in SEQ ID NOS: 33, 34, and 35, respectively, and/or the VH region comprises the sequence of amino acids set forth in SEQ ID NO: 13. In some of any of such embodiments, the VH region comprises the CDR-H1, CDR-H2, and CDR-H3 set forth in SEQ ID NOS: 27, 28, and 29, respectively, and/or the VH region comprises the sequence of amino acids set forth in SEQ ID NO: 15.
In any of such provided embodiments, the antibody or antigen-binding fragment is a heavy chain only, a VH-only, and/or does not include a VL or antigen-binding portion thereof and/or the antigen-binding site of the antibody or fragment includes residues from the heavy chain only and/or does not include residues from a light chain.
In some of any such embodiments, the antibody or fragment does not contain a light chain variable (VL) region, does not contain a CDR-L1, CDR-L2, and/or CDR-L3, and/or is a single-domain antibody (sdAb) containing only the VH region. In some embodiments, the antibody or fragment is an sdAb that only contains a VH region from any as described.
In some embodiments of any of the antibodies or fragments containing any of the above VH region sequences, the antibody or fragment further contains a light chain variable (VL) region. In some such embodiments, the VL region has at least 90% sequence identity to the VL region amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the VL region amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248. In some such embodiments, the VL region has at least 90% sequence identity to the VL region amino acid sequence set forth in SEQ ID NO: 14, 16, or 18, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the VL region amino acid sequence set forth in SEQ ID NO: 14, 16, or 18.
In some of any such embodiments, he VL region comprises a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence set forth in SEQ ID NO: 25, 32, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48, 49, 232 or 233. In some of any such embodiments, the VL region contains a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence set forth in SEQ ID NO: 25, 32, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48 or 49.
In some of any such embodiments, he VL region contains a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO: 23, 30, 36, 210, 211, 212, 213, 214, 215, 217, 216, 218, 219 or 220 and/or a CDR-L1 contained within the VL sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248; and/or a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence set forth in SEQ ID NO: 24, 31, 37, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230 or 231 and/or a CDR-L2 contained within the VL sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248. In some of any such embodiments, the VL region contains a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence set forth in SEQ ID NO: 23, 30, or 36 and/or a CDR-L1 contained within the VL sequence set forth in SEQ ID NO: 14, 16, or 18; and/or a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence set forth in SEQ ID NO: 24, 31, or 37 and/or a CDR-L2 contained within the VL sequence set forth in SEQ ID NO: 14, 16, or 18.
In some of any such embodiments, the VL region contains a CDR-L1 containing the amino acid sequence set forth in SEQ ID NO: 23, 30, 36, 210, 211, 212, 213, 214, 215, 217, 216, 218, 219 or 220; a CDR-L2 containing the amino acid sequence set forth in SEQ ID NO: 24, 31, 37, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230 or 231; and a CDR-L3 containing the amino acid sequence set forth in SEQ ID NO: 25, 32, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48, 49, 232 or 233. In some of any such embodiments, the VL region contains a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 23, 30, or 36; a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 24, 31, or 37; and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 25, 32, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48, or 49.
In some of any such embodiments, the CDR-L3 has the amino acid sequence set forth in SEQ ID NO: 25, 32 or 38.
In some of any such embodiments, the antibody includes a VL region that includes the CDR-L1, CDR-L2, and CDR-L3 comprising the sequences of SEQ ID NOs: 23, 24, and 25, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprising the sequences of SEQ ID NOs: 30, 31, and 32, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprising the sequences of SEQ ID NOs: 36, 37, and 38, respectively; the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 216, 227, and 40, respectively; the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 218, 229, and 39, respectively; the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 219, 230, and 43, respectively; the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 220, 231, and 46, respectively; the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 210, 221, and 49, respectively; the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 210, 221, and 233, respectively; the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 211, 222, and 48, respectively; the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 212, 223, and 42, respectively; the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 214, 225, and 232, respectively; the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 215, 226, and 44, respectively; the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 217, 228, and 41, respectively; the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 36, 37, and 38, respectively; or the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 213, 224, and 47, respectively. In some of any such embodiments, the antibody or fragment includes a VL region that includes the CDR-L1, CDR-L2, and CDR-L3 comprising the sequences of SEQ ID NOs: 23, 24, and 25, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprising the sequences of SEQ ID NOs: 30, 31, and 32, respectively; or the CDR-L1, CDR-L2, and CDR-L3 comprising the sequences of SEQ ID NOs: 36, 37, and 38, respectively.
In some of any such embodiments, the VL region contains the CDR-L1, CDR-L2, and CDR-L3, respectively, contained within the VL region amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248. In some of any such embodiments, the VL region contains the CDR-L1, CDR-L2, and CDR-L3, respectively, contained within the VL region amino acid sequence set forth in SEQ ID NO: 14, 16, or 18.
In some of any such embodiments, the VL region contains a framework region 1 (FR1), a FR2, a FR3, and/or a FR4 having at least 90% sequence identity, respectively, to the FR1, FR2, FR3, and/or FR4 of the amino acid sequence set forth in SEQ ID NO: 14, 16, or 18. In some embodiments, the VL region contains a framework region 1 (FR1), a FR2, a FR3, and/or a FR4 sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% sequence identity, respectively, to a FR1, FR2, FR3, and/or FR4 of the amino acid sequence set forth in SEQ ID NO: 14, 16, or 18. In some embodiments, the VL region contains a framework region 1 (FR1), a FR2, a FR3, and a FR4 sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99% sequence identity, respectively, to a FR1, FR2, FR3, and FR4 of the amino acid sequence set forth in SEQ ID NO: 14, 16, or 18.
In some of any such embodiments, the VL region has the amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248. In some of any such embodiments, the VL region has the amino acid sequence set forth in SEQ ID NO: 14, 16, or 18.
Provided are antibodies or antigen-binding fragments thereof that contain the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 sequences contained within the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209; and/or contain the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 sequences contained within the light chain variable (VL) region amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248. In some embodiments, provided are antibodies or antigen-binding fragments thereof that contain the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 sequences contained within the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19; and/or contain the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 sequences contained within the light chain variable (VL) region amino acid sequence set forth in SEQ ID NO: 14, 16, or 18.
Provided are antibodies or antigen-binding fragments thereof that include the VH and VL regions having amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 19 and 18, respectively; VH and VL regions having amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 17 and 18, respectively; VH and VL regions having amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 15 and 16, respectively; VH and VL regions having amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 13 and 14, respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 182 and 242 respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 182 and 246, respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 182 and 247, respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 185 and 248, respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 186 and 248, respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 175 and 234, respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 175 and 235, respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 176 and 236, respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 176 and 237, respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 177 and 238, respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 179 and 240 respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 180 and 241, respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 181 and 241, respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 183 and 243 respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 183 and 244 respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 184 and 243, respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 184 and 244 respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 183 and 245, respectively; a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 184 and 245, respectively; or a VH and VL regions comprising amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs: 178 and 239, respectively.
In some embodiments, provided are antibodies or antigen-binding fragments thereof that include the VH and VL regions having amino acid sequences set forth in SEQ ID NOs: 19 and 18, respectively; VH and VL regions having amino acid sequences set forth in SEQ ID NOs: 17 and 18, respectively; VH and VL regions having amino acid sequences set forth in SEQ ID NOs: 15 and 16, respectively; VH and VL regions having amino acid sequences set forth in SEQ ID NOs: 13 and 14, respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 182 and 242 respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 182 and 246, respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 182 and 247, respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 185 and 248, respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 186 and 248, respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 175 and 234, respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 175 and 235, respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 176 and 236, respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 176 and 237, respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 177 and 238, respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 179 and 240 respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 180 and 241, respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 181 and 241, respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 183 and 243 respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 183 and 244 respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 184 and 243, respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 184 and 244 respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 183 and 245, respectively; a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 184 and 245, respectively; or a VH and VL regions comprising amino acid sequences set forth in SEQ ID NOs: 178 and 239, respectively.
In some of any such embodiments, the VH and VL regions include the amino acid sequences of SEQ ID NOs: 19 and 18, respectively; the VH and VL regions include the amino acid sequences of SEQ ID NOs: 17 and 18, respectively; the VH and VL regions include the amino acid sequences of SEQ ID NOs: 15 and 16 respectively; or the VH and VL regions include the amino acid sequences of SEQ ID NOs: 13 and 14 respectively.
In some of any such embodiments, the antibody or fragment specifically binds to a ROR1 protein. In some embodiments, the antibody or fragment has a binding affinity for a ROR1 protein with an EC50 that is from or from about 0.1 nM to 100 nM, 0.5 nM to 50 nM or 1 nM to 10 nM. In some embodiments, the antibody or fragment has a binding affinity for a ROR1 protein with an EC50 that is less than or less than about 100 nM, less than or less than about 50 nM, less than or less than about 10 nM or less than or less than about 1 nM.
In some of any such embodiments, the antibody or fragment has a binding affinity for a ROR1 protein, such as a human ROR1 protein, that is at least as high or substantially as high as the binding affinity for the same ROR1 protein of the corresponding form of the anti-ROR1 antibody R12 or antigen-binding fragment thereof, which is optionally an scFv fragment of R12.
In some of any such embodiments, the antibody or fragment specifically binds to the same or an overlapping epitope of a ROR1 protein, such as a human ROR1 protein, as the epitope specifically bound by the anti-ROR1 antibody R12 or an antigen-binding fragment thereof, which is optionally an scFv fragment of R12. In some embodiments, the antibody or fragment inhibits the binding of the anti-ROR1 antibody R12 or an antigen-binding fragment thereof, which is optionally an scFv fragment of R12, to a ROR1 protein, such as a human ROR1 protein, by greater than or greater than about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, such as greater than or greater than about 80% or greater than or greater than about 90%.
In some of any such embodiments, the ROR1 protein is a human ROR1 protein and the antibody or fragment binds to a human ROR1 protein. In some embodiments, the human ROR1 protein has the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, provided are anti-human ROR1 antibodies.
In some of any such embodiments, the antibody or fragment binds an epitope within an extracellular portion of the ROR1 protein, such as a human ROR1 protein. In some embodiments, the extracellular portion of ROR1 contains the amino acids 1-377 of SEQ ID NO: 103. In some of any such embodiments, the antibody or fragment binds to an epitope of a ROR1 protein, such as a human ROR1 protein, containing residues within the fz domain or the Ig domain of the ROR1 protein. In some embodiments, the epitope contains a residue within the fz domain of the ROR1 protein and a residue within the Ig domain of the ROR1 protein. In some embodiments, the Ig domain is one that contains residues 13-118 of the amino acid sequence set forth in SEQ ID NO: 103; and/or the fz domain contains residues 136-270 of the amino acid sequence set forth in SEQ ID NO:103.
In some embodiments, the ROR1 protein is a mouse ROR1 protein and the antibody or fragment binds to a mouse ROR1 protein. In some embodiments, the mouse ROR1 protein has the amino acid sequence set forth in SEQ ID NO:106. In some embodiments, the antibody or fragment binds to an overlapping epitope of a human ROR1 protein as the epitope specifically bound by the anti-ROR1 antibody R12 or an antigen-binding fragment thereof, which is optionally an scFv fragment of R12 and the antibody or fragment also binds to mouse ROR1.
In some of any such embodiments, the antibody or fragment does not specifically bind to a mouse ROR1 protein or does not specifically bind to a protein having the amino acid sequence set forth in SEQ ID NO: 106.
In some of any such embodiments, the antibody or fragment does not contain the CDR-H1, CDR-H2, CDR-H3 and/or CDR-L1, CDR-L2, CDR-L3 sequences of the anti-ROR1 antibody, R12, and/or of the anti-ROR1 antibody 2A2. In some embodiments, the antibody or fragment does not contain a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and/or CDR-L3 sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the CDR-H1, CDR-H2, CDR-H3 and/or CDR-L1, CDR-L2, CDR-L3 sequences of the anti-ROR1 antibody, R12, and/or of the anti-ROR1 antibody 2A2.
In some of any such embodiments, the antibody or fragment is human.
Provided are human antibodies or antigen-binding fragments thereof that specifically bind to the same or an overlapping epitope of a ROR1 protein, such as a human ROR1 protein, as the epitope specifically bound by any of the above provided antibodies or antigen-binding fragments thereof or by the anti-ROR1 antibody designated R12 or an antigen-binding fragment thereof. Also provided are human antibodies or antigen-binding fragments thereof that specifically bind to ROR1 and competes for binding to ROR1, such as a human ROR1 protein, with any of the above provided antibodies or antigen-binding fragments thereof or by the anti-ROR1 antibody designated R12 or antigen-binding fragment thereof. In some such embodiments, the human antibody or fragment contains a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and/or CDR-L3 that is distinct from a corresponding CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and/or CDR-L3 present in an antibody designated R12 and/or 2A2.
In some of any such embodiments, the human antibody or fragment contains a VH region with a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain V segment, a portion with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain D segment, and/or a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain J segment. In some of any such embodiments, the human antibody or fragment contains vale region with a portion with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain V segment, and/or a portion with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain J segment.
In some of any such embodiments, the human antibody or fragment contains a CDR-H1 and/or CDR-H2 that has a sequence 100% identical or with no more than one amino acid difference as compared to an amino acid sequence of a CDR-H1 and/or CDR-H2, respectively, within a sequence encoded by a germline nucleotide human heavy chain V segment; and/or contains a CDR-L1 and/or CDR-L2 that has a sequence 100% identical or with no more than one amino acid difference as compared to an amino acid sequence of a CDR-L1 and/or CDR-L2, respectively, within a sequence encoded by a germline nucleotide human kappa or lambda v segment.
In some of any such embodiments, the antibody or fragment is recombinant. In some of any such embodiments, the antibody or fragment is monoclonal.
In some of any such embodiments, the antibody or fragment is an antigen-binding fragment. In some embodiments, the antibody is a single chain fragment. In some embodiments, the antibody is a single domain antibody, such as a single domain antibody containing only a VH region. In some embodiments, the antibody contains both a VH region and a VL region and is a fragment in which the antibody variable regions are joined by a flexible linker.
In some of any such embodiments, the fragment is an scFv. In some embodiments, the scFv contains a linker having the sequence set forth SEQ ID NO: 91. In some embodiments, the scFv contains the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 12, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267 or 268, or a sequence of amino acids that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 12, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267 or 268. In some embodiments, the scFv contains the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, or 12, or a sequence of amino acids that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, or 12. In some embodiments, the scFv contains the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, or 12.
In some aspects, the scFv contains the VH, linker and VL as set forth in SEQ ID NO: 2, 4, 6, 12, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267 or 268, or a sequence at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such a sequence, but in which the VH and VL are configured in the opposite orientation, i.e. VL-VH, as compared to such sequence.
In some embodiments, the antibody or fragment comprises the VH region set forth in SEQ ID NO: 19 and the VL region set forth in SEQ ID NO: 18, and/or the antibody or fragment comprises the sequence of amino acids set forth in SEQ ID NO: 12. In some embodiments, the antibody or fragment comprises the VH region set forth in SEQ ID NO: 13 and the VL region set forth in SEQ ID NO: 14, and/or the antibody or fragment comprises the sequence of amino acids set forth in SEQ ID NO: 6. In some embodiments, the antibody or fragment comprises the VH region set forth in SEQ ID NO: 15 and the VL region set forth in SEQ ID NO: 16, and/or the antibody or fragment comprises the sequence of amino acids set forth in SEQ ID NO: 4. In some embodiments, the antibody or fragment comprises the VH region set forth in SEQ ID NO: 17 and the VL region set forth in SEQ ID NO: 18, and/or the antibody or fragment comprises the sequence of amino acids set forth in SEQ ID NO: 2.
Provided is a single chain cell-surface protein containing any of the provided single chain antibody fragment. In some embodiments, the single chain cell surface protein contains any of the provided single domain antibodies. In some embodiments, the single chain cell surface protein contains the VH sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209. In some embodiment, the single chain cell surface protein contains the VH region sequence having the sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19. In some embodiments, the single chain cell surface protein contains any of the provided scFv antibody fragments. In some embodiments, the single chain cell surface protein contains the scFv sequence of SEQ ID NO: 2, 4, 6, 12, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267 or 268. In some embodiment, the single chain cell surface protein contains the scFv sequence of SEQ ID NO: 2, 4, 6 or 12. In some embodiments, the single chain cell surface protein contains the scFv sequence of SEQ ID NO: 2, 4, 6, 12, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267 or 268, but in which the VH and VL are configured in the opposite orientation, i.e. VL-VH, as compared to such sequence.
In some of any such embodiments, the antibody or fragment further contains at least a portion of an immunoglobulin constant region. In some embodiments, the antibody or fragment further contains a spacer. In some embodiments, the antibody or fragment contains a spacer set forth in SEQ ID NO: 108. In some embodiments, the antibody or fragment contains a spacer set forth in SEQ ID NO: 142. In some embodiments, the antibody or fragment contains a spacer set forth in SEQ ID NO: 143. In some embodiments, the antibody or fragment contains an immunoglobulin constant region containing an Fc region. In some embodiments, the Fc region is an Fc region of a human IgG.
Also provided is a conjugate containing any of the provided antibodies or fragments. Also provided is a conjugate containing any of the provided single chain cell-surface proteins. In some embodiments, the conjugate contains a heterologous moiety that is linked directly or indirectly to the antibody or antigen-binding fragment. In some embodiments, the conjugate can be a fusion protein or chimeric molecule. In some embodiments, the heterologous moiety is an effect domain.
In some embodiments, provided is a conjugate, fusion protein or chimeric molecule containing a) any of the provided antibodies or antigen-binding fragments and b) an effector domain. In some embodiments, the effector domain is not naturally associated in a single polypeptide chain with the antibody or antigen-binding fragment thereof. In some embodiments, the effector domain is a chemotherapeutic agent or toxin. In some embodiments, the effector domain contains a signaling domain that is capable of delivering a signal to a cell, such as an immune cell, such as a lymphocyte, for example, an activating signal, a costimulatory signal, a suppressive signal and/or an inhibitory signal.
Also provided is a chimeric antigen receptor (CAR) including an extracellular portion containing any of the provided antibodies or antigen-binding fragments and an intracellular signaling domain. In some embodiments, the antibody or fragment is an scFv and the intracellular signaling domain contains an ITAM. In some embodiments, the intracellular signaling domain contains a signaling domain of a zeta chain of a CD3-zeta (CD3) chain.
In some of any such embodiments, the CAR also contains a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the transmembrane domain contains a transmembrane portion of a costimulatory molecule, such as a T cell costimulatory molecule, e.g., CD28 and/or 41BB. In some embodiments, the T cell costimulatory molecule is CD28 or 41BB. In some embodiments, the intracellular signaling domain also includes an intracellular domain of a T cell costimulatory molecule. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.
In some embodiments of any of the provided CARs, the CAR contains an antibody or antigen-binding fragment as provided herein, a transmembrane domain that is a portion of CD28 or variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some such embodiments, the receptor further includes a spacer containing an Ig hinge, e.g., an IgG4 hinge, such as a hinge-only spacer, between the antibody or antigen-binding fragment and the transmembrane domain.
In some embodiments of any of the provided CARs, the CAR contains an antibody or antigen-binding fragment as provided herein, a transmembrane domain that is a portion of CD28 or variant thereof, and an intracellular signaling domain containing a signaling portion of a 4-1BB or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some such embodiments, the receptor further includes a spacer containing an Ig hinge, e.g., an IgG4 hinge, such as a hinge-only spacer, between the antibody or antigen-binding fragment and the transmembrane domain.
Provided are nucleic acids encoding any of the provided antibodies or fragments thereof, any of the provided single chain cell-surface proteins, any of the provided conjugates, or any of the provided CARs. In some embodiments, the nucleic acid molecule encoding an antibody or antigen-binding fragment comprises the sequence of nucleotides set forth in SEQ ID NO:7 or 9 or a sequence of nucleotides that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO:7 or 9. In some embodiments, the nucleic acid molecule encoding an antibody or antigen-binding fragment comprises the sequence of nucleotides set forth in SEQ ID NO: 1, 3, 5 or 11 or a sequence of nucleotides that exhibits at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence set forth in SEQ ID NO: 1, 3, 5 or 11.
Also provided are cells that contain any of the provided antibodies or fragments, any of the provided single chain cell-surface proteins, any of the provided conjugates, or any of the provided CARs. In some embodiments, the cell is an engineered cell expressing a receptor and contains any of the provided antibodies or fragments, any of the single chain cell-surface proteins, any of the provided conjugates, or any of the provided CARs. In some embodiments, the cell or engineered cell is a T cell.
Provided are compositions or pharmaceutical compositions containing any of the provided antibodies or fragments, any of the provided single chain cell-surface proteins, any of the provided conjugates, any of the provided CARs or any of the provided cells. In some embodiments, the composition or pharmaceutical composition contains a pharmaceutically acceptable excipient.
Provided are methods of treatments that include administering any of the provided compositions or pharmaceutical compositions to a subject having a disease or disorder associated with ROR1. Provided are methods of treatment that include administering any of the provided antibodies or fragments, any of the provided single chain cell-surface proteins, any of the provided conjugates, any of the provided CARs or any of the provided cell to a subject having a disease or disorder associated with ROR1.
Provided are any of the provided compositions or pharmaceutical compositions for use in treating a disease or disorder associated with ROR1. Provided is use of any of the provided compositions or pharmaceutical compositions for the manufacture of a medicament for treating a disease or disorder associated with ROR1.
In some of any such embodiments, the disease or disorder is a ROR-1-expressing cancer. In some embodiments, the ROR-1-expressing cancer is a B cell leukemia, lymphoma, B cell chronic lymphocytic leukemia (CLL), AML, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), Burkett's Lymphoma, mantle cell lymphoma (MCL), non-small cell lung cancer (NSCLC), neuroblastoma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell cancer, melanoma, myeloma, stomach cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, uterine cancer, adrenal cancer or head and neck cancer.
In some embodiments, administration of the antibody or receptor, or conjugate or cell containing the same, is associated with a lower degree of immunogenicity as compared to administration of a reference antibody (or receptor containing the reference antibody) that competes for binding with the antibody or binds to an overlapping epitope. In some aspects, the reference antibody is a humanized, chimeric, or non-human antibody.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows cytolytic activity of primary human CD8+ T cells expressing various anti-ROR1 specific CARs against ROR1-expressing cells. tEGFR alone is a negative control; R12 is R12 scFv CAR. FIG. 1B shows cytokine secretion of primary human CD8+ T cells expressing various anti-ROR1 specific CARs after co-culture with ROR1-expressing cells. tEGFR alone is a negative control; R12 is R12 scFv CAR.
FIG. 2 shows cytokine secretion of primary human CD8+ T cells expressing various anti-ROR1 specific CARs after co-culture with human ROR1-expressing cells (hROR1) or mouse ROR1-expressing cells (mROR1). tEGFR alone is a negative control; R12 is R12 scFv CAR.
DETAILED DESCRIPTION
Provided are ROR1-binding molecules, including antibodies (including antigen-binding antibody fragments, such as heavy chain variable (VH) regions and single chain fragments, including scFvs) and recombinant receptors, including chimeric receptors containing such antibodies and fragments, nucleic acids encoding such antibodies and fragments, and cells, such as recombinant cells, expressing and for production of these antibodies and fragments. The invention also provides methods of making and using the antibodies and fragments as well as cells expressing or containing the antibodies and fragments.
I. ROR1 Binding Molecules
Provided in some aspects are ROR1 binding molecules, such as ROR1-binding polypeptides. Such binding molecules include antibodies (including antigen-binding fragments) that specifically bind to ROR1 proteins, such as human ROR1 protein. Also among the binding molecules are polypeptides containing such antibodies, including single chain cell surface proteins, e.g., recombinant receptors such as chimeric antigen receptors, containing such antibodies.
A. ROR1 Antibodies
Provided are anti-ROR1 antibodies, including functional antibody fragments. In some embodiments, the antibodies include those that are single domain antibodies, containing a variable heavy chain that without pairing with a light chain antigen-binding site and/or without any additional antibody domain or binding site are capable of specifically binding to ROR1. Also among the antibodies are multi-domain antibodies, such as those containing VH and VL domains, comprised of the VH domain or antigen-binding site thereof of the single-domain antibody. In some embodiments, the antibodies include a variable heavy chain and a variable light chain, such as scFvs. The antibodies include antibodies that specifically bind to ROR1, e.g., human ROR1. Among the provided anti-ROR1 antibodies are human antibodies. The antibodies include isolated antibodies. The molecules include isolated molecules. Also provided are molecules containing such antibodies, e.g., single-chain proteins, fusion proteins, and/or recombinant receptors such as chimeric receptors, including antigen receptors.
The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab′)2 fragments, Fab′ fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
The terms “complementarity determining region,” and “CDR,” synonymous with “hypervariable region” or “HVR,” are known in the art to refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and/or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). “Framework regions” and “FR” are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).
The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745,” (“Contact” numbering scheme), Martin et al., Proc. Natl. Acad. Sci., 86:9268-9272 (1989) (“AbM” numbering scheme), Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme), and Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (“Aho” numbering scheme).
The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
Table 1, below, lists exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FR-L1 located between CDR-L1 and CDR-L2, and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.
TABLE 1
CDR
Kabat
Chothia
AbM
Contact
CDR-L1
L24-L34
L24-L34
L24-L34
L30-L36
CDR-L2
L50-L56
L50-L56
L50-L56
L46-L55
CDR-L3
L89-L97
L89-L97
L89-L97
L89-L96
CDR-H1
H31-H35B
H26-
H26-H35B
H30-H35B
(Kabat
H32 . . . 34
Numbering1)
CDR-H1
H31-H35
H26-H32
H26-H35
H30-H35
(Chothia
Numbering2)
CDR-H2
H50-H65
H52-H56
H50-H58
H47-H58
CDR-H3
H95-H102
H95-H102
H95-H102
H93-H101
1Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD
2Al-Lazikani et al., (1997) JMB 273, 927-948
Thus, unless otherwise specified, a “CDR” or “complementary determining region,” or individual specified CDRs (e.g., “CDR-H1, CDR-H2), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the aforementioned schemes. For example, where it is stated that a particular CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given VH or VL amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the aforementioned schemes. In some embodiments, specific CDR sequences are specified.
Likewise, unless otherwise specified, a FR or individual specified FR(s) (e.g., FR-H1, FR-H2), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) framework region as defined by any of the known schemes. In some instances, the scheme for identification of a particular CDR, FR, or FRs or CDRs is specified, such as the CDR as defined by the Kabat, Chothia, or Contact method. In other cases, the particular amino acid sequence of a CDR or FR is given.
The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
Among the provided antibodies are antibody fragments. An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; variable heavy chain (VH) regions, single-chain antibody molecules such as scFvs and single-domain VH single antibodies; and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody.
Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some aspects, the antibody fragments are scFvs.
A “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
Among the provided anti-ROR1 antibodies are human antibodies. A “human antibody” is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences, including human antibody libraries. The term excludes humanized forms of non-human antibodies comprising non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human. The term includes antigen-binding fragments of human antibodies.
Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated. Human antibodies also may be derived from human antibody libraries, including phage display and cell-free libraries, containing antibody-encoding sequences derived from a human repertoire.
Among the provided antibodies are monoclonal antibodies, including monoclonal antibody fragments. The term “monoclonal antibody” as used herein refers to an antibody obtained from or within a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible variants containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. The term is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be made by a variety of techniques, including but not limited to generation from a hybridoma, recombinant DNA methods, phage-display and other antibody display methods.
The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains and other peptides, e.g., linkers and ROR1-binding peptides, may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
Exemplary Antibodies
In some embodiments, the antibody, e.g., the anti-ROR1 antibody, e.g., antigen-binding antibody fragment, contains a heavy and/or light chain variable (VH or VL) region sequence as described, or a sufficient antigen-binding portion thereof. In some embodiments, the anti-ROR1 antibody, e.g., antigen-binding antibody fragment, contains a VH region sequence or sufficient antigen-binding portion thereof that contains a CDR-H1, CDR-H2 and/or CDR-H3 as described. In some embodiments, the anti-ROR1 antibody, e.g., antigen-binding antibody fragment, contains a VL region sequence or sufficient antigen-binding portion that contains a CDR-L1, CDR-L2 and/or CDR-L2 as described. In some embodiments, the anti-ROR1 antibody, e.g., antigen-binding antibody fragment, contains a VH region sequence that contains a CDR-H1, CDR-H2 and/or CDR-H3 as described and contains a VL region sequence that contains a CDR-L1, CDR-L2 and/or CDR-L2 as described. Also among the provided antibodies are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such a sequence.
In some embodiments, the antibody, e.g., antigen-binding fragment thereof, has a heavy chain variable (VH) region having the amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17 or 19, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19, or contains a CDR-H1, CDR-H2, and/or CDR-H3 present in such a VH sequence.
In some embodiments, the VH region of the anti-ROR1 antibody is one that includes a heavy chain complementarity determining region 3 (CDR-H3) that contains the amino acid sequence: X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16 (SEQ ID NO: 92), wherein X1 is D, V, Q, I, T, G, A, or E; X2 is S, F, N, R, G, T, Y, D, Q, M, K, I, L, or P; X3 is S, N, G, E, D, P, I, V, R, F, N, or L; X4 is Y, R, V, G, S, P, E, A, or L; X5 is D, E, W, R, S, L, A, V, G, or P; X6 is A, W, S, E, G, Y, R, F, H, V, D, S, or Q; X7 is F, Y, A, W, L, G, D, V, N, S, K, or R; X8 is F, Y, H, E, L, W, V, N, I, D, or null; X9 is F, L, V, S, E, W, G, P, Y, or null; X10 is S, E, L, Y, W, F, N, G, P, or null; X11 is Y, V, L, F, I, W, N, or null; X12 is F, Y, W, or null; X13 is Y, F, T, or null; X14 is F or null; X15 is D or Q; and X16 is I, Y, or P. In some such embodiments, in said CDR-H3, X1 is D or V; X2 is S or F; X3 is S, N, G, or E; X4 is Y, R, or V; X5 is D, E, W, or R; X6 is A, Y, S, or E; X7 is F, Y, or A; X8 is F, Y, H, or null; X9 is F, L, or null; X10 is S or null; X11 is Y or null; X12 is F or null; X13 is null; X14 is null; X15 is D; and X16 is I or Y. In some such embodiments, in said CDR-H3, X1 is V or D; X2 is S or F; X3 is N or G; X4 is Y or R; X5 is E or W; X6 is Y or G; X7 is Y; X8 is F or Y; X9 is F or null; X10 is null; X11 is null; X12 is null; X13 is null; X14 is null; X15 is D; and X16 is Y.
In some embodiments, the VH region of the anti-ROR1 antibody is one that includes a CDR-H3 that contains the amino acid sequence: X1X2X3X4X5X6X7X8X9X10X11X12DX14 (SEQ ID NO: 93), wherein X1 is D or V; X2 is S or F; X3 is S, N, G, or E; X4 is Y, R, or V; X5 is D, E, W, or R; X6 is A, Y, S, or E; X7 is F, Y, or A; X8 is F, Y, H, or null; X9 is F, L, or null; X10 is S or null; X11 is Y or null; X12 is F or null; and X14 is I or Y.
In some embodiments, the VH region of the anti-ROR1 antibody is one that includes a CDR-H3 that contains the amino acid sequence: X1X2X3X4X5X6YX8X9DY (SEQ ID NO: 94), wherein X1 is V or D; X2 is S or F; X3 is N or G; X4 is Y or R; X5 is E or W; X6 is Y or G; X8 is F or Y; and X9 is F or null.
In some embodiments, the VH region of the anti-ROR1 antibody is one that includes a CDR-H3 that contains the amino acid sequence: X1SX3YX5X6 X7X8DX10 (SEQ ID NO: 109), wherein X1 is V or D; X3 is S or N; X5 is D or E; X6 is Y or A; X7 is F or Y; X8 is F or null; and X10 is I or Y.
In some embodiments, the VH region of the anti-ROR1 antibody is one that includes a CDR-H3 that contains the amino acid sequence: VX2X3X4EYYFDY (SEQ ID NO: 110), wherein X2 is S or N or R; X3 is N or G; X4 is Y or G or S.
In some embodiments, the provided antibody contains a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 22, 29, 35, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 174. In any of such examples, the provided antibody can contain a VH region sequence set forth in any of SEQ ID NOs: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209 in which the corresponding CDR-H3 sequence contained therein (e.g. corresponding to amino acid residues H95-H102 by Kabat numbering) is replaced by the CDR-H3 sequence set forth in SEQ ID NO: 22, 29, 35, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 174.
In some embodiments, the provided antibody contains a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 22, 29, 35, 45, or 52. In some embodiments, the provided antibody contains a CDR-H3 set forth in SEQ ID NO: 22, 29 or 35. In some embodiments, the provided antibody contains a CDR-H3 set forth in SEQ ID NO: 29 or 35. In some embodiments, the provided antibody contains a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 29 or 52. In some embodiments, the provided antibody contains a CDR-H3 contained within the CDR-H3 of the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209. In some embodiments, the provided antibody contains a CDR-H3 contained within the CDR-H3 of the VH region amino acid sequence set forth in SEQ ID NO: 8 or 10. In some embodiments, the provided antibody contains a CDR-H3 contained within the CDR-H3 of the VH region amino acid sequence set forth in SEQ ID NO: 10, 13, 15, 17 or 19. In some embodiments, the provided antibody contains a CDR-H3 contained within the CDR-H3 of the VH region amino acid sequence set forth in SEQ ID NO: 13, 15 or 19. In some embodiments, the provided antibody contains a CDR-H3 contained within the CDR-H3 of the VH region amino acid sequence set forth in SEQ ID NO: 13 or 15.
In some embodiments, the VH region of the anti-ROR1 antibody is one that includes a heavy chain complementarity determining region 1 (CDR-H1) that contains the amino acid sequence: X1X2X3MX5 (SEQ ID NO: 95), wherein X1 is S, D, or N; X2 is Y or A; X3 is Y, A, or W; and X5 is H or S. In some embodiments, in said CDR-H1, X1 is D or N; X2 is Y or A; X3 is A or W; and X5 is S.
In some embodiments, the VH region of the anti-ROR1 antibody is one that includes a CDR-H1 that contains the amino acid sequence: X1X2X3MS (SEQ ID NO: 97), wherein X1 is D or N; X2 is Y or A; and X3 is A or W.
In some embodiments, the provided antibody contains a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 20, 27, 33, 155, 156, 157, 158, 159, 160, 161, 162 or 163. In some embodiments, the provided antibody contains a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 269, 270, 271, 272, 273, 274, 275, 276, 277, 278 or 279. In some embodiments, the provided antibody contains a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 75, 77, 79, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289 or 290. In some embodiments, the provided antibody contains a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 20, 27, 33, 75, 77, 79, 269, 270 or 271. In some embodiments, the provided antibody contains a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 27, 33, 77, 79, 270 or 271. In some embodiments, the provided antibody contains a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 74, 76 or 78. In some embodiments, the provided antibody contains a CDR-H1 contained within the CDR-H1 of the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209. In some embodiments, the provided antibody contains a CDR-H1 contained within the CDR-H1 of the VH region amino acid sequence set forth in SEQ ID NO: 8 or 10. In some embodiments, the provided antibody contains a CDR-H1 contained within the CDR-H1 of the VH region amino acid sequence set forth in SEQ ID NO: 10, 13, 15, 17 or 19. In some embodiments, the provided antibody contains a CDR-H1 contained within the CDR-H1 of the VH region amino acid sequence set forth in SEQ ID NO: 13, 15 or 19. In some embodiments, the provided antibody contains a CDR-H1 contained within the CDR-H1 of the VH region amino acid sequence set forth in SEQ ID NO: 13 or 15.
In some embodiments, the VH region of the anti-ROR1 antibody is one that includes a heavy chain complementarity determining region 2 (CDR-H2) that contains the amino acid sequence: X1IX3X4X5X6X7GX9X10TX12X13AX15X16X17X18G (SEQ ID NO: 96), wherein X1 is I, S, or R; X3 is N, S, or K; X4 is P, G, or S; X5 is K or null; X6 is T or null; X7 is S, D, or N; X9 is G or R; X10 is S or T; X12 is S or D; X13 is Y or H; X15 is Q, D, or A; X16 is K, Y, or P; X17 is F or V; and X18 is Q or K. In some embodiments in said CDR-H2, X1 is S or R; X3 is S or K; X4 is G or S; X5 is K or null; X6 is T or null; X7 is S or D; X9 is G or R; X10 is S or T; X12 is D; X13 is Y or H; X15 is D or A; X16 is Y or P; X17 is F; and X18 is K
In some embodiments, the VH region of the anti-ROR1 antibody is one that includes a CDR-H2 that contains the amino acid sequence: X1IX3X4X5X6X7GX9X10TX12X13AX15X16X17X18G (SEQ ID NO: 98), wherein X1 is I, S, or R; X3 is N, S, or K; X4 is P, G, or S; X5 is K or null; X6 is T or null; X7 is S, D, or N; X9 is G or R; X10 is S or T; X12 is S or D; X13 is Y or H; X15 is Q, D, or A; X16 is K, Y, or P; X17 is F or V; and X18 is Q or K.
In some embodiments, the provided antibody contains a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 21, 26, 28, 34, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173 or 318. In some embodiments, the provided antibody contains a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302 or 303. In some embodiments, the provided antibody contains a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316 or 317. In some embodiments, the provided antibody contains a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 21, 26, 28, 34, 291, 292, 293, 294, 304, 305, 306, 307. In some embodiments, the provided antibody contains a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 26, 28, 34, 291, 292, 294, 304, 305 or 307. In some embodiments, the provided antibody contains a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 28, 34, 291, 292, 304 or 305. In some embodiments, the provided antibody contains a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 80, 81, 82 or 83. In some embodiments, the provided antibody contains a CDR-H2 contained within the CDR-H2 of the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209. In some embodiments, the provided antibody contains a CDR-H2 contained within the CDR-H2 of the VH region amino acid sequence set forth in SEQ ID NO:8 or 10. In some embodiments, the provided antibody contains a CDR-H2 contained within the CDR-H2 of the VH region amino acid sequence set forth in SEQ ID NO: 10, 13, 15, 17 or 19. In some embodiments, the provided antibody contains a CDR-H2 contained within the CDR-H2 of the VH region amino acid sequence set forth in SEQ ID NO: 13, 15 or 19. In some embodiments, the provided antibody contains a CDR-H2 contained within the CDR-H2 of the VH region amino acid sequence set forth in SEQ ID NO: 13 or 15.
In some embodiments, the provided antibody contains a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 20, 27, 33, 155, 156, 157, 158, 159, 160, 161, 162 or 163; a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 21, 26, 28, 34, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173 or 318; and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 22, 29, 35, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 174. In some embodiments, the provided antibody contains a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 269, 270, 271, 272, 273, 274, 275, 276, 277, 278 or 279; a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302 or 303; and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 22, 29, 35, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 174. In some embodiments, the provided antibody contains a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 75, 77, 79, 280, 281, 282, 283, 284, 285, 286, 287, 288 or 289; a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316 or 317; and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 22, 29, 35, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 174.
In some embodiments, the provided antibody contains a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 20, 27, 33, 75, 77, 79, 269, 270, or 271; a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 21, 26, 28, 34, 291, 292, 293, 294, 304, 305 306 or 307; and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 22, 29, 35, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 174. In some embodiments, the provided antibody contains a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 20, 27, 33, 75, 77, 79, 269, 270, or 271; a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 21, 26, 28, 34, 291, 292, 293, 294, 304, 305 306 or 307; and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 22, 29, 35 or 52. In some embodiments, the provided antibody contains a CDR-H1 having the amino acid sequence set forth in SEQ ID NO: 27, 33, 77, 79, 269, 271; a CDR-H2 having the amino acid sequence set forth in SEQ ID NO: 28, 34, 291, 292, 304 or 305; and a CDR-H3 having the amino acid sequence set forth in SEQ ID NO: 29 or 352.
In some embodiments, the CDR-H1, CDR-H2 and CDR-H3 contain the sequences of SEQ ID NOS: 20, 21, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 20, 26, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 27, 28, and 29, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 33, 34, and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 33, 34, and 52, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 27, 164 and 45, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 27, 164 and 68, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 27, 164 and 64, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 27, 164 and 66, respectively; the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 33, 318, and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 33, 34 and 70, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 33, 34 and 55, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 33, 34 and 53, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 33, 34 and 56, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 33, 34 and 61, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 33, 34 and 59, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 33, 171 and 60, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 155, 34 and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 156, 34 and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 162, 170 and 50, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 162, 170 and 51, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 161, 169 and 54, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs:159, 167 and 57, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 160, 168 and 58, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 158, 166 and 62, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 158, 166 and 63, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 158, 166 and 65, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 157, 165 and 67, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 163, 173 and 69, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 160, 172, 71, respectively; or the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 160, 172, 174, respectively.
In some embodiments, the CDR-H1, CDR-H2 and CDR-H3 contain the sequences of SEQ ID NOS: 269, 293, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 269, 294, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 270, 292, and 29, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 271, 291, and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 276, 291, and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 271, 291, and 52, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 276, 291, and 52, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 270, 296 and 45, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 270, 296 and 68, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 270, 296 and 64, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 270, 296 and 66, respectively; the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 271, 295, and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 276, 295, and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 271, 291 and 70, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 276, 291 and 70, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 271, 291 and 55, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 276, 291 and 55, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 271, 291 and 53, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 276, 291 and 53, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 271, 291 and 56, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 276, 291 and 56, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 271, 291 and 61, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 276, 291 and 61, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 271, 291 and 59, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 276, 291 and 59, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 271, 291 and 60, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 276, 291 and 60, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 274, 291 and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 275, 291 and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 273, 302 and 50, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 273, 302 and 51, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 279, 301 and 54, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs:277, 299 and 57, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 278, 300 and 58, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 273, 298 and 62, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 273, 298 and 63, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 273, 298 and 65, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 272, 297 and 67, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 273, 303 and 69, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 278, 300, 71, respectively; or the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 278, 300, 174, respectively.
In some embodiments, the CDR-H1, CDR-H2 and CDR-H3 contain the sequences of SEQ ID NOS: 75, 306, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 75, 307, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 77, 305, and 29, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 79, 304, and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 79, 304, and 52, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 77, 309 and 45, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 77, 309 and 68, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 77, 309 and 64, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 77, 309 and 66, respectively; the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 79, 308, and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 79, 304 and 70, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 79, 304 and 55, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 284, 304 and 55, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 79, 304 and 53, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 79, 304 and 56, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 79, 304 and 61, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 79, 304 and 59, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 79, 304 and 60, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 280, 304 and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 283, 304 and 35, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 288, 315 and 50, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 288, 315 and 51, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 287, 314 and 54, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs:285, 312 and 57, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 286 or 289, 313 and 58, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 282, 311 and 62, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 282, 311 and 63, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 282, 311 and 65, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 281, 310 and 67, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 290, 317 and 69, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 286 or 289, 316, 71, respectively; or the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 286 or 289, 316, 174, respectively.
In some embodiments, the CDR-H1, CDR-H2 and CDR-H3 contain the sequences of SEQ ID NOS: 20, 80, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 20, 83, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 27, 81, and 29, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 33, 82, and 35, respectively; or the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 33, 82, and 52, respectively.
In some embodiments, the CDR-H1, CDR-H2 and CDR-H3 contain the sequences of SEQ ID NOS: 269, 293, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 269, 294, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 270, 292, and 29, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 271, 291, and 35, respectively; or the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 271, 291, and 52, respectively.
In some embodiments, the CDR-H1, CDR-H2 and CDR-H3 contain the sequences of SEQ ID NOS: 75, 306, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 75, 307, and 22, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 77, 305, and 29, respectively; the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 79, 304, and 35, respectively; or the CDR-H1, CDR-H2, and CDR-H3 contain the sequences of SEQ ID NOs: 79, 304, and 52, respectively.
In some embodiments, the provided antibody contains a CDR-H1, CDR-H2 and CDR-H3, respectively, contained within the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209. In some embodiments, the provided antibody contains a CDR-H1, CDR-H2 and CDR-H3, respectively, contained within the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19. In some embodiments, the provided antibody contains a CDR-H1, CDR-H2 and CDR-H3, respectively, contained within the VH region amino acid sequence set forth in SEQ ID NO: 8 or 10. In some embodiments, the provided antibody contains a CDR-H1, CDR-H2 and CDR-H3, respectively, contained within the VH region amino acid sequence set forth in SEQ ID NO: 10, 13, 15, 17 or 19. In some embodiments, the provided antibody contains a CDR-H1, CDR-H2 and CDR-H3, respectively, contained within the VH region amino acid sequence set forth in SEQ ID NO: 13, 15 or 19. In some embodiments, the provided antibody contains a CDR-H1, CDR-H2 and CDR-H3, respectively, contained within the VH region amino acid sequence set forth in SEQ ID NO: 13 or 15.
In some embodiments, the VH region contains any of the CDR-H1, CDR-H2 and CDR-H3 as described and contains a framework region that contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework region set forth in the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209. In some embodiments, the anti-ROR1 antibody can contain a CDR-H1, CDR-H2 and CDR-H3, respectively, contained within the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209, and a frame work region that contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework region of the amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209.
In some embodiments, the VH region contains any of the CDR-H1, CDR-H2 and CDR-H3 as described and contains a framework region that contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework region set forth in the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19. For example, the anti-ROR1 antibody can contain a CDR-H1, CDR-H2 and CDR-H3, respectively, contained within the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19, and a frame work region that contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework region of the amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19.
In some embodiments, the provided antibody contains a VH region that has the sequence of amino acids set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209. In some embodiments, the provided antibody contains a VH region that has the sequence of amino acids set forth in SEQ ID NO: 8, 10, 13, 15, 17 or 19. In some embodiments, the provided antibody contains a VH region that has the sequence of amino acids set forth in SEQ ID NO: 10, 13, 15 or 19. In some embodiments, the provided antibody contains a VH region that has the sequence of amino acids set forth in SEQ ID NO: 13, 15 or 19. In some embodiments, the provided antibody contains a VH region that has the sequence of amino acids set forth in SEQ ID NO: 13 or 15.
Also provided are antibodies having sequences at least at or about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.
In some embodiments, the antibody is a single domain antibody (sdAb) containing only the VH region sequence, such as any of the above described VH region sequences, or a sufficient antigen-binding portion thereof.
In some embodiments, the anti-ROR1 antibody containing a VH region further contains a light chain or a sufficient antigen binding portion thereof. For example, in some embodiments, the antibody contains a VH region and a VL region, or a sufficient antigen-binding portion of a VH and VL region. In such embodiments, a VH region sequence can be any of the above described VH sequence. In some such embodiments, the antibody is an antigen-binding fragment, such as a Fab or a scFv. In some such embodiments, the antibody is a full-length or intact antibody that also contains a constant region.
In some embodiments, the antibody, e.g., antigen-binding fragment thereof, has a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VH region amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248. In some embodiments, the antibody, e.g., antigen-binding fragment thereof, has a light chain variable (VL) region having the amino acid sequence set forth in SEQ ID NO: 14, 16, or 18, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VH region amino acid sequence set forth in SEQ ID NO: 14, 16, or 18.
In some embodiments, the VL region of the anti-ROR1 antibody is one that includes a light chain complementarity determining region 3 (CDR-L3) that contains the amino acid sequence: X1X2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 99), wherein X1 is Q, A, or K; X2 is Q, V, A, or S; X3 is Y, W, or L; X4 is E, D, K, or N; X5 is S, D, N, or G; X6 is L, T, S, D, R, or Y; X7 is P, G, L, S, N, or T; X8 is D, S, N, or null; X9 is H, G, L, or null; X10 is Y, P, V, R, H, or L; and X11 is T, V, S, or M. In some embodiments, in said CDR-L3, X1 is Q or A; X2 is Q, V, or A; X3 is Y or W; X4 is E or D; X5 is S or D; X6 is L, T, or S; X7 is P, G, or L; X8 is D, S, or null; X9 is H, G, or null; X10 is Y, P, or V; and X11 is T or V.
In some embodiments, the VL region of the anti-ROR1 antibody is one that includes a light chain complementarity determining region 3 (CDR-L3) that contains the amino acid sequence: X1X2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 100), wherein X1 is Q or A; X2 is Q, V, or A; X3 is Y or W; X4 is E or D; X5 is S or D; X6 is L, T, or S; X7 is P, G, or L; X8 is D, S, or null; X9 is H, G, or null; X10 is Y, P, or V; and X11 is T or V.
In some embodiments, the provided antibody contains a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 25, 32, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48, 49, 232 or 233. In some embodiments, the provided antibody can contain a VL region sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248 in which the corresponding CDR-L3 sequence contained therein (e.g. corresponding to amino acid residues L89 to L97 by Kabat numbering) is replaced by the CDR-L3 sequence set forth in SEQ ID NO: 25, 32, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48, 49, 232 or 233. In some embodiments, the provided antibody can contain a VL region sequence set forth in SEQ ID NO: 14, 16, or 18 in which the corresponding CDR-L3 sequence contained therein (e.g. corresponding to amino acid residues L89 to L97 by Kabat numbering) is replaced by the CDR-L3 sequence set forth in SEQ ID NO: 25, 32, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48, and 49.
In some embodiments, the provided antibody contains a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 25, 32, or 38. In some embodiments, the provided antibody contains a CDR-L3 contained within the CDR-L3 of the VL region amino acid sequence set forth in SEQ ID NO: 14, 16, or 18. In some embodiments, the provided antibody contains a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 32, or 38. In some embodiments, the provided antibody contains a CDR-L3 contained within the CDR-L3 of the VL region amino acid sequence set forth in SEQ ID NO: 14 or 16.
In some embodiments, the VL region of the anti-ROR1 antibody is one that includes a light chain complementarity determining region 1 (CDR-L1) that contains the amino acid sequence: X1X2SX4X5X6X7X8SX10X11X12X13 (SEQ ID NO: 101), wherein X1 is Q, G, or S; X2 is A or G; X4 is Q, N, or S; X5 is S or null; X6 is N or null; X7 is D or I; X8 is I or G; X10 is N or E; X11 is Y or S; X12 is L or V; and X13 is N or Y.
In some embodiments, the provided antibody contains a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 23, 30, 36, 210, 211, 212, 213, 214, 215, 217, 216, 218, 219 or 220. In some embodiments, the provided antibody contains a CDR-L1 contained within the CDR-L1 of the VL region amino acid sequence set forth in SEQ ID NO: 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248. In some embodiments, the provided antibody contains a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 23, 30, or 36. In some embodiments, the provided antibody contains a CDR-L1 contained within the CDR-L1 of the VL region amino acid sequence set forth in SEQ ID NO: 14, 16, or 18.
In some embodiments, the VL region of the anti-ROR1 antibody is one that includes a light chain complementarity determining region 2 (CDR-L2) that contains the amino acid sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 102), wherein X1 is D or R; X2 is A, T, or N; X3 is S, T, or N; X4 is Y, D, or Q; X5 is L or R; X6 is E or P; and X7 is T or S.
In some embodiments, the provided antibody contains a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 24, 31, 37, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230 or 231. In some embodiments, the provided antibody contains a CDR-L2 contained within the CDR-L2 of the VL region amino acid sequence set forth in SEQ ID NO: 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248. In some embodiments, the provided antibody contains a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 24, 31, or 37. In some embodiments, the provided antibody contains a CDR-L2 contained within the CDR-L2 of the VL region amino acid sequence set forth in SEQ ID NO: 14, 16, or 18.
In some embodiments, the provided antibody contains a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 23, 30, 36, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219 or 220; a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 24, 31, 37, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230 or 231; and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 25, 32, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48, 49 or 233. In some embodiments, the provided antibody contains a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 23, 30, or 36; a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 24, 31, or 37; and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 25, 32, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48 49 or 233. In some embodiments, the provided antibody contains a CDR-L1 having the amino acid sequence set forth in SEQ ID NO: 23, 30, or 36; a CDR-L2 having the amino acid sequence set forth in SEQ ID NO: 24, 31, or 37; and a CDR-L3 having the amino acid sequence set forth in SEQ ID NO: 25, 32 or 38.
In some embodiments, the CDR-L1, CDR-L2 and CDR-L3 contain the sequences of SEQ ID NOs: 23, 24, and 25, respectively; the CDR-L1, CDR-L2, and CDR-L3 contain the sequences of SEQ ID NOs: 30, 31, and 22, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprising the sequences of SEQ ID NOs: 36, 37, and 38, respectively; the CDR-L1, CDR-L2 and CDR-L3 contain the sequences of SEQ ID NOs: 216, 227, and 40, respectively; the CDR-L1, CDR-L2 and CDR-L3 contain the sequences of SEQ ID NOs: 218, 229, and 39, respectively; the CDR-L1, CDR-L2 and CDR-L3 contain the sequences of SEQ ID NOs: 219, 230, and 43, respectively; the CDR-L1, CDR-L2 and CDR-L3 contain the sequences of SEQ ID NOs: 220, 231, and 46, respectively; the CDR-L1, CDR-L2 and CDR-L3 contain the sequences of SEQ ID NOs: 210, 221, and 49, respectively; the CDR-L1, CDR-L2 and CDR-L3 contain the sequences of SEQ ID NOs: 210, 221, and 233, respectively; the CDR-L1, CDR-L2 and CDR-L3 contain the sequences of SEQ ID NOs: 211, 222, and 48, respectively; the CDR-L1, CDR-L2 and CDR-L3 contain the sequences of SEQ ID NOs: 212, 223, and 42, respectively; the CDR-L1, CDR-L2 and CDR-L3 contain the sequences of SEQ ID NOs: 214, 225, and 232, respectively; the CDR-L1, CDR-L2 and CDR-L3 contain the sequences of SEQ ID NOs: 215, 226, and 44, respectively; the CDR-L1, CDR-L2 and CDR-L3 contain the sequences of SEQ ID NOs: 217, 228, and 41, respectively; the CDR-L1, CDR-L2 and CDR-L3 contain the sequences of SEQ ID NOs: 36, 37, and 38, respectively; or the CDR-L1, CDR-L2 and CDR-L3 contain the sequences of SEQ ID NOs: 213, 224, and 47, respectively.
In some embodiments, the provided antibody contains a CDR-L1, CDR-L2, and CDR-L3, respectively, contained within the VL region amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245 246, 247 or 248. In some embodiments, the provided antibody contains a CDR-L1, CDR-L2, and CDR-L3, respectively, contained within the VL region amino acid sequence set forth in SEQ ID NO: 14, 16, or 18. In some embodiments, the provided antibody contains a CDR-L1, CDR-L2, and CDR-L3, respectively, contained within the VL region amino acid sequence set forth in SEQ ID NO: 14 or 16.
In some embodiments, the VL region contains any of the CDR-L1, CDR-L2 and CDR-L3 as described and contains a framework region that contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework region of the amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245 246, 247 or 248. For example, the anti-ROR1 antibody can contain a CDR-L1, CDR-L2 and CDR-L3, respectively, contained within the VL region amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245 246, 247 or 248, and a frame work region that contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework region of the amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245 246, 247 or 248.
In some embodiments, the VL region contains any of the CDR-L1, CDR-L2 and CDR-L3 as described and contains a framework region that contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework region of the amino acid sequence set forth in SEQ ID NO: 14, 16, or 18. For example, the anti-ROR1 antibody can contain a CDR-L1, CDR-L2 and CDR-L3, respectively, contained within the VL region amino acid sequence set forth in SEQ ID NO: 14, 16, or 18, and a frame work region that contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework region of the amino acid sequence set forth in SEQ ID NO: 14, 16, or 18.
In some embodiments, the provided antibody contains a VL region that has the sequence of amino acids set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245 246, 247 or 248. In some embodiments, the provided antibody contains a VL region that has the sequence of amino acids set forth in SEQ ID NO: 14, 16, or 18. In some embodiments, the provided antibody contains a VL region that has the sequence of amino acids set forth in SEQ ID NO: 14 or 16.
Also provided are antibodies having sequences at least at or about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.
In some embodiments, the VH region of the antibody or fragment comprises the amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209 and the VL region of the antibody or fragment comprises the amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245 246, 247 or 248. In some embodiments, the VH region of the antibody or fragment comprises the amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17 or 19 and the VL region of the antibody or fragment comprises the amino acid sequence set forth in SEQ ID NO: 14, 16, or 18. In some embodiments, the VH region of the antibody or fragment comprises the amino acid sequence set forth in SEQ ID NO: 10, 13, 15 or 19 and the VL region of the antibody or fragment comprises the amino acid sequence set forth in SEQ ID NO: 14, 16, or 18. In some embodiments, the VH region of the antibody or fragment comprises the amino acid sequence set forth in SEQ ID NO: 13, 15 or 19 and the VL region of the antibody or fragment comprises the amino acid sequence set forth in SEQ ID NO: 14, 16, or 18. In some embodiments, the VH region of the antibody or fragment comprises the amino acid sequence set forth in SEQ ID NO: 13 or 15 and the VL region of the antibody or fragment comprises the amino acid sequence set forth in SEQ ID NO: 14 or 16.
Also provided are antibodies having sequences at least at or about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.
In some embodiments, the VH region of the antibody contains the CDR-H1, CDR-H2, and CDR-H3, respectively contained within the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209; and contains the CDR-L1, CDR-L2, and CDR-L3 sequences contained within the light chain variable (VL) region amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245 246, 247 or 248. In some embodiments, the VH region of the antibody contains the CDR-H1, CDR-H2, and CDR-H3, respectively contained within the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19; and contains the CDR-L1, CDR-L2, and CDR-L3 sequences contained within the light chain variable (VL) region amino acid sequence set forth in SEQ ID NO: 14, 16, or 18. In some embodiments, the VH region of the antibody contains the CDR-H1, CDR-H2, and CDR-H3, respectively contained within the VH region amino acid sequence set forth in SEQ ID NO: 10, 13, 15, 17, or 19; and contains the CDR-L1, CDR-L2, and CDR-L3 sequences contained within the light chain variable (VL) region amino acid sequence set forth in SEQ ID NO: 14, 16, or 18. In some embodiments, the VH region of the antibody contains the CDR-H1, CDR-H2, and CDR-H3, respectively contained within the VH region amino acid sequence set forth in SEQ ID NO: 13, 15 or 19; and contains the CDR-L1, CDR-L2, and CDR-L3 sequences contained within the light chain variable (VL) region amino acid sequence set forth in SEQ ID NO: 14, 16, or 18. In some embodiments, the VH region of the antibody contains the CDR-H1, CDR-H2, and CDR-H3, respectively contained within the VH region amino acid sequence set forth in SEQ ID NO: 13 or 15; and contains the CDR-L1, CDR-L2, and CDR-L3 sequences contained within the light chain variable (VL) region amino acid sequence set forth in SEQ ID NO: 14 or 16.
In some embodiments, the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 19 and 18, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 17 and 18, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 15 and 16 respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 13 and 14, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 182 and 242 respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 182 and 246, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 182 and 247, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 185 and 248, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 186 and 248, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 175 and 234, respectively; the Vu and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 175 and 235, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 176 and 236, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 176 and 237, respectively; the Vu and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 177 and 238, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 179 and 240 respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 180 and 241, respectively; the Vu and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 181 and 241, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 183 and 243 respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 183 and 244 respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 184 and 243, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 184 and 244 respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 183 and 245, respectively; the Vu and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 184 and 245, respectively; or the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 178 and 239, respectively.
Also provided are antibodies having sequences at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.
In some embodiments, the antibody is a single-chain antibody fragment, such as an scFv or diabody or a VH-only single domain antibody. In some embodiments, the single-chain antibody includes one or more linkers joining two antibody domains or regions, such as a variable heavy chain (VH) region and a variable light chain (VL). The linker typically is a peptide linker, e.g., a flexible and/or soluble peptide linker. Among the linkers are those rich in glycine and serine and/or in some cases threonine. In some embodiments, the linkers further include charged residues such as lysine and/or glutamate, which can improve solubility. In some embodiments, the linkers further include one or more proline.
Accordingly, the provided anti-ROR1 antibodies include single-chain antibody fragments, such as scFvs and diabodies, particularly human single-chain fragments, typically comprising linker(s) joining two antibody domains or regions, such VH and VL domains. The linker typically is a peptide linker, e.g., a flexible and/or soluble peptide linker, such as one rich in glycine and serine.
In some aspects, the linkers rich in glycine and serine (and/or threonine) include at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% such amino acid(s). In some embodiments, they include at least at or about 50%, 55%, 60%, 70%, or 75%, glycine, serine, and/or threonine. In some embodiments, the linker is comprised substantially entirely of glycine, serine, and/or threonine. The linkers generally are between about 5 and about 50 amino acids in length, typically between at or about 10 and at or about 30, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and in some examples between 10 and 25 amino acids in length. Exemplary linkers include linkers having various numbers of repeats of the sequence GGGGS (4GS; SEQ ID NO:111) or GGGS (3GS; SEQ ID NO:112), such as between 2, 3, 4, and 5 repeats of such a sequence. Exemplary linkers include those having or consisting of a sequence set forth in SEQ ID NO: 91 (GGGGSGGGGSGGGGS). Exemplary linkers further include those having or consisting of the sequence set forth in SEQ ID NO: 113 (GSTSGSGKPGSGEGSTKG).
Accordingly, in some embodiments, the provided embodiments include single-chain fragments, e.g., scFvs, comprising one or more of the aforementioned linkers, such as glycine/serine rich linkers, including linkers having repeats of GGGS or GGGGS, such as the linker set forth as SEQ ID NO: 91.
In some embodiments, the linker has an amino acid sequence containing the sequence set forth SEQ ID NO: 91. The fragment, e.g., scFv, may include a VH region or portion thereof, followed by the linker, followed by a VL or portions thereof. The fragment, e.g., the scFv, may include the VL, followed by the linker, followed by the VH.
In some embodiments, the scFv has the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 12, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267 or 268, or has a sequence at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 12, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267 or 268. In some aspects, the scFv has the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, or 12, or has a sequence at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, or 12. In some aspects, the scFv has the amino acid sequence set forth in SEQ ID NO: 4 or 6, or has a sequence at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 4 or 6.
In some aspects, the scFv contains the VH, linker and VL as set forth in SEQ ID NO: 2, 4, 6, 12, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267 or 268, or a sequence at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such a sequence, but in which the VH and VL are configured in the opposite orientation, i.e. VL-VH, as compared to such sequence. In some aspects, the scFv has the amino acid sequence set forth in SEQ ID NO: 2, 4, 6 or 12, or has a sequence at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6 or 12, but in which the VH and VL are configured in the opposite orientation, i.e. VL-VH, as compared to such sequence. In some embodiments, the scFv has the amino acid sequence set forth in SEQ ID NO:12, but in which the VH and VL are configured in the opposite orientation, i.e. VL-VH, as compared to such sequence. In some embodiments, the scFv has the amino acid sequence set forth in SEQ ID NO:6, but in which the VH and VL are configured in the opposite orientation, i.e. VL-VH, as compared to such sequence.
Also provided are human anti-ROR1 antibodies, e.g., antigen-binding antibody fragments that specifically bind to the same or an overlapping epitope of a ROR1 protein, such as a human ROR1 protein, as the epitope specifically bound by a reference antibody. Such antibody may be any of the above described antibodies or the anti-ROR1 antibody designated R12 or an antigen-binding fragment thereof.
Also provided are human anti-ROR1 antibodies, e.g., antigen-binding antibody fragments, that specifically bind to a ROR1 protein, such as a human ROR1 protein, and compete for binding to the ROR1 protein with a reference antibody that is any of the above described antibodies or is the anti-ROR1 antibody designated R12 or an antigen-binding fragment thereof.
In some such embodiments, the reference antibody can be an scFv that comprises the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 12, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267 or 268, or that has a sequence at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 12, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267 or 268. In some such embodiments, the reference antibody can be an scFv that comprises the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, or 12, or that has a sequence at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, or 12. In some embodiments, the reference antibody can be an antibody that contains the VH and VL of an scFv that comprises the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 12, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267 or 268, such as set forth in SEQ ID NO: 2, 4, 6, or 12.
In some embodiments of a provided human anti-ROR1 antibody, the human antibody contains a VH region that contains a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain V segment, a portion with at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence encoded by a germline nucleotide human heavy chain D segment, and/or a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence encoded by a germline nucleotide human heavy chain J segment; and/or contains a VL region that contains a portion with at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain V segment, and/or a portion with at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain J segment. In some embodiments, the portion of the VH region corresponds to the CDR-H1, CDR-H2 and/or CDR-H3. In some embodiments, the portion of the VH region corresponds to the framework region 1 (FR1), FR2, FR2 and/or FR4. In some embodiments, the portion of the VL region corresponds to the CDR-L1, CDR-L2 and/or CDR-L3. In some embodiments, the portion of the VL region corresponds to the FR1, FR2, FR2 and/or FR4.
In some embodiments, the human antibody contains a CDR-H1 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-H1 region within a sequence encoded by a germline nucleotide human heavy chain V segment. For example, the human antibody in some embodiments contains a CDR-H1 having a sequence that is 100% identical or with no more than one, two or three amino acid differences as compared to the corresponding CDR-H1 region within a sequence encoded by a germline nucleotide human heavy chain V segment.
In some embodiments, the human antibody contains a CDR-H2 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-H2 region within a sequence encoded by a germline nucleotide human heavy chain V segment. For example, the human antibody in some embodiments contains a CDR-H2 having a sequence that is 100% identical or with no more than one, two or three amino acid difference as compared to the corresponding CDR-H2 region within a sequence encoded by a germline nucleotide human heavy chain V segment.
In some embodiments, the human antibody contains a CDR-H3 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-H3 region within a sequence encoded by a germline nucleotide human heavy chain V segment, D segment and J segment. For example, the human antibody in some embodiments contains a CDR-H3 having a sequence that is 100% identical or with no more than one, two or three amino acid differences as compared to the corresponding CDR-H3 region within a sequence encoded by a germline nucleotide human heavy chain V segment, D segment and J segment.
In some embodiments, the human antibody contains a CDR-L1 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-L1 region within a sequence encoded by a germline nucleotide human light chain V segment. For example, the human antibody in some embodiments contains a CDR-L1 having a sequence that is 100% identical or with no more than one, two or three amino acid differences as compared to the corresponding CDR-L1 region within a sequence encoded by a germline nucleotide human light chain V segment.
In some embodiments, the human antibody contains a CDR-L2 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-L2 region within a sequence encoded by a germline nucleotide human light chain V segment. For example, the human antibody in some embodiments contains a CDR-L2 having a sequence that is 100% identical or with no more than one, two or three amino acid difference as compared to the corresponding CDR-L2 region within a sequence encoded by a germline nucleotide human light chain V segment.
In some embodiments, the human antibody contains a CDR-L3 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-L3 region within a sequence encoded by a germline nucleotide human light chain V segment and J segment. For example, the human antibody in some embodiments contains a CDR-L3 having a sequence that is 100% identical or with no more than one, two or three amino acid differences as compared to the corresponding CDR-L3 region within a sequence encoded by a germline nucleotide human light chain V segment and J segment.
In some embodiments, the human antibody contains a framework region that contains human germline gene segment sequences. For example, in some embodiments, the human antibody contains a VH region in which the framework region, e.g. FR1, FR2, FR3 and FR4, has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a framework region encoded by a human germline antibody segment, such as a V and/or J segment. In some embodiments, the human antibody contains a VL region in which the framework region e.g. FR1, FR2, FR3 and FR4, has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a framework region encoded by a human germline antibody segment, such as a V and/or segment. For example, in some such embodiments, the framework sequence of the VH and/or VL sequence differs by no more than 10 amino acids, such as no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid, compared to the framework region encoded by a human germline antibody segment.
The antibody, e.g., antibody fragment, may contain at least a portion of an immunoglobulin constant region, such as one or more constant region domain. In some embodiments, the constant regions include a light chain constant region and/or a heavy chain constant region 1 (CH1). In some embodiments, the antibody includes a CH2 and/or CH3 domain, such as an Fc region. In some embodiments, the Fc region is an Fc region of a human IgG, such as an IgG1 or IgG4.
Also provided are nucleic acids encoding the antibodies and/or portions, e.g., chains, thereof. Among the provided nucleic acids are those encoding the anti-ROR antibodies described herein. The nucleic acids may include those encompassing natural and/or non-naturally occurring nucleotides and bases, e.g., including those with backbone modifications. The terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide” may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide.
Also provided are vectors containing the nucleic acids, host cells containing the vectors, e.g., for producing the antibodies. Also provided are methods for producing the antibodies. The nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In another such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody.
Also provided are methods of making the anti-ROR1 antibodies (including antigen-binding fragments). For recombinant production of the anti-ROR1 antibody, nucleic acid encoding an antibody, e.g., as described above, may be isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). In some embodiments, a method of making the anti-ROR1 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been modified to mimic or approximate those in human cells, resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lec13 CHO cells, and FUT8 CHO cells; PER.C6® cells; and NSO cells. In some embodiments, the antibody heavy chains and/or light chains may be expressed in yeast. See, e.g., U.S. Publication No. US 2006/0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and/or light chains. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.
In some embodiments, the antibody is produced in a cell-free system. Exemplary cell-free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713 (2003).
The provided embodiments further include vectors and host cells and other expression systems for expressing and producing the antibodies and other binding proteins, including eukaryotic and prokaryotic host cells, including bacteria, filamentous fungi, and yeast, as well as mammalian cells such as human cells, as well as cell-free expression systems.
Exemplary Features
In some aspects, the provided antibodies have one or more specified functional features, such as binding properties, including binding to particular epitopes, such as epitopes that are similar to or overlap with those specifically bound by other antibodies such as reference antibodies, the ability to compete for binding with other antibodies such as reference antibodies, and/or particular binding affinities.
In some embodiments, the antibodies specifically bind to ROR1 protein. In some embodiments of any of the embodiments herein, ROR1 refers to human ROR1. The observation that an antibody or other binding molecule binds to ROR1 or specifically binds to ROR1 does not necessarily mean that it binds to ROR1 of every species. For example, in some embodiments, features of binding to ROR1, such as the ability to specifically bind thereto and/or to compete for binding thereto with a reference antibody, and/or to bind with a particular affinity or compete to a particular degree, in some embodiments, refers to the ability with respect to a human ROR1 protein and the antibody may not have this feature with respect to a ROR1 of another species such as mouse. In some embodiments, the antibody binds to human ROR1 and binds to ROR1 of another species, such as mouse.
In some embodiments, the antibodies specifically bind to human ROR1, such as to an epitope or region of human ROR1, such as the human ROR1 set forth in SEQ ID NO:103 (GenBank No. NP_005003.2, such as encoded by nucleotides set forth in GenBank No. NM_005012.3), or an allelic variant or splice variant thereof. In one embodiment, human ROR1 is a transcript variant or isoform that has the sequence of amino acids forth in SEQ ID NO:104 or SEQ ID NO:105.
In some embodiments, the antibody binds to non-human ROR1, such as monkey, rabbit, rat, mouse, or other species of ROR1. In some embodiments, the antibody binds to mouse ROR1, such as to an epitope or region of mouse ROR1, such as the mouse ROR1 set forth in SEQ ID NO: 106 (GenBank No. NP_038873, such as encoded by nucleotides set forth in GenBank No. NM_013845). In some embodiments, the antibody binds to human ROR1 and binds to mouse ROR1. In some embodiments, the extent of binding of an anti-ROR1 antibody to a non-human ROR1, such as mouse ROR1, is at least or about at least 75%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150% or more of the binding of the antibody to ROR1.
In some embodiments, the antibodies do not bind to mouse ROR1, such as the mouse ROR1 set forth in SEQ ID NO:106 (GenBank No. NP_038873, such as encoded by nucleotides set forth in GenBank No. NM_013845).
In one embodiment, the extent of binding of an anti-ROR1 antibody to an unrelated, non-ROR1 protein or to anon-human ROR1 or other non-ROR1 protein, is less than at or about 10% of the binding of the antibody to human ROR1 as measured, e.g., by a radioimmunoassay (MA). In some embodiments, among provided antibodies are antibodies in which binding to mouse ROR1 is less than or at or about 10% of the binding of the antibody to human ROR1. In some embodiments, among provided antibodies are antibodies in which binding to a ROR2, such as a human ROR2, is less than or at or about 10% of the binding of the antibody to human ROR1.
In some embodiments, the provided antibodies are capable of binding ROR1, such as human ROR1 and/or mouse ROR1, with at least a certain affinity, as measured by any of a number of known methods. In some embodiments, the affinity is represented by a dissociation constant (Kd). In some embodiments, the affinity is represented by EC50.
In some embodiments, the binding affinity (EC50) and/or the dissociation constant of the antibody to ROR1, such as human ROR1 or mouse ROR1, is from or from about 0.1 nM to 500 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, 0.1 nM to 1 nM, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 10 nM, 10 nM to 500 nM, 10 nM to 100 nM, 10 nM to 50 nM, 50 nM to 500 nM, 50 nM to 100 nM or 100 nM to 500 nM. In certain embodiments, the binding affinity (EC50) and/or the dissociation constant of the antibody to ROR1, such as human ROR1 or mouse ROR1, is at or about or less than at or about 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the antibodies bind to ROR1, such as human ROR1 or mouse ROR1, with a sub-nanomolar binding affinity, for example, with a binding affinity less than 1 nM, such as less than 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or 0.1 nM.
In some embodiments, the antibodies, such as the human antibodies, specifically bind to a particular epitope or region of ROR1, such as generally an extracellular epitope or region. ROR1 is a type I membrane protein that contains an extracellular region containing an immunoglobulin (Ig) domain, a frizzled (Fz) domain and a kringle (Kr) domain followed by a transmembrane domain. With reference to human ROR1 set forth in SEQ ID NO:103, the extracellular region corresponds to amino acids 1-377, whereby amino acids 13-118 correspond to the Ig domain, amino acids 136-270 correspond to the Fz domain and amino acids 283-362 correspond to the Kr domain. In some embodiments, the antibodies, such as human antibodies, bind to an epitope comprising residues within the Ig domain, the Fz domain and/or the Kr domain. In some embodiments, the antibodies, such as human antibodies, bind to an epitope comprising residues with the Ig domain and/or Fz domain. In some embodiments, the antibodies, such as human antibodies, bind to an epitope comprising residues within both the Ig and Fz domains.
In some embodiments, properties or features of the provided antibodies are described in relation to properties observed for another antibody, e.g., a reference antibody. In some embodiments, the reference antibody is a non-human anti-ROR1 antibody, such as a rabbit or chimeric or humanized anti-ROR1 antibody. In some aspects, the reference antibody is the chimeric rabbit/human IgG1 antibody designated R12 (see, e.g., Yang et al. (2011) PloS ONE, 6:e21018; U.S. Patent Application No. US 2013/0251642), and/or a fragment derived therefrom such as an scFv fragment thereof, and/or an antibody containing the VH and VL sequences of such an antibody and/or the heavy and light chain CDRs of such an antibody. A chimeric antigen receptor (CAR) containing an antigen-binding scFv fragment of R12 has been demonstrated to effectively promote antitumor reactivity in a CAR therapy (Hudecek et al. (2013) Clin. Cancer Res., 19:3153; International published PCT Appl. No. WO2014031687).
For example, in some embodiments, the reference antibody has a VH region containing the sequence set forth in SEQ ID NO: 85, or comprises CDR1, CDR2, and/or CDR3 within such a sequence, and/or has a VL containing the sequence set forth in SEQ ID NO: 86, or comprises CDR1, CDR2, and/or CDR3 within such a sequence. For example, the reference antibody can be an antibody that contains a CDR-H1 sequence of AYYMS (SEQ ID NO:87), a CDR-H2 sequence of TIYPSSGKTYYATWVNG (set forth in SEQ ID NO:88), a CDR-H3 sequence of DSYADDGALFN (SEQ ID NO:72), a CDR-L1 sequence of TLSSAHKTDTID (SEQ ID NO:89), a CDR-L2 sequence of GSYTKRP (SEQ ID NO:90) and/or a CDR-L3 sequence of GADYIGGYV (SEQ ID NO:73). In some embodiments, the reference antibody is an scFv that comprises the sequence of amino acids set forth in SEQ ID NO:84.
In some embodiments, the reference antibody is the mouse anti-human ROR1 antibody designated 2A2, and/or a fragment derived therefrom such as an scFv fragment thereof, and/or an antibody containing the VH and VL sequences of such an antibody and/or the heavy and light chain CDRs of such an antibody (see, e.g., Baskar et al. (2012) I, 4:349-361; published U.S. Patent Appl. No. US2012/20058051).
For example, in some embodiments, the reference antibody has a VH region containing the sequence set forth in SEQ ID NO: 114, or comprises CDR1, CDR2, and/or CDR3 within such a sequence, and/or has a VL containing the sequence set forth in SEQ ID NO: 115, or comprises CDR1, CDR2, and/or CDR3 within such a sequence. For example, the reference antibody can be an antibody that contains a CDR-H1 sequence of DYEMH (SEQ ID NO:116), a CDR-H2 sequence of AIDPETGGTAYNQKFKG (set forth in SEQ ID NO:117), a CDR-H3 sequence of YYDYDSFTY (SEQ ID NO:118), a CDR-L1 sequence of KASQNVDAAVA (SEQ ID NO:119), a CDR-L2 sequence of SASNRYT (SEQ ID NO:120) and/or a CDR-L3 sequence of QQYDIYPYT (SEQ ID NO:121). In some embodiments, the reference antibody is an scFv form of antibody 2A2.
In some embodiments, the reference antibody is a human or humanized anti-ROR1 antibody. Exemplary humanized anti-ROR1 antibodies are described in International PCT Appl. No. WO2014/031174. In some embodiments, the reference antibody is a humanized variant of an antibody designated 99961. In some embodiments, the reference antibody has a VH region containing the sequence set forth in SEQ ID NO: 122, 123, 124 or 125, or comprises CDR1, CDR2, and/or CDR3 within such a sequence, and/or has a VL containing the sequence set forth in SEQ ID NO: 126, 127, 128 or 129, or comprises CDR1, CDR2, and/or CDR3 within such a sequence. In some embodiments, the reference antibody can be an antibody that contains a CDR-H1 sequence set forth in SEQ ID NO:130 or 133, a CDR-H2 sequence set forth in SEQ ID NO:131 or 134, a CDR-H3 sequence set forth in SEQ ID NO:132 or 135, a CDR-L1 sequence set forth in SEQ ID NO:136 or 139, a CDR-L2 sequence set forth in SEQ ID NO:137 or 140 and/or a CDR-L3 sequence set forth in SEQ ID NO:138 or 141.
In some embodiments, the provided antibody contains heavy and light chain CDRs that are distinct from the CDRs present in the reference antibody or antibodies. In some embodiments, the provided antibody contains heavy and light chain CDRs that are distinct from the CDRs present in the antibody designated R12, such as present in the VH region set forth in SEQ ID NO: 85 and/or the VL region set forth in SEQ ID NO: 86. In some embodiments, the provided antibody contains heavy and light chain CDRs that are distinct from the CDRs present in the antibody designated 2A2, such as present in the VH region set forth in SEQ ID NO: 104 and/or the VL region set forth in SEQ ID NO: 105. In some embodiments, the provided antibody contains heavy and light chain CDRs that are distinct from the CDRs present in a humanized variant of antibody designated 99961, such as present in the VH region set forth in SEQ ID NO: 122, 123, 124 or 125 and/or the VL region set forth in SEQ ID NO: 126, 127, 128 or 129.
Among the provided antibodies are those that compete for binding with and/or bind to the same or overlapping epitopes of ROR1 as those bound by a reference antibody or antibody, such as R12, but nonetheless contain distinct CDRs, e.g., distinct heavy and/or light chain CDR1, CDR2, and CDR3.
In some embodiments, the reference antibody has a sequence present in an antibody or portion thereof as described herein, such as any of the provided exemplary antibodies. For example, in some embodiments, the reference antibody contains a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 20, 27, or 33; a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 21, 26, 28, or 34; a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 22, 29, 35, 46, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 174; a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 23, 30, or 36; CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 24, 31, or 37; and/or a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 25, 32, 38, 39, 40, 41, 42, 43, 44, 45, 47, 48, and 49. For example, in some embodiments, the reference antibody has a light chain variable (VL) region amino acid sequence set forth in SEQ ID NO: 14, 16, or 18 and/or has a heavy chain variable (VH) region set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19. In some such embodiments, the antibody has heavy and/or light chain CDRs 1, 2, and/or 3 as present in such an antibody.
In some embodiments, the antibody has an affinity, e.g., EC50 or Kd, about the same as or lower than that of the corresponding form of the reference antibody, e.g., no more than about 1.5-fold or no more than about 2-fold greater, no more than 3-fold greater, and/or no more than 10-fold greater, than the EC50 or Kd of the corresponding form of the reference antibody. In some embodiments, the antibody has an affinity, e.g., EC50 or Kd, that is greater than or greater than about 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 250-fold or greater than the EC50 or Kd of the corresponding form of the reference antibody.
In some embodiments, the antibodies display a binding preference for ROR1-expressing cells as compared to ROR1-negative cells, such as particular cells known and/or described herein to express ROR1 and known not to express ROR1. In some embodiments, the binding preference is observed where a significantly greater degree of binding is measured to the ROR1-expressing, as compared to the non-expressing, cells. In some embodiments, the fold change in degree of binding detected, for example, as measured by mean fluorescence intensity in a flow cytometry-based assay and/or dissociation constant or EC50, to the ROR1-expressing cells as compared to the non-ROR1-expressing cells, is at least at or about 1.5, 2, 3, 4, 5, 6, or more, and/or is about as great, about the same, at least as great or at least about as great, or greater, than the fold change observed for the corresponding form of the reference antibody. In some cases, the total degree of observed binding to ROR1 or to the ROR1-expressing cells is approximately the same, at least as great, or greater than that observed for the corresponding form of the reference antibody.
In some aspects, the affinity is at or about the same degree or substantially the same degree of affinity compared to the corresponding form of the reference antibody, such as rabbit ROR1 antibody. In some aspects, the affinity is at least 80, 85, 90, 95, or 99% the same as that of the corresponding form of the reference antibody.
In some embodiments, the antibody specifically binds to an epitope that overlaps with the epitope of ROR1 bound by a reference antibody. In some aspects, among such antibodies are antibodies that bind to the same or a similar epitope as the reference antibody. In some embodiments, two antibodies specifically bind to the same epitope and/or an overlapping epitope if all or essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.
In some embodiments, the antibodies bind to the same or a similar epitope or an epitope within the same region or containing residues within the same region of ROR1 as a reference antibody, such as anti-ROR1 antibody R12 or scFv fragment thereof (set forth in SEQ ID NO: 84; see e.g. Yang et al. (2011) PloS ONE, 6:e21018). In some such embodiments, the antibodies bind to an epitope that contains amino acids within the Ig domain and the Fz domain of a ROR1, such as a human ROR1, such as an epitope comprising residues from both such domains and/or at the conjunction of the Ig and Fz domains. In some embodiments, the antibodies bind to an epitope of the ROR1 that overlaps with the epitope specifically bound by R12 and/or an scFv fragment thereof and/or compete for binding with such an antibody.
In some embodiments, the antibody inhibits binding to and/or competes for binding to ROR1, such as human ROR1, with the reference antibody. In some embodiments, the antibody inhibits binding to and/or competes for binding to ROR1, such as human ROR1, with R12 or an IgG or antigen-binding fragment thereof.
An antibody “competes for binding” to ROR1 with a reference antibody if it competitively inhibits binding of the reference antibody to ROR1, and/or if the reference antibody competitively inhibits binding of the antibody to ROR1. An antibody competitively inhibits binding of a reference antibody to an antigen if the presence of the antibody in excess detectably inhibits (blocks) binding of the other antibody to its antigen. A particular degree of inhibition may be specified.
Competitive inhibition assays are known and include ELISA-based, flow cytometry-based assays, and MA-based assays. In some aspects, competitive inhibition assays are carried out by incorporating an excess of an unlabeled form of one of the antibodies and assessing its ability to block binding of the other antibody, which is labeled with a detectable marker, such that degree of binding and reduction thereof can be assessed by detection of the label or marker.
In some embodiments, addition of the provided antibody in excess, e.g., 1-, 2-, 5-, 10-, 50- or 100-fold excess, as compared to the amount or concentration of the reference antibody, inhibits binding to the antigen by the reference antibody (or vice versa). In some embodiments, the inhibition of binding is by at least 50%, and in some embodiments by at least 75%, 90% or 99%. In some aspects, the competitive inhibition is as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).
In some embodiments, where the reference antibody is present at a concentration of or of about 2 nM, the provided antibody inhibits binding of the reference antibody with an IC50 of less than at or about 200 nM, 150 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, or 10 nM, or less than at or about 9 nM, 8 nM, 7 nM, 6 nM, or 5 nM. In some embodiments, where the provided antibody is present at a concentration of or about 2 nM, the reference antibody inhibits binding of the provided antibody with an IC50 of less than at or about 200 nM, 150 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, or 10 nM, or less than at or about 9 nM, 8 nM, 7 nM, 6 nM, or 5 nM.
In some embodiments, competitive inhibition of the reference antibody's binding by the provided antibody (or vice versa) is at or about or least at or about the same degree as the degree of competitive inhibition of the reference antibody's binding by the reference antibody itself, e.g., unlabeled reference antibody. In some embodiments, the provided antibody inhibits binding of the reference antibody, such as binding of R12 scFv, to human ROR1 by at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
Anti-ROR1 antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various known assays. In one aspect, the antibody is tested for its antigen binding activity, e.g., by known methods such as ELISA, Western blotting, and/or flow cytometric assays, including cell-based binding assays, for example, assessing binding of the antibody (e.g., conjugated to a fluorescent marker or tagged) to a cell expressing the target antigen, e.g., ROR1, in some cases compared to results using cells that do not express the target antigen, e.g., ROR1. Binding affinity may be measured as Kd or EC50.
Competition assays may be used to identify an antibody that competes with any of the antibodies described herein. Assays for mapping epitopes bound by the antibodies and reference antibodies also may be used and are known.
Immunoconjugates
In some embodiments, the antibody is or is part of an immunoconjugate, in which the antibody is conjugated to one or more heterologous molecule(s), such as, but not limited to, a cytotoxic or an imaging agent. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents (e.g., methotrexate, Adriamycin® (doxorubicin), vinca alkaloids (vincristine, vinblastine, etoposide), melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins. In some embodiments, the antibody is conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
Among the immunoconjugates are antibody-drug conjugates (ADCs), in which an antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an auristatin such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.
Also among the immunoconjugates are those in which the antibody is conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
Also among the immunoconjugates are those in which the antibody is conjugated to a radioactive atom to form a radioconjugate. Exemplary radioactive isotopes include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu.
Conjugates of an antibody and cytotoxic agent may be made using any of a number of known protein coupling agents, e.g., linkers, (see Vitetta et al., Science 238:1098 (1987)), WO94/11026. The linker may be a “cleavable linker” facilitating release of a cytotoxic drug in the cell, such as acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, and disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020).
Multispecific Antibodies
In certain embodiments, the ROR1-binding molecules, e.g., antibodies or polypeptides such as chimeric receptors containing the same, are multispecific. Among the multispecific binding molecules are multispecific antibodies, including, e.g. bispecific. Multi specific binding partners, e.g., antibodies, have binding specificities for at least two different sites, which may be in the same or different antigens. In certain embodiments, one of the binding specificities is for ROR1 and the other is for another antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of ROR1. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express ROR1. Bispecific antibodies can be prepared as full length antibodies or antibody fragments. Among the multispecific antibodies are multispecific single-chain antibodies, e.g., diabodies, triabodies, and tetrabodies, tandem di-scFvs, and tandem tri-scFvs. Also provided are multispecific chimeric receptors, such as multispecific CARs, containing the antibodies. Also provided are multispecific cells containing the antibodies or polypeptides including the same, such as cells containing a cell surface protein including the anti-ROR1 antibody and an additional cell surface protein, such as an additional chimeric receptor, which binds to a different antigen or a different epitope on ROR1.
Exemplary additional antigens include B cell specific antigens, other tumor-specific antigens, such as antigens expressed specifically on or associated with B cell leukemia, lymphoma, B cell chronic lymphocytic leukemia (CLL), AML, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), Burkett's Lymphoma, mantle cell lymphoma (MCL), non-small cell lung cancer (NSCLC), neuroblastoma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell cancer, melanoma, myeloma, stomach cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, uterine cancer, adrenal cancer and/or head and neck cancer, and antigens expressed on T cells. Exemplary antigens include CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC-1, Ia, HM1.24, HLA-DR, tenascin, an angiogenesis factor, VEGF, PIGF, ED-B fibronectin, an oncogene, an oncogene product, CD66a-d, necrosis antigens, Ii, IL-2, T101, TAC, IL-6, TRAIL-R1 (DR4) and TRAIL-R2 (DR5).
Variants
In certain embodiments, the antibodies include one or more amino acid variations, e.g., substitutions, deletions, insertions, and/or mutations, compared to the sequence of an antibody described herein. Exemplary variants include those designed to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
In certain embodiments, the antibodies include one or more amino acid substitutions, e.g., as compared to an antibody sequence described herein and/or compared to a sequence of a natural repertoire, e.g., human repertoire. Sites of interest for substitutional mutagenesis include the CDRs and FRs. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, improved half-life, and/or improved effector function, such as the ability to promote antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).
In some embodiments, one or more residues within a CDR of a parent antibody (e.g. a humanized or human antibody) is/are substituted. In some embodiments, the substitution is made to revert a sequence or position in the sequence to a germline sequence, such as an antibody sequence found in the germline (e.g., human germline), for example, to reduce the likelihood of immunogenicity, e.g., upon administration to a human subject.
In some embodiments, alterations are made in CDR “hotspots,” residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. Such alterations may, for example, be outside of antigen contacting residues in the CDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.
Modifications
In certain embodiments, the antibody is altered to increase or decrease the extent to which the antibody is glycosylated, for example, by removing or inserting one or more glycosylation sites by altering the amino acid sequence and/or by modifying the oligosaccharide(s) attached to the glycosylation sites, e.g., using certain cell lines.
In some embodiments, an N-linked glycosylation, which is a glycosylation site that occurs at asparagines in the consensus sequence -Asn-Xaa-Ser/Thr is removed or inserted. For example, an exemplary N-linked consensus sequence corresponds to residues N52/P52A/553, by Kabat numbering, of the exemplary heavy chain set forth in SEQ ID NO: 17. In some embodiments, one or more residues corresponding to N52, P52A and/or S53 are replaced with another amino acid to remove the glycosylation site. Exemplary of a modification is S53N, such as is provided in the exemplary heavy chain set forth in SEQ ID NO: 19.
Exemplary modifications, variants, and cell lines are described, e.g., in Patent Publication Nos. US 2003/0157108, US 2004/0093621, US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107); WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.); WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
Among the modified antibodies are those having one or more amino acid modifications in the Fc region, such as those having a human Fc region sequence or other portion of a constant region (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
Such modifications can be made, e.g., to improve half-life, alter binding to one or more types of Fc receptors, and/or alter effector functions.
Also among the variants are cysteine engineered antibodies such as “THIOMABS™” and other cysteine engineered variants, in which one or more residues of an antibody are substituted with cysteine residues, in order to generate reactive thiol groups at accessible sites, e.g., for use in conjugation of agents and linker-agents, to produce immunoconjugates. Cysteine engineered antibodies are described, e.g., in U.S. Pat. Nos. 7,855,275 and 7,521,541.
In some embodiments, the antibodies are modified to contain additional non proteinaceous moieties, including water soluble polymers. Exemplary polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
B. Recombinant Receptors
Among the provided binding molecules, e.g., ROR1 binding molecules, are recombinant receptors, such as those that include one of the provided antibodies. The receptors include antigen receptors and other chimeric receptors that specifically bind to ROR1, such as receptors containing the provided anti-ROR1 antibodies, e.g., antibody fragments. Among the antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Also provided are cells expressing the recombinant receptors and uses thereof in adoptive cell therapy, such as treatment of diseases and disorders associated with ROR1 expression.
Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in international patent application publication numbers WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the antigen receptors include a CAR as described in U.S. Pat. No. 7,446,190, and those described in International Patent Application Publication No.: WO/2014055668 A1. Exemplary of the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190, 8,389,282, e.g., and in which the antigen-binding portion, e.g., scFv, is replaced by an antibody, e.g., as provided herein.
Among the chimeric receptors are chimeric antigen receptors (CARs). The chimeric receptors, such as CARs, generally include an extracellular antigen binding domain that includes, is, or is comprised within, one of the provided anti-ROR1 antibodies. Thus, the chimeric receptors, e.g., CARs, typically include in their extracellular portions one or more ROR1-binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable domains, and/or antibody molecules, such as those described herein. In some embodiments, the CAR includes a ROR1-binding portion or portions of the antibody molecule, such as a variable heavy (VH) chain region and/or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment.
ROR1-targeting CARs are described, for example, by Hudecek et al., Clin Cancer Res, 19(12), 3153-3164 (2013) and Baskar et al. MAbs. 4(3): 349-361 (2012). See also WO2014031687.
In some embodiments, the recombinant receptor such as the CAR, such as the antibody portion thereof, further includes a spacer, which may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region, e.g., an IgG4 hinge region, and/or a CH1/CL and/or Fc region. In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgG1. In some aspects, the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. In some examples, the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153 or international patent application publication number WO2014031687. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 108, and is encoded by the sequence set forth in SEQ ID NO: 107. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 142. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 143.
In some embodiments, the constant region or portion is of IgD. In some embodiments, the spacer has the sequence set forth in SEQ ID NO:144. In some embodiments, the spacer has a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 108, 142, 143 or 144.
The antigen recognition domain generally is linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and/or signal via another cell surface receptor. Thus, in some embodiments, the ROR1-specific binding component (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and/or transmembrane domain(s).
Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
The receptor, e.g., the CAR, generally includes at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the ROR1-binding antibody is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25 or CD16.
In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling domain of the CAR activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functional capability.
In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.
T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.
In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from TCR or CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.
In some embodiments, the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR includes both the activating and costimulatory components.
In some embodiments, the activating domain is included within one CAR, whereas the costimulatory component is provided by another CAR recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, and costimulatory CARs, both expressed on the same cell (see WO2014/055668). In some aspects, the ROR1-targeting CAR is the stimulatory or activating CAR; in other aspects, it is the costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than ROR1, whereby an activating signal delivered through the ROR1-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.
In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.
In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.
In some embodiments, the CAR or other antigen receptor further includes a marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor, such as a truncated version of a cell surface receptor, such as truncated EGFR (tEGFR). In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. See WO2014031687. In some embodiments, introduction of a construct encoding the CAR and EGFRt separated by a T2A ribosome switch can express two proteins from the same construct, such that the EGFRt can be used as a marker to detect cells expressing such construct. In some embodiments, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence. An exemplary polypeptide for a truncated EGFR (e.g. tEGFR) comprises the sequence of amino acids set forth in SEQ ID NO: 154 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 154. An exemplary T2A linker sequence comprises the sequence of amino acids set forth in SEQ ID NO: 153 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 153.
In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.
In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self” by the immune system of the host into which the cells will be adoptively transferred.
In some embodiments, the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive transfer and encounter with ligand.
In some cases, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR in some aspects is one that includes multiple costimulatory domains of different costimulatory receptors.
In some embodiments, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment includes an scFv or a single-domain VH antibody and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain includes a signaling domain of a zeta chain of a CD3-zeta (CD3) chain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain.
In some aspects, the transmembrane domain contains a transmembrane portion of CD28. The extracellular domain and transmembrane can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule, such as between the transmembrane domain and intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.
In some embodiments, the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some embodiments, the CAR contains an antibody, e.g., antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4-1BB or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some such embodiments, the receptor further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4 hinge, such as a hinge-only spacer.
In some embodiments, the transmembrane domain of the receptor, e.g., the CAR is a transmembrane domain of human CD28 or variant thereof, e.g., a 27-amino acid transmembrane domain of a human CD28 (Accession No.: P10747.1), or is a transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 145 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:145; in some embodiments, the transmembrane-domain containing portion of the recombinant receptor comprises the sequence of amino acids set forth in SEQ ID NO: 146 or a sequence of amino acids having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.
In some embodiments, the intracellular signaling domain comprises an intracellular costimulatory signaling domain of human CD28 or functional variant or portion thereof, such as a 41 amino acid domain thereof and/or such a domain with an LL to GG substitution at positions 186-187 of a native CD28 protein. In some embodiments, the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 147 or 148 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 147 or 148. In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of 41BB or functional variant or portion thereof, such as a 42-amino acid cytoplasmic domain of a human 4-1BB (Accession No. Q07011.1) or functional variant or portion thereof, such as the sequence of amino acids set forth in SEQ ID NO: 149 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 149.
In some embodiments, the intracellular signaling domain comprises a human CD3 zeta stimulatory signaling domain or functional variant thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human CD3 (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. No. 7,446,190 or 8,911,993. In some embodiments, the intracellular signaling domain comprises the sequence of amino acids set forth in SEQ ID NO: 150, 151 or 152 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 150, 151 or 152
In some aspects, the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgG1, such as the hinge only spacer set forth in SEQ ID NO:108. In other embodiments, the spacer is an Ig hinge, e.g., and IgG4 hinge, linked to a CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains, such as set forth in SEQ ID NO:143. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a CH3 domain only, such as set forth in SEQ ID NO:142. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers.
For example, in some embodiments, the CAR includes a ROR-1 antibody or fragment, such as any of the human ROR1 antibodies, including sdAbs (e.g. containing only the VH region) and scFvs, described herein, a spacer such as any of the Ig-hinge containing spacers, a CD28 transmembrane domain, a CD28 intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR includes the ROR-1 antibody or fragment, such as any of the human ROR1 antibodies, including sdAbs and scFvs described herein, a spacer such as any of the Ig-hinge containing spacers, a CD28 transmembrane domain, a CD28 intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, such CAR constructs further includes a T2A ribosomal skip element and/or a tEGFR sequence, e.g., downstream of the CAR.
C. Engineered Cells
Also provided are cells such as cells that contain an engineered antigen receptor, e.g., that contains an extracellular domain including the anti-ROR1 antibody or fragment, described herein. Also provided are populations of such cells, compositions containing such cells and/or enriched for such cells, such as in which cells expressing the ROR1 binding molecule make up at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or more percent of the total cells in the composition or cells of a certain type such as T cells or CD8+ or CD4+ cells. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.
Thus also provided are genetically engineered cells expressing the recombinant receptors containing the antibodies, e.g., cells containing the CARs. The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, as described herein, and re-introducing them into the same patient, before or after cryopreservation.
Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naïve T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
In some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.
Vectors and Methods for Genetic Engineering
Also provided are methods, nucleic acids, compositions, and kits, for expressing the binding molecules, including receptors comprising the antibodies, and for producing the genetically engineered cells expressing such binding molecules. The genetic engineering generally involves introduction of a nucleic acid encoding the recombinant or engineered component into the cell, such as by retroviral transduction, transfection, or transformation.
In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.
In some contexts, overexpression of a stimulatory factor (for example, a lymphokine or a cytokine) may be toxic to a subject. Thus, in some contexts, the engineered cells include gene segments that cause the cells to be susceptible to negative selection in vivo, such as upon administration in adoptive immunotherapy. For example in some aspects, the cells are engineered so that they can be eliminated as a result of a change in the in vivo condition of the patient to which they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell II: 223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).
In some aspects, the cells further are engineered to promote expression of cytokines or other factors. Various methods for the introduction of genetically engineered components, e.g., antigen receptors, e.g., CARs, are well known and may be used with the provided methods and compositions. Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.
In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557.
In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
Methods of lentiviral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.
In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).
Other approaches and vectors for transfer of the nucleic acids encoding the recombinant products are those described, e.g., in international patent application, Publication No.: WO2014055668, and U.S. Pat. No. 7,446,190.
Among additional nucleic acids, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.
Preparation of Cells for Engineering
In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for introduction of the ROR1-binding molecule, e.g., CAR, may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, or pig.
In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.
In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the COBE® 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++/Mg++ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.
In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques.
For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (markerhigh) on the positively or negatively selected cells, respectively.
In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.
In embodiments, memory T cells are present in both CD62L+ and CD62L− subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L−CD8+ and/or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.
In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA or ROR1, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.
CD4+ T helper cells are sorted into naïve, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naïve CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L− and CD45RO−.
In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher© Humana Press Inc., Totowa, N.J.).
In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as DYNABEADS® or MACS® beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.
In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.
The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.
In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.
In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.
In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS®) (Miltenyi Biotech, Auburn, Calif.). Magnetic Activated Cell Sorting (MACS®) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS® operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.
In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Number WO2009/072003, or US 20110003380 A1.
In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.
In some aspects, the separation and/or other steps is carried out using CliniMACS® system (Miltenyi Biotic), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.
The CliniMACS® system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.
In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy® system (Miltenyi Biotec). The CliniMACS Prodigy® system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy® system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood may be automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy® system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.
In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.
In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to −80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.
In some embodiments, the provided methods include cultivation, incubation, culture, and/or genetic engineering steps. For example, in some embodiments, provided are methods for incubating and/or engineering the depleted cell populations and culture-initiating compositions.
Thus, in some embodiments, the cell populations are incubated in a culture-initiating composition. The incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.
In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agents include IL-2 and/or IL-15, for example, an IL-2 concentration of at least about 10 units/mL.
In some aspects, incubation is carried out in accordance with techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, the T cells are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.
In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.
In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigen-specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.
II. Compositions, Methods and Uses
Also provided are compositions including the ROR1 binding molecules and engineered cells, including pharmaceutical compositions and formulations, and methods of using and uses of the molecules and compositions, such as in the treatment of diseases, conditions, and disorders in which ROR1 is expressed, and/or detection, diagnostic, and prognostic methods.
A. Pharmaceutical Compositions and Formulations
Provided are pharmaceutical formulations including the ROR1-binding molecule, e.g., antibody or chimeric receptor, and/or the engineered cells expressing the molecules. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.
The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
In some aspects, the choice of carrier is determined in part by the particular cell, binding molecule, and/or antibody, and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).
Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
Formulations of the antibodies can include lyophilized formulations and aqueous solutions.
The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the binding molecules or cells, preferably those with activities complementary to the binding molecule or cell, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the cells or antibodies are administered in the form of a salt, e.g., a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p-toluenesulphonic acid.
Active ingredients may be entrapped in microcapsules, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. In certain embodiments, the pharmaceutical composition is formulated as an inclusion complex, such as cyclodextrin inclusion complex, or as a liposome. Liposomes can serve to target the host cells (e.g., T-cells or NK cells) to a particular tissue. Many methods are available for preparing liposomes, such as those described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
The pharmaceutical composition in some aspects can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Many types of release delivery systems are available and known. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.
The pharmaceutical composition in some embodiments contains the binding molecules and/or cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
In certain embodiments, in the context of genetically engineered cells containing the binding molecules, a subject is administered the range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges, and/or such a number of cells per kilogram of body weight of the subject.
The may be administered using standard administration techniques, formulations, and/or devices. Provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. Administration of the cells can be autologous or heterologous. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, intracranial, intrathoracic, and intraperitoneal administration. In some embodiments, the cell populations are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
Sterile injectable solutions can be prepared by incorporating the binding molecule in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.
Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
B. Therapeutic and Prophylactic Methods and Uses
Also provided are methods of administering and uses, such as therapeutic and prophylactic uses, of the ROR1 binding molecules, including the anti-ROR1 antibodies, e.g., antibody fragments and proteins containing the same such as the chimeric receptors, and/or engineered cells expressing the recombinant receptors. Such methods and uses include therapeutic methods and uses, for example, involving administration of the molecules, cells, or compositions containing the same, to a subject having a disease, condition, or disorder expressing or associated with ROR1 expression, and/or in which cells or tissues express, e.g., specifically express, ROR1. In some embodiments, the molecule, cell, and/or composition is administered in an effective amount to effect treatment of the disease or disorder. Uses include uses of the binding molecules, CARS, antibodies, and cells in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods are carried out by administering the antibodies or cells, or compositions comprising the same, to the subject having, having hand, or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject.
As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.
As used herein, “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.
“Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided molecules and compositions are used to delay development of a disease or to slow the progression of a disease.
As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, an antibody or composition or cell which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody or composition or cell.
An “effective amount” of an agent, e.g., a pharmaceutical formulation, binding molecule, antibody, or cells, or composition, in the context of administration, refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.
A “therapeutically effective amount” of an agent, e.g., a pharmaceutical formulation, antibody, or cells, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered. In some embodiments, the provided methods involve administering the molecules, cells, and/or compositions at effective amounts, e.g., therapeutically effective amounts.
A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human.
Among the diseases to be treated are any ROR1-associated disease or condition or disease or condition in which ROR1 is specifically expressed. In certain diseases and conditions, ROR1 is expressed on malignant cells and cancers. In some embodiments, the disease or condition is a ROR1-expressing cancer. Among the ROR1-associated diseases or conditions that can be treated include, but are not limited to, B cell leukemia, lymphoma, B cell chronic lymphocytic leukemia (CLL), AML, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), Burkett's Lymphoma, mantle cell lymphoma (MCL), non-small cell lung cancer (NSCLC), neuroblastoma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell cancer, melanoma, myeloma, stomach cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, uterine cancer, adrenal cancer and head and neck cancer.
In some embodiments, the methods may identify a subject who has, is suspected to have, or is at risk for developing a ROR1-associated disease or disorder. Hence, provided are methods for identifying subjects with diseases or disorders associated with elevated ROR1 expression and selecting them for treatment with a provided ROR1 binding molecule, including any of the anti-ROR1 antibodies, e.g., antibody fragments and proteins containing the same such as the chimeric receptors, and/or engineered cells expressing the recombinant receptors.
For example, a subject may be screened for the presence of a disease or disorder associated with elevated ROR1 expression, such as a ROR1-expressing cancer. In some embodiments, the methods include screening for or detecting the presence of a ROR1-associated disease, e.g. a tumor. Thus, in some aspects, a sample may be obtained from a patient suspected of having a disease or disorder associated with elevated ROR1 expression and assayed for the expression level of ROR1. In some aspects, a subject who tests positive for a ROR1-associated disease or disorder may be selected for treatment by the present methods, and may be administered a therapeutically effective amount of a ROR1 antibody, CAR expressing ROR1, cells containing a CAR or a pharmaceutical composition thereof as described herein. In some embodiments, the methods can be used to monitor the size or density of a ROR1-expressing tissue, e.g. tumor, over time, e.g., before, during, or after treatment by the methods.
In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another ROR1-specific antibody and/or cells expressing a ROR1-targeting chimeric receptor and/or other therapy, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another ROR1-targetetd therapy. In some embodiments, the subject has not relapsed but is determined to be at risk for relapse, such as at a high risk of relapse, and thus the compound or composition is administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.
In some embodiments, the treatment does not induce an immune response by the subject to the therapy, and/or does not induce such a response to a degree that prevents effective treatment of the disease or condition. In some aspects, the degree of immunogenicity and/or graft versus host response is less than that observed with a different but comparable treatment. For example, in the case of adoptive cell therapy using cells expressing CARs including the provided anti-ROR1 antibodies, the degree of immunogenicity in some embodiments is reduced compared to CARs including a different antibody that binds to a similar, e.g., overlapping epitope and/or that competes for binding to ROR1 with the provided antibody, such as a mouse or rabbit or humanized antibody.
In some embodiments, the methods include adoptive cell therapy, whereby genetically engineered cells expressing the provided anti-ROR1-containing receptors (e.g., ROR1-targeted CARs) are administered to subjects. Such administration can promote activation of the cells (e.g., T cell activation) in a ROR1-targeted manner, such that the cells of the disease or disorder are targeted for destruction.
Thus, the provided methods and uses include methods and uses for adoptive cell therapy. In some embodiments, the methods include administration of the cells or a composition containing the cells to a subject, tissue, or cell, such as one having, at risk for, or suspected of having the disease, condition or disorder. In some embodiments, the cells, populations, and compositions are administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells or compositions are administered to the subject, such as a subject having or at risk for the disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptom of the disease or condition, such as by lessening tumor burden in a ROR1-expressing cancer.
Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.
In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.
In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.
In some embodiments, the subject, to whom the cells, cell populations, or compositions are administered is a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent. In some examples, the patient or subject is a validated animal model for disease, adoptive cell therapy, and/or for assessing toxic outcomes such as cytokine release syndrome (CRS).
The ROR1-binding molecules, such as antibodies and chimeric receptors containing the antibodies and cells expressing the same, can be administered by any suitable means, for example, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjunctival injection, subconjunctival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intracranial, intrathoracic, or subcutaneous administration. Dosing and administration may depend in part on whether the administration is brief or chronic. Various dosing schedules include but are not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion.
For the prevention or treatment of disease, the appropriate dosage of the binding molecule or cell may depend on the type of disease to be treated, the type of binding molecule, the severity and course of the disease, whether the binding molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the binding molecule, and the discretion of the attending physician. The compositions and molecules and cells are in some embodiments suitably administered to the patient at one time or over a series of treatments.
Depending on the type and severity of the disease, dosages of antibodies may include about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg), about 1 μg/kg to 100 mg/kg or more, about 0.05 mg/kg to about 10 mg/kg, 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg. Multiple doses may be administered intermittently, e.g. every week or every three weeks. An initial higher loading dose, followed by one or more lower doses may be administered.
In certain embodiments, in the context of genetically engineered cells containing the binding molecules, a subject is administered the range of about one million to about 100 billion cells and/or that amount of cells per kilogram of body weight, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges and/or per kilogram of body weight. Again, dosages may vary depending on attributes particular to the disease or disorder and/or patient and/or other treatments.
In some embodiments, the cells or antibodies are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as another antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.
The cells or antibodies in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells or antibodies are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells or antibodies are administered after to the one or more additional therapeutic agents.
Once the cells are administered to a mammal (e.g., a human), the biological activity of the engineered cell populations and/or antibodies in some aspects is measured by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells also can be measured by assaying expression and/or secretion of certain cytokines, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
In certain embodiments, engineered cells are modified in any number of ways, such that their therapeutic or prophylactic efficacy is increased. For example, the engineered CAR or TCR expressed by the population in some embodiments are conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating compounds, e.g., the CAR or TCR, to targeting moieties is known in the art. See, for instance, Wadwa et al., J. Drug Targeting 3: 1 1 1 (1995), and U.S. Pat. No. 5,087,616.
C. Diagnostic and Detection Methods
Also provided are methods involving use of the provided binding molecules, e.g., antibodies, in detection of ROR1, for example, in diagnostic and/or prognostic methods in association with a ROR1-expressing disease or condition. The methods in some embodiments include incubating a biological sample with the antibody and/or administering the antibody to a subject. In certain embodiments, a biological sample includes a cell or tissue, such as tumor or cancer tissue. In certain embodiments, the contacting is under conditions permissive for binding of the anti-ROR1 antibody to ROR1, and detecting whether a complex is formed between the anti-ROR1 antibody and ROR1. Such a method may be an in vitro or in vivo method. In one embodiment, an anti-ROR1 antibody is used to select subjects eligible for therapy with an anti-ROR1 antibody or engineered antigen receptor, e.g. where ROR1 is a biomarker for selection of patients.
In some embodiments, a sample, such as a cell, tissue sample, lysate, composition, or other sample derived therefrom is contacted with the anti-ROR1 antibody and binding or formation of a complex between the antibody and the sample (e.g., ROR1 in the sample) is determined or detected. When binding in the test sample is demonstrated or detected as compared to a reference cell of the same tissue type, it may indicate the presence of an associated disease or condition. In some embodiments, the sample is from human tissues.
Various methods known in the art for detecting specific antibody-antigen binding can be used. Exemplary immunoassays include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). An indicator moiety, or label group, can be attached to the subject antibodies and is selected so as to meet the needs of various uses of the method which are often dictated by the availability of assay equipment and compatible immunoassay procedures. Exemplary labels include radionuclides (e.g. I251, I131, 35S, 3H, or 32P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or β-galactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot™ nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, Calif.). General techniques to be used in performing the various immunoassays noted above are known to those of ordinary skill in the art.
For purposes of diagnosis, the antibodies can be labeled with a detectable moiety including but not limited to radioisotopes, fluorescent labels, and various enzyme-substrate labels know in the art. Methods of conjugating labels to an antibody are known in the art.
In some embodiments, antibodies need not be labeled, and the presence thereof can be detected using a labeled antibody which binds to the antibodies.
The provided antibodies in some embodiments can be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).
The antibodies and polypeptides can also be used for in vivo diagnostic assays, such as in vivo imaging. Generally, the antibody is labeled with a radionuclide (such as 111In, 99Tc, 14C, 131I, 125I, or 3H) so that the cells or tissue of interest can be localized in vivo following administration to a subject.
The antibody may also be used as staining reagent in pathology, e.g., using known techniques.
III. Articles of Manufacture
Also provided are articles of manufacture containing the provided binding molecules, e.g., antibodies and CARs and/or genetically engineered cells, and/or compositions. The articles of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container in some embodiments holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition. In some embodiments, the container has a sterile access port. Exemplary containers include an intravenous solution bags, vials, including those with stoppers pierceable by a needle for injection. The label or package insert may indicate that the composition is used for treating the ROR1-expressing or -associated disease or condition. The article of manufacture may include (a) a first container with a composition contained therein, wherein the composition includes the antibody or engineered antigen receptor; and (b) a second container with a composition contained therein, wherein the composition includes a further agent, such as a cytotoxic or otherwise therapeutic agent. The article of manufacture may further include a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further include another or the same container comprising a pharmaceutically-acceptable buffer. It may further include other materials such as other buffers, diluents, filters, needles, and/or syringes.
As used herein, reference to a “corresponding form” of an antibody means that when comparing a property or activity of two antibodies, the property is compared using the same form of the antibody. For example, if it is stated that an antibody has greater activity compared to the activity of the corresponding form of a first antibody, that means that a particular form, such as a scFv of that antibody, has greater activity compared to the scFv form of the first antibody.
“Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.
The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).
An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
“Isolated nucleic acid encoding an anti-ROR1 antibody” refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
As used herein, “percent (%) amino acid sequence identity” and “percent identity” when used with respect to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence (e.g., the subject antibody or fragment) that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MegAlign (DNASTAR™) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
An amino acid substitution may include replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1. Amino acid substitutions may be introduced into a binding molecule, e.g., antibody, of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
Amino acids generally can be grouped according to the following common side-chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro;
(6) aromatic: Trp, Tyr, Phe.
Non-conservative amino acid substitutions will involve exchanging a member of one of these classes for another class.
The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include “consisting” and/or “consisting essentially of” aspects and variations.
Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.
The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.
As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
As used herein, a statement that a cell or population of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.
As used herein, a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.
All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
IV. Exemplary Embodiments
Among the embodiments provided herein are:
1. An antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable (VH) region comprising:
a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence set forth in SEQ ID NO: 22, 29, 35, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 174; or
a CDR-H3 contained within the heavy chain variable (VH) sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209.
2. An antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable (VH) region comprising at least 90% sequence identity to the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209.
3. The antibody or antigen-binding fragment thereof of embodiment 2, said antibody or antigen-binding fragment comprising a heavy chain variable (VH) region comprising at least 90% sequence identity to the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19.
4. The antibody or fragment of embodiment 2 or embodiment 3, wherein the VH region comprises a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence set forth in SEQ ID NO: 92, 93, 94, 109 or 110.
5. The antibody or fragment of any of embodiments 2-4, wherein the VH region comprises:
a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence set forth in SEQ ID NO: 22, 29, 35, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 174; or
a CDR-H3 contained within the VH sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209.
6. The antibody or fragment of any of embodiments 2-5, wherein the VH region comprises:
a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence set forth in SEQ ID NO: 22, 29, 35, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 174; or
a CDR-H3 contained within the VH sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19.
7. The antibody or fragment of any of embodiments 2-6, wherein the VH region comprises:
a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence set forth in SEQ ID NO: 22, 29, 35 or 52; or
a CDR-H3 contained within the VH sequence set forth in SEQ ID NO: 10, 13, 15 or 19.
8. The antibody or fragment of embodiment 2 or embodiment 3, wherein the VH region comprises a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 72 or a CDR-H3 contained within the VH sequence set forth in SEQ ID NO: 85.
9. The antibody or fragment of any of embodiments 1-8, wherein the VH region comprises:
a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO:95 or 97; and/or
a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence set forth in SEQ ID NO: 96 or 98.
10. The antibody or fragment of any of embodiments 1-9, wherein the VH region comprises:
a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO: 20, 27, 33, 155, 156, 157, 158, 159, 160, 161, 162 or 163 and/or comprising the amino acid sequence set forth in SEQ ID NO: 269, 270, 271, 272, 273, 274, 275, 276, 278 or 279 and/or comprising the amino acid sequence set forth in SEQ ID NO: 75, 77, 79, 280, 281, 282, 283, 284, 285, 286, 287, 288 or 289 and/or a CDR-H1 contained within the VH sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209; and/or
a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence set forth in SEQ ID NO: 21, 26, 28, 34, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, or 318 and/or comprising the amino acid sequence set forth in SEQ ID NO: 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302 or 303 and/or comprising the amino acid sequence set forth in SEQ ID NO: 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316 or 317 and/or a CDR-H2 contained within the VH sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209.
11. The antibody or fragment of any of embodiments 1-10, wherein the VH region comprises:
a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO: 20, 27, or 33 and/or a CDR-H1 contained within the VH sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19; and/or
a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence set forth in SEQ ID NO: 21, 26, 28, or 34 and/or a CDR-H2 contained within the VH sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19.
12. The antibody or fragment of any of embodiments 1-11, wherein the VH region comprises:
a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO: 20, 27, or 33 and/or a CDR-H1 contained within the VH sequence set forth in SEQ ID NO: 10, 13, 15 or 19; and/or
a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence set forth in SEQ ID NO: 26, 28, or 34 and/or a CDR-H2 contained within the VH sequence set forth in SEQ ID NO: 10, 13, 15 or 19.
13. An antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) region comprising a heavy chain complementarity determining region 1 (CDR-H1), CDR-H2, and CDR-H3, wherein:
the CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 20, 27, 33, 75, 77, 79, 155, 156, 157, 158, 159, 160, 161, 162, 163, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278 or 279;
the CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 21, 26. 28. 34, 80, 81, 82, 83, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303 or 318; and/or
the CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 22, 29, 35, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 174.
14. The antibody or antigen-binding fragment of embodiment 11, wherein:
the CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO: 20, 27, or 33;
the CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 21, 26, 28, or 34; and/or
the CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO: 22, 29, 35, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or 71.
15. The antibody or fragment of any of embodiments 1, 4-7 and 9-14, wherein the CDR-H3 comprises the amino acid sequence VSNYEYYFDY (SEQ ID NO: 29), DFGRWGYYFDY (SEQ ID NO: 52), DFGRWSYYFDY (SEQ ID NO:35) or DSSYDAFDI (SEQ ID NO:22).
16. The antibody or fragment of any of embodiments 1-7 and 9-15, comprising a heavy chain variable (VH) region comprising a heavy chain complementarity determining region 1 (CDR-H1), CDR-H2, and CDR-H3, wherein:
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 20, 21, and 22, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 20, 26, and 22, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 27, 28, and 29, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 34, and 35, respectively; or
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 34, and 52, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 27, 164 and 45, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 27, 164 and 68, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 27, 164 and 64, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 27, 164 and 66, respectively;
the CDR-H1, CDR-H2, and CDR-H3 having the sequences of SEQ ID NOs: 33, 318, and 35, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 34 and 70, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 34 and 55, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 34 and 53, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 34 and 56, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 34 and 61, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 34 and 59, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 171 and 60, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 155, 34 and 35, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 156, 34 and 35, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 162, 170 and 50, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 162, 170 and 51, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 161, 169 and 54, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs:159, 167 and 57, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 160, 168 and 58, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 158, 166 and 62, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 158, 166 and 63, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 158, 166 and 65, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 157, 165 and 67, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 163, 173 and 69, respectively; or
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 160, 172, 71, respectively; or
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 160, 172, 174, respectively.
17. The antibody or fragment of any of embodiments 1-7 and 9-16, comprising a heavy chain variable (VH) region comprising a heavy chain complementarity determining region 1 (CDR-H1), CDR-H2, and CDR-H3, wherein:
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 20, 21, and 22, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 20, 26, and 22, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 27, 28, and 29, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 34, and 35, respectively; or
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 33, 34, and 52, respectively.
18. An antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) region comprising a heavy chain complementarity determining region 1 (CDR-H1), a CDR-H2, and a CDR-H3, respectively, comprising the amino acid sequences of a CDR-H1, a CDR-H2, and a CDR-H3 contained within the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209.
19. The antibody or antigen-binding fragment of embodiment 18, wherein the CDR-H1, CDR-H2, and CDR-H3, respectively, comprise the amino acid sequences of a CDR-H1, a CDR-H2, and a CDR-H3 contained within the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19.
20. The antibody or fragment of any of embodiments 1-19, wherein the VH region comprises a framework region 1 (FR1), a FR2, a FR3, and/or a FR4 comprising at least 90% sequence identity, respectively, to a FR1, FR2, FR3, and/or FR4 of the amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209.
21. The antibody or fragment of any of embodiments 1-20, wherein the VH region comprises a framework region 1 (FR1), a FR2, a FR3, and/or a FR4 comprising at least 90% sequence identity, respectively, to a FR1, FR2, FR3, and/or FR4 of the amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17 or 19.
22. The antibody or fragment of any of embodiments 1-7 and 9-21, wherein the Vu region comprises the sequence of amino acids set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209.
23. The antibody or fragment of any of embodiments 1-7 and 9-22, wherein the Vu region comprises the sequence of amino acids set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19.
24. The antibody or fragment of embodiment 23, wherein the VH region comprises the sequence of amino acids set forth in SEQ ID NO: 8 or 10.
25. The antibody or fragment of any of embodiments 1-7 and 9-24, wherein the Vu region comprises the CDR-H1, CDR-H2, and CDR-H3 set forth in SEQ ID NOS: 20, 26, and 22, respectively, and/or the VH region comprises the sequence of amino acids set forth in SEQ ID NO: 19.
26. The antibody or fragment of any of embodiments 1-7 and 9-24, wherein the Vu region comprises the CDR-H1, CDR-H2, and CDR-H3 set forth in SEQ ID NOS: 33, 34, and 52, respectively, and/or the VH region comprises the sequence of amino acids set forth in SEQ ID NO: 10.
27. The antibody or fragment of any of embodiments 1-7 and 9-24, wherein the Vu region comprises the CDR-H1, CDR-H2, and CDR-H3 set forth in SEQ ID NOS: 33, 34, and 35, respectively, and/or the VH region comprises the sequence of amino acids set forth in SEQ ID NO: 13.
28. The antibody or fragment of any of embodiments 1-7 and 9-24, wherein the Vu region comprises the CDR-H1, CDR-H2, and CDR-H3 set forth in SEQ ID NOS: 27, 28, and 29, respectively, and/or the VH region comprises the sequence of amino acids set forth in SEQ ID NO: 15.
29. The antibody or fragment of any of embodiments 1-28, wherein the antibody or fragment does not comprise a light chain variable (VL) region, does not comprise a CDR-L1, CDR-L2, and/or CDR-L3, and/or is a single-domain antibody (sdAb) comprising only the VH region.
30. The antibody or fragment of any of embodiments 1-29 that is a sdAb comprising only the VH region.
31. The antibody or fragment of any of embodiments 1-28, wherein the antibody or fragment further comprises a light chain variable (VL) region.
32. The antibody or fragment of embodiment 31, wherein the VL region comprises at least 90% sequence identity to the VL region amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248.
33. The antibody or fragment of embodiment 31 or embodiment 32, wherein the VL region comprises at least 90% sequence identity to the VL region amino acid sequence set forth in SEQ ID NO: 14, 16, or 18.
34. The antibody of fragment of any of embodiments 31-33, wherein the VL region comprises a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence set forth in SEQ ID NO: 99 or 100.
35. The antibody or fragment of any of embodiments 31-34, wherein the VL region comprises a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence set forth in SEQ ID NO: 25, 32, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48, 49, 232 or 233.
36. The antibody or fragment of any of embodiments 31-35, wherein the VL region comprises a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence set forth in SEQ ID NO: 25, 32, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48 or 49.
37. The antibody or fragment of any of embodiments 31-36, wherein the VL region comprises a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO: 101 and/or a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence set forth in SEQ ID NO: 102.
38. The antibody or fragment of any of embodiments 31-38, wherein the VL region comprises:
a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO: 23, 30, 36, 210, 211, 212, 213, 214, 215, 217, 216, 218, 219 or 220 and/or a CDR-L1 contained within the VL sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248; and/or
a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence set forth in SEQ ID NO: 24, 31, 37, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230 or 231 and/or a CDR-L2 contained within the VL sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248.
39. The antibody or fragment of any of embodiments 31-38, wherein the VL region comprises:
a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO: 23, 30, or 36 and/or a CDR-L1 contained within the VL sequence set forth in SEQ ID NO: 14, 16, or 18; and/or
a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence set forth in SEQ ID NO: 24, 31, or 37 and/or a CDR-L2 contained within the VL sequence set forth in SEQ ID NO: 14, 16, or 18.
40. The antibody or fragment of any of embodiments 31-39, wherein the VL region comprises:
a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO: 23, 30, 36, 210, 211, 212, 213, 214, 215, 217, 216, 218, 219 or 220;
a light chain complementarity determining region 2 (CDR-L2), comprising the amino acid sequence set forth in SEQ ID NO: 24, 31, 37, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230 or 231; and
a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence set forth in SEQ ID NO: 25, 32, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48, 49, 232 or 233.
41. The antibody or fragment of any of embodiments 31-40, wherein the VL region comprises:
a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO: 23, 30, or 36;
a light chain complementarity determining region 2 (CDR-L2), comprising the amino acid sequence set forth in SEQ ID NO: 24, 31, or 37; and
a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence set forth in SEQ ID NO: 25, 32, 38, 39, 40, 41, 42, 43, 44, 46, 47, 48, or 49.
42. The antibody or fragment of any of embodiments 36-41, wherein the CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 25, 32 or 38.
43. The antibody or fragment of any of embodiments 31-42, wherein the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 selected from:
the CDR-L1, CDR-L2, and CDR-L3 comprising the sequences of SEQ ID NOs: 23, 24, and 25, respectively;
the CDR-L1, CDR-L2, and CDR-L3 comprising the sequences of SEQ ID NOs: 30, 31, and 32, respectively;
the CDR-L1, CDR-L2, and CDR-L3 comprising the sequences of SEQ ID NOs: 36, 37, and 38, respectively;
the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 216, 227, and 40, respectively;
the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 218, 229, and 39, respectively;
the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 219, 230, and 43, respectively;
the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 220, 231, and 46, respectively;
the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 210, 221, and 49, respectively;
the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 210, 221, and 233, respectively;
the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 211, 222, and 48, respectively;
the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 212, 223, and 42, respectively;
the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 214, 225, and 232, respectively;
the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 215, 226, and 44, respectively;
the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 217, 228, and 41, respectively; or
the CDR-L1, CDR-L2 and CDR-L3 comprising the sequences of SEQ ID NOs: 213, 224, and 47, respectively.
44. The antibody or fragment of any of embodiments 31-43, wherein the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 selected from:
the CDR-L1, CDR-L2, and CDR-L3 comprising the sequences of SEQ ID NOs: 23, 24, and 25, respectively;
the CDR-L1, CDR-L2, and CDR-L3 comprising the sequences of SEQ ID NOs: 30, 31, and 32, respectively; or
the CDR-L1, CDR-L2, and CDR-L3 comprising the sequences of SEQ ID NOs: 36, 37, and 38, respectively.
45. The antibody or fragment of any of embodiments 31-44, wherein the VL region comprises light chain complementarity determining region 1 (CDR-L1), CDR-L2, and CDR-L3, respectively, contained within the VL region amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248.
46. The antibody or fragment of any of embodiments 31-45, wherein the VL region comprises light chain complementarity determining region 1 (CDR-L1), CDR-L2, and CDR-L3, respectively, contained within the VL region amino acid sequence set forth in SEQ ID NO: 14, 16, or 18.
47. The antibody or fragment of any of embodiments 31-46, wherein the VL region comprises a framework region 1 (FR1), a FR2, a FR3, and/or a FR4 comprising at least 90% sequence identity, respectively, to the FR1, FR2, FR3, and/or FR4 of the amino acid sequence set forth in SEQ ID NO: 14, 16, or 18.
48. The antibody or fragment of any of embodiments 31-47, wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248.
49. The antibody or fragment of any of embodiments 31-48, wherein the VL region comprises the amino acid sequence set forth in SEQ ID NO: 14, 16, or 18.
50. An antibody or antigen-binding fragment thereof, comprising:
a heavy chain complementarity determining regions 1, 2, and 3 (CDR-H1, CDR-H2, and CDR-H3), respectively comprising the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 sequences contained within the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209; and/or
a light chain complementarity determining regions 1, 2, and 3 (CDR-L1, CDR-L2, and CDR-L3), respectively comprising the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 sequences contained within the light chain variable (VL) region amino acid sequence set forth in SEQ ID NO: 14, 16, 18, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247 or 248.
51. The antibody or antigen-binding fragment of embodiment 50, wherein:
the CDR-H1, CDR-H2, and CDR-H3, respectively, comprise the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 sequences contained within the VH region amino acid sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19; and/or
the CDR-L1, CDR-L2, and CDR-L3, respectively, comprise the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 sequences contained within the light chain variable (VL) region amino acid sequence set forth in SEQ ID NO: 14, 16, or 18.
52. An antibody or antigen-binding fragment thereof, comprising:
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 19 and 18, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 17 and 18, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 15 and 16, respectively; or a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 13 and 14, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 182 and 242 respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 182 and 246, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 182 and 247, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 185 and 248, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 186 and 248, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 175 and 234, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 175 and 235, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 176 and 236, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 176 and 237, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 177 and 238, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 179 and 240 respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 180 and 241, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 181 and 241, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 183 and 243 respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 183 and 244 respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 184 and 243, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 184 and 244 respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 183 and 245, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 184 and 245, respectively; or
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 178 and 239, respectively.
53. The antibody or antigen-binding fragment of embodiment 52, comprising:
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 19 and 18, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 17 and 18, respectively;
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 15 and 16, respectively; or
a VH and VL regions comprising amino acid sequences having at least 90% identity to SEQ ID NOs: 13 and 14, respectively.
54. The antibody or fragment of embodiment 52 or embodiment 53, wherein:
the VH and VL regions comprise the amino acid sequences of SEQ ID NOs: 19 and 18, respectively;
the VH and VL regions comprise the amino acid sequences of SEQ ID NOs: 17 and 18, respectively;
the VH and VL regions comprise the amino acid sequences of SEQ ID NOs: 15 and 16 respectively; or
the VH and VL regions comprise the amino acid sequences of SEQ ID NOs: 13 and 14 respectively.
55. The antibody or fragment of any of embodiments 1-54, wherein said antibody or fragment specifically binds to a ROR1 protein.
56. The antibody or fragment of embodiment 55, wherein the ROR1 protein is a human ROR1 protein.
57. The antibody or fragment of embodiment 55, wherein the ROR1 protein is a mouse ROR1 protein.
58. The antibody or fragment of any of embodiments 1-57, wherein:
said antibody or fragment has a binding affinity for a ROR1 protein with an EC50 that is from or from about 0.1 nM to 100 nM, 0.5 nM to 50 nM or 1 nM to 10 nM; or
said antibody or fragment has a binding affinity for a ROR1 protein with an EC50 that is less than or less than about 100 nM, less than or less than about 50 nM, less than or less than about 10 nM or less than or less than about 1 nM.
59. The antibody or fragment of any of embodiments 1-58, wherein said antibody or fragment has a binding affinity for a human ROR1 protein that is at least as high or substantially as high as the binding affinity for the ROR1 protein of the corresponding form of the anti-ROR1 antibody R12 or antigen-binding fragment thereof, which is optionally an scFv fragment of R12.
60. The antibody or fragment of any of embodiments 1-59, wherein said antibody or fragment specifically binds to the same or an overlapping epitope of a human ROR1 protein as the epitope specifically bound by the anti-ROR1 antibody R12 or an antigen-binding fragment thereof, which is optionally an scFv fragment of R12.
61. The antibody or fragment of any of embodiments 1-60, wherein said antibody or fragment competes for binding to a human ROR1 protein with anti-ROR1 antibody R12 or an antigen-binding fragment thereof, which is optionally an scFv fragment of R12.
62. The antibody or fragment of any of embodiments 1-61, wherein said antibody or fragment inhibits the binding of the anti-ROR1 antibody R12 or an antigen-binding fragment thereof, which is optionally an scFv fragment of R12, to a human ROR1 protein by greater than or greater than about 80% or greater than or greater than about 90%.
63. The antibody or fragment of any of embodiments 1-62, wherein the antibody or fragment binds to an overlapping epitope of a human ROR1 protein as the epitope specifically bound by the anti-ROR1 antibody R12 or an antigen-binding fragment thereof, which is optionally an scFv fragment of R12 and the antibody or fragment binds to mouse ROR1.
64. The antibody or antigen-binding fragment of any of embodiments 55, 56 and 58-63, wherein said human ROR1 protein comprises an amino acid sequence set forth in SEQ ID NO: 103.
65. The antibody or antigen-binding fragment of any of embodiments 55, 56 and 58-64, which
binds an epitope within an extracellular portion of the ROR1 protein; and/or
binds to an epitope of said ROR1 protein comprising residues within the fz domain or the Ig domain of the ROR1 protein.
66. The antibody or antigen-binding fragment of embodiment 65, wherein the epitope comprises a residue within the fz domain of the ROR1 protein and a residue within the Ig domain of the ROR1 protein.
67. The antibody or antigen-binding fragment thereof of embodiment 65 or embodiment 66, wherein said Ig domain comprises residues 13-118 of the amino acid sequence set forth in SEQ ID NO: 103; and/or said fz domain comprises residues 136-270 of the amino acid sequence set forth in SEQ ID NO:103.
68. The antibody or antigen binding fragment of embodiment 65, wherein the extracellular portion comprises amino acids 1-377 of SEQ ID NO:103.
69. The antibody or fragment thereof of any of embodiments 1-56, 58-62 and 64-68, wherein the antibody does not specifically bind to a mouse ROR1 protein or does not specifically bind to a protein having the amino acid sequence set forth in SEQ ID NO: 106.
70. The antibody or fragment of any of embodiments 1-7 and 9-69, which does not comprise the CDR-H1, CDR-H2, CDR-H3 and/or CDR-L1, CDR-L2, CDR-L3 sequences of the anti-ROR1 antibody, R12, and/or of the anti-ROR1 antibody 2A2.
71. The antibody or fragment of any of embodiments 1-7 and 9-70, which does not comprise CDR-H1, CDR-H2, CDR-H3 and/or CDR-L1, CDR-L2, CDR-L3 sequences having at least 90% identity to the CDR-H1, CDR-H2, CDR-H3 and/or CDR-L1, CDR-L2, CDR-L3 sequences of the anti-ROR1 antibody, R12, and/or of the anti-ROR1 antibody 2A2.
72. The antibody or fragment of any of embodiments 1-71, wherein the antibody or fragment is human.
73. A human antibody or antigen-binding fragment thereof that specifically binds to the same or an overlapping epitope of a ROR1 protein as the epitope specifically bound by a reference antibody, wherein the reference antibody is the antibody or fragment thereof of any of embodiments 1-72 or is the anti-ROR1 antibody, R12, or antigen binding fragment thereof, said human antibody or fragment comprising heavy and light chain CDRs that are distinct from the CDRs present in R12 and/or 2A2.
74. A human antibody or antigen-binding fragment thereof that specifically binds to ROR1 and competes for binding to ROR1 with a reference antibody, wherein the reference antibody is the antibody or fragment of any of embodiments 1-72 or is the anti-ROR1 antibody R12 or an antigen binding fragment thereof, said human antibody or fragment comprising heavy and light chain CDRs that are distinct from the CDRs present in R12 and/or 2A2.
75. The human antibody or fragment of any of embodiments 72-74, wherein:
the antibody comprises a heavy chain variable (VH) region, said VH region comprises a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain V segment, a portion with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain D segment, and/or a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain J segment; and/or
the antibody comprises a light chain variable (VL) region, said VL region comprises a portion with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain V segment, and/or a portion with at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain J segment.
76. The human antibody or fragment of any of embodiments 72-75, wherein:
the CDR-H1 and/or CDR-H2 comprises a sequence 100% identical or with no more than one amino acid difference as compared to an amino acid sequence of a CDR-H1 and/or CDR-H2, respectively, within a sequence encoded by a germline nucleotide human heavy chain V segment; and/or
the CDR-L1 and/or CDR-L2 comprises a sequence 100% identical or with no more than one amino acid difference as compared to an amino acid sequence of a CDR-L1 and/or CDR-L2, respectively, within a sequence encoded by a germline nucleotide human kappa or lambda v segment.
77. The antibody or fragment of any of embodiments 1-76, wherein the antibody or fragment is recombinant.
78. The antibody or fragment of any of embodiments 1-77, which is monoclonal. 79. The antibody or fragment of any of embodiments 1-78 that is an antigen-binding fragment.
80. The antibody or fragment of any of any of embodiments 1-79, which is a single chain fragment.
81. The antibody or fragment of any of embodiments 31-80, which is a fragment comprising antibody VH and VL regions joined by a flexible linker.
82. The antibody or fragment of embodiment 80 or embodiment 81, wherein the fragment comprises an scFv.
83. The antibody or fragment of embodiment 82, wherein the scFv comprises a linker comprising the sequence set forth SEQ ID NO: 91.
84. The antibody or fragment of embodiment 82 or embodiment 83, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 12, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267 or 268, or a sequence of amino acids that exhibits at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 12, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267 or 268.
85. The antibody or fragment of any of embodiments 82-84, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, or 12, or a sequence of amino acids that exhibits at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, or 12.
86. The antibody or fragment of any of embodiments 82-85, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, or 12.
87. The antibody or fragment of any of embodiments 31-86, wherein the antibody or fragment comprises the VH region set forth in SEQ ID NO: 19 and the VL region set forth in SEQ ID NO: 18, and/or the antibody or fragment comprises the sequence of amino acids set forth in SEQ ID NO: 12.
88. The antibody or fragment of any of embodiments 31-86, wherein the antibody or fragment comprises the VH region set forth in SEQ ID NO: 13 and the VL region set forth in SEQ ID NO: 14, and/or the antibody or fragment comprises the sequence of amino acids set forth in SEQ ID NO: 6.
89. The antibody or fragment of any of embodiments 31-86, wherein the antibody or fragment comprises the VH region set forth in SEQ ID NO: 15 and the VL region set forth in SEQ ID NO: 16, and/or the antibody or fragment comprises the sequence of amino acids set forth in SEQ ID NO: 4.
90. The antibody or fragment of any of embodiments 31-86, wherein the antibody or fragment comprises the VH region set forth in SEQ ID NO: 17 and the VL region set forth in SEQ ID NO: 18, and/or the antibody or fragment comprises the sequence of amino acids set forth in SEQ ID NO: 2.
91. A single chain cell-surface protein, comprising the antibody or fragment of any of embodiments 1-90.
92. A single chain cell surface protein comprising the scFv sequence of SEQ ID NO: 2, 4, 6, 12, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267 or 268 or comprising the VH sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, 19, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208 or 209.
93. The single chain cell surface protein of embodiment 92, comprising the scFv sequence of SEQ ID NO: 2, 4, 6 or 12 or comprising the VH sequence set forth in SEQ ID NO: 8, 10, 13, 15, 17, or 19.
94. The antibody or fragment of any of embodiments 1-90, which further comprises at least a portion of an immunoglobulin constant region.
95. The antibody or fragment of embodiment 94, further comprising a spacer set forth in SEQ ID NO:108.
96. The antibody or fragment of embodiment 94, wherein the at least a portion of an immunoglobulin constant region comprises an Fc region.
97. The antibody or fragment of embodiment 96, wherein the Fc region is an Fc region of a human IgG.
98. A conjugate, comprising the antibody or fragment of any of embodiments 1-97 and a heterologous molecule or moiety.
99. A chimeric antigen receptor (CAR) comprising an extracellular portion comprising the antibody or fragment of any of embodiments 1-97 and an intracellular signaling domain.
100. The chimeric antigen receptor of embodiment 99, wherein the antibody or fragment comprises a VH sdAb, an scFv and the intracellular signaling domain comprises an ITAM.
101. The chimeric antigen receptor of embodiment 99 or 100, wherein the intracellular signaling domain comprises a signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain.
102. The chimeric antigen receptor of any of embodiments 99-101, further comprising a transmembrane domain linking the extracellular domain and the intracellular signaling domain.
103. The chimeric antigen receptor of embodiment 102, wherein the transmembrane domain comprises a transmembrane portion of CD28.
104. The chimeric antigen receptor of any of embodiments 99-103, further comprising an intracellular signaling domain of a T cell costimulatory molecule.
105. The chimeric antigen receptor of embodiment 104, wherein the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB.
106. A nucleic acid encoding the antibody or fragment thereof of any of embodiments 1-97, conjugate of embodiment 98 or the chimeric antigen receptor of any of embodiments 99-105.
107. An engineered cell expressing a receptor comprising the antibody or fragment of any of embodiments 1-97, conjugate of embodiment 98 or the chimeric antigen receptor of any of embodiments 99-105.
108. The engineered cell of embodiment 107, which is a T cell.
109. A composition comprising the antibody or fragment thereof of any of embodiments 1-97, conjugate of embodiment 98, the CAR of any of embodiments 99-105, or the cell of embodiment 107 or 108.
110. The composition of embodiment 109, further comprising a pharmaceutically acceptable excipient.
111. A method of treatment, comprising administering the composition of embodiment 109 or embodiment 110 to a subject having a disease or disorder associated with ROR1.
112. A method of treatment, comprising administering an antibody or fragment of any of embodiments 1-97, conjugate of embodiment 98, a CAR of any of embodiments 99-103 or the cell of embodiment 107 or 108 to a subject having a disease or disorder associated with ROR1.
113. A method of treatment, comprising administering the cell of embodiment 107 or embodiment 108 to a subject having a disease or disorder associated with ROR1.
114. The method of any of embodiments 112-113, wherein the disease or disorder is a ROR1-expressing cancer.
115. The method of embodiment any of embodiments 112-114, wherein the ROR1-expressing cancer is selected from the group consisting of B cell leukemia, lymphoma, B cell chronic lymphocytic leukemia (CLL), AML, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), Burkett's Lymphoma, mantle cell lymphoma (MCL), non-small cell lung cancer (NSCLC), neuroblastoma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell cancer, melanoma, myeloma, stomach cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, uterine cancer, adrenal cancer and head and neck cancer.
116. A composition of embodiment 109 or embodiment 110 for use in treating a disease or disorder associated with ROR1.
117. Use of a composition of embodiment 109 or embodiment 110 for the manufacture of a medicament for treating a disease or disorder associated with ROR1.
118. The composition for use of embodiment 116 or the use of embodiment 117, wherein the disease or disorder is a ROR1-expressing cancer.
119. The composition for use or use of embodiment 118, wherein the ROR1-expressing cancer is selected from the group consisting of B cell leukemia, lymphoma, B cell chronic lymphocytic leukemia (CLL), AML, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), Burkett's Lymphoma, mantle cell lymphoma (MCL), non-small cell lung cancer (NSCLC), neuroblastoma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell cancer, melanoma, myeloma, stomach cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, uterine cancer, adrenal cancer and head and neck cancer.
V. EXAMPLES
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1: Generation and Assessment of Anti-ROR1 Antibodies (VHs)
Exemplary anti-ROR1 antibodies, variable heavy (VH) chains that specifically bound to ROR1, even in the absence of a variable light (VL) chain, were generated and assessed. The anti-ROR1 antibodies (VHs) resulted from selections based on ability to specifically bind to ROR1-expressing cells, and/or to compete for binding with a rabbit-derived anti-ROR1 reference antibody.
1A. Library Selection, Antibody Generation
Exemplary anti-ROR1 antibodies (VHs) were generated through a series of selection steps carried out on dsDNA-encoded human naïve antibody VH libraries displayed in a cell-free system. Members of a VH library were selected for binding to live cells through five successive rounds, enriching for members that bound specifically to stably-transfected ROR1-expressing CHOK1, K562, and/or HEK293 cells, but not parental K562 and/or CHOK1 cells that did not express ROR1. To recover VH binders, a further selection was performed. In branch (a), surface stripping was applied to all 5 rounds of selection to recover ROR1 VH binders that cover broad epitopes and affinity ranges. In another branch (b), after two rounds of surface stripping, competitive elution was applied using a rabbit anti-ROR1 antibody, R12 scFv (having the sequence set forth in SEQ ID NO: 84, including the VH and VL sequences from the rabbit-human antibody designated R12), to enrich for binders that competed with R12 scFv for binding to ROR1, such as to the R12 scFv epitope.
Certain resulting clones were sequenced using forward and reverse primers to determine amino acid sequences. Table 2A lists sequence identifiers of the VH sequence of exemplary clones and the corresponding exemplary CDR-H1, CDR-H2 and CDR-H3 therein. Table 2B lists sequence identifiers corresponding to exemplary CDR-H3 (Kabat) amino acid sequences for exemplary clones. A VH sequence for clone V70 is set forth in SEQ ID NO: 8, which had CDR-H1 and CDR-H2 (Kabat) sequences as set forth in SEQ ID NOs: 27 and 28, respectively. A VH sequence for clone V353 is set forth in SEQ ID NO: 10, which had CDR-H1 and CDR-H2 (Kabat) sequences set forth in SEQ ID NOs: 33 and 34, respectively. Table 2B also lists a CDR-H3 (Kabat) sequence of a rabbit-derived reference antibody R12 (which has a VH sequence set forth in SEQ ID NO: 85), an scFv fragment of which was used as a control in this study. Clone V355 had a heavy chain framework with a sequence derived from a VH1 sequence; clones V331 and V345 had heavy chain frameworks with sequences derived from human VH4 sequences. All other clones (aside from R12) listed in Table 2A and 2B contained frameworks with sequences derived from human VH3 sequences.
TABLE 2A
Sequences for Exemplary Clones (SEQ ID NO.)
CDR-H
Heavy Chain
Heavy Chain Variable (VH)
(1, 2, 3)
Framework
Region
(Kabat)
Derived
Clone #
(Amino Acid)
(nucleotide)
(Amino Acid)
From
V70
8
7
27, 28, 29
VH3
V420
206
162, 170, 50
VH3
V282
203
162, 170, 51
VH3
V353
10
9
33, 34, 52
VH3
V163
193
33, 34, 53
VH3
V355
201
161, 169, 54
VH1
V18
179
33, 34, 35
VH3
V224
192
33, 34, 55
VH3
V95
194
33, 34, 56
VH3
V316
198
159, 167, 57
VH3
V331
199
160, 168, 58
VH4
V357
202
33, 34, 59
VH3
V397
205
33, 171, 60
VH3
V312
197
33, 34, 61
VH3
V278
200
158, 166, 62
VH3
V86
191
158, 166, 63
VH3
V102
195
27, 164, 64
VH3
V365
204
158, 166, 65
VH3
V181
196
27, 164, 66
VH3
V128
188
156, 34, 35
VH3
V71
190
157, 165, 67
VH3
V68
189
27, 164, 68
VH3
V336
208/209
163, 173,
VH3
69/174
V9
187
33, 34, 70
VH3
V345
207
160, 172, 71
VH4
TABLE 2B
Clone#
CDR-H3 Sequence (Kabat)
SEQ ID NO:
V70
VSNYEYYFDY
29
V420
VNGGEYYFDY
50
V282
VRGSEYYFDY
51
V353
DFGRWGYYFDY
52
V163
QGDSSSWYVEVYYFDY
53
V355
ITPPDAFDI
54
V18
DFGRWSYYFDY
35
V224
TYSSSWYESLLFDY
55
V95
GSGELRFLESYYFDY
56
V316
VDSERFLEWYYFDY
57
V331
GQIAAHVWGWFDP
58
V357
DMVGAWLVLSYFDY
59
V397
AKGLWFGESYYFDY
60
V312
TSRGRFLEWLLFDY
61
V278
ERSRWGDNWFDP
62
V86
VIFGVVNIPDY
63
V102
VGPSWDYYFDY
64
V365
GIGYSSSWYEIWTFDY
65
V181
DFEVREAHLSYFDY
66
V128
DFGRWSYYFDY
35
V71
VLRSGFLEWNLFDY
67
V68
DFEVRGAHLSYFDY
68
V336
VYGYDYRDFGWFDP
69
V9
GSNERFLEWLNFDY
70
V345
GPLRPQKVLPFQI
71
R12 scFv
DSYADDGALFNI
72
reference
antibody
1B. Binding Affinities, Competition with Reference Antibody
Exemplary ROR1 binding VH clones, generated in the selection process described in Example 1A, were further assessed. Clones were purified and titrated, and their binding affinities (EC50) to ROR1-expressing K562 and CHOK1 cells assessed by flow cytometry, with EC50 of the reference antibody (R12 scFv) serving as a positive control.
Assays also were performed to assess competition of the clones with the reference antibody (R12 scFv), for binding to ROR1-expressing cells. VH clones were titrated against 2 nM Myc-R12 on ROR1/K562 cells and competition assessed with an anti-Myc-Alexa647 secondary antibody, and the half maximal inhibitory concentration (IC50) determined for respective clones.
Additionally, percent (%) maximum (max) binding to ROR1-expressing cells and % max competition with the reference antibody (R12 scFv) were calculated. Percent max binding and competition values were calculated based on predicted plateaus (measured by GraphPad Prism® using four parameter non-linear regression), relative to values for R12 scFv binding and inhibition, respectively, assigned as 100% in each study.
Results are presented in Table 2C, listing mean EC50 (binding affinity), IC50 (competition), % max binding, and % max competition values, based on the indicated number of experiments for various clones. As expected, the reference antibody (R12 scFv) was confirmed in this study to specifically bind to ROR1 and compete with itself. The results demonstrated ROR1-specific binding and competition with the R12 scFv reference antibody, to varying degrees for various clones. Among the clones generated were those exhibiting similar affinity (EC50) for ROR1 and similar levels of competition for binding to ROR1 with the reference antibody.
Two preps of clone V9 were tested in a single R12 scFv competition assay and found not to have any detectable competitive binding activity.
TABLE 2C
Results from Various Binding and Competition Assays
Binding affinity to
ROR1-Expressing Cells
% Max
Competition for binding with R12 scFv
Clone #
EC50 (nM)
Binding
IC50 (nM)
% Max Competition
V70
3.1 ± 0.3
(n = 7)
92 ± 6
38.2 ± 4.8
(n = 8)
92 ± 12
V420
8.0
(n = 1)
74
71.5
(n = 1)
58
V282
13.1
(n = 1)
64
94.3
(n = 1)
48
V353
0.7
(n = 2)
35 ± 5
.4
(n = 2)
62
V163
92.9
(n = 1)
122
154.2
(n = 1)
58
V355
0.9
(n = 1)
54
1.9 ± 0.4
(n = 2)
53 ± 8
V18
0.2 ± 0.1
(n = 2)
37 ± 12
3.4 ± 0.8
(n = 3)
54 ± 6
V224
46.2
(n = 1)
74
94.6 ± 56.7
32 ± 17
V95
35.5 ± 2.1
(n = 2)
61 ± 14
19.0 ± 2.1
(n = 2)
36 ± 7
V316
28.7
(n = 1)
78
28.8 ± 4.9
(n = 2)
37 ± 14
V331
18.8
(n = 1)
70
32.4
(n = 1)
31
V357
46.0
(n = 1)
22
74.0
(n = 1)
44
V397
49.7
(n = 1)
18
25.2
(n = 1)
18
V312
23.7
(n = 1)
29
5.6
(n = 1)
19
V278
31.5
(n = 1)
22
ND
(n = 2)
V86
97.7 ± 62.1
(n = 2)
ND
(n = 2)
V102
128.9 ± 46.0
(n = 2)
ND
(n = 1)
V365
>200
(n = 1)
8
ND
(n = 1)
V181
>200
(n = 1)
>200
(n = 1)
76
V128
>200
(n = 1)
>200
(n = 1)
V71
>200
(n = 2)
ND
(n = 2)
V68
>200
(n = 2)
ND
(n = 2)
V336
>200
(n = 1)
72
ND
(n = 1)
V345
ND
(n = 1)
ND
(n = 1)
R12 scFv reference antibody
0.8 ± 0.1
(n = 20)
100
3.3 ± 0.2
(n = 17)
100
ND = not detected
Example 2: Generation and Assessment of Anti-ROR1 Antibodies (scFvs)
Exemplary anti-ROR1 antibodies (scFvs) that specifically bound to ROR1 were generated and assessed, including those that competed for binding with the R12 scFv reference antibody.
2A. Library Selection, Antibody Generation
Exemplary anti-ROR1 scFv antibodies were generated through various selection approaches, carried out on dsDNA-encoded human naïve antibody libraries displayed in a cell-free system. In some approaches, VL library members were paired with ROR1-specific VH-only antibodies generated in the study described in Example 1.
In one approach, a VH library was enriched through three successive rounds for members that bound specifically to stably-transfected ROR1-expressing CHOK1, K562, and/or HEK293 cells, but not parental ROR1-negative cells. The resulting enriched VH libraries were converted to scFv libraries by shuffling with a naïve human VL library, in VH-(G4S)3-VL format. The shuffled scFv libraries then were enriched in a fourth and fifth round of selection for specific binding to ROR1-expressing (but not parental) CHOK1 cells, with elution by surface stripping. In a sixth round, negative selection enriched for members that did not bind parental cells (K562, CHOK1), followed by positive selection for binding to ROR1-expressing K562 and/or CHOK1 cells. Elution pools were generated by either (a) surface stripping, or (b) R12 competitive elution.
In another approach, de novo selection was carried out by enriching a naïve human scFv library for ROR1-specific binding with ROR1-HEK293, ROR1-CHO, and ROR1-K562 cells and not HEK293, CHO-K1, and K562 parental cells over multiple rounds by alternating cell lines each round, followed by a combination of surface stripping (R1), competitive elution with R12 scFv (R2-4) and immunoprecipitation (R5) to recover scFv binders.
In another approach, ROR1-specific VH-only antibodies generated in the study described in Example 1B were non-covalently paired with members of a human naïve VL library (constructed from the same set of donors as naïve VH library) by dsDNA display, followed by selection for ROR1 binding for 3 successive rounds with ROR1-HEK293 and ROR1-CHO cells. Sequences of selected VLs were used to generate scFv clones.
For scFv clones selected in these various approaches, specific binding to ROR1-expressing K562 cells, as compared to control cells not expressing ROR1, was assessed by flow cytometry either with in vitro translated crude cell lysate or with bacterially-produced supernatant. Certain clones displaying binding preference for ROR1 were further analyzed.
Clones were sequenced using forward and reverse primers. Exemplary CDR-H3 (Kabat) amino acid sequences for exemplary clones and respective sequence identifiers are listed Table 3A. Clone “83B,” a de-glycosylation variant of clone 83, was generated by a introducing a serine (S) to asparagine (N) substitution at VH position 53 (Kabat) to remove the N-glycosylation motif of clone 83. Table 3B lists sequence identifiers for VH and VL sequences and exemplary CDR-H 1, 2, and 3 (Kabat) and CDR-L sequences of exemplary clones. Table 3A also lists the respective light chain human v-segment from which framework sequences of individual antibodies were derived. Each antibody in this table included a heavy chain framework sequence derived from a human VH3 sequence.
TABLE 3A
Exemplary Clones
Light Chain
Clone/
SEQ ID
SEQ ID
Framework
Antibody
CDR-H3
NO.
CDR-L3
NO:
Derived From
83B
DSSYDAFDI
22
QQYESLPYT
25
Vk1
83
DSSYDAFDI
22
QQYESLPYT
25
Vk1
305
VSNYEYYFDY
29
QVWDNDSDHRV
40
Vλ3
298
VSNYEYYFDY
29
QVWDDTGDHPV
32
Vλ3
350
VSNYEYYFDY
29
QSYDSSNHV
41
Vλ6
20
DFGRWSYYFDY
35
QQLKSRPLS
42
Vk1
16
DFGRWSYYFDY
35
QQLNSYPLT
43
Vk1
48
DFGRWSYYFDY
35
AAWDDSLSGVV
39
Vλ3
43
DFGRWSYYFDY
35
AAWDDSLSGVV
39
Vλ1
366
VSNYEYYFDY
29
AAWDDSLNGYV
44
Vλ1
40
DFGRWSYYFDY
35
QSYDGRNLM
45
Vλ6
461
DFEVREAHLSYFDY
46
QVWDSSSDHRV
47
Vλ3
65
DFGRWSYYFDY
35
KSWDSSGSLYV
48
Vλ3
81
DFGRWSYYFDY
35
QVWDSSSDHYV
49
Vλ3
7
DFGRWSYYFDY
35
QAWDSSTVV
50
Vλ3
R12 scFv
DSYADDGALFN
73
GADYIGGYV
74
Rb
reference
antibody
TABLE 3B
Sequences for Exemplary Clones and Reference Antibody (SEQ ID NO:)
Heavy
Light
Chain
Chain
Variable
Variable
CDR-H
Heavy
Light
(VH)
(VL)
ScFv
(1, 2, 3)
CDR-L
Chain
Chain
Region
Region
Sequence
(Kabat)
(1, 2, 3)
Framework
Framework
(Amino
(Amino
Amino Acid
(Amino
(Kabat)
Derived
Derived
Clone #
Acid)
Acid)
(Nucleotide)
Acid)
(Amino Acid)
From
From
83
17
18
2 (1)
20, 21, 22
23, 24, 25
VH3
Vk1
83B
19
18
12 (11)
20, 26, 22
23, 24, 25
VH3
Vk1
305
182
246
265
27, 28, 29
218, 229, 39
VH3
Vλ3
298
15
16
4 (3)
27, 28, 29
30, 31, 32
VH3
Vλ3
350
182
242
258
27, 28, 29
216, 227, 40
VH3
Vλ6
20
183/184
243/244
259/260/261/
33, 34, 35
217, 228, 41
VH3
Vk1
262
16
177
238
253
33, 34, 35
212, 223, 42
VH3
Vk1
48
13
14
6 (5)
33, 34, 35
36, 37, 38
VH3
Vλ3
43
183/184
245
263/264
33, 34, 35
36, 37, 38
VH3
Vλ1
366
182
247
266
27, 28, 29
219, 230, 43
VH3
Vλ1
40
180/181
241
256/257
33, 34, 35
215, 226, 44
VH3
Vλ6
461
185/186
248
267/268
27, 164, 45
220, 231, 46
VH3
Vλ3
65
178
239
254
155, 34, 35
213, 224, 47
VH3
Vλ3
81
176
236/237
251/252
33, 34, 35
211, 222, 48
VH3
Vλ3
7
175
234/235
249/250
33, 318, 35
210, 221, 49/233
VH3
Vλ3
R12 scFv
85
86
84
87, 88, 72
89, 90, 73
Rb
Rb
reference
antibody
2B. Binding Affinities, Competition with Reference Antibody
Clones were purified and titrated, and their binding affinities (EC50) to ROR1-expressing K562 and CHOK1 cells assessed by flow cytometry. EC50 for the R12 scFv reference antibody in this study served as a positive control.
Assays also were performed to assess competition (measured as half maximal inhibitory concentration (IC50)) of the clones with the R12 scFv reference antibody for binding to ROR1-expressing cells, as described in Example 1. Percent (%) max ROR1 and % max competition, as compared to the R12 scFv reference antibody, were determined as described in Example 1.
Results are presented in Table 3C, listing mean EC50 (binding affinity), IC50 (competition), % max binding, and % max competition values for various clones, as determined over a plurality of experiments. As expected, the reference antibody was confirmed to exhibited ROR1-specific binding and competition with itself in this study. The results demonstrated varying degrees of ROR1-specific binding affinities and competition with the R12 scFv reference antibody among the various clones. Among the assayed clones were those with similar ROR1 binding affinity as compared to the reference antibody and those that competed for binding with the reference antibodies to similar degrees as compared to the reference antibody itself.
TABLE 3C
Results from Various Binding and Competition Assays
Binding affinity to ROR1-
Competition for
Expressing Cells
binding with R12 scFv
Clone#
EC50 (nM)
% Max
IC50 (nM)
% Max
83B
6.4
(n = 1)
140
15.0
(n = 1)
103
83
2.5 ± 0.3
(n = 2)
70 ± 7
11.1 ± 3.4
(n = 3)
103 ± 3
305
5.1
(n = 1)
86
62.5
(n = 1)
97
298
5.9
(n = 1)
96
41.3
(n = 1)
88
350
22.9
(n = 1)
137
49.1
(n = 1)
80
20
2.6 ± 1.1
(n = 9)
97 ± 18
4.7 ± 2.2
(n = 5)
77 ± 7
16
3.7 ± 1.3
(n = 6)
91 ± 23
4.2 ± 1.8
(n = 4)
71 ± 8
48
9.2 ± 1.9
(n = 4)
69 ± 24
53.7 ± 18.7
(n = 3)
55 ± 11
43
10.6 ± 3.4
(n = 4)
66 ± 14
56.1 ± 28.4
56 ± 17
366
10.0
(n = 1)
104
53.9
(n = 1)
71
40
3.2 ± 0.8
(n = 4)
81 ± 20
22.8 ± 11.2
(n = 4)
70 ± 11
461
22.8
(n = 1)
63
99.8
(n = 1)
48
65
18.4 ± 11.6
(n = 2)
ND
(n = 3)
81
>200
(n = 2)
not assayed
R12 scFv reference
0.8 ± 0.1
(n = 20)
100
3.3 ± 0.2
(n = 17)
100
antibody
ND = none detected
Example 3: Generation of Chimeric Antigen Receptors (CARs) Against ROR1, Engineering of Cells Expressing Such CARs, and Assessment of Effector Functions of Such CAR-Expressing Cells
Various exemplary chimeric antigen receptors (CARs) were generated, with antigen-binding regions containing human anti-ROR1 antigen-binding domains as described in Example 1. Specifically, nucleic acid molecules were generated that encoded CARs containing either an scFv antigen-binding domains in the VH-VL format (Clone 83B, Clone 298 or Clone 48) or a VH only antigen-binding domain (Clone V70 and Clone V353). For exemplary scFv antigen-binding domains, constructs encoding a CAR having the same VH and VL sequences, but present in the reverse orientation (VL-VH), also were generated. A CAR containing a rabbit anti-ROR1 scFv (R12) (in the VH-VL orientation) was used as a control. Each CAR further contained an Ig-derived hinge only spacer, a human CD28-derived transmembrane domain, a human 4-1BB-derived intracellular signaling domain, a human CD3 zeta-derived signaling domain, and a truncated EGFR (tEGFR) sequence for use as a transduction marker, separated from the CAR sequence by a self-cleaving T2A sequence.
Primary human T cell populations expressing the various CARs were generated. Nucleic acid molecules encoding each CAR were individually cloned into a lentiviral vector, which was used to transduce CD8+ T cells in populations isolated from human PBMC samples obtained from healthy donors (essentially as described by Yam et al. (2002) Mol. Ther. 5:479; WO2015/095895). As a control, a lentiviral vector containing nucleic acid encoding only the tEGFR sequence was used to generate control cells expressing the tEGFR only. The genetically engineered human CD8+ T cells expressing various CARs containing human anti-ROR1 antigen-binding domains, produced as described above, were assessed for various responses following co-culture with ROR1-expressing cells.
A. Cytolytic Activity
ROR1-expressing target cells were incubated with CD8+ T cells expressing the various CARs or tEGFR alone (negative control). Following incubation, lysis of target cells was monitored. Specifically, lysis of ROR1-transduced K562 cells (K562/ROR1), JeKo-1 (mantle cell lymphoma line) cells, and non-transduced K562 control cells (negative control) (FIG. 1A) were tested.
The target cells (K562/ROR1, JeKo-1, or non-transduced K562 control cells) were labeled overnight with 51Cr. Labeled cells were washed and incubated with effector T cells (CAR-expressing and negative control CD8+ cells) at a single effector to target (E:T) ratio. To measure spontaneous lysis, target cells were incubated with an equal volume of media but without effector cells and maximum lysis was determined following incubation of target cells with detergent to completely lyse the target cells. Supernatants were harvested for γ-counting after a 4 hour incubation. The percent specific lysis for the experimental conditions was calculated as: [(Experimental Release−Spontaneous Release)/(Maximum Release−Spontaneous Release)]×100.
The results are set forth in FIG. 1A. As shown in FIG. 1A, engineered CD8+ T cells expressing various human anti-ROR1 antigen-binding domain-containing CARs exhibited antigen-specific cytolytic activity against K562/ROR1 cells, as did cells expressing the CAR containing the rabbit anti-ROR1 (R12) scFv. This cytotoxic activity was generally not observed against control K562 cells not expressing ROR1. The results showed that cells engineered with a CAR containing the exemplary V353 clone containing only the VH domain exhibited comparable cytolytic activity to CARs containing an antigen-binding domain in the scFv format. The degree of cytolytic activity observed for cells expressing a CAR with a given human scFv in the VH-VL orientation was generally greater than that observed for cells expressing a CAR with the corresponding scFv in the reverse VL-VH orientation (LH).
B. Cytokine Release
Cytokine release was assessed following incubation of the CAR-expressing cells with antigen-expressing and control target cells. Transduced CD8+ T cells were co-cultured with target cells (K562, K562/ROR1, JeKo-1) at a single effector to target (E:T) ratio. The co-cultured cells were incubated for about 24 hours, and then supernatants were collected for measurement of IFN-γ using a cytokine immunoassay.
The results are set forth in FIG. 1B. Engineered CD8+ T cells expressing various human anti-ROR1 antigen-binding domain-containing CARs were observed to secrete IFN-γ in an antigen-specific manner following incubation with K562/ROR1 cells, as did cells expressing the CAR containing the rabbit anti-ROR1 (R12) scFv. The cytokine secretion was generally not observed following incubation with control K562 cells not expressing ROR1. The results showed that cells engineered with a CAR containing the exemplary V353 clone containing only the VH domain exhibited comparable levels of IFN-γ secretion to CARs containing an antigen-binding domain in the scFv format. The degree of IFNγ secretion observed for cells expressing a CAR with a given human scFv in the VH-VL orientation was generally greater that observed for cells expressing a CAR with the corresponding scFv in the reverse (VL-VH) orientation.
Example 4: Assessment of Effector Functions of CAR-Expressing Cells Against Cells Expressing Human or Mouse ROR1
Various exemplary chimeric antigen receptors (CARs) were generated and used to generate CAR-expressing CD8+ T cells as described in Example 3.
Cytokine release was assessed following incubation of the CAR-expressing cells with either K562/human ROR1 (hROR1) or K562/mouse ROR1 (mROR1) antigen-expressing cells. Transduced CD8+ T cells were co-cultured with antigen-expressing cells at a single effector to target (E:T). The co-cultured cells were incubated for about 24 hours, and then supernatants were collected for measurement of IFN-γ using a cytokine immunoassay.
The results are set forth in FIG. 2. Engineered CD8+ T cells expressing the various human anti-ROR1 antigen-binding domain-containing CARs were observed to secrete IFN-γ at comparable levels following incubation with either hROR1-expressing cells or mROR1-expressing cells. In contrast, cells engineered with the rabbit anti-ROR1 (R12) scFv were observed to secrete IFN-γ only following incubation with hROR-1-expressing cells and not following incubation with mROR-1-expressing cells. This result is consistent with the reported specificity of R12 for human and not mouse ROR1 (Yang et al. (2011) PLoS ONE, 6:e21018). Thus, together with the results in Example 2, the results showed that the tested human anti-ROR1 antigen-binding binding domains of the CARs bind to an epitope in human ROR1 that may overlap with R12, but also bind to an epitope in mouse ROR1 that is not recognized by R12.
The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.
SEQUENCES
SEQ
ID
NO:
Sequence
Description
1
CAGGTCCAGCTTGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCT
Clone 83 scFv
CAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTA
TATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGG
AATAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAG
GGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATG
GAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGA
GAGATAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATGGT
CACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGT
GGCGGATCGGCCATCCAGTTGACCCAATCTCCTTCCACCCTGTCTGCATC
TGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATCAGC
AATTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCC
TGATCAACGATGCATCCTATTTGGAGACAGGGGTCCCATCAAGGTTCAG
TGGAAGTGGATCTGGGACAGATTTTACTTTAACCATCAGCAGCCTGCAG
CCTGAAGATATTGCAACATATTACTGTCAACAGTATGAAAGTCTCCCGT
ACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA
2
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWM
Clone 83 scFv
GIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDS
SYDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSAIQLTQSPSTLSASVGDR
VTITCQASQDISNYLNWYQQKPGKAPKLLINDASYLETGVPSRFSGSGSGTD
FTLTISSLQPEDIATYYCQQYESLPYTFGQGTKLEIK
3
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGG
Clone 298
TCCCTGAGACTCTCCTGTACAGCTTCTGGATTCACCTTTGGTGATTATGC
scFv
GATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTC
AAGTATTAGTGGTAGTGGTCGTAGCACAGACCACGCAGACTACGTGAAG
GGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGGTATATCTGC
AAATGAACAGGCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCAA
AAGTCAGTAACTACGAGTATTACTTTGACTACTGGGCCCAGGGAACCCT
GGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGC
GGTGGCGGATCGGAAATTGTGCTGACTCAGTCTCCCTCGGTGTCAGTGG
CCCCAGGACAGACGGCCAGAATTACCTGTGGGGGAAGCAACATTGGAT
CTGAGAGTGTCAACTGGTACCAGTGGAAGTCGGGCCAGGTTCCTGTCTT
GGTCGTCTCTGACACTACCGACCGACCCTCAGGGATCCCTGGGCGATTC
ACTGGCACCCGGTCTGGGACCACGGCCACCTTGACCATCAGTGGGGTCG
AAGCCGGGGATGAGGCCGACTATCACTGTCAGGTGTGGGATGACACTG
GTGATCATCCTGTCTTCGGCGGAGGGACCAAGCTGACCGTCCTA
4
EVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAMSWVRQAPGKGLEWVSS
Clone 298
ISGSGRSTDHADYVKGRFTISRDNSKNTVYLQMNRLRAEDTAVYYCAKVS
scFv
NYEYYFDYWAQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPSVSVAPGQ
TARITCGGSNIGSESVNWYQWKSGQVPVLVVSDTTDRPSGIPGRFTGTRSGT
TATLTISGVEAGDEADYHCQVWDDTGDHPVFGGGTKLTVL
5
CAGGTCACCTTGAAGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGG
Clone 48 scFv
TCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTG
GATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGG
CCGTATTAAAAGCAAAACTGATGGTGGGACAACAGACTACGCTGCACCC
GTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTGT
ATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTG
TGCGAGAGATTTCGGACGATGGAGCTACTACTTTGACTACTGGAGCCAG
GGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTG
GCTCTGGCGGTGGCGGATCGCAGTCTGTGCTGACTCAGCCATCCTCAGT
GTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGC
TCCAACATCGGAAGTAATTATGTATACTGGTACCAGCAGCTCCCAGGAA
CGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGT
CCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCA
TCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATG
GGATGACAGCCTGAGTGGTGTGGTATTCGGCGGAGGGACCAAGCTCACC
GTCCTA
6
QVTLKESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone 48 scFv
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
(aa)
DFGRWSYYFDYWSQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPSSVSGT
PGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSG
SKSGTSASLAISGLRSEDEADYYCAAWDDSLSGVVFGGGTKLTVL
7
CAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGT
Clone V70 VH
CCCTGAGACTCTCCTGTACAGCTTCTGGATTCACCTTTGGTGATTATGCG
ATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCA
AGTATTAGTGGTAGTGGTCGTAGCACAGACCACGCAGACTACGTGAAGG
GCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGGTATATCTGCA
AATGAACAGGCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCAAA
AGTCAGTAACTACGAGTATTACTTTGACTACTGGGCCCAGGGAACCCTG
GTCACCGTCTCCTCA
8
QVQLLESGGGLVQPGRSLRLSCTASGFTFGDYAMSWVRQAPGKGLEWVSS
Clone V70 VH
ISGSGRSTDHADYVKGRFTISRDNSKNTVYLQMNRLRAEDTAVYYCAKVS
NYEYYFDYWAQGTLVTVSS
9
CAGGTCACCTTGAAGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGG
Clone V353
TCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTG
VH
GATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGG
CCGTATTAAAAGCAAAACTGATGGTGGGACAACAGACTACGCTGCACCC
GTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTGT
ATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTG
TGCGAGAGATTTCGGACGATGGGGCTACTACTTTGACTACTGGAGCCAG
GGAACCCTGGTCACCGTCTCCTCA
10
QVTLKESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone V353
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
VH
DFGRWGYYFDYWSQGTLVTVSS
11
CAGGTCCAGCTTGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCT
Clone 83B
CAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTA
scFv
TATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGG
AATAATCAACCCTAATGGTGGTAGCACAAGCTACGCACAGAAGTTCCAG
GGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATG
GAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGA
GAGATAGCAGCTATGATGCTTTTGATATCTGGGGCCAAGGGACAATGGT
CACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGT
GGCGGATCGGCCATCCAGTTGACCCAATCTCCTTCCACCCTGTCTGCATC
TGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATCAGC
AATTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCC
TGATCAACGATGCATCCTATTTGGAGACAGGGGTCCCATCAAGGTTCAG
TGGAAGTGGATCTGGGACAGATTTTACTTTAACCATCAGCAGCCTGCAG
CCTGAAGATATTGCAACATATTACTGTCAACAGTATGAAAGTCTCCCGT
ACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA
12
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWM
Clone 83B
GIINPNGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARD
scFv
SSYDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSAIQLTQSPSTLSASVGD
RVTITCQASQDISNYLNWYQQKPGKAPKLLINDASYLETGVPSRFSGSGSGT
DFTLTISSLQPEDIATYYCQQYESLPYTFGQGTKLEIK
13
QVTLKESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone 48 VH
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
DFGRWSYYFDYWSQGTLVTVSS
14
QSVLTQPSSVSGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKWYRN
Clone 48 VL
NQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGVVFGG
GTKLTVL
15
EVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAMSWVRQAPGKGLEWVSS
Clone 298 VH
ISGSGRSTDHADYVKGRFTISRDNSKNTVYLQMNRLRAEDTAVYYCAKVS
NYEYYFDYWAQGTLVTVSS
16
EIVLTQSPSVSVAPGQTARITCGGSNIGSESVNWYQWKSGQVPVLVVSDTT
Clone 298 VL
DRPSGIPGRFTGTRSGTTATLTISGVEAGDEADYHCQVWDDTGDHPVFGGG
TKLTVL
17
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWM
Clone 83 VH
GIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDS
SYDAFDIWGQGTMVTVSS
18
AIQLTQSPSTLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLINDAS
Clone 83, 83B
YLETGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQYESLPYTFGQGTKLEI
VL
K
19
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWM
Clone 83B VH
GIINPNGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARD
SSYDAFDIWGQGTMVTVSS
20
SYYMH
Clone 83, 83B
CDR-H1
21
IINPSGGSTSYAQKFQG
Clone 83
CDR-H2
22
DSSYDAFDI
Clone 83, 83B
CDR-H3
23
QASQDISNYLN
Clone 83, 83B
CDR-L1
24
DASYLET
Clone 83, 83B
CDR-L2
25
QQYESLPYT
Clone 83, 83B
CDR-L3
26
IINPNGGSTSYAQKFQG
Clone 83B
CDR-H2
27
DYAMS
Clone 298,
V70, 350, 305,
366, 461, V68,
V102, V181
CDR-H1
28
SISGSGRSTDHADYVKG
Clone 298,
V70, 350, 305,
366 CDR-H2
29
VSNYEYYFDY
Clone 298,
305, 350, 366,
V70 CDR-H3
30
GGSNIGSESVN
Clone 298
CDR-L1
31
DTTDRPS
Clone 298
CDR-L2
32
QVWDDTGDHPV
Clone 298
CDR-L3
33
NAWMS
Clone 48,
V353, 7, 81,
16, V18, 40,
20, 43, V9,
V224, V163,
V95, V312,
V357, V397
CDR-H1
34
RIKSKTDGGTTDYAAPVKG
Clone 48,
V353, 81, 16,
65, V18, 40,
20, 43, V9,
V128, V224,
V163, V95,
V312, V357
CDR-H2
35
DFGRWSYYFDY
Clones 16, 20,
43, 48, 40, 65,
81, 7, V128,
V18 CDR-H3
36
SGSSSNIGSNYVY
Clone 48, 43
CDR-L1
37
RNNQRPS
Clone 48, 43
CDR-L2
38
AAWDDSLSGVV
Clones 43, 48
CDR-L3
39
QVWDNDSDHRV
Clone 305
CDR-L3
40
QSYDSSNHV
Clone 350
CDR-L3
41
QQLKSRPLS
Clone 20
CDR-L3
42
QQLNSYPLT
Clone 16
CDR-L3
43
AAWDDSLNGYV
Clone 366
CDR-L3
44
QSYDGRNLM
Clone 40
CDR-L3
45
DFEVREAHLSYFDY
Clone 461
CDR-H3
46
QVWDSSSDHRV
Clone 461
CDR-L3
47
KSWDSSGSLYV
Clone 65
CDR-L3
48
QVWDSSSDHYV
Clone 81
CDR-L3
49
QAWDSSTVV
Clone 7 CDR-
L3
50
VNGGEYYFDY
Clone V420
CDR-H3
51
VRGSEYYFDY
Clone V282
CDR-H3
52
DFGRWGYYFDY
Clone V353
CDR-H3
53
QGDSSSWYVEVYYFDY
Clone V163
CDR-H3
54
ITPPDAFDI
Clone V355
CDR-H3
55
TYSSSWYESLLFDY
Clone V224
CDR-H3
56
GSGELRFLESYYFDY
Clone V95
CDR-H3
57
VDSERFLEWYYFDY
Clone V316
CDR-H3
58
GQIAAHVWGWFDP
Clone V331
CDR-H3
59
DMVGAWLVLSYFDY
Clone V357
CDR-H3
60
AKGLWFGESYYFDY
Clone V397
CDR-H3
61
TSRGRFLEWLLFDY
Clone V312
CDR-H3
62
ERSRWGDNWFDP
Clone V278
CDR-H3
63
VIFGVVNIPDY
Clone V86
CDR-H3
64
VGPSWDYYFDY
Clone V102
CDR-H3
65
GIGYSSSWYEIWTFDY
Clone V365
CDR-H3
66
DFEVREAHLSYFDY
Clone V181
CDR-H3
67
VLRSGFLEWNLFDY
Clone V71
CDR-H3
68
DFEVRGAHLSYFDY
Clone V68
CDR-H3
69
VYGYDYRDFGWFDP
Clone V336
CDR-H3
70
GSNERFLEWLNFDY
Clone V9
CDR-H3
71
GPLRPQKVLPFQI
Clone V345
CDR-H3
72
DSYADDGALFNI
R12 CDR-H3
73
GADYIGGYV
R12 CDR-L3
74
GYTFTS
Clone 83, 83B
CDR-H1
75
GYTFTSYYMH
Clone 83, 83B
CDR-H1
76
GFTFGD
Clone 298,
V70, 350, 305,
366, 461, V68,
V102, V181
CDR-H1
77
GFTFGDYAMS
Clone 298,
V70, 350, 305,
366, 461, V68,
V102, V181
CDR-H1
78
GFTFSN
Clone 48,
V353, 7, 81,
16, V18, 40,
20, 43, V9,
V163, V95,
V312, V357,
V397
CDR-H1
79
GFTFSNAWMS
Clone 48,
V353, 7, 81,
16, V18, 40,
20, 43, V9,
V163, V95,
V312, V357,
V397
CDR-H1
80
IINPSGGS
Clone 83
CDR-H2
81
SISGSGRS
Clone 298,
V70 CDR-H2
82
RIKSKTDGGT
Clone 48,
V353 CDR-
H2
83
IINPNGGS
Clone 83B
CDR-H2
84
QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATI
R12 scFv
YPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSY
ADDGALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGS
PAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFS
GSSSGADRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTG
85
QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATI
R12 VH
YPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSY
ADDGALFNIWGPGTLVTISS
86
ELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQ
R12 VL
SDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDEADYYCGADYIGGYVF
GGGTQLTVTG
87
AYYMS
R12 CDR-H1
88
TIYPSSGKTYYATWVNG
R12 CDR-H2
89
TLSSAHKTDTID
R12 CDR-L1
90
GSYTKRP
R12 CDR-L2
91
GGGGSGGGGSGGGGS
Linker
92
X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16
Consensus
X1 = D, V, Q, I, T, G, A, or E;
CDR-H3
X2 = S, F, N, R, G, T, Y, D, Q, M, K, I, L, or P;
X3 = S, N, G, E, D, P, I, V, R, F, N, or L;
X4 = Y, R, V, G, S, P, E, A, or L;
X5 = D, E, W, R, S, L, A, V, G, or P;
X6 = A, W, S, E, G, Y, R, F, H, V, D, S, or Q;
X7 = F, Y, A, W, L, G, D, V, N, S, K, or R;
X8 = F, Y, H, E, L, W, V, N, I, D, or null;
X9 = F, L, V, S, E, W, G, P, Y, or null;
X10 = S, E, L, Y, W, F, N, G, P, or null;
X11 = Y, V, L, F, I, W, N, or null;
X12 = F, Y, W, or null;
X13 = Y, F, T, or null;
X14 = F or null;
X15 = D or Q;
X16 = I, Y, or P
93
X1X2X3X4X5X6X7X8X9X10X11X12DX14
Consensus
X1 = D or V;
CDR-H3
X2 = S or F;
X3 = S, N, G, or E;
X4 = Y, R, or V;
X5 = D, E, W, or R;
X6 = A, Y, S, or E;
X7 = F, Y, or A;
X8 = F, Y, H, or null;
X9 = F, L, or null;
X10 = S or null;
X11 = Y or null;
X12 = F or null;
X14 = I or Y
94
X1X2X3X4X5X6YX8X9DY
Consensus
X1 = V or D;
CDR-H3
X2 = S or F;
X3 = N or G;
X4 = Y or R;
X5 = E or W;
X6 = Y or G;
X8 = F or Y;
X9 = F or null
95
X1X2X3MX5
Consensus
X1 = S, D, or N;
CDR-H1
X2 = Y or A;
X3 = Y, A, or W;
X5 = H or S
96
X1IX3X4X5X6X7GX9X10TX12X13AX15X16X17X18G
Consensus
X1 = I, S, or R;
CDR-H2
X3 = N, S, or K;
X4 = P, G, or S;
X5 = K or null;
X6 = T or null;
X7 = S, D, or N;
X9 = G or R;
X10 = S or T;
X12 = S or D;
X13 = Y or H;
X15 = Q, D, or A;
X16 = K, Y, or P;
X17 = F or V;
X18 = Q or K
97
X1X2X3MS
Consensus
X1 = D or N;
CDR-H1
X2 = Y or A;
X3 = A or W
98
X1IX3X4X5X6X7GX9X10TX12X13AX15X16X17X18G
Consensus
X1 = I, S, or R;
CDR-H2
X3 = N, S, or K;
X4 = P, G, or S;
X5 = K or null;
X6 = T or null;
X7 = S, D, or N;
X9 = G or R;
X10 = S or T;
X12 = S or D;
X13 = Y or H;
X15 = Q, D, or A;
X16 = K, Y, or P;
X17 = F or V;
X18 = Q or K
99
X1X2X3X4X5X6X7X8X9X10X11X12X13X14
Consensus
X1 = Q, A, or K;
CDR-L3
X2 = Q, V, A, or S;
X3 = Y, W, or L;
X4 = E, D, K, or N;
X5 = S, D, N, or G;
X6 = L, T, S, D, R, or Y;
X7 = P, G, L, S, N, or T;
X8 = D, S, N, or null;
X9 = H, G, L, or null;
X10 = Y, P, V, R, H, or L;
X11 = T, V, S, or M
100
X1X2X3X4X5X6X7X8X9X10X11X12X13X14
Consensus
X1 = Q or A;
CDR-L3
X2 = Q, V, or A;
X3 = Y or W;
X4 = E or D;
X5 = S or D;
X6 = L, T, or S;
X7 = P, G, or L;
X8 = D, S, or null;
X9 = H, G, or null;
X10 = Y, P, or V;
X11 = T or V
101
X1X2SX4X5X6X7X8SX10X11X12X13
Consensus
X1 = Q, G, or S;
CDR-L1
X2 = A or G;
X4 = Q, N, or S;
X5 = S or null;
X6 = N or null;
X7 = D on;
X8 = I or G;
X10 = N or E;
X11 = Y or S;
X12 = L or V;
X13 = N or Y
102
X1X2X3X4X5X6X7
Consensus
X1 = D or R;
CDR-L2
X2 = A, T, or N;
X3 = S, T, or N;
X4 = Y, D, or Q;
X5 = L or R;
X6 = E or P;
X7 = T or S
103
QETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKV
Human ROR1;
SGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVAT
GenBank No.
NGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRT
NP_005003.2
VYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDET
SSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESP
EAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYP
HTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDS
KDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSAPVQRQPKHV
RGQNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECAFGKIYKGHLYLPG
MDHAQLVAIKTLKDYNNPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQ
PVCMLFEYINQGDLHEFLIMRSPHSDVGCSSDEDGTVKSSLDHGDFLHIAIQI
AAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQSK
SLLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIE
MVRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLSS
HTSSTTPSGGNATTQTTSLSASPVSNLSNPRYPNYMFPSQGITPQGQIAGFIG
PPIPQNQRFIPINGYPIPPGYAAFPAAHYQPTGPPRVIQHCPPPKSRSPSSASGS
TSTGHVTSLPSSGSNQEANIPLLPHMSIPNHPGGMGIT
VFGNKSQKPYKIDSKQASLLGDANIHGHTESMISAEL
104
QETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKV
ROR1 Isoform
SGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVAT
short
NGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRT
VYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDET
SSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESP
EAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYP
HTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDS
KDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSAPVQRQPKHV
RGQNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECAFGKIYKGHLYLPG
MDHAQLVAIKTLKDYNNPQQWTEFQQEASLMAELHEIPNIVCLLGAVTQEQ
PVC
105
QETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKV
ROR1 isoform
SGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVAT
3
NGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRT
VYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDET
SSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESP
EAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYP
HTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACGK
106
QETELSVSAELVPTSSWNTSSEIDKGSYLTLDEPMNNITTSLGQTAELHCKV
Mouse ROR1;
SGNPPPSIRWFKNDAPVVQEPRRISFRATNYGSRLRIRNLDTTDTGYFQCVA
GenBank No.
TNGKKVVSTTGVLFVKFGPPPTASPGSSDEYEEDGFCQPYRGIACARFIGNR
NP_038873
TVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDE
TSSVPKPRDLCRDECEVLENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPE
SPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQ
YPHTHSFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPAC
DSKDSKEKNKMEILYILVPSVAIPLAIAFLFFFICVCRNNQKSSSPPVQRQPKP
VRGQNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECTFGKIYKGHLYLP
GMDHAQLVAIKTLKDYNNPQQWTEFQQEASLMAELHEIPNIVCLLGAVTQE
QPVCMLFEYMNQGDLHEFLIMRSPHSDVGCSSDEDGTVKSSLDHGDFLHIA
IQIAAGMEYLSSHFFVHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQ
SKSSLPIRWMPPEAIMYGKFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVI
EMVRKRQLLPCSEDCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWEGLS
SHTSSTTPSGGNATTQTTSLSASPVSNLSNPRFPNYMFPSQGITPQGQIAGFIG
PAIPQNQRFIPINGYPIPPGYAAFPAAHYQPAGPPRVIQHCPPPKSRSPSSASG
STSTGHVASLPSSGSNQEANVPLLPHMSIPNHPGGMGITVFGNKSQKPYKID
SKQSSLLGDSHIHGHTESMISAEV
107
GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT
spacer
(IgG4hinge)
108
ESKYGPPCPPCP
spacer
(IgG4hinge)
109
X1SX3YX5X6X7X8DX10
Consensus
X1 = V or D;
CDR-H3
X3 = S or N;
X5 = D or E;
X6 = Y or A;
X7 = F or Y;
X8 = F or null;
X10 = I or Y;
110
VX2X3X4EYYFDY
Consensus
X2 = S, N, or R;
CDR-H3
X3 = N or G;
X4 = Y, G, or S;
111
GGGGS
4GS linker
112
GGGS
3GS linker
113
GSTSGSGKPGSGEGSTKG
Linker
114
QVQLQQSGAELVRPGASVTLSCKASGYTFSDYEMHWVIQTPVHGLEWIGAI
2A2 VH
DPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTGYYD
YDSFTYWGQGTLVTVSA
115
DIVMTQSQKIMSTTVGDRVSITCKASQNVDAAVAWYQQKPGQSPKWYSA
2A2 VL
SNRYTGVPDRFTGSGSGTDFTLTISNMQSEDLADYFCQQYDIYPYTFGGGT
KLEIK
116
DYEMH
2A2 CDR-H1
117
AIDPETGGTAYNQKFKG
2A2 CDR-H2
118
YYDYDSFTY
2A2 CDR-H3
119
KASQNVDAAVA
2A2 CDR-L1
120
SASNRYT
2A2 CDR-L2
121
QQYDIYPYT
2A2 CDR-L3
122
QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWVRQAPGQGLEWMGS
99961
FDPYDGGSSY
humanized VH
NQKFKDRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGWYYFDYWGH
GTLVTVSS
123
QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWVRQAPGQGLEWMGS
99961
FDPYDGGSSY
humanized VH
NQKFKDRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGWYYFDYWGH
GTLVTVSS
124
QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWIRQPPGKGLEWIGSFD
99961
PYDGGSSY
humanized VH
NQKFKDRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGWYYFDYWGH
GTLVTVSS
125
QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWIRQPPGKGLEWIGSFD
99961
PYDGGSSY
humanized VH
NQKFKDRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGWYYFDYWGH
GTLVTVSS
126
DIVMTQTPLSLPVTPGEPASISCRASKSISKYLAWYQQKPGQAPRLLIYSGST
99961
LQSGIPP
humanized VL
RFSGSGYGTDFTLTINNIESEDAAYYFCQQHDESPYTFGEGTKVEIK
127
DVVMTQSPLSLPVTLGQPASISCRASKSISKYLAWYQQKPGKAPKWYSGS
99961
TLQSGIPP
humanized VL
RFSGSGYGTDFTLTINNIESEDAAYYFCQQHDESPYTFGEGTKVEIK
128
DIVMTQTPLSLPVTPGEPASISCRASKSISKYLAWYQQKPGQAPRLLIYSGST
99961
LQSGIPP
humanized VL
RFSGSGYGTDFTLTINNIESEDAAYYFCQQHDESPYTFGEGTKVEIK
129
DVVMTQSPLSLPVTLGQPASISCRASKSISKYLAWYQQKPGKAPKWYSGS
99961
TLQSGIPP
humanized VL
RFSGSGYGTDFTLTINNIESEDAAYYFCQQHDESPYTFGEGTKVEIK
130
SGYAFTAYNIHWVRQ
99961
humanized
CDR-H1
131
GSFDPYDGGSSYNQKFKD
99961
humanized
CDR-H2
132
YYCARGWYYFDYWGHGTLVTVSS
99961
humanized
CDR-H3
133
GYAFTAYN
99961
humanized
CDR-H1
134
FDPYDGGS
99961
humanized
CDR-H2
135
GWYYFDY
99961
humanized
CDR-H3
136
CRASKSISKYLAWY
99961
humanized
CDR-L1
137
LLIYSGSTLQSG
99961
humanized
CDR-L2
138
CQQHDESPYTFGEGTK
99961
humanized
CDR-L3
139
KSISKY
99961
humanized
CDR-L1
140
SGS
99961
humanized
CDR-L2
141
QQHDESPY
99961
humanized
CDR-L3
142
ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE
Hinge-CH3
WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE
spacer
ALHNHYTQKSLSLSLGK
143
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDP
Hinge-CH2-
EVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK
CH3 spacer
CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF
SCSVMHEALHNHYTQKSLSLSLGK
144
RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEK
IgD-hinge-Fc
EEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKD
AHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTC
TLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSP
PNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPAT
YTCVVSHEDSRTLLNASRSLEVSYVTDH
145
FWVLVVVGGVLACYSLLVTVAFIIFWV
CD28 (amino
acids 153-179
of Accession
No. P10747)
146
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP
CD28 (amino
FWVLVVVGGVLACYSLLVTVAFIIFWV
acids 114-179
of Accession
No. P10747)
147
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
CD28 (amino
acids 180-220
of P10747)
148
RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
CD28 (LL to
GG)
149
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
4-1BB (amino
acids 214-255
of Q07011.1)
150
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP
CD3 zeta
RRKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY
QGLSTATKDTYDALHMQALP PR
151
RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
CD3 zeta
RKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY
QGLSTATKDTYDALHMQALP PR
152
RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP
CD3 zeta
RRKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY
QGLSTATKDTYDALHMQALP PR
153
LEGGGEGRGSLLTCGDVEENPGPR
T2A
154
MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSI
tEGFR
SGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLH
AFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYA
NTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDC
VSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRG
PDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNC
TYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM
155
XaaRLDE
Clone 65
CDR-H1
156
TFWMS
Clone V128
CDR-H1
157
TSAMS
Clone V71
CDR-H1
158
SYAMS
Clone V86,
V278, V365
CDR-H1
159
RTWMS
Clone V316
CDR-H1
160
GYYWS
Clone V331,
V345 CDR-H1
161
SYAIS
Clone V355
CDR-H1
162
SYWMS
Clone V420,
V282 CDR-H1
163
SYAMH
Clone V336
CDR-H1
164
FIRSKAYGGTTEYAASVKG
Clone 461,
V68, V102,
V181 CDR-H2
165
TISTDGATTWYADSVRG
Clone V71
CDR-H2
166
AISGSGGSTYYADSVKG
Clone V86,
V278, V365
CDR-H2
167
SINDDGSEKYYVDSVKG
Clone V316
CDR-H2
168
KINHSGSTNYNPSLKS
Clone V331
CDR-H2
169
GIIPIFGTANYAQKFQG
Clone V355
CDR-H2
170
NIKQDGSEKYYVDSVKG
Clone V282,
V420 CDR-H2
171
RIKSKTDGGTTDYAAPLKG
Clone V397
CDR-H2
172
EINHSGSTNYNPSLKS
Clone V345
CDR-H2
173
VISYDGSTNYNPSLXaaS
Clone V336
CDR-H2
174
VYGYDYXaaDFGWFDP
Clone V336
CDR-H3
175
QITLKESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGR
Clone 7 VH
IRSKTDGGTTDYAALVKGRFTISRDDSENTLYLQMNSLKTEDTAVYYCARD
FGRWSYYFDYWSQGTLVTVSS
176
QITLKESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGR
Clone 81 VH
IKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARD
FGRWSYYFDYWSQGTLVTVSS
177
RSPXaaRSLGEAXaaVKPGGSLRLSCAASGFTFSNAWMSGSAQAPGKGLEWV
Clone 16 VH
GRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCA
RDFGRWSYYFDYWSQGTLVTVSS
178
QVTLKESGGGLVKPGGSLRLSCAASGFTFQXaaRLDEWVRQAPGKGLEWVG
Clone 65 VH
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
DFGRWSYYFDYWSQGTLVTVSS
179
QVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone V18 VH
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR
DFGRWSYYFDYWGQGTLVTVSS
180
QVTLKESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone 40 VH
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLRTEDTAVYYCAR
DFGRWSYYFDYWSQGTLVTVSS
181
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone 40 VH
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLRTEDTAVYYCAR
DFGRWSYYFDYWSQGTLVTVSS
182
EVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAMSWVRQAPGKGLEWVSS
Clone 350,
ISGSGRSTDHADYVKGRFTISRDNSKNTVYLQMNRLRAEDTAVYYCAKVS
305, 366 VH
NYEYYFDYWAQGTLVTVSS
183
QVTLKESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone 20,43
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
VH
DFGRWSYYFDYWSQGTLVTVSS
184
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone 20,43
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
VH
DFGRWSYYFDYWSQGTLVTVSS
185
QVQLVESGGGLVKPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGF
Clone 461 VH
IRSKAYGGTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAKD
FEVREAHLSYFDYWGQGTLVTVSS
186
EVQLVESGGGLVKPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGF
Clone 461 VH
IRSKAYGGTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAKD
FEVREAHLSYFDYWGQGTLVTVSS
187
QVTLKESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKELEWVG
Clone V9 VH
RIKSKTDGGTTDYAAPVKGRFTISRDDSKKTLYLQMNSLKTEDTAVYYCAR
GSNERFLEWLNFDYWGQGTLVTVSS
188
EVQLLESGGGVVQPGGSLRLSCAASGFTFDTFWMSWVRQAPGKGLEWVG
Clone V128
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
VH
DFGRWSYYFDYWSQGTLVTVSS
189
QITLKESGGGLVKPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFI
Clone V68 VH
RSKAYGGTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAKDF
EVRGAHLSYFDYWGQGTLVTVSS
190
QVQLVESGGGLVQPGGSLRLSCAASGFTFSTSAMSWVRQAPGKGLEWVSTI
Clone V71 VH
STDGATTWYADSVRGRFSVSRDNSKNTLYLQMTGLRAEDTAVYYCARVL
RSGFLEWNLFDYWGQGTLVTVSS
191
QVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA
Clone V86 VH
ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVIF
GVVNIPDYWGQGTLVTVSS
192
QVQLVESGGGLVKPGGSLRLSCAASGFTFINAWMSWVRQAPGKGLEWVG
Clone V224
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
VH
TYSSSWYESLLFDYWGQGTLVTVSS
193
QVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone V163
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
VH
QGDSSSWYVEVYYFDYWGQGTLVTVSS
194
QVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone V95 VH
RIKSKTDGGTTDYAAPVKGRFTISRDDSKKTLYLQMNSLKTEDTAVYYCAR
GSGELRFLESYYFDYWGQGTLVTVSS
195
QVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAMSWVRQAPGKGLEWVG
Clone V102
FIRSKAYGGTTEYAASVKGRFTISRDDSQSIAYLQMDSLKTEDTAVYYCAK
VH
VGPSWDYYFDYWGQGTLVTVSS
196
QITLKESGGGLVKPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFI
Clone V181
RSKAYGGTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAKDF
VH
EVREAHLSYFDYWGQGTLVTVSS
197
EVQLVESGGGLVKPRGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone V312
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
VH
TSRGRFLEWLLFDYWGQGTLVTVSS
198
EVQLVESGGGLVRPGGSLRLSCAASGLTFSRTWMSWVRQAPGKGLEWVAS
Clone V316
INDDGSEKYYVDSVKGRFTISRDNARNSLYLQMNRLRAEDTAVYYCARVD
VH
SERFLEWYYFDYWGQGTLVTVSS
199
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGK
Clone V331
INHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGQIAA
VH
HVWGWFDPWGQGTLVTVSS
200
QITLKESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAI
Clone V278
SGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKERSR
VH
WGDNWFDPWGQGTLVTVSS
201
EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGG
Clone V355
IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARIYPPD
VH
AFDIWGQGTMVTVSS
202
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone V357
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
VH
DMVGAWLVLSYFDYWGQGTLVTVSS
203
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVA
Clone V282
NIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARV
VH
RGSEYYFDYWGQGTLVTVSS
204
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA
Clone V365
ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGIG
VH
YSSSWYEIWTFDYWGQGTLVTVSS
205
QITLKESGGGLVQPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGR
Clone V397
IKSKTDGGTTDYAAPLKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARA
VH
KGLWFGESYYFDYWGQGTLVTVSS
206
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVA
Clone V420
NIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARV
VH
NGGEYYFDYWGQGTLVTVSS
207
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEI
Clone V345
NHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAEDTAVYYCARGPLRPQ
VH
KVLPFQIGAQGTLVTVSS
208
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVA
Clone V336
VISYDGSTNYNPSLXaaSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVYG
VH
YDYRDFGWFDPWGQGTLVTVSS
209
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVA
Clone V336
VISYDGSTNYNPSLXaaSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVYG
VH
YDYXaaDFGWFDPWGQGTLVTVSS
210
SGDKLGDKYAC
Clone 7 CDR-
L1
211
GGNNIGTKSVH
Clone 81
CDR-L1
212
RASQGISSYLA
Clone 16
CDR-L1
213
PRRQPQKTIMQ
Clone 65
CDR-L1
214
GGNNLGSKNVH
Clone V18
CDR-L1
215
TRSSGNIASNFVQ
Clone 40
CDR-L1
216
TRSSGSIASNYVQ
Clone 350
CDR-L1
217
RASQSINKWLA
Clone 20
CDR-L1
218
GGNNIGSTSVH
Clone 305
CDR-L1
219
SGSRFNIGSNTVN
Clone 366
CDR-L1
220
GGNRIGTKAVH
Clone 461
CDR-L1
221
QDSKRPS
Clone 7 CDR-
L2
222
DESDRPS
Clone 81
CDR-L2
223
AASTLQS
Clone 16
CDR-L2
224
GKNDRPS
Clone 65
CDR-L2
225
DDDNRPS
Clone V18
CDR-L2
226
EDTQRPS
Clone 40
CDR-L2
227
EDNQRPS
Clone 350
CDR-L2
228
DASTLES
Clone 20
CDR-L2
229
DDSDRPS
Clone 305
CDR-L2
230
SNNQRPS
Clone 366
CDR-L2
231
DDTDRPS
Clone 461
CDR-L2
232
QVWDSSSRHVV
Clone V18
CDR-L3
233
XaaAWDSSTVV
Clone 7 CDR-
L3
234
SYELTQPPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDS
Clone 7 VL
KRPSGIPERFSGSNSGNTATLTISGTQAMDEADYXaaXaaXaaAWDSSTVVFG
GGTKLTVL
235
SYELTQPPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPVLVIYQDS
Clone 7 VL
KRPSGIPERFSGSNSGNTATLTISGTQAMDEADYXaaXaaQAWDSSTVVFGG
GTKLTVL
236
QSVLTQPPSVSVAPGQTARITCGGNNIGTKSVHWYQQKPGQAPVLVLYDES
Clone 81 VL
DRPSGIPERFSGSXaaSGNTATLTISRVEAGDEADXaaLLSGVGXaaXaaXaaXaaSLC
FGSGTKLTVL
237
QSVLTQPPSVSVAPGQTARITCGGNNIGTKSVHWYQQKPGQAPVLVLYDES
Clone 81 VL
DRPSGIPERFSGSXaaSGNTATLTISRVEAGDEADXaaLLQVWDSSSDHYVFGS
GTKLTVL
238
EIVMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAAS
Clone 16 VL
TLQSGSHQGFSGSGSGTEFTLTISSLQPEDFATYYC
239
QSALTQDPAVSVALGTDSQDHMPRRQPQKTIMQWYQQKPGQAPALVIYGK
Clone 65 VL
NDRPSGIPDRFSGSTSGNTASLTITGAQAEDEADYYXaa
240
VLTQPPSVSVAPGKTARITCGGNNLGSKNVHWYQQKPGQAPVLVIYDDDN
Clone V18 VL
RPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSRHVVFGGGT
KLTVL
241
QPVLTQPHSVSESPGKTVTISCTRSSGNIASNFVQWYQRRPDGATTNVIYED
Clone 40 VL
TQRPSGVSGRFSGSIDRSSNSASLTISGLRAEDEADYYCQSYDGRNLMFGGG
TKVTVL
242
QPVLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTTVIYED
Clone 350 VL
NQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNHVFGTGT
KLTVL
243
NIQMTQSPSSLSASVGDRVTITCRASQSINKWLAWYQQKPGKAPKLLIHDA
Clone 20 VL
STLESGVPSRFSGSGSGTEFTLTISSLHPDDSATYYCQQLKSPPAQFGEGTKL
EIK
244
NIQMTQSPSSLSASVGDRVTITCRASQSINKWLAWYQQKPGKAPKLLIHDA
Clone 20 VL
STLESGVPSRFSGSGSGTEFTLTISSLHPDDSATYYCQQLKSRPLSFGEGTKL
EIK
245
QSVLTQPSSVSGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRN
Clone 43 VL
NQRPSGVPDRFSGSKSGTSASLGHQWAPGPRMRLIITAAWDDSLSGVVFGG
GTKLTVL
246
QSVLTQPPSVSVAPGQTARITCGGNNIGSTSVHWFQQKPGQAPVLVVYDDS
Clone 305 VL
DRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDNDSDHRVFGGG
TKLTVL
247
QSVLTQPPSVPGTPGQRVTITCSGSRFNIGSNTVNWYQQLPGTAPKWYSN
Clone 366 VL
NQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGYVFGT
GTKLTVL
248
QSVLTQPPSVSVAPGQTARITCGGNRIGTKAVHWYQQKSGQAPVLVVRDD
Clone 461 VL
TDRPSGSLRDSLAPTLGTXaaATLTISGVEAGDEADYYQVWDSSSDHRVFGG
GTKLTVL
249
QITLKESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGR
Clone 7 scFv
IRSKTDGGTTDYAALVKGRFTISRDDSENTLYLQMNSLKTEDTAVYYCARD
FGRWSYYFDYWSQGTLVTVSSSYELTQPPSVSVSPGQTASITCSGDKLGDK
YACWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAMDE
ADYXaaXaaXaaAWDSSTVVFGGGTKLTVL
250
QITLKESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGR
Clone 7 scFv
IRSKTDGGTTDYAALVKGRFTISRDDSENTLYLQMNSLKTEDTAVYYCARD
FGRWSYYFDYWSQGTLVTVSSSYELTQPPSVSVSPGQTASITCSGDKLGDK
YACWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAMDE
ADYXaaXaaQAWDSSTVVFGGGTKLTVL
251
QITLKESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGR
Clone 81 scFv
IKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARD
FGRWSYYFDYWSQGTLVTVSSQSVLTQPPSVSVAPGQTARITCGGNNIGTK
SVHWYQQKPGQAPVLVLYDESDRPSGIPERFSGSXaaSGNTATLTISRVEAGD
EADXaaLLSGVGXaaXaaXaaXaaSLCFGSGTKLTVL
252
QITLKESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGR
Clone 81 scFv
IKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARD
FGRWSYYFDYWSQGTLVTVSSQSVLTQPPSVSVAPGQTARITCGGNNIGTK
SVHWYQQKPGQAPVLVLYDESDRPSGIPERFSGSXaaSGNTATLTISRVEAGD
EADXaaLLQVWDSSSDHYVFGSGTKLTVL
253
RSPXaaRSLGEAXaaVKPGGSLRLSCAASGFTFSNAWMSGSAQAPGKGLEWV
Clone 16 scFv
GRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCA
RDFGRWSYYFDYWSQGTLVTVSSEIVMTQSPSFLSASVGDRVTITCRASQGI
SSYLAWYQQKPGKAPKLLIYAASTLQSGSHQGFSGSGSGTEFTLTISSLQPE
DFATYYC
254
QVTLKESGGGLVKPGGSLRLSCAASGFTFQ*RLDEWVRQAPGKGLEWVGR
Clone 65 scFv
IKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCARD
FGRWSYYFDYWSQGTLVTVSSQSALTQDPAVSVALGTDSQDHMPRRQPQK
TIMQWYQQKPGQAPALVIYGKNDRPSGIPDRFSGSTSGNTASLTITGAQAED
EADYYXaa
255
QVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone V18
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR
scFv
DFGRWSYYFDYWGQGTLVTVSSVLTQPPSVSVAPGKTARITCGGNNLGSK
NVHWYQQKPGQAPVLVIYDDDNRPSGIPERFSGSNSGNTATLTISRVEAGD
EADYYCQVWDSSSRHVVFGGGTKLTVL
256
QVTLKESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone 40 scFv
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLRTEDTAVYYCAR
DFGRWSYYFDYWSQGTLVTVSSQPVLTQPHSVSESPGKTVTISCTRSSGNIA
SNFVQWYQRRPDGATTNVIYEDTQRPSGVSGRFSGSIDRSSNSASLTISGLR
AEDEADYYCQSYDGRNLMFGGGTKVTVL
257
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone 40 scFv
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLRTEDTAVYYCAR
DFGRWSYYFDYWSQGTLVTVSSQPVLTQPHSVSESPGKTVTISCTRSSGNIA
SNFVQWYQRRPDGATTNVIYEDTQRPSGVSGRFSGSIDRSSNSASLTISGLR
AEDEADYYCQSYDGRNLMFGGGTKVTVL
258
EVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAMSWVRQAPGKGLEWVSS
Clone 350
ISGSGRSTDHADYVKGRFTISRDNSKNTVYLQMNRLRAEDTAVYYCAKVS
scFv
NYEYYFDYWAQGTLVTVSSQPVLTQPHSVSESPGKTVTISCTRSSGSIASNY
VQWYQQRPGSAPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDE
ADYYCQSYDSSNHVFGTGTKLTVL
259
QVTLKESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone 20 scFv
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
DFGRWSYYFDYWSQGTLVTVSSNIQMTQSPSSLSASVGDRVTITCRASQSIN
KWLAWYQQKPGKAPKLLIHDASTLESGVPSRFSGSGSGTEFTLTISSLHPDD
SATYYCQQLKSPPAQFGEGTKLEIK
260
QVTLKESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone 20 scFv
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
DFGRWSYYFDYWSQGTLVTVSSNIQMTQSPSSLSASVGDRVTITCRASQSIN
KWLAWYQQKPGKAPKLLIHDASTLESGVPSRFSGSGSGTEFTLTISSLHPDD
SATYYCQQLKSRPLSFGEGTKLEIK
261
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone 20 scFv
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
DFGRWSYYFDYWSQGTLVTVSSNIQMTQSPSSLSASVGDRVTITCRASQSIN
KWLAWYQQKPGKAPKLLIHDASTLESGVPSRFSGSGSGTEFTLTISSLHPDD
SATYYCQQLKSPPAQFGEGTKLEIK
262
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone 20 scFv
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
DFGRWSYYFDYWSQGTLVTVSSNIQMTQSPSSLSASVGDRVTITCRASQSIN
KWLAWYQQKPGKAPKLLIHDASTLESGVPSRFSGSGSGTEFTLTISSLHPDD
SATYYCQQLKSRPLSFGEGTKLEIK
263
QVTLKESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone 43 scFv
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
DFGRWSYYFDYWSQGTLVTVSSQSVLTQPSSVSGTPGQRVTISCSGSSSNIG
SNYVYWYQQLPGTAPKWYRNNQRPSGVPDRFSGSKSGTSASLGHQWAP
GPRMRLIITAAWDDSLSGVVFGGGTKLTVL
264
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVG
Clone 43 scFv
RIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAR
DFGRWSYYFDYWSQGTLVTVSSQSVLTQPSSVSGTPGQRVTISCSGSSSNIG
SNYVYWYQQLPGTAPKWYRNNQRPSGVPDRFSGSKSGTSASLGHQWAP
GPRMRLIITAAWDDSLSGVVFGGGTKLTVL
265
EVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAMSWVRQAPGKGLEWVSS
Clone 305
ISGSGRSTDHADYVKGRFTISRDNSKNTVYLQMNRLRAEDTAVYYCAKVS
scFv
NYEYYFDYWAQGTLVTVSSQSVLTQPPSVSVAPGQTARITCGGNNIGSTSV
HWFQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEA
DYYCQVWDNDSDHRVFGGGTKLTVL
266
EVQLVESGGGLVQPGRSLRLSCTASGFTFGDYAMSWVRQAPGKGLEWVSS
Clone 366
ISGSGRSTDHADYVKGRFTISRDNSKNTVYLQMNRLRAEDTAVYYCAKVS
scFv
NYEYYFDYWAQGTLVTVSSQSVLTQPPSVPGTPGQRVTITCSGSRFNIGSNT
VNWYQQLPGTAPKWYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEA
DYYCAAWDDSLNGYVFGTGTKLTVL
267
QVQLVESGGGLVKPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGF
Clone 461
IRSKAYGGTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAKD
scFv
FEVREAHLSYFDYWGQGTLVTVSSQSVLTQPPSVSVAPGQTARITCGGNRI
GTKAVHWYQQKSGQAPVLVVRDDTDRPSGSLRDSLAPTLGTXaaATLTISGV
EAGDEADYYQVWDSSSDHRVFGGGTKLTVL
268
EVQLVESGGGLVKPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGF
Clone 461
IRSKAYGGTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAKD
scFv
FEVREAHLSYFDYWGQGTLVTVSSQSVLTQPPSVSVAPGQTARITCGGNRI
GTKAVHWYQQKSGQAPVLVVRDDTDRPSGSLRDSLAPTLGTXaaATLTISGV
EAGDEADYYQVWDSSSDHRVFGGGTKLTVL
269
GYTFTSY
Clone 83, 83B
CDR-H1
270
GFTFGDY
Clone 298,
V70, 350, 305,
366, 461, V68,
V102, V181
CDR-H1
271
GFTFSNA
Clone 48,
V353, 7, 81,
16, V18, 40,
20, 43, V9,
V163, V95,
V312, V357,
V397
CDR-H1
272
GFTFSTS
Clone V71
CDR-H1
273
GFTFSSY
Clone V86,
V278, V282,
V365, V420,
V336 CDR-H1
274
GFTFQXaaR
Clone 65
CDR-H1
275
GFTFDTF
Clone V128
CDR-H1
276
GFTFINA
Clone V224
CDR-H1
277
GLTFSRT
Clone V316
CDR-H1
278
GGSFSGY
Clone V331,
V345 CDR-H1
279
GGTFSSY
Clone V355
CDR-H1
280
GFTFQXaaRLDE
Clone 65
CDR-H1
281
GFTFSTSAMS
Clone V71
CDR-H1
282
GFTFSSYAMS
Clone 86,
V278, V365
CDR-H1
283
GFTFDTFWMS
Clone V128
CDR-H1
284
GFTFINAWMS
Clone V224
CDR-H1
285
GLTFSRTWMS
Clone V316
CDR-H1
286
GGSFSGYYWS
Clone V331
CDR-H1
287
GGTFSSYAIS
Clone V355
CDR-H1
288
GFTFSSYWMS
Clone V282,
V420 CDR-H1
289
GGSFSGYYWS
Clone V345
CDR-H1
290
GFTFSSYAMH
Clone V336
CDR-H1
291
KSKTDGGT
Clone V353,
V48, V81,
V16, V65,
V18, V40,
V20, V43, 9,
128, V224,
V163, V95,
V312, V357,
V397 CDR-H2
292
SGSGRS
Clone V298,
V70, V350,
V305, V366
CDR-H2
293
NPSGGS
Clone V83
CDR-H2
294
NPNGGS
Clone V83B
CDR-H2
295
RSKTDGGT
Clone 7
CDR-H2
296
RSKAYGGT
Clone V68,
V102, 461,
181 CDR-H2
297
STDGAT
Clone V71
CDR-H2
298
SGSGGS
Clone V86,
V278, V365
CDR-H2
299
NDDGSE
Clone V316
CDR-H2
300
NHSGS
Clone V331,
V345 CDR-H2
301
IPIFGT
Clone V355
CDR-H2
302
KQDGSE
Clone V282,
V420 CDR-H2
303
SYDGS
Clone V336
CDR-H2
304
RIKSKTDGGTTD
Clone V353,
V48, V81,
V16, V65,
V18, V40,
V20, V43, 9,
128, V224,
V163, V95,
V312, V357,
V397 CDR-H2
305
SISGSGRSTD
Clone 298,
V70, V350,
V305, V366
CDR-H2
306
IINPSGGSTS
Clone V83
CDR-H2
307
IINPNGGSTS
Clone V83B
CDR-H2
308
RIRSKTDGGTTD
Clone 7 CDR-
H2
309
FIRSKAYGGTTE
Clone V68,
V102, V181,
461 CDR-H2
310
TISTDGATTW
Clone V71
CDR-H2
311
AISGSGGSTY
Clone V86,
V278, V365
CDR-H2
312
SINDDGSEKY
Clone V316
CDR-H2
313
KINHSGSTN
Clone V331
CDR-H2
314
GIIPIFGTAN
Clone V355
CDR-H2
315
NIKQDGSEKY
Clone V282,
V420 CDR-H2
316
EINHSGSTN
Clone V345
CDR-H2
317
VISYDGSTN
Clone V336
CDR-H2
318
RIRSKTDGGTTDYAALVKG
Clone 7 CDR-
H2
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15997635
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juno therapeutics, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Mar 30th, 2022 06:04PM
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Mar 30th, 2022 06:04PM
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Bristol-Myers Squibb
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:bmy
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Juno Therapeutics
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Jul 30th, 2019 12:00AM
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Jan 12th, 2016 12:00AM
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https://www.uspto.gov?id=US10363269-20190730
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Modified hepatitis post-transcriptional regulatory elements
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Provided are polynucleotides containing a modified PRE having a variant X gene that includes one or more stop codons not present in an unmodified, such as wild-type, hepatitis X gene. Also provided are polynucleotides containing a modified PRE having a variant X gene that includes one or more degradation sequences not present in an unmodified, such as wild-type, hepatitis X gene. The modified PRE can be operably linked to a nucleic acid encoding a recombinant protein. Also provided are expression cassettes, viral vectors and cells containing the polynucleotides, and compositions and methods of use thereof.
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10363269
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1. A polynucleotide, comprising a modified post-transcriptional regulatory element (PRE), said modified PRE comprising a variant of a wild-type hepatitis virus X gene, said variant X gene comprising a plurality of stop codons not present in the wild-type X gene, wherein the variant X gene comprises a stop codon in each reading frame present in said variant X gene.
2. The polynucleotide of claim 1, further comprising a nucleic acid encoding a recombinant protein operably linked to the modified PRE.
3. A vector comprising the polynucleotide of claim 2.
4. A cell comprising the polynucleotide of claim 2.
5. A polynucleotide, comprising a modified post-transcriptional regulatory element (PRE), said modified PRE comprising a variant of a wild-type hepatitis virus X gene, said variant X gene comprising a plurality of stop codons not present in the wild-type X gene; and,
a nucleic acid encoding a recombinant protein that is a recombinant receptor operably linked to the modified PRE.
6. The polynucleotide of claim 5, wherein the stop codon comprises at least one stop codon selected from among:
a stop codon beginning at a position within or within at least 9, 12, 15, or 18 nucleotides in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the X protein open reading frame; and/or
a stop codon beginning at a position within or within at least 9, 12, 15, or 18 nucleotides in the 3′ direction from a position in the variant X gene corresponding to residue 411 of WHV post-transcriptional regulatory element (WPRE) sequence set forth in SEQ ID NO: 1 and/or residue 1503 of the WHV sequence set forth as SEQ ID NO: 2.
7. The polynucleotide of claim 5, wherein the variant X gene further comprises a sequence encoding a post-translational modification signal not present in the wild-type hepatitis virus X gene.
8. The polynucleotide of claim 5, wherein said variant X gene is a variant of a wild-type mammalian hepatitis virus X gene.
9. The polynucleotide of claim 8, wherein said wild-type mammalian hepatitis virus X gene is a wild-type woodchuck hepatitis virus (WHV) X gene.
10. The polynucleotide of claim 5, wherein the modified PRE comprises nucleotide modifications compared to a wild-type or unmodified hepatitis virus PRE that is a mammalian hepatitis PRE.
11. The polynucleotide of claim 10, wherein said wild-type or unmodified mammalian hepatitis PRE is a wild-type woodchuck hepatitis virus PRE (WPRE).
12. The polynucleotide of claim 10, wherein the wild-type or unmodified hepatitis virus PRE comprises:
a) the sequence of nucleotides set forth in SEQ ID NO:1 or 125 or a sequence of nucleotides that exhibits at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1 or 125; or
b) a portion of the sequence of nucleotides of a), wherein the portion exhibits post-transcriptional activity.
13. The polynucleotide of claim 5, wherein the modified PRE is selected from among:
a) a modified PRE comprising a sequence of nucleotides that exhibits at least 95% sequence identity to SEQ ID NO:1 or 125, said modified PRE containing a variant X gene comprising at least one stop codon not present in SEQ ID NOS:1 or 125; and
b) a modified PRE comprising a portion of the sequence of nucleotides of a), said portion comprising a variant X gene comprising the plurality of stop codons, wherein the portion exhibits post-transcriptional activity.
14. The polynucleotide of claim 5, wherein the variant X gene comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 44-54 or 141-151 and/or the modified PRE comprises the sequence of nucleotides set forth in any of SEQ ID NOS:29-39 or 126-136.
15. The polynucleotide of claim 5, wherein said variant X gene further comprises a variant of a start codon comprising one or more nucleotide differences compared to a wild-type hepatitis virus X gene start codon and/or compared to the start codon corresponding to nucleotide positions 411-413 of SEQ ID NO: 1, wherein the one or more nucleotide differences results in restricted or prevented translation initiation from said start codon.
16. The polynucleotide of claim 5, wherein said variant X gene comprises a variant promoter operably linked to said variant X gene, said variant promoter comprising one or more nucleotide differences compared to a wild-type hepatitis virus X gene promoter and/or compared to a promoter of SEQ ID NO: 11, wherein said one or more differences results in restricted or prevention of transcription from said promoter.
17. The polynucleotide of claim 5, wherein:
upon introduction into a eukaryotic cell, no polypeptide of a length greater than 12, 11, 10, 9, or 8 amino acids in length encoded by said variant X gene is produced; and/or
said polynucleotide is incapable of producing a polypeptide of a length greater than 12, 11, 10, 9, or 8 amino acids in length encoded by said variant X gene.
18. The polynucleotide of claim 5, wherein the variant X gene comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotide changes.
19. The polynucleotide of claim 5, wherein said modified PRE encodes an RNA that promotes nuclear RNA export and/or increases mRNA stability.
20. The polynucleotide of claim 5, further comprising a viral nucleic acid comprising a variant Flap, wherein the variant Flap contains a deletion of all or a portion of the nucleotides corresponding to the central polypurine tract (cPPT) and/or the central termination sequence (CTS) regions of a wild-type or unmodified Flap sequence.
21. The polynucleotide of claim 20, wherein the variant Flap comprises deletion of all or a contiguous portion of nucleotides corresponding to nucleotides in the cPPT region set forth in SEQ ID NO:123 and comprises deletion of all or a contiguous portion of nucleotides corresponding to nucleotides in the CTS region set forth in SEQ ID NO:124.
22. The polynucleotide of claim 20, comprising:
a variant Flap comprising the sequence of SEQ ID NO: 122 or a sequence having at least at or about 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:122; and
a modified PRE comprising the sequence of SEQ ID NO:29 or 126 or a sequence having at least at or about 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:29 or 126.
23. The polynucleotide of claim 5, wherein the recombinant receptor is an antigen receptor and/or a chimeric receptor.
24. An expression cassette comprising the polynucleotide of claim 5 and a promoter operably linked to the nucleic acid encoding the recombinant protein.
25. The polynucleotide of claim 5, wherein said stop codon is selected from among:
a stop codon beginning at a nucleotide position corresponding to position 420 in the sequence set forth in SEQ ID NO:1;
a stop codon beginning at a nucleotide position corresponding to position 424 in the sequence set forth in SEQ ID NO:1;
a stop codon beginning at a nucleotide position corresponding to position 428 in the sequence set forth in SEQ ID NO:1; and
a stop codon beginning at a nucleotide position corresponding to position 432 in the sequence set forth in SEQ ID NO:1.
26. The polynucleotide of claim 25, wherein said stop codon is an amber (TAG) or opal (TGA) stop codon.
27. A vector comprising the polynucleotide of claim 5.
28. The vector of claim 27, which is a viral vector.
29. A cell comprising the polynucleotide of claim 5.
30. A virus particle, comprising the vector of claim 27.
31. A pharmaceutical composition comprising the cell of claim 29 and a pharmaceutically effective carrier.
32. A method, comprising introducing the vector of claim 27 to a cell, under conditions whereby expression of the recombinant protein is effected in the cell.
33. A polynucleotide, comprising a modified post-transcriptional regulatory element (PRE), said modified PRE comprising a variant of a wild-type hepatitis virus X gene, said variant X gene comprising a plurality of stop codons not present in the wild-type X gene, wherein said stop codon is selected from among:
a stop codon beginning at a nucleotide position corresponding to position 420 in the sequence set forth in SEQ ID NO:1;
a stop codon beginning at a nucleotide position corresponding to position 424 in the sequence set forth in SEQ ID NO:1;
a stop codon beginning at a nucleotide position corresponding to position 428 in the sequence set forth in SEQ ID NO:1; and
a stop codon beginning at a nucleotide position corresponding to position 432 in the sequence set forth in SEQ ID NO:1.
34. The polynucleotide of claim 33, wherein said stop codon is an amber (TAG) or opal (TGA) stop codon.
35. The polynucleotide of claim 33, further comprising a nucleic acid encoding a recombinant protein operably linked to the modified PRE.
36. A vector comprising the polynucleotide of claim 35.
37. A cell comprising the polynucleotide of claim 35.
38. A pharmaceutical composition comprising the cell of claim 37 and a pharmaceutically effective carrier.
39. A polynucleotide, comprising a modified post-transcriptional regulatory element (PRE), said modified PRE comprising a variant of a wild-type hepatitis virus X gene, said variant X gene comprising a sequence encoding a post-translational modification signal not present in the wild-type hepatitis virus X gene.
40. The polynucleotide of claim 39, further comprising a nucleic acid encoding a recombinant protein operably linked to the modified PRE.
41. A vector comprising the polynucleotide of claim 40.
42. A cell comprising the polynucleotide of claim 40.
43. A pharmaceutical composition comprising the cell of claim 42 and a pharmaceutically effective carrier.
44. A polynucleotide, comprising a viral nucleic acid comprising a variant Flap, wherein the variant Flap contains a deletion of all or a portion of the nucleotides corresponding to the central polypurine tract (cPPT) and/or the central termination sequence (CTS) regions of a wild-type or unmodified Flap sequence.
45. The polynucleotide of claim 44, further comprising a nucleic acid encoding a recombinant protein operably linked to the variant Flap.
46. A cell comprising the polynucleotide of claim 45.
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46
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S. provisional application No. 62/102,569 filed Jan. 12, 2015, entitled “Modified Hepatitis Post-transcriptional Regulatory Elements,” the contents of which is incorporate by reference in its entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042001800SeqList.txt, created on Jan. 12, 2016, which is 152,051 bytes in size. The information in electronic format of the Sequence Listing is incorporated by reference in its entirety.
FIELD
The present disclosure relates to methods for expressing recombinant molecules and polynucleotides for use in such methods. In particular, the disclosure relates to polynucleotides containing post-transcriptional regulatory elements (PREs) that can enhance expression of recombinant molecules operably linked thereto, such as cis-acting post-transcriptional regulatory elements and modified variants thereof. The modified post-transcriptional regulatory elements include those derived from hepatitis viruses, such as the woodchuck hepatitis virus (WHV), including modified variants of a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The modified PREs typically include one or more modifications that reduce the potential for oncogenesis and/or immunogenicity upon introduction into a subject for expression of the recombinant molecule, for example, by preventing expression of certain proteins. In some embodiments, such modifications include one or more stop codon included within a sequence within the PRE encoding a viral X protein or portion thereof. In some embodiments, the polynucleotide includes one or more sequences encoding a site to promote post-translational modification, such as degradation, e.g., a ubiquitination site.
BACKGROUND
Viral vectors can provide an effective delivery system for transferring genetic material into a cell. Nucleic acids within many viral vectors, such as gamma-retroviral and lentiviral vectors, can integrate into the genome of a host cell. Cis-acting elements, such as viral post-transcriptional regulatory elements (PREs), including hepatitis virus PREs and variants thereof, have been used in viral vectors to enhance gene expression. Nucleic acids, expression cassettes, and vectors containing such elements are available. There is a need for improved PRE-containing nucleic acids, cassettes and vectors. Provided are embodiments that address such needs.
SUMMARY
Provided are polynucleotides containing a modified post-transcriptional regulatory element (PRE) that contains a variant X gene that includes a stop codon and/or post-translational degradation sequence that is not present in an unmodified or wild-type hepatitis virus X gene, such as not present in an X gene from a wild-type mammalian hepatitis virus, such as a WHV or HBV. Among the provided polynucleotides are those that contain a modified WPRE containing a variant X gene that includes a stop codon and/or post-translational degradation sequence that is not present in an unmodified or wild-type WHV X gene. The provided polynucleotides include expression cassettes and viral vectors containing such modified PREs. In particular embodiments, the modified PRE is operably linked to a nucleic acid encoding a recombinant protein, e.g. a heterologous protein.
Provided are polynucleotides containing a modified PRE that includes a variant of a wild-type hepatitis virus X gene in which the variant X gene contains a stop codon, such as at least one stop codon, not present in the wild-type X gene. In some embodiments, at least one stop codon, e.g., the first stop codon or each of a plurality of stop codons present in the variant X gene, begins at a position within 36 or 24 nucleotides in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the wild-type X protein open reading frame. In some embodiments, at least one stop codon, e.g., the first stop codon or one or more of a plurality of stop codons present in the variant X gene, begins at a position within 36 or 24 nucleotides in the 3′ direction from a position corresponding to residue 411 of WHV PRE sequence set forth in SEQ ID NO:1 or SEQ ID NO:125 and/or residue 1503 of the sequence set forth in SEQ ID NO:2. In some such embodiments, the variant X gene does not contain an open reading frame of greater than at or about 39 nucleotides in length or greater than at or about 27 nucleotides in length.
Provided are polynucleotides containing a modified PRE that contains a variant of a wild-type hepatitis virus X gene in which the variant X gene contains a stop codon, such as at least one stop codon, not present in the wild-type X gene and beginning at a position within 32 or 30 nucleotides of a start codon of the X gene. In some embodiments, the at least one stop codon, e.g. the first stop codon or one or more of a plurality of stop codons present in the variant X gene, begins at a position within 32 or 30 nucleotides in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the wild-type X protein open reading frame. In some embodiments, the at least one stop codon, e.g. the first stop codon or one or more of a plurality of stop codons present in the variant X gene, begins at a position within 32 or 30 nucleotides in the 3′ direction from a position corresponding to residue 411 of WHV PRE sequence set forth in SEQ ID NO:1 or SEQ ID NO:125 and/or residue 1503 of the sequence set forth in SEQ ID NO:2. In some such embodiments, the variant X gene has an open reading frame of less than or equal to 33 nucleotides in length.
In any of such embodiments of a modified PRE, the at least one stop codon not present in the wild-type X gene includes a plurality of stop codons. Provided are polynucleotides comprising a modified PRE that contains a variant of a wild-type hepatitis virus X gene in which the variant X gene contains a plurality of stop codons not present in the wild-type X gene. In any of such embodiments, the modified PRE contains 2, 3, 4, 5 or 6 stop codons. In some embodiments, the plurality of stop codons comprises at least one stop codon in each reading frame present in the variant X gene. In some embodiments, the plurality of stop codons comprises at least two stop codons in the same reading frame.
In any of such embodiments of a modified PRE containing a plurality of stop codons, the first stop codon among the plurality or one or more of a plurality of stop codons begins at a position within 36, 30 or 24 nucleotides in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the wild-type X protein open reading frame. In some embodiments of a modified PRE containing a plurality of stop codons, the first stop codon among the plurality or one or more of a plurality of stop codons begins at a position within 36, 30 or 24 nucleotides in the 3′ direction from a position in the variant X gene corresponding to residue 411 of WHV post-transcriptional regulatory element (WPRE) sequence set forth in SEQ ID NO: 1 or SEQ ID NO:125 and/or residue 1503 of the WHV sequence set forth as SEQ ID NO: 2. In some such embodiments, the variant X gene does not comprise an open reading frame of greater than at or about 39 nucleotides in length, greater than at or about 30 nucleotides in length or greater than at or about 27 nucleotides in length.
In any of such embodiments, the provided polynucleotides also can include a nucleic acid encoding a recombinant protein. In particular embodiments, the modified PRE is operably linked to the nucleic acid encoding the recombinant protein.
In some embodiments of any of the provided polynucleotides, the at least one stop codon, e.g., the first stop codon or one or more of a plurality of stop codons present in the variant X gene, begins at a position within or within at least or at 9, 12, 15, 18 or 21 nucleotides in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the wild-type X protein open reading frame. In some embodiments of any of the provided polynucleotides, the at least one stop codon, e.g., the first stop codon or one or more of a plurality of stop codons present in the variant X gene, begins at a position within or within at least or at 9, 12, 15, 18, 21 nucleotides in the 3′ direction from a position in the variant X gene corresponding to residue 411 of WHV post-transcriptional regulatory element (WPRE) sequence set forth in SEQ ID NO: 1 or SEQ ID NO:125 and/or residue 1503 of the WHV sequence set forth as SEQ ID NO: 2. In some embodiments, the modified PRE contains at least one stop codon, such as 1, 2, 3, 4 or 5 stop codons, in which one or more stop codons of the plurality begins at a position within or within at least or at a nucleotide position corresponding to position 420, 423, 426, 429 or 432 of the sequence set forth in SEQ ID NO:1 or 125. In some such embodiments, the variant X gene does not comprise an open reading frame of greater than or at or about 24 nucleotides in length, greater than at or about 21 nucleotides in length, greater than at or about 18 nucleotides in length, greater than at or about 15 nucleotides in length, or greater than at or about 12 nucleotides in length.
In some embodiments of any of the provided polynucleotides, the at least one stop codon, e.g., the first stop codon or one or more of a plurality of stop codons present in the variant X gene, begins at a position within or within at least or at 9, 13, 17 or 21 nucleotides in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the wild-type X protein open reading frame. In some embodiments of any of the provided polynucleotides, the at least one stop codon, e.g., the first stop codon or one or more of a plurality of stop codons present in the variant X gene, begins at a position within or within at least or at 9, 13, 17 or 21 nucleotides in the 3′ direction from a position in the variant X gene corresponding to residue 411 of WHV post-transcriptional regulatory element (WPRE) sequence set forth in SEQ ID NO: 1 or SEQ ID NO:125 and/or residue 1503 of the WHV sequence set forth as SEQ ID NO: 2. In some embodiments, the modified PRE contains at least one stop codon, such as 1, 2, 3 or 4 stop codons, in which one or more of a plurality of stop codons begins at a position within or within at least or at a nucleotide position corresponding to position 420, 424, 428 or 432 of the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:125. In some such embodiments, the variant X gene does not comprise an open reading frame of greater than at or about 24 nucleotides in length, greater than at or about 21 nucleotides in length, greater than at or about 16 nucleotides in length, or greater than at or about 12 nucleotides in length.
In some embodiments, the unmodified or wild-type X gene comprises the sequence of nucleotides set forth in SEQ ID NO: 221, and the variant X gene contains at least one stop codon, such as 1, 2, 3, 4 or 5 stop codons introduced therein. In some embodiments, a plurality of stop codons are introduced, where at least one stop codon is introduced in each reading frame therein. In some embodiments, a plurality of stop codons is introduced where at least two stop codons are introduced in the same reading frame present therein. In some embodiments, the modified PRE contains a variant X gene that contains at least one stop codon, such as 1, 2, 3 or 4 stop codons, in which one or more begins at a position within or within at least or at position 9, 13, 17 or 21 in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of the ATG start codon set forth in SEQ ID NO:221. In some embodiments, the modified PRE contains a variant X gene that comprises the sequence of nucleotides set forth in SEQ ID NO:28.
In some embodiments of any of the provided polynucleotides, the modified PRE contains a beta stem loop corresponding to nucleotide residues 448-470 of SEQ ID NO:1 or SEQ ID NO:125. In some embodiments of any of the provided polynucleotides, the modified PRE does not contain a nucleotide change in a position within the beta stem loop corresponding to nucleotides 448-470 of SEQ ID NO:1 or SEQ ID NO:125. In some embodiments of any of the provided polynucleotides, the stop codon or stop codons does not comprise a nucleotide in a position within the beta stem loop corresponding to one or more of nucleotides positions 448-470 of SEQ ID NO:1 or SEQ ID NO:125.
In any of such embodiments, the modified PRE contains a stop codon selected from among: a stop codon beginning at position 9 in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the wild-type X protein open reading frame and/or at a nucleotide position corresponding to position 420 in the sequence set forth in SEQ ID NO:1 or SEQ ID NO:125; a stop codon beginning at position 13 in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the wild-type X protein open reading frame and/or at a nucleotide position corresponding to position 424 in the sequence set forth in SEQ ID NO:1 or SEQ ID NO:125; a stop codon beginning at position 17 in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the wild-type X protein open reading frame and/or at a nucleotide position corresponding to position 428 in the sequence set forth in SEQ ID NO:1 or SEQ ID NO:125; and/or a stop codon beginning at position 21 in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the wild-type X protein open reading frame and/or at a nucleotide position corresponding to position 432 in the sequence set forth in SEQ ID NO:1 or SEQ ID NO:125.
In any of such embodiments, the stop codon can be an amber (TAG), ochre (TAA) or opal (TGA) stop codon. In some embodiments, the stop codon can be an amber (TAG) or opal (TGA) stop codon. In any of such embodiments, the stop codon, such as each of a plurality of stop codons, is introduced by nucleotide substitution, deletion or insertion.
In some embodiments of any of such polynucleotides, the variant X gene in the modified PRE is no more than 180 nucleotides in length or is no more than at or about 210 nucleotides in length. In some embodiments of any of such polynucleotides, the variant X gene is at least at or about 90 nucleotides in length or at least about 120 nucleotides in length or is at least at or about 180 nucleotides in length.
In some embodiments of any of such polynucleotides, the variant X gene can be a variant of a wild-type or unmodified mammalian hepatitis X gene or a partial or truncated portion thereof. In some embodiments, the wild-type or unmodified X gene can include a partial X gene encoding a truncated X protein, such as is present in a wild-type or unmodified PRE. In some embodiments, the wild-type or unmodified mammalian hepatitis X gene or partial X gene is or is derived from a wild-type woodchuck hepatitis virus (WHV) X gene. In some embodiments, the wild-type or unmodified WHV X gene contains the nucleotide sequence of SEQ ID NO:9 or is a partial X gene thereof. In some embodiments, the wild-type or unmodified WHV X gene contains the sequence of nucleotides set forth as nucleotides 411-592 of any of SEQ ID NOS: 1 and 12-20 or nucleotides 411-589 of SEQ ID NO:125.
In some embodiments of any of such polynucleotides, the modified PRE can contain at least two or at least three cis-acting post-transcriptional regulatory subelements of a wild-type hepatitis virus PRE or functional variant(s) thereof. In some such embodiments, the at least two or at least three subelements comprise a wild-type PRE alpha subelement or functional variant thereof, a functional variant of a wild-type PRE beta subelement, and/or a wild-type PRE gamma subelement or functional variant thereof. In some embodiments, the modified PRE comprises an alpha subelement of a wild-type hepatitis virus PRE or functional variant thereof and a functional variant of a wild-type PRE beta subelement. In any of such embodiments, the alpha subelement can comprise the sequence of SEQ ID NO: 3, SEQ ID NO:4 or SEQ ID NO:5 or a variant thereof, the beta subelement can comprise a variant of the sequence of SEQ ID NO: 6 or SEQ ID NO:7, and/or the gamma subunit can comprise the sequence of SEQ ID NO: 8 or variant thereof.
In some embodiments of any of such polynucleotides, the wild-type or unmodified hepatitis virus PRE can be a wild-type mammalian hepatitis PRE. In some embodiments, the wild-type or unmodified mammalian hepatitis virus PRE contains the sequence of nucleotides set forth in any of SEQ ID NOS: 1 and 12-27 or is a functional portion thereof that exhibits post-transcriptional activity. In some cases, one or more N- or C-terminal amino acid residues can be deleted or removed without substantial effect on the activity of the PRE. In some embodiments, the wild-type or unmodified hepatitis virus PRE has the sequence set forth in SEQ ID NO:125. In some embodiments, the wild-type mammalian hepatitis PRE is a wild-type woodchuck hepatitis virus PRE (WPRE).
In any of such embodiments of the provided polynucleotides, the modified PRE can be modified with reference to a wild-type or unmodified hepatitis PRE that comprises the sequence of nucleotides set forth in SEQ ID NO:1 or SEQ ID NO:125, a sequence of nucleotides that exhibits at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:125, or a functionally active portion thereof that exhibits post-transcriptional activity, so long as the variant X gene contains at least one stop codon not present in the corresponding wild-type hepatitis PRE.
In any of such embodiments of the provided polynucleotides, the wild-type or unmodified hepatitis PRE can contain the sequence of nucleotides set forth in any of SEQ ID NOS: 1, 12-20 or 125. In some embodiments, the wild-type or unmodified hepatitis PRE can contain the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the wild-type or unmodified hepatitis PRE can contain the nucleotide sequence of SEQ ID NO: 125.
In some embodiments of any of the polynucleotides, the variant X gene can contain a start codon beginning at a position corresponding to position 411 of SEQ ID NO: 1 or SEQ ID NO:125. In some embodiments, the start codon is an ATG start codon.
In some embodiments of any of the polynucleotides, the variant X gene can further contain a promoter operably linked to the variant X gene. In some embodiments, the promoter is a wild-type X-gene promoter comprising the sequence set forth in SEQ ID NO: 11 or a wild-type WHV X gene promoter sequence.
In some embodiments of any of the polynucleotides, the modified PRE can be selected from: a) a modified PRE comprising a sequence of nucleotides that exhibits at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:125, in which the modified PRE contains a variant X gene having at least one stop codon not present in SEQ ID NO:1 or SEQ ID NO:125; or b) a modified PRE comprising a portion of the sequence of nucleotides of a) in which the portion contains a variant X gene comprising the at least one stop codon and the portion exhibits post-transcriptional activity. In some embodiments, the variant X gene contains at least 2 stop codons, at least 3 stop codons or at least 4 stop codons. In some embodiments, the variant X gene comprises a stop codon in each reading frame present in said variant X gene.
In some embodiments, the variant X gene comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 44-58 or 141-155. In some embodiments, the modified PRE comprises the sequence of nucleotides set forth in any of SEQ ID NOS:29-43 or 126-140.
In some embodiments of any of the provided polynucleotides, the modified PRE does not contain any modifications in addition to the at least one stop codon not present in the wild-type or unmodified PRE, such as not present in SEQ ID NO:1 or SEQ ID NO:125. In some embodiments of any of the provided polynucleotides, the modified PRE contains additional modifications in addition to the at least one stop codon as compared to the sequence of the wild-type or unmodified PRE, such as compared to SEQ ID NO:1 or SEQ ID NO:125.
In some embodiments of any of the provided polynucleotides, the variant X gene further contains a variant start codon comprising one or more nucleotide differences compared to a wild-type hepatitis virus X gene start codon. In some embodiments, the variant start codon contains one or more nucleotide differences compared to the start codon corresponding to nucleotide positions 411-413 of SEQ ID NO: 1 or SEQ ID NO:125. In some such embodiments, the one or more differences results in restricted or prevented translation initiation from said start codon. In some such embodiments, the variant X gene comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 74-88 or 171-185 and/or the modified PRE comprises the sequence of nucleotides set forth in any of SEQ ID NOS:59-73 or 156-170.
In some embodiments of any of the provided polynucleotides, the variant X gene contains a variant promoter operably linked to the variant X gene in which the variant promoter comprises one or more nucleotide differences compared to a wild-type hepatitis virus X gene promoter. In some embodiments, the variant promoter contains one or more nucleotide differences compared to the promoter set forth as SEQ ID NO: 11. In some embodiments, the one or more differences results in restricted or prevention of transcription from said promoter. In some such embodiments, the modified PRE comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 89-118 or 186-215.
In some embodiments of any of the provided polynucleotides, upon introduction of the polynucleotide into a eukaryotic cell, no polypeptide of a length greater than 12, 11, 10, 9, or 8 amino acids in length encoded by said variant X gene is produced. In some embodiments of any of the provided polynucleotides, the polynucleotide is incapable of producing a polypeptide of a length greater than 12, 11, 10, 9, or 8 amino acids in length encoded by said variant X gene.
In some embodiments of any of the provided polynucleotides, the modified PRE encodes an RNA that promotes nuclear RNA export and/or increases mRNA stability. In some embodiments of any of the provided polynucleotides, the modified PRE encodes an RNA polynucleotide that promotes nuclear RNA export and/or increases mRNA stability, wherein said promotion of nuclear RNA export and/or mRNA stability increases expression of the recombinant protein. In some embodiments of any of the provided polynucleotides, the modified PRE retains the post-transcriptional activity of the corresponding wild-type or unmodified hepatitis PRE and/or the PRE set forth in SEQ ID NO:1, SEQ ID NO:125, SEQ ID NO:119, SEQ ID NO:120 and/or SEQ ID NO:216. In some such embodiments, the modified PRE exhibits at least 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120% or 130% or more of the post-transcriptional activity of the corresponding wild-type or unmodified hepatitis PRE and/or the PRE set forth in SEQ ID NO:1, SEQ ID NO:125, SEQ ID NO:119, SEQ ID NO:120 and/or SEQ ID NO:216. In some embodiments, the post-transcriptional activity of the modified PRE can be assessed by monitoring gene expression of an operably linked nucleic acid encoding a recombinant protein.
In some embodiments of any of the provided polynucleotides, the variant X gene further contains a sequence encoding a post-translational modification signal not present in the wild-type hepatitis virus X gene.
Also provided are polynucleotides containing a modified PRE that includes a variant of a wild-type or unmodified hepatitis virus X gene in which the variant X gene contains a sequence encoding a post-translational modification signal not present in the wild-type or unmodified hepatitis virus X gene. In some embodiments of any of the provided polynucleotides, the post-translational modification signal contains a ubiquitination site.
In some embodiments of any of the provided polynucleotides, the post-translational modification signal contains a first codon beginning at a position within or within at least 2, 3, 4, 5 or 6 nucleotides in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the X protein open reading frame, wherein the first codon encodes a glycine, arginine, glutamic acid, phenylalanine, aspartate, cysteine, lysine, asparagine, serine, tyrosine, tryptophan, histidine, or leucine residue according to the N-end rule; and optionally a second codon beginning at a position within or within at least 2, 3, 4, 5 or 6 nucleotides in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the X protein open reading frame, wherein the second codon encodes a glutamic acid or asparagine residue according to the N-end rule.
In some embodiments of any of the provided polynucleotides, the post-translational modification signal contains one or more PEST sequences.
In some embodiments of any of the provided polynucleotides, the polynucleotide contains a nucleic acid encoding a recombinant protein operably linked to the modified PRE and a variant X gene containing a sequence encoding a post-translational modification signal not present in the wild-type or unmodified hepatitis virus X gene.
In some embodiments of any of the provided polynucleotides, the variant X gene contains a sequence encoding a post-translational modification signal containing a beta stem loop corresponding to nucleotide residues 448-470 of SEQ ID NO:1 or SEQ ID NO:125. In some embodiments of any of the provided polynucleotides, the variant X gene contains a sequence encoding a post-translational modification signal that does not contain a nucleotide change in a position within the beta stem loop corresponding to nucleotides 448-470 of SEQ ID NO:1 or SEQ ID NO:125.
In some embodiments of any of the provided polynucleotides, the variant X gene is no more than 180 nucleotides in length or is no more than at or about 210 nucleotides in length. In some embodiments of any of the provided polynucleotides, the variant X gene is at least at or about 90 nucleotides in length or at least about 120 nucleotides in length or is at least at or about 180 nucleotides in length.
In some embodiments of any of the provided polynucleotides, the variant X gene is a variant of a wild-type or unmodified mammalian hepatitis X gene or a partial or truncated portion thereof. In some embodiments, the wild-type or unmodified X gene can include a partial X gene encoding a truncated X protein, such as is present in a wild-type or unmodified PRE. In some embodiments of any of the provided polynucleotides, the wild-type or unmodified mammalian hepatitis X gene or partial X gene is or is derived from a wild-type woodchuck hepatitis virus (WHV) X gene. In some embodiments, the wild-type or unmodified WHV X gene contains the nucleotide sequence of SEQ ID NO:9, the sequence of nucleotides set forth as nucleotides 1503-1928 of SEQ ID NO: 2 or is a partial X gene thereof. In some embodiments of any of the provided polynucleotides, the wild-type or unmodified WHV X gene contains the sequence of nucleotides set forth as nucleotides 411-592 of any of SEQ ID NOS: 1 and 12-20 or nucleotides 411-589 of SEQ ID NO:125.
In some embodiments of any of the provided polynucleotides, the modified PRE contains at least two or at least three cis-acting post-transcriptional regulatory subelements of a wild-type hepatitis virus PRE or functional variant(s) thereof. In some embodiments of any of the provided polynucleotides, the at least two or at least three subelements contain a wild-type PRE alpha subelement or functional variant thereof, a functional variant of a wild-type PRE beta subelement, and/or a wild-type PRE gamma subelement or functional variant thereof. In some embodiments of any of the provided polynucleotides, the modified PRE contains an alpha subelement of a wild-type hepatitis virus PRE or functional variant thereof and a functional variant of a wild-type PRE beta subelement. In some embodiments of any of the provided polynucleotides, the alpha subelement contains the sequence of SEQ ID NO: 3, SEQ ID NO:4 or SEQ ID NO:5 or a variant thereof, the beta subelement contains a variant of the sequence of SEQ ID NO: 6 or SEQ ID NO:7, and/or the gamma subunit contains the sequence of SEQ ID NO: 8 or variant thereof.
In some embodiments of any of the provided polynucleotides, the wild-type or unmodified hepatitis virus PRE is a wild-type mammalian hepatitis PRE. In some embodiments of any of the provided polynucleotides, the wild-type or unmodified mammalian hepatitis virus PRE contains the sequence of nucleotides set forth in any of SEQ ID NOS: 1, 12-27 or 125. In some embodiments of any of the provided polynucleotides, the wild-type mammalian hepatitis PRE is a wild-type woodchuck hepatitis virus PRE (WPRE).
In some embodiments of any of the provided polynucleotides, the modified PRE is selected from among: a) a modified PRE comprising a variant X gene of a wild-type or unmodified hepatitis PRE, the wild-type or unmodified hepatitis PRE containing the sequence of nucleotides set forth in SEQ ID NO:1 or SEQ ID NO:125 or a sequence of nucleotides that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:125, wherein the variant X gene contains a sequence encoding a post-translational modification signal not present in the wild-type or unmodified hepatitis virus X gene; and b) a modified PRE containing a portion of the sequence of nucleotides of a), the portion containing a variant X gene containing the sequence encoding a post-translational modification signal, wherein the portion exhibits post-transcriptional activity.
In some embodiments of any of the provided polynucleotides, the variant X gene contains a start codon beginning at a position corresponding to position 411 of SEQ ID NO: 1 or SEQ ID NO:125. In some embodiments of any of the provided polynucleotides, the start codon is an ATG start codon. In some embodiments of any of the provided polynucleotides, the variant X gene contains a promoter operably linked to said variant X gene. In some embodiments of any of the provided polynucleotides, the promoter is a wild-type X-gene promoter containing the sequence set forth in SEQ ID NO: 11 or a wild-type WHV X gene promoter sequence.
In some embodiments of any of the provided polynucleotides, the modified PRE is selected from among: a) a modified PRE comprising a sequence of nucleotides that exhibits at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:125, the modified PRE containing a variant X gene containing a sequence encoding a post-translational modification signal not present in SEQ ID NO:1 or SEQ ID NO:125; and b) a modified PRE containing a portion of the sequence of nucleotides of a), the portion containing a variant X gene containing the sequence encoding the post-translational modification, wherein the portion exhibits post-transcriptional activity.
In some embodiments of any of the provided polynucleotides, the variant X gene contains up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotide changes.
In some embodiments of any of the provided polynucleotides, the modified PRE does not contain any modifications in addition to the sequence encoding a post-translational modification signal. In some embodiments of any of the provided polynucleotides, the modified PRE contains additional modification(s) in addition to the sequence encoding a post-translational modification signal not present in the wild-type or unmodified hepatitis virus X gene. In some embodiments of any of the provided polynucleotides, the additional modification(s) are in the variant X gene. In some embodiments of any of the provided polynucleotides, the additional modification(s) results in a variant X gene encoding an inactive X protein and/or a truncated X protein.
In some embodiments of any of the provided polynucleotides, the modified PRE encodes an RNA that promotes nuclear RNA export and/or increases mRNA stability. In some embodiments of any of the provided polynucleotides, the promotion of nuclear RNA export and/or mRNA stability increases expression of the recombinant protein.
In some embodiments of any of the provided polynucleotides, the modified PRE retains the post-transcriptional activity of the corresponding wild-type or unmodified hepatitis PRE and/or the PRE set forth in SEQ ID NO:1, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 125 or SEQ ID NO:216. In some such embodiments, the modified PRE exhibits at least 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120% or 130% or more of the post-transcriptional activity of the corresponding wild-type or unmodified hepatitis PRE and/or the PRE set forth in SEQ ID NO:1, SEQ ID NO:125, SEQ ID NO:119, SEQ ID NO:120 and/or SEQ ID NO:216. In some embodiments, the post-transcriptional activity of the modified PRE can be assessed by monitoring gene expression of an operably linked nucleic acid encoding a recombinant protein.
In some embodiments, any of the provided polynucleotides can further contain viral nucleic acid containing a variant Flap, wherein the variant Flap contains a deletion of all or a portion of the nucleotides corresponding to the central polypurine tract (cPPT) and/or the central termination sequence (CTS) regions of a wild-type or unmodified Flap sequence. In such embodiments, the polynucleotides contain a modified PRE and a variant Flap. In some embodiments, such elements are operably linked to nucleic acid encoding a recombinant protein and/or result in an increase in efficiency of gene transfer of a heterologous nucleic acid encoding a recombinant protein compared to the absence of such elements.
Also provided are polynucleotides containing a variant Flap, wherein the variant Flap contains a deletion of all or a portion of the nucleotides corresponding to the central polypurine tract (cPPT) and/or the central termination sequence (CTS) regions of a wild-type or unmodified Flap sequence. In some embodiments, the polynucleotide containing a variant Flap can further contain nucleic acid encoding a recombinant protein. In some embodiments, the presence of a polynucleotide containing a variant Flap, while deleted for all or a portion of the cPPT and CTS that are involved in forming the Flap structure, nevertheless can successfully transduce or transfer into cells with the nucleic acid encoding the recombinant protein.
In some embodiments of any of the provided polynucleotides containing a variant Flap, the variant Flap contains a deletion of all or a portion of the nucleotide corresponding to the cPPT and the CTS. In some embodiments, all of the nucleotide residues corresponding to cPPT and all of the nucleotide residues corresponding to the CTS are deleted and/or the variant Flap does not contain nucleotide residues corresponding to the cPPT or nucleotide residues corresponding to the CTS. In some embodiments of any of the provided polynucleotides, the variant Flap contains deletion of all or a contiguous portion of nucleotides corresponding to nucleotides in the cPPT region set forth in SEQ ID NO: 123. In some embodiments of any of the provided polynucleotides, the variant Flap contains deletion of all or a contiguous portion of nucleotides corresponding to nucleotides in the CTS region set forth in SEQ ID NO: 124. In some embodiments of any of the provided polynucleotides, the variant Flap contains deletion of all or a contiguous portion of nucleotides corresponding to nucleotides in the cPPT region set forth in SEQ ID NO:123 and deletion of all or a contiguous portion of nucleotides corresponding to nucleotides in the CTS region set forth in SEQ ID NO:124.
In any of such embodiments of a polynucleotide containing a variant Flap, the variant Flap is modified or comprises modifications as compared to a sequence containing a wild-type or unmodified Flap. In some embodiments, the wild-type or unmodified Flap sequence contains from or from about 80 to 200 contiguous nucleotides that includes the cPPT or CTS regions of a retrovirus, which optionally is a lentivirus. In some embodiments, the retrovirus is a lentivirus. In some embodiments, the lentivirus is HIV-1. In some embodiments, the wild-type or unmodified Flap contains a) the sequence of nucleotides set forth in SEQ ID NO:121; b) a sequence of nucleotides comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of nucleotides set forth in SEQ ID NO:121 that contains the cPPT and CTS regions; or c) a contiguous portion of a) or b) that includes the cPPT and CTS regions.
In some embodiments, the viral nucleic acid containing the variant Flap is or comprises a sequence of nucleotides that exhibits at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:121, said variant Flap lacking all or a portion of the cPPT and CTS regions. In some embodiments of any of the provided polynucleotides, the viral nucleic acid is or contains the sequence set forth in SEQ ID NO:122.
In some embodiments of any of the provided polynucleotides, the polynucleotide contains a) a variant Flap that is or comprises the sequence of SEQ ID NO: 122 or a sequence having at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:122, said variant Flap lacking all or a portion of the cPPT and CTS regions of the corresponding wild-type or unmodified Flap or of the sequence set forth in SEQ ID NO:121; and b) a modified PRE that is or comprises the sequence of SEQ ID NO:29 or 126 or a sequence having at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:29 or 126, said modified PRE containing at least one stop codon in the variant X gene of the modified PRE not present in a wild-type or unmodified X gene or not present in a wild-type or unmodified PRE containing an X gene, such as not present in SEQ ID NO:1 or SEQ ID NO:125.
In some embodiments of any of the provided polynucleotides, the modified WPRE and/or variant Flap is operably linked to nucleotides encoding a recombinant protein that is or contains a recombinant receptor. In some embodiments of any of the provided polynucleotides, the recombinant receptor is an antigen receptor and/or a chimeric receptor. In some embodiments of any of the provided polynucleotides, the recombinant receptor is a functional non-TCR antigen receptor or a transgenic TCR. In some embodiments of any of the provided polynucleotides, the recombinant receptor is a chimeric antigen receptor (CAR).
In some embodiments, also provided is an expression cassette that contains any of the provided polynucleotides and a promoter operably linked to a nucleic acid encoding a recombinant protein. In some embodiments, also provided is a vector that contains any of the provided polynucleotides or expression cassettes. In some embodiments of any of the provided vectors, the vector is a viral vector. In some embodiments of any of the provided vectors, the vector is a retroviral vector. In some embodiments of any of the provided vectors, the vector is a lentiviral vector. In some embodiments, the viral vector is a lentiviral vector that is derived from HIV-1.
In some embodiments, also provided is a cell that contains any of the provided polynucleotides, expression cassettes or any of the provided vectors. In some embodiments of any of the provided cells, the cell is a T cell, a natural killer (NK) cell, an iPS cell, or an iPS-derived cell.
In some embodiments, also provided is a virus particle that contains any of the provided vectors.
In some embodiments, also provided are methods that include introducing any of the provided polynucleotides, expression cassettes, vectors, or virus particles into a cell, under conditions whereby expression of the recombinant protein is effected in the cell. In some embodiments of any of the provided methods, the introduction is effected by transducing the cell with the vector or virus particle. In some embodiments, the introduction is effected by transfecting the cell with the vector; and/or the introduction is effected by electroporation of the cell with the vector. In some embodiments of any of the provided methods, the recombinant protein is expressed at a level that is increased compared to that achieved by introducing the vector in the absence of the modified PRE or a corresponding vector that does not contain the modified PRE or a PRE.
Also provided is a cell or cells produced by any of the provided methods.
Also provided is a pharmaceutical composition that contains a cell of any of the provided embodiments and a pharmaceutically effective carrier.
Also provided are methods of treatment that include administering to a subject having a disease or condition any of the provided polynucleotides, vectors, virus particles, cells, or pharmaceutical compositions.
In some embodiments of any of the provided methods of treatment, the method results in expression of the recombinant protein encoded by the polynucleotide. In some embodiments, the recombinant protein comprises a recombinant receptor that specifically binds to a ligand expressed by the disease or condition or a cell or tissue thereof. In some embodiments of any of the provided methods, the receptor is an antigen receptor and the ligand is an antigen specific for and/or associated with the disease or condition. In some embodiments of any of the provided methods, the disease or condition is a cancer, and autoimmune disorder, or an infectious disease.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts the nucleic acid sequence of an exemplary WPRE set forth in SEQ ID NO:1. Nucleic acids corresponding to reported PRE subelements (Donello et al. (1998) J. Virol., 72:5085; Smith et al. (1998) Nucleic Acids Research, 26:4818) are indicated as follows: a gamma subelement, alpha subelement, and beta subelement are indicated by solid lines with arrows indicating the beginning and end positions; nucleotides corresponding to an exemplary stem loop-forming portions of the alpha and beta subelements, respectively, are indicated with a dashed line and italics; nucleotides corresponding to a WHX protein promoter are indicated with a dashed and dotted line and by italics; and nucleotides corresponding to the truncated X gene contained within this exemplary WPRE are underlined. It is understood that the description of elements thereof are theoretically or empirically derived. Thus, the exact locus can vary (e.g. longer of shorter), and a corresponding element is not necessarily the same for each PRE, such as for each species or subspecies of PRE.
FIGS. 2A-2E depict exemplary alignments of the WPRE sequence set forth in SEQ ID NO:1 with other exemplary hepatitis PREs, and identifies corresponding residues. The symbol “I” between two aligned nucleotides indicates that the aligned nucleotides are identical. The absence of a “I” between two aligned nucleotides indicates that the aligned nucleotides are not identical. The symbol “-” indicates a gap in the alignment. Exemplary, non-limiting positions corresponding to an X gene start codon are indicated with bold and italicized text. Exemplary, non-limiting positions within an X gene corresponding to positions into which a stop codon may be introduced are boxed. Such positions correspond to an exemplary beginning residue (e.g. 3′ position of a stop codon) for positioning of an introduced stop codon. For example, FIG. 2A depicts the alignment of the exemplary WPRE sequence set forth in SEQ ID NO:1 with the nucleic acids corresponding to the exemplary WPRE sequence set forth in SEQ ID NO:12. FIG. 2B depicts the alignment of the exemplary WPRE sequence set forth in SEQ ID NO:1 with the nucleic acids corresponding to the exemplary WPRE sequence set forth in SEQ ID NO:13. FIG. 2C depicts the alignment of the exemplary WPRE sequence set forth in SEQ ID NO:1 with the nucleic acids corresponding to the exemplary modified WPRE sequence set forth in SEQ ID NO:119. FIG. 2D depicts the alignment of the exemplary WPRE sequence set forth in SEQ ID NO:1 with the nucleic acids corresponding to an exemplary Ground Squirrel Hepatitis B virus PRE sequence set forth in SEQ ID NO:27. FIG. 2E depicts the alignment of the exemplary WPRE sequence set forth in SEQ ID NO:1 with the nucleic acids corresponding to an exemplary Human hepatitis B virus (HBV) PRE sequence set forth in SEQ ID NO:21.
DETAILED DESCRIPTION
I. Polynucleotides and Viral Vectors Containing Modified Post-Transcriptional Regulatory Element (PRE)
Provided are polynucleotides useful in enhancing the expression of recombinant molecules, such as recombinant proteins. The polynucleotides contain modified post-transcriptional regulatory elements (modified PREs), such as cis-acting post-transcriptional regulatory elements, including modified versions of viral post-transcriptional regulatory elements, such as modified versions of PREs derived from hepatitis viruses, such as the woodchuck hepatitis virus (WHV) and hepatitis B virus (HBV).
In some embodiments, the modified PREs include modified versions of wild-type WHV post-transcriptional regulatory elements (WPREs) or modified versions of HBV post-transcriptional regulatory elements (HBVPRE), including those with modifications reducing the risk that protein(s) or polypeptide(s), particularly those having oncogenic or immunogenic potential to host subjects, will be expressed from coding sequence within the PRE and/or that, if produced, such proteins will be retained. In general, the sequence of wildtype PREs contain an open-reading frame (ORF) encoding a partial X gene encoding a truncated X protein, which, in some cases, may be involved in tumorigenesis when expressed. Also, since PREs are generally used to enhance transgene expression from an expression vector, delivery of a vector containing a PRE may also result in exogenous expression of the truncated X protein that may contribute to immunogenicity when administered to a subject.
The provided modified PREs include a variant X-gene, the variant X-gene including one or more nucleotide modifications compared to an unmodified or wild-type hepatitis virus X gene. Such modifications generally reduce the potential for oncogenesis and/or immunogenicity upon introduction into a subject of a polynucleotide (e.g. viral vector) containing such modified PRE operably linked to nucleic acid encoding a recombinant molecule for expression of the recombinant molecule, for example, by preventing expression of certain proteins encoded by the PRE. For example, the modification(s) generally include those designed to prevent or reduce the likelihood of expression of polypeptides from the variant X gene of the modified PRE. The modifications are such that the modified PRE retains at least some post-transcriptional regulatory activity, such as all or a portion of the post-transcriptional regulatory activity of a corresponding wild-type PRE. In some embodiments, at least or at least about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the post-transcriptional regulatory activity is retained.
Whereas viral vectors can provide an efficient system for the introduction of genetic material into cells, the use of certain viral vectors may not always produce the desired expression levels of such genetic material. Insertion of introns is used to enhance expression in some contexts, but may not always be appropriate in certain viral vectors. Hepatitis post-transcriptional regulatory elements (HPREs), such as woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), have been employed to enhance gene expression.
Hepatitis virus-derived PREs, including WPREs and HBVPREs, generally promote, e.g., enhance, the expression of transgenes operably linked thereto, by facilitating post-transcriptional RNA export from the nucleus. Secondary and tertiary structures formed by cis-acting sequences or elements contained within the PREs can promote such functions. For example, wild-type hepatitis virus-derived PREs generally include an alpha subelement and beta subelement, which each independently form stem loop structures, that affect and/or are involved in full PRE post-transcriptional activity (Smith et al. (1998) Nucleic Acids Research, 26:4818-4827). Some wild-type non-human hepatitis virus PREs, such as WPRE, also includes a gamma subelement that can further enhance post-transcriptional activity. Such subelements may have or encode RNAs having structures that promote RNA export from the nucleus, for example, via interaction with CRM1-dependent and/or independent export machinery, provide binding sites for cellular proteins, increase the total amount of RNA, e.g. recombinant and/or heterologous RNA, transcripts, increase RNA stability, increase the number of poly-adenylated transcripts and/or augment the size of the poly-adenylated tails in such transcripts.
In addition to providing expression-enhancing function via these cis-acting sequences, wild-type and unmodified hepatitis-virus PREs, including mammalian hepatitis virus-derived PREs, such as HBVPRE or WPRE, contain a portion of a viral X gene, including at least a partial open reading frame encoding at least a truncated X protein. This region generally overlaps at least in part with the cis-acting regulatory subelements. X-proteins, however—even certain truncated X-proteins—can exhibit oncogenic potential when included in vectors for gene expression in subjects. Mutations made in the X-gene region of PREs, e.g., at the start site and/or promoter region, are not necessarily entirely satisfactory in addressing these concerns. For example, even a mutant promoter with diminished activity may permit some degree of expression of the X protein of the PRE or other polypeptide encoded by the region. Reversion or further mutation at one or more positions in the X gene could restore or increase expression levels of the X protein, promoting oncogenicity. Even aside from oncogenic potential, the risk of expression of any polypeptide, particularly of at least a certain length (e.g., at least 8, 9, 10, 11, or 12 amino acids in length or more), from the X protein or other reading frame within this region or operably linked with the promoter, may promote immunogenicity when administered to a subject. Provided are nucleic acids with modified PREs addressing these problems.
Typically, the X region of a PRE includes a partial reading frame that encodes a truncated X protein that is less than the full length hepatitis X protein, for example, that is less than 141 amino acids in length, such as less than 130 nucleotides, less than 120 nucleotides, less than 110 nucleotides, less than 100 nucleotides, less than 90 nucleotides, less than 80 nucleotides, less than 70 nucleotides, and generally less than 60, 50, 40, 30, 20, 10 or less nucleotides in length. As exemplified in the exemplary wild-type WPRE shown in FIG. 1, the X promoter and partial reading frame overlap with the alpha and beta cis-acting sequences. Translation of such a partial open reading frame can result in an encoded truncated X protein, which may have the potential to promote tumorigenesis and/or an immunogenic reaction upon introduction into a subject.
For example, with reference to the exemplary WPRE set forth in SEQ ID NO:1 (corresponding to nucleotides 1093-1684 of SEQ ID NO:2 or GenBank Acc. No. J04514.1), nucleotides 411-592 correspond to a partial X protein open reading frame under the operable control of an X promoter set forth as nucleotides 391-410. Translation from transcripts initiated from the X promoter encodes a truncated X protein. While generally not fully represented in a PRE, a corresponding exemplary full length X protein open reading frame is 425 base pairs and corresponds to nucleotides 1503-1928 of the exemplary hepatitis virus sequence set forth in SEQ ID NO:2 under the operable control of the X promoter corresponding to nucleotides 1483-1502. Other wild-type or unmodified mammalian hepatitis virus PREs can also contain a partial X protein open reading frame. Exemplary unmodified or wild-type mammalian hepatitis virus PREs, and the corresponding partial X protein open reading frame, are set forth in Table 1. Residues in any PRE, such as any mammalian PRE, that correspond to residues in the exemplary WPRE set forth in SEQ ID NO:1 or SEQ ID NO:125 (which contains nucleotides 1-589 of SEQ ID NO:1), can be identified by alignment of the full-length sequence of the PRE of each as exemplified in FIG. 2A-2E for exemplary sequences. Non-limiting examples of corresponding residues that can be identified include, for example, residues of any cis-acting element or portion thereof (e.g. alpha, beta or gamma or portion thereof), residues of X protein open reading frame, residues of a start codon, residues for introduction of a stop codon.
TABLE 1
Exemplary Mammalian Hepatitis Virus PRE
X region in
REFERENCE
SEQ ID NO
SEQ ID
(PRE
X gene
PRE
NO
nucleotides)
promoter
ORF
WPRE
1
GenBank No.
391-410
411-592
(WHV8)
J04514.1
(1093-1684)
WPRE
125
GenBank No.
391-410
411-589
J04514.1
(1093-1681)
WPRE
12
GenBank No.
391-410
411-592
J02442.1
(1093-1684)
WPRE
13
GenBank No.
391-410
411-592
(WHV2)
M11082.1
(1093-1684)
WPRE
14
GenBank No.
391-410
411-592
(WHV7)
M18752.1
(1093-1684)
WPRE
15
GenBank No.
391-410
411-592
(WHV59)
M19183.1
(1093-1684)
WPRE
16
GenBank No.
391-410
411-592
(WHV34)
KF874491.1
(1093-1684)
WPRE
17
GenBank No.
391-410
411-592
AY628100.1
(1093-1684)
WPRE
18
GenBank No.
391-410
411-592
AY628099.1
(1093-1684)
WPRE
19
GenBank No.
391-410
411-592
KF874493.1
(1093-1684)
WPRE
20
GenBank No.
391-410
411-592
GU734791.1
(1093-1684)
HBVPRE
21
GenBank No.
412-722
D00329.1
(963-1684)
HBVPRE
22
GenBank No.
412-722
AF100309.1
(965-1686)
Orangutan
23
GenBank No.
412-722
hepatitis
AF193863.1
b virus
(963-1684)
Chimpanzee
24
GenBank No.
411-721
hepatitis
D00220.2
b virus
(964-1684)
Gorilla
25
GenBank No.
411-721
hepatitis
AJ131567.1
b virus
(2333-3053)
artic ground
26
GenBank No.
401-582
squirrel
U29144.1
hepatitis
(2475-3056)
b virus
ground
27
GenBank No.
411-592
squirrel
NC_001484.1
hepatitis
(2474-3065)
b virus
promoter/
119
nucleotides 2058-2649
391-410
411-592
start codon
of “Cloning vector
mutant
pLV.MCS.WHVPRE,
WPRE
complete sequence”
(GenBank: JN622008.1;
GI:373501904);
nucleotides 5700-6291
of SEQ ID NO: 5 from
U.S. Pat. No. 7,384,738
Promotor/
120
U.S. Pat. No. 7,419,829
391-410
411-592
start codon
mutant
WPRE
In embodiments of the provided modified PREs, the variant X gene contained therein are variants of an unmodified or wild-type X gene. The unmodified X gene can be any that includes a partial X gene reading frame, such as an X gene open reading frame present in an unmodified or wild-type PRE. For example, the variants of the X gene of the modified PRE include portions of mammalian hepatitis virus X genes, which can contain a partial X protein open reading frame and encode a truncated X protein, typically further modified to include one or more stop codon or other modification to reduce the risk of oncogenicity and/or immunogenicity. For example, the variant X genes include variants of a portion of the wild-type X gene. In some embodiments, the variant X gene may contain at least 180 nucleotides in length but fewer nucleotides than contained in a full-length hepatitis virus X gene, e.g., fewer than 425 nucleotides in length. Generally, the modified PRE in which is contained the variant X gene contains a sufficient portion of the cis-regulating sequences (e.g. one or more of an alpha, beta and/or gamma subelements, portion(s) thereof and/or functional variant(s) thereof) to regulate post-transcriptional activity of an RNA transcript.
The nucleotide modifications in a variant X gene of a modified PRE can include nucleotide deletions, insertions, and/or substitutions. Such modifications can include nucleotide changes in a region of the PRE encoding the viral X protein or portion thereof that include, but are not limited to, nucleotide changes resulting in a stop codon, a sequence that targets a protein for degradation, and combinations thereof. The modifications typically are such that the modified PRE retains at least some post-transcriptional regulatory activity, such as all or a portion of the post-transcriptional regulatory activity of a corresponding wild-type PRE or an unmodified PRE (e.g. SEQ ID NO:1 or SEQ ID NO:125), or another modified PRE known in the art to have a sufficient degree of activity for use in viral vector transduction, such as the PRE having the sequence set forth in SEQ ID NO: 119 or SEQ ID NO:120. In some embodiments, the modified PREs are provided in an expression vector (e.g. viral vector) operably linked to nucleic acid encoding a recombinant protein, such as a heterologous protein. By heterologous in this context refers to a protein that is not normally expressed from a virus and/or not encoded by a viral genome. In some embodiments, the heterologous protein is not expressed by the hepatitis virus from which the PRE is derived. In any of such embodiments, the provided modified PREs may enhance expression of nucleic acid encoding a recombinant protein, for example, compared to expression of the recombinant protein from an expression vector not containing a PRE.
In some embodiments, the provided modified PREs exhibit post-transcriptional activity, for example to enhance expression of an operably linked nucleic acid, while avoiding or reducing the likelihood of oncogenesis upon introduction and/or expression in a subject. For example, unwanted expression of functional full-length or truncated X proteins encoded by PREs can promote oncogenesis. In some embodiments, the modified PREs contain a variant X gene that includes modifications that prevent or reduce the likelihood of expression of an unwanted protein, such as a viral X protein or functional portion thereof, thereby reducing the risk of oncogenesis. For example, such modifications can include nucleotide changes that introduce one or more stop codons in the variant X gene, such as stop codon(s) not present in the unmodified or wild-type X gene, so that an unwanted protein, such as an X protein or functional portion thereof sufficient to cause oncogenesis, and/or any peptide with oncogenic potential, is not expressed. In other examples, such modifications include nucleotide changes that introduce a post-translational modification signal, such as a ubiquitination site, in the variant X gene not present in the unmodified or wild-type X gene, such that the encoded X protein is targeted for degradation.
In some embodiments, the provided modified PREs further include modification(s) (e.g. a nucleotide insertion, substitution or replacement or deletion) in the X gene promoter and/or start codon as compared to an unmodified or wild-type PRE. In some embodiments, the provided modified PRE does not include a modification in the X gene promoter. In some embodiments, the provided modified PRE does not include a modification in the X gene start codon. In some embodiments, the provided modified PRE does not include modifications in the X gene promoter and the X gene start codon. In other embodiments, the X gene promoter and/or start codon is wild-type.
In some embodiments, more than one modification affecting expression of the X protein encoded by the variant X gene in the modified PRE is present. For example, in some embodiments, the modified PRE contains a variant X gene that contains a plurality of stop codons not present in the corresponding unmodified or wild-type PRE, such as at least 2, 3, 4, 5, 6 or more stop codons. Due to the presence of multiple nucleotide changes, such as to effect introduction of a plurality of stop codons, the provided modified PREs can reduce the likelihood of X protein expression resulting from reversion to the unmodified sequence, such as a wild-type sequence. In some embodiments, a plurality of stop codons can include a stop codon in each reading frame of the variant X gene, and/or multiple stop codons in a single reading frame, thereby reducing the likelihood of unwanted protein expression. In some embodiments, at least 3 stop codons are included, e.g., to cover all three reading frames, which, in some embodiments, can be sequential.
The provided modified PREs contain features that can reduce the risk of oncogenicity due to reversion to wild-type sequence of the X-gene present in the PRE or start site or promoter thereof and/or the expression of other oncogenic polypeptides due to other mutations. Such features can provide advantages over available modified post-transcriptional regulatory sequences, such as those containing modifications only in the X protein promoter and/or start codon (See U.S. Pat. No. 7,419,829). For example, even mutated promoters may not necessarily result in a complete absence of protein expression from the promoter. By incorporating multiple modifications to produce multiple stop codons in the variant X gene, the provided modified PREs in some embodiments prevent or reduce the likelihood of oncogenic or immunogenic peptides, even in the context of such leaky promoters.
In some embodiments, the provided modified PREs containing a variant X gene can reduce the likelihood of X protein immunogenicity upon introduction into a subject. For example, to reduce the chance of creating an immunogenic epitope from an expressed variant X protein, the one or a plurality of stop codons can begin close enough to the start codon of the X protein open reading frame to prevent expression of a polypeptide containing an epitope that could induce an unwanted immune response. In some embodiments, such features ensure or increase the likelihood that, upon introduction into a subject, the variant X gene in the modified PRE does not result in the expression of any polypeptide having a potentially immunogenic epitope. In some embodiments, the polynucleotide containing a modified PRE includes at least one stop codon, e.g. the first stop codon or each of a plurality of introduced stop codons present in the variant X gene of the modified PRE, beginning at a position within at least 18 or 21 or 24 or 27 or 30 or 33 or 36 nucleotides of a start codon of the variant X gene (e.g., of the first position of such a start codon) or of a codon corresponding to a start codon in the corresponding wild-type or unmodified sequence. In some examples of such embodiments, such features ensure that no polypeptide longer than 6 or 7 or 8 or 9 or 10 or 11 or 12 amino acids in length encoded by the PRE is produced, e.g., even in the case of a wild-type or unmodified start codon or promoter or reversion thereto or other mutation resulting in a functional start codon and/or promoter. In some embodiments, the polynucleotide containing a modified PRE includes a stop codon, e.g., the first stop codon or each of a plurality of stop codons present in the variant X gene of the modified PRE, beginning at a position within at least 9, 12 or 15 nucleotides of a start codon of the variant X gene (e.g., of the first position of such a start codon) or of a codon corresponding to a start codon in the corresponding wild-type or unmodified sequence.
The provided polynucleotides include those with modified PREs designed to promote safety advantages, e.g., reduction in oncogenesis and/or immunogenicity, while maintaining a sufficient degree of post-transcriptional activity, such as expression-enhancing function. Hence, the modified PREs of the provided polynucleotides generally also are designed to retain a sufficient amount of the post-transcriptional activity, such as expression-enhancing function, of the corresponding unmodified or wild-type PRE. As describe above, the region of a hepatitis virus, such as WHV, that encodes the X-protein generally overlaps with structural features of a PRE of such virus that are important for its expression-enhancing function. In some embodiments, the sites and/or nature of modifications in the modified PREs, such as those that introduce the stop codon(s) and/or post-translational signal-encoding sequence(s) as described are designed to minimize alterations to certain secondary or tertiary structure encoded by the PRE. In some embodiments, such modifications are introduced in regions of the PRE other than those encoding a stem-loop structure of a PRE subelement, such as a stem-loop structure of an alpha and/or beta subelement of a PRE. In some embodiments, no stop codon is introduced or modification is made within a minimal alpha subelement encoding the alpha stem-loop structure or within a portion of the beta subelement encoding the beta stem-loop structure. In some embodiments, if a modification is introduced into a stem-loop structure, the modifications are made by selecting nucleobases that are different from the corresponding wild-type or unmodified nucleobases and yet maintain the secondary or tertiary structure(s) of the PRE. For example, in some embodiments, where a modification to a certain position within a stem-loop forming structure is modified, e.g., to introduce a stop codon, a complementary modification may be made at another position to permit the formation of a secondary or tertiary structure similar to or the same as that in the wild-type or unmodified sequence, e.g., a stem-loop structure.
Also among the provided polynucleotides are polynucleotides containing a variant Flap sequence (“variant Flap” polynucleotides). The variant Flap polynucleotides include polynucleotides, such as those containing one or more viral nucleic acid, in which one or more Flap sequence or portion thereof has been deleted. Such variant Flap polynucleotides include expression cassettes and vectors, such as viral vectors, for the expression of recombinant molecules, such as recombinant, e.g., heterologous proteins. In some embodiments, the polynucleotides comprising the modified PREs further comprise variations in Flap sequences and/or are variant Flap polynucleotides. In general, the variation(s) in the Flap are such that they still permit and/or do not substantially disrupt viral delivery or expression of the recombinant molecule, e.g., in a host cell.
Also provided are expression cassettes containing the polynucleotides, generally further including a promoter operably linked to the sequence encoding the recombinant protein, such as heterologous protein, and vectors containing such polynucleotides and expression cassettes, including retroviral vectors such as lentiviral and gamma-retroviral vectors. Also provided are viruses and cells containing such polynucleotides, cassettes, and/or vectors, including packaging cells and host cells, such as T cells and compositions containing the same. Also provided are methods and uses of such embodiments, including therapeutic methods and uses, such as those involving the administration of the virus, vector, and/or cells to a subject in an amount effective to treat or prevent a disease or condition.
A. Modified PREs
The modified PREs in the provided polynucleotides contain variant X genes. The variant X genes are variants of unmodified, typically wild-type, hepatitis X genes. The variant X genes include one and typically more than one modification as compared to the wild-type or other unmodified X gene. In particular, the variant X gene generally includes one or more modifications designed to prevent or reduce the likelihood that an unwanted protein or peptide encoded by a sequence within the X gene and/or PRE will be expressed and/or maintained, e.g., upon introduction into a subject. The modifications may include nucleotide deletion(s), substitutions, and/or insertions as compared to the unmodified, e.g., wild-type sequence.
In some embodiments, the modifications include mutations that result in the presence of a stop codon in the variant X gene of the modified PRE not present in the corresponding wild-type or otherwise unmodified X gene, such as not present in the corresponding wild-type or otherwise unmodified PRE containing a partial X gene. In some embodiments, such modifications shorten the X protein-coding open reading frame as compared to a wild-type X gene or X gene fragment within a wild-type or unmodified PRE. In some embodiments, the modifications result in a modified PRE in which the variant X gene contains an X protein open reading frame that is no longer than 9, 12, 15, 18, or 21 nucleotides in length. In some embodiments, the modifications in the variant X gene of the modified PRE prevent the expression of any protein from the X protein reading frame contained therein that is longer than 3, 4, 5, 6, or 7 amino acids in length or that may be immunogenic to a subject or have oncogenic potential or activity. In some embodiments, if the variant X gene also contains a mutated or modified start site or promoter, the stop codon modifications prevent such expression even in the event of a mutation that results in reversion to a functional promoter or start site.
In some embodiments, the modifications in the variant X gene of the modified PRE prevent the expression of any unwanted polypeptides, whether in the X protein-coding reading frame or not. For example, in some embodiments, the modifications in the variant X gene of the modified PRE prevent the expression of any peptide(s) or peptide(s) from the X gene contained therein that are longer than a certain length, such as longer than 3, 4, 5, 6, or 7 amino acids in length and/or that may be immunogenic upon expression in a subject.
In some embodiments, the modifications include those which introduce coding sequences to introduce or result in post-translation degradation amino acid sequences that target expressed protein(s) encoded by the variant X gene or portion thereof of the modified PRE for degradation. Exemplary degradation sequences include ubiquitination signals. Thus, the modifications in the variant X gene of the modified PRE may further include those resulting in a post-translational degradation sequence, such as a ubiquitination sequence, not present in the corresponding protein encoded by an unmodified or wild-type X gene, such as not present in the corresponding wild-type or unmodified PRE containing a partial X gene.
The variant X gene of the modified PRE generally contains a portion of an X gene that includes fewer nucleobases in length than a corresponding wild-type full-length X gene present in a corresponding wild-type hepatitis virus. In some embodiment, the portion of the X gene lacks X gene nucleotides that do not overlap with and/or that are not important or essential for post-transcriptional regulatory function(s) of the PRE. In some embodiments, the portion of the X gene lacks X gene residues of a wild-type hepatitis virus not within the alpha, beta, and/or gamma subelement(s) of the PRE and/or functional regions thereof. Thus, the modified PREs herein generally do not include full-length X genes or full-length variants thereof. In some such embodiments, the variant X gene within the modified PRE generally includes fewer than at or about 425, 400, 300, 200, or 180 nucleobases in length.
The modified PRE generally contains one or more of an alpha, beta and/or gamma subelement, which, in some cases, can be a variant of such a subelement of a wild-type or unmodified PRE or a functional portion thereof, e.g., one that is sufficient for post-transcriptional activity and/or formation of a stem-loop or other secondary or tertiary structure. In some cases, at least some of such portion(s) generally overlap with the variant X gene portion of the modified PRE. Thus, in some cases, a portion of the variant X gene may be contained within a portion of the alpha subelement and/or a portion of the variant X gene may be contained within or overlaps with the beta subelement. In some embodiments, the modifications in the variant X gene of the provided modified PREs as compared to a wild-type or unmodified X gene or PRE do not ablate, reduce and/or interfere with the post-transcriptional activity to control expression of an operably linked nucleic acid (e.g. recombinant molecule or transgene) mediated by the alpha and/or beta and/or gamma subelement(s) of the modified PRE, and/or do not do so substantially, as compared to the post-transcriptional activity mediated by the subelement(s) of a corresponding wild-type or unmodified PRE not having the modification(s) or compared to another modified PRE known to retain sufficient activity, such as that having the sequence of SEQ ID NO: 119, 120 or 216.
In one embodiment, the nucleotide changes that introduce stop codons and/or post-translation degradation sequence are such that, on the RNA level, they do not alter the secondary and/or tertiary structure of the cis-element sequences within the PRE and/or they retain a substantial degree of the secondary and/or tertiary structure of the cis-element sequences within the PRE, as compared to a corresponding wild-type or unmodified PRE and/or PRE not containing such modifications. In another embodiment, the nucleotide changes that introduce stop codons and/or a post-translation degradation sequence, are such that, on the RNA level, the nucleotide changes do not alter the activity of the PRE, e.g., its ability to promote RNA export from the nucleus, for example, via interaction with CRM1-dependent and/or independent export machinery, provide binding sites for cellular proteins, increase the total amount of RNA, e.g. transgene RNA, transcripts, increase RNA stability, increase the number of poly-adenylated transcripts and/or augment the size of the poly-adenylated tails in such transcripts, as compared with a corresponding wild-type PRE and/or PRE not containing such modifications, and/or compared to another modified PRE known in the art to have a sufficient degree of activity for use in viral vector transduction or accepted for use therein and/or accepted for such uses in the context of gene therapy. In some embodiments, the another unmodified PRE has the sequence set forth in SEQ ID NO: 119, SEQ ID NO: 120 or SEQ ID NO:216.
In some embodiments, a beta subelement within the modified PRE includes at least a portion of the variant X gene. In some such embodiments, the beta subelement is a functional variant or portion thereof of a beta subelement of a wild-type or unmodified PRE, such that the modified PRE containing the functional variant or portion of the beta subelement exhibits post-transcriptional activity. In some embodiments, the functional variant of the beta subelement is a variant of a beta subelement set forth in SEQ ID NO:6 or a functional portion thereof. In some embodiments, a functional variant or portion of a beta sub-element contains nucleotide residues sufficient to form a beta stem-loop. For example, in some embodiments, a functional variant or portion of a beta sub-element contains nucleotide residues corresponding to nucleotide residues 448-470 of SEQ ID NO:1 or SEQ ID NO:125, such as the sequence of nucleotides set forth in SEQ ID NO:7. In aspects of the modified PRE provided herein, the variant X gene does not contain a nucleotide change within any nucleotides corresponding to nucleotides 448-470 of SEQ ID NO:1 or SEQ ID NO:125 and/or in a region of nucleotides corresponding to nucleotides set forth in SEQ ID NO:7. In some embodiments, where a modification, e.g., to introduce a stop codon, is at a nucleotide base corresponding to one or more nucleotides 448-470 of SEQ ID NO:1 or SEQ ID NO:125 and/or one or more nucleotides corresponding to nucleotides set forth in SEQ ID NO:7, a complementary modification may be made at another position to permit the formation of a secondary or tertiary structure similar to or the same as a beta-stem loop of the wild-type or unmodified sequence.
In some embodiments of a modified PRE containing an alpha subelement, the modified alpha subelement can be from an unmodified hepatitis PRE, such as a wild-type hepatitis PRE, or can be a functional variant thereof such that the modified PRE containing the functional variant of the alpha subelement exhibits post-transcriptional activity. In some embodiments, the alpha subelement is set forth in SEQ ID NO:3 or is set forth in SEQ ID NO:4, or is a functional variant or portion of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, a functional variant or portion of an alpha sub-element contains nucleotide residues sufficient to form an alpha stem-loop. For example, in some embodiments, a functional variant of an alpha sub-element contains nucleotide residues corresponding to nucleotide residues 329-366 of SEQ ID NO:1 or SEQ ID NO:125, such as the sequence of nucleotides set forth in SEQ ID NO:5. In aspects of the modified PRE provided herein, the modified PRE does not contain a nucleotide change within any nucleotides corresponding to nucleotides 329-366 of SEQ ID NO:1 or SEQ ID NO:125 and/or in a region of nucleotides corresponding to nucleotides set forth in SEQ ID NO:5. In some embodiments, where a modification is at a nucleotide base corresponding to one or more nucleotides 329-366 of SEQ ID NO:1 or SEQ ID NO:125 and/or one or more nucleotides corresponding to nucleotides set forth in SEQ ID NO:5, a complementary modification may be made at another position to permit the formation of a secondary or tertiary structure similar to or the same as an alpha-stem loop of the wild-type or unmodified sequence.
In one embodiment, the modified PRE at least contains an alpha and beta subelement, such as a functional variant of an alpha and beta subelement, at least some portion of which generally overlaps with the variant X gene. In some embodiments, the modified PRE contains a gamma subelement or functional variant thereof. In embodiments of a modified PRE containing a gamma subelement, the modified gamma subelement can be from an unmodified hepatitis PRE, such as a wild-type hepatitis PRE, or can be a functional variant thereof such that the modified PRE containing the functional variant of the gamma subelement retains post-transcriptional activity. In some embodiments, the gamma subelement is set forth in SEQ ID NO:8, or is a functional variant or portion of SEQ ID NO:8.
The nucleotide modifications, such as those within the variant X gene, of a modified PRE can include nucleotide deletions, insertions, and/or substitutions. The variant X gene can contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 nucleotide changes compared to the X gene of the unmodified PRE, such as a wild-type PRE. In some embodiments, the variant X gene contains no more than 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotide changes. In some embodiments, the variant X gene has a sequence of nucleotides that exhibits at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the X gene set forth in SEQ ID NO: 10, to the sequence of nucleotides 391-592 of any of SEQ ID NOS:1, 12-20, 119 or 120 or to the sequence of nucleotides 391-589 of SEQ ID NO:125 or 216, in which the variant X gene contains at least one stop codon not present therein and/or a sequence encoding a post-translational modification signal or degradation sequence not present therein. For example, in some embodiments, the variant X gene has a sequence of nucleotides that exhibits at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the X gene set forth in SEQ ID NO: 10, to the sequence of nucleotides 391-592 of SEQ ID NO:1 or to the sequence of nucleotides 391-589 of SEQ ID NO:125, in which the variant X gene contains at least one stop codon not present therein and/or a sequence encoding a post-translational modification signal or degradation sequence not present therein. In some embodiments, the modified PRE has a sequence of nucleotides that exhibits at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the PRE set forth in any of SEQ ID NOS:1, 12-20, 119, 120, 125 or 216, in which the modified PRE contains at least one stop codon not present therein and/or a sequence encoding a post-translational modification signal or degradation sequence not present therein. For example, in some embodiments, the modified PRE has a sequence of nucleotides that exhibits at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the PRE set forth in SEQ ID NO:1 or SEQ ID NO:125, in which the modified PRE contains at least one stop codon not present therein and/or a sequence encoding a post-translational modification signal or degradation sequence not present therein.
In some embodiments, the variant X gene is a partial X gene, such as a portion of a wild-type X genes, having a sequence that is less than the full length sequence of the X gene of an hepatitis virus, such as a wild-type hepatitis virus, such as a mammalian hepatitis virus. For example, in some embodiments, the modified PRE contains a variant X gene that is less than or no more than at or about 250 nucleotides in length, such as less than or no more than at or about 240 nucleotides, 230 nucleotides, 220 nucleotides, 210 nucleotides, 200 nucleotides, 190 nucleotides, 180 nucleotide, 170 nucleotides, 160 nucleotides, 150 nucleotides, 140 nucleotides, 130 nucleotides, 120 nucleotides, 110 nucleotides, 100 nucleotides, 90 nucleotides, 80 nucleotides or 70 nucleotides in length. In some embodiments, the modified PRE contains a variant X gene that is at least 60 nucleotides in length but is less than the full length sequence of the X gene of a hepatitis virus, such as a wild-type hepatitis virus, such as a mammalian hepatitis virus. For example, in some embodiments, the modified PRE contains a variant X gene that is at least 60 nucleotides in length but is less than or no more than at or about 250 nucleotides in length, such as less than or no more than at or about 240 nucleotides, 230 nucleotides, 220 nucleotides, 210 nucleotides, 200 nucleotides, 190 nucleotides, 180 nucleotide, 170 nucleotides, 160 nucleotides, 150 nucleotides, 140 nucleotides, 130 nucleotides, 120 nucleotides, 110 nucleotides, 100 nucleotides, 90 nucleotides, 80 nucleotides or 70 nucleotides in length. In some embodiments, the modified PRE contains a variant X gene that is at least or about at least 180 nucleotides in length.
In some embodiments, the modified PRE containing the variant X gene is a variant of an unmodified PRE, such as a wild-type hepatitis PRE, that contains an X gene or portion thereof. For example, the variant X gene can be a variant of a wild-type hepatitis PRE that has an X gene that is less than the full-length sequence of the corresponding X gene of the hepatitis virus, such as a wild-type hepatitis virus, such as a mammalian hepatitis virus. The unmodified or wild-type X gene can contain an X gene that is less than or no more than at or about 250 nucleotides in length, such as less than or no more than at or about 240 nucleotides, 230 nucleotides, 220 nucleotides, 210 nucleotides, 200 nucleotides, 190 nucleotides, 180 nucleotide, 170 nucleotides, 160 nucleotides, 150 nucleotides, 140 nucleotides, 130 nucleotides, 120 nucleotides, 110 nucleotides, 100 nucleotides, 90 nucleotides, 80 nucleotides or 70 nucleotides in length.
In some embodiments, the unmodified PRE, such as wild-type hepatitis PRE, is one that contains an X gene having an open reading frame encoding a functional X protein, e.g., a truncated X protein. In such an embodiment, the variant X gene contains a start codon for initiating translation of the X protein. In some embodiments, the start codon is an ATG start codon. In some embodiments, the variant X gene contains a start codon corresponding to a start codon beginning at a position corresponding to position 411 of SEQ ID NO:1 or SEQ ID NO:125. In some embodiments, the unmodified PRE, such as a wild-type PRE, can contain an open reading frame encoding an X protein that is at least 30 amino acids in length, such as at least 40 amino acids, 50 amino acids, 60 amino acids, 70 amino acids, or 80 amino acids.
The modified PRE generally contains a variant X gene that contains a modification, for example one or more nucleotide modifications that introduces one or more stop codon and/or post-translational degradation sequence not present in an unmodified hepatitis X gene, such as not present in a wild-type hepatitis X gene or corresponding truncated version thereof (e.g. as present in a PRE). In some embodiments, the variant X gene contains a modification not present in a wild-type mammalian hepatitis X gene or corresponding truncated version thereof (e.g. as present in a PRE). In some embodiments, the unmodified or wild-type hepatitis X gene or the truncated version thereof is an X gene that exhibits at least or about at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more sequence identity to SEQ ID NO:9 or SEQ ID NO:10 or to nucleotides 411-592 of SEQ ID NO:1 or to nucleotides 411-589 of SEQ ID NO:125. In some embodiments, the wild-type or unmodified mammalian hepatitis X gene or corresponding truncated version thereof has the sequence of nucleotides corresponding to nucleotides 411-592 of any of SEQ ID NOS: 1 or 12-20, nucleotides 411-589 of SEQ ID NO:125, nucleotides 412-722 of any of SEQ ID NO:21-23, nucleotides 411-721 of SEQ ID NO:25, nucleotides 401-582 of SEQ ID NO:26 or nucleotides 411-592 of SEQ ID NO:27. In some embodiments, the unmodified or wild-type hepatitis X gene is a WHV X gene or corresponding truncated version thereof that exhibits at least or about at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:9, nucleotides 411-592 of SEQ ID NO:1 or to nucleotides 411-589 of SEQ ID NO:125. In some embodiments, the unmodified or wild-type WHV X gene or corresponding truncated version thereof has the sequence of nucleotides corresponding to nucleotides 411-592 of any of SEQ ID NOS: 1, 12-20, 119 or 120 or to nucleotides 411-589 of SEQ ID NO:125 or 216. In some embodiments, the hepatitis X gene is a WHV X gene set forth in SEQ ID NO:9 or a corresponding truncated version thereof set forth in SEQ ID NO:10.
In some embodiments, the variant X gene of the modified PRE contains difference(s) compared to an X gene or portion thereof contained within an unmodified hepatitis PRE, such as a wild-type hepatitis PRE. In some embodiments, such a wild-type hepatitis PRE is a mammalian hepatitis PRE. In some embodiments, the wild-type or unmodified mammalian PRE has a sequence of nucleotides with at least or about at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more sequence identity to SEQ ID NO:1 or SEQ ID NO:125. In some embodiments, the wild-type or unmodified mammalian hepatitis PRE has the sequence of nucleotides set forth in any of SEQ ID NOS: 1 or 12-27. In some embodiments, the unmodified or wild-type hepatitis PRE is a WPRE that exhibits at least or about at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1 or SEQ ID NO:125. For example, in some embodiments, the unmodified or wild-type WPRE has the sequence of nucleotides set forth in any of SEQ ID NOS: 1, 12-20, 119, 120, 125 or 216.
In some embodiments, the variant X gene is operably coupled to a promoter. In some embodiments, the promoter is an unmodified or wild-type X gene promoter. For example, the promoter can have the sequence of nucleotides set forth in SEQ ID NO: 11 or a functional variant or portion thereof. The promoter can have the sequence of nucleotides 7-20 of SEQ ID NO:11. In some embodiments, the promoter is a mutant or modified X gene promoter, such as described in subsection I.A.3 below.
In some embodiments, the modified PRE exhibits post-transcriptional activity to mediate, such as to enhance, the expression of a nucleic acid encoding a recombinant protein, such as a heterologous protein, operably linked thereto, e.g., as compared to expression of a nucleic acid that is not operably linked to the PRE and/or not linked to any cis-regulatory sequence. In some embodiments, the modified PRE exhibits activity to promote RNA export from the nucleus, provide binding sites for cellular proteins, increase the total amount of RNA transcripts, increase RNA stability, increase the number of poly-adenylated transcripts and/or augment the size of the poly-adenylated tails in such transcripts. In some embodiments, the modified PRE retains the activity or a substantial and/or sufficient degree of activity of the corresponding unmodified hepatitis PRE, such as wild-type hepatitis PRE, and/or of another modified PRE known in the art to have a sufficient degree of activity for use in viral vector transduction, such as the PRE having the sequence set forth in SEQ ID NO: 119, SEQ ID NO:120 or SEQ ID NO:216. In some embodiments, the modified PRE exhibits at least or about at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the post-transcriptional activity of an unmodified or wild-type PRE set forth in any of SEQ ID NOS: 1, 12-27, 119, 120, 125 or 216. In some embodiments, the modified PRE exhibits at least or about at least or about 80%, 85%, 90%, 95%, or 100% of the activity of the WPRE set forth in SEQ ID NO:1 or SEQ ID NO:125.
In some such embodiments, the relevant activity (e.g., of the modified PRE and/or other PRE, such as unmodified or wild-type PRE to which it is being compared), is the enhancement or promotion of expression of a recombinant protein encoded by a nucleic acid operably linked to the modified PRE. In some embodiments, such enhancement or promotion is assessed by measuring the level of expression of a recombinant protein operably linked to the modified PRE within a vector, following transduction or other form of transfer of the vector into a host cell. The enhancement may be measured by determining a relative degree of such expression as compared to a vector not containing the respective PRE. The question of whether a modified PRE has the activity at the same level or degree or a substantial or acceptable degree thereof as compared to an unmodified PRE may be assessed using vectors containing the respective PREs. A number of well-known methods for assessing expression level of recombinant molecules may be used, such as detection by affinity-based methods, e.g., immunoaffinity-based methods, e.g., in the context of cell surface proteins, such as by flow cytometry. In some examples, the expression is measured by detection of a transduction marker and/or reporter construct. In some embodiments, nucleic acid encoding a truncated surface protein is included within the vector and used as a marker of expression and/or enhancement thereof by the modified PRE.
1. Stop Codons
The stop codon in the modified PREs, e.g., within the variant X gene (e.g., one not present at a corresponding position in a corresponding wild-type or unmodified X gene or PRE) can be an amber (TAG), ochre (TAA), or opal (TGA) stop codon. In one embodiment, the stop codon is an amber (TAG) stop codon. In one embodiment, the stop codon is an opal (TGA) stop codon.
The stop codon can be introduced by nucleotide substitution, deletion or insertion. In one embodiment, the stop codon is introduced by nucleotide substitution. Nucleotide substitution minimizes the nucleotide changes, such as frame shifts, that can occur by deletion or insertion of nucleotide residues. Hence, nucleotide substitutions can minimize structural changes to the cis-regulatory sequences involved in PRE activity, such as the beta-element or a functional portion thereof. In some embodiments, the stop codon is formed by substitution of only a single nucleotide, such that two positions within the stop codon represent unmodified nucleotides present in the corresponding positions in the wild-type or unmodified sequence. In some embodiments, the stop codon is formed by substitution of two nucleotides, such that only one position within the stop codon represent unmodified nucleotides present in the corresponding position in the wild-type or unmodified sequence. In some embodiments, the stop codon is formed by substitution of three nucleotides, such that all positions within the stop codon differ compared to the corresponding codon of the wild-type or unmodified sequence.
The variant X gene can contain at least 1, 2, 3, 4, 5, 6 or more stop codons not present in the unmodified or wild-type X gene or not present in the unmodified or wild-type PRE. Generally, at least one stop codon is in-frame with the X protein reading frame. In some embodiments, the variant X gene contains a plurality of stop codons, such as at least 2, 3, 4, 5 or 6 stop codons.
In some embodiments, the plurality of stop codons includes at least two stop codons in which one is in-frame with the X protein reading frame and the other is in a different reading frame. In other embodiments, the plurality of stop codons includes at least three stop codons in each reading frame present in the variant X gene.
In some embodiments, the plurality of stop codons includes at least two stop codons in the same reading frame. Typically, the at least two stop codons are in the same reading frame as the X protein reading frame.
In some embodiments, each of the at least one stop codon(s) begins at a position within or no more than 36 nucleotides in the 3′ direction from a position corresponding to the 5′ position of a start codon of the X protein open reading frame, e.g. a codon corresponding to a start codon in a corresponding wild-type or unmodified X protein open reading frame, such as corresponding to a start codon in the X protein open reading frame set forth in SEQ ID NO:1 or SEQ ID NO:125 that begins position 411. Typically, the first stop codon, and, in some cases, each of the plurality of introduced stop codons, present in the variant X gene after the 5′ position of a start codon of the X protein open reading frame begins within or no more than 36 nucleotides in the 3′ direction from a position corresponding to the 5′ position of the start codon. For example, in some embodiments, each of the at least one stop codon, e.g., the first stop codon or one or more of a plurality of stop codons present in the variant X gene after a position corresponding to the 5′ position of a start codon of the X protein open reading frame, begins at a position within or no more than 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or 9 nucleotides in the 3′ direction from a position corresponding to the 5′ position of a start codon of the X protein open reading frame. In some embodiments, each of the at least one stop codon begins at a position within no more than 9, 12, 15, 18, or 21 nucleotides in the 3′ direction from a position corresponding to the 5′ position of a start codon of the X protein open reading frame, such as no more than 21 nucleotides from such position.
With reference to the exemplary WPRE set forth in SEQ ID NO:1 or SEQ ID NO:125, the position corresponding to the 5′ position of a start codon of the X protein open reading frame corresponds to residue 411 (corresponding to residue 1503 of the WHV sequence set forth in SEQ ID NO:2). In some embodiments, the at least one stop codon, e.g. the first stop codon or one or more of a plurality of stop codons present in the variant X gene after a position corresponding to the 5′ position of a start codon of the X protein open reading frame, begins at a position within or no more than 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or 9 nucleotides in the 3′ direction from a position corresponding to residue 411 in the WPRE set forth in SEQ ID NO:1 or SEQ ID NO:125, e.g., no more than 9, 12, 15, 18, or 21, e.g., no more than 21, nucleotides in the 3′ direction from such position. It is readily within the level of a skilled artisan to identify a residue in another hepatitis PRE, such another WPRE, that corresponds to residue 411 to identify a 5′ position of a codon that corresponds to a start codon of the exemplary wild-type PRE set forth in SEQ ID NO:1 or SEQ ID NO:125 (which is a portion of SEQ ID NO:1 containing residues 1-589 or SEQ ID NO:1). FIG. 2A-2E exemplifies the identification of corresponding residues in exemplary hepatitis PREs.
In some embodiments of a modified PRE containing a variant X gene with a plurality of stop codons, the stop codons can be present sequentially in each reading frame of the X gene. In some embodiments of a modified PRE containing a variant X gene with a plurality of stop codons, the stop codons can be present sequentially in the same reading frame.
In some embodiments of a modified PRE, the variant X gene does not contain an open reading frame of greater than at or about 39 nucleotides in length, such as greater than at or about 36 nucleotides, 33 nucleotides, 30 nucleotides, 27 nucleotides, 24 nucleotides, 21 nucleotides, 18 nucleotides, 15 nucleotides, or 12 nucleotides in length.
In some embodiments, the at least one stop codon, e.g. the first stop codon or one or more of a plurality of stop codons present in the variant X gene after a position corresponding to the 5′ position of a start codon of the X protein open reading frame, begins at a position that is in-frame with the X protein reading frame. For example, in some embodiments, the at least one stop codon, e.g., the first stop codon or one or more of a plurality of stop codons present in the variant X gene after a position corresponding to the 5′ position of a start codon of the X protein open reading frame, begins at a position within or no more than 36, 33, 30, 27, 24, 21, 18, 15, 12, or 9 nucleotides in the 3′ direction from a position corresponding to the 5′ position of a start codon of the X protein open reading frame, e.g., no more than 9, 12, 15, 18, or 21, e.g., no more than 21, nucleotides in the 3′ direction from such position. In some embodiments, the at least one stop codon, e.g. the first stop codon or one or more of a plurality of stop codons present in the variant X gene after a position corresponding to the 5′ position of a start codon of the X protein open reading frame, begins at a position within or no more than 36, 33, 30, 27, 24, 21, 18, 15, 12, or 9 nucleotides in the 3′ direction from a position corresponding to residue 411 in the WPRE set forth in SEQ ID NO:1 or SEQ ID NO:125.
In some embodiments, the modified PRE contains a variant X gene that contains a stop codon or a plurality of stop codons beginning at positions 9, 13, 17 and/or 21 in the 3′ direction from a position corresponding to the 5′ position of a start codon of the X protein open reading frame. With reference to the exemplary WPRE set forth in SEQ ID NO:1 or SEQ ID NO:125, the modified PRE contains a variant X gene that contains a stop codon or a plurality of stop codons beginning at a nucleotide position corresponding to position 420, 424, 428 and/or 432 in the sequence set forth in SEQ ID NO:1 or SEQ ID NO:125. In such embodiments, one, two, three or all four positions can be the beginning of a stop codon.
In some embodiments, the modified PRE contains a variant X gene that contains four stop codon beginning at each of positions 9, 13, 17 and 21 in the 3′ direction from a position corresponding to the 5′ position of a start codon of the X protein open reading frame. With reference to the exemplary WPRE set forth in SEQ ID NO:1 or SEQ ID NO:125, the modified PRE contains a variant X gene that contains a stop codon beginning at a nucleotide position corresponding to position 420, 424, 428 and 432 in the sequence set forth in SEQ ID NO:1 or SEQ ID NO:125.
In some embodiments, the modified PRE comprises a sequence of nucleotides that exhibits at least 65% sequence identity, such as at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, to SEQ ID NO:1 or SEQ ID NO:125, so long as the modified PRE contains a variant X gene containing at least one stop codon not present in the X gene set forth in SEQ ID NO:1 or SEQ ID NO:125. In some embodiments, the modified PRE differs from SEQ ID NO:1 or SEQ ID NO:125 only by nucleotide substitution. In other embodiments, the modified PRE differs from SEQ ID NO:1 or 125 by nucleotide insertion or deletion. In some embodiments, the modified PRE can be longer or shorter than SEQ ID NO:1 or SEQ ID NO:125.
In some embodiments, the modified PRE does not contain any modifications in addition to the at least one stop codon not present in the wild-type or unmodified PRE.
Tables 2A and 2B set forth exemplary positions for introduction of at least one stop codons in a variant X gene or modified PRE, respectively. The corresponding SEQ ID NOs for sequences of the exemplary polynucleotides also are set forth. In some embodiments, provided are polynucleotides comprising a modified PRE containing a variant X gene comprising the sequence of nucleotides set forth in any of SEQ ID NOS: 44-58 or 141-155. In some embodiments, provided are polynucleotides comprising a modified PRE that have the sequence of nucleotides set forth in any of SEQ ID NOS: 29-43 or 126-140. In some embodiments, provided are polynucleotides containing such PREs operably linked to nucleic acids encoding recombinant molecules, such as recombinant antigen receptors, CARs, TCRs, chimeric receptors, immunomodulators, immunostimulatory molecules, and/or transduction or expression markers, and expression cassettes and vectors containing the same.
TABLE 2A
Exemplary Variant X gene with at least one introduced stop codon
5′ Position of
Introduced Stop Codon(s)
Relative to
3′ Position
SEQ ID
of X Gene
X gene
NOs
Start Codon
Start Codon
44, 141
9, 13, 17, 21
Unmodified
45, 142
9, 13, 17
Unmodified
46, 143
9, 13, 21
Unmodified
47, 144
9, 17, 21
Unmodified
48, 145
13, 17, 21
Unmodified
49, 146
9, 13
Unmodified
50, 147
9, 17
Unmodified
51, 148
9, 21
Unmodified
52, 149
13, 17
Unmodified
53, 150
13, 21
Unmodified
54, 151
17, 21
Unmodified
55, 152
9
Unmodified
56, 153
13
Unmodified
57, 154
17
Unmodified
58, 155
21
Unmodified
TABLE 2B
Exemplary Modified PRE with at least one introduced stop codon
5′ Position of
Introduced Stop Codon(s)
Relative to
Residue
3′ Position
corresponding
SEQ ID
of X Gene
to SEQ ID
X gene
Promoter
NOs
Start Codon
NOs: 1 or 125
Start Codon
Sequence
29, 126
9, 13, 17, 21
420, 424, 428,
Unmodified
Unmodified
432
30, 127
9, 13, 17
420, 424, 428
Unmodified
Unmodified
31, 128
9, 13, 21
420, 424, 432
Unmodified
Unmodified
32, 129
9, 17, 21
420, 428, 432
Unmodified
Unmodified
33, 130
13, 17, 21
424, 428, 432
Unmodified
Unmodified
34, 131
9, 13
420, 424
Unmodified
Unmodified
35, 132
9, 17
420, 428
Unmodified
Unmodified
36, 133
9, 21
420, 432
Unmodified
Unmodified
37, 134
13, 17
424, 428
Unmodified
Unmodified
38, 135
13, 21
424, 432
Unmodified
Unmodified
39, 136
17, 21
428, 432
Unmodified
Unmodified
40, 137
9
420
Unmodified
Unmodified
41, 138
13
424
Unmodified
Unmodified
42, 139
17
428
Unmodified
Unmodified
43, 140
21
432
Unmodified
Unmodified
2. Degradation Sequences
In some embodiments, the truncated X gene comprises a post-translational modification signal, which can include degradation sequences known to increase the rate by which a protein is degraded by the proteasome, thereby decreasing the protein's half-life. Non-limiting examples of such sequences include the incorporation of nucleotides encoding PEST sequences, ubiquitin binding domains, or amino acids following the N-end rule.
Pest sequences are amino acid regions rich in proline (P), glutamic acid (E), serine (S), and threonine (T). (Rogers S, Wells R, Rechsteiner M (1986). “Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis”. Science (journal) 234 (4774): 364-8. doi:10.1126/science.2876518. The presence of PEST regions can result in the rapid intracellular degradation of the proteins containing them. Thus, in some embodiments the variant X gene comprises nucleic acids encoding PEST regions.
In some embodiments, the variant X gene comprises nucleic acids encoding ubiquitin binding domains. Ubiquitin-binding domains (UBDs) are a collection of modular protein domains that non-covalently bind to ubiquitin. These motifs interpret and transmit information conferred by protein ubiquitination to control various cellular events, including proteasomal degradation. Thus, in some embodiments, the variant X gene can be operably linked to nucleic acids encoding ubiquitin binding domains such that ubiquitin ligases target the variant X protein for ubiquitin labeling, and thus degradation by the proteasome, more rapidly. Non-limiting examples of ubiquitin binding domains include CUE, GAT, GLUE, NZF, PAZ, UBA, UEV, UIM, and VHS. See Hicke, Ubiquitin-Binding Domains, doi:10.1038/nrm1701.
In some embodiments, the variant X gene comprises mutations corresponding to amino acid substitutions that decrease X protein stability according to the N-end rule. In the N-end rule, the identity of the amino acid that is exposed at the N-terminus of a polypeptide following the removal of the initiation methionine can impact the polypeptide's metabolic stability and half-life. (Varshaysky, A, 1996 The N-end rule: functions, mysteries, uses. Proc Natl Acad. Sci. USA 93(22):12142-12149. This amino acid can be called a destabilizing amino acid. Thus, in some embodiments, the modified PRE comprises mutations in the variant X gene in which the start codon is immediately followed by a codon encoding a destabilizing amino acid. Non-limiting examples of destabilizing amino acids include glycine, arginine, glutamic acid, phenylalanine, aspartate, cysteine, lysine, asparagine, serine, tyrosine, tryptophan, histidine, and leucine. See Varshaysky 1996; see also Gonda et al. (1989). “Universality and Structure of the N-end Rule.”. Journal of Biological Chemistry 264 (28): 16700-16712. In some embodiments, the modified PRE comprises mutations in the variant X gene in which the start codon is immediately followed by codons encoding a primary destabilizing amino acid and a secondary destabilizing amino acid. These mutations may correspond to pairs of amino acids, such as arginine-glutamic acid, or arginine-asparagine. In some embodiments, the arginine is the primary destabilizing amino acid and the glutamic acid or asparagine is the secondary destabilizing amino acid.
3. Other Modifications
Provided are polynucleotides containing a modified PRE that contains additional modifications in addition to any described above. In some embodiments, the additional modifications can be any modification of the X gene contained in the modified PRE that reduce or prevent one of more of the expression, degradation, stability, oncogenicity and/or immunogenicity of the X gene or encoded X protein. In some embodiments, the additional modifications alter, such as reduce, the transcription of the variant X gene and/or translation of the X protein encoded by the variant X gene.
In one embodiment, the modified PRE additionally contains a variant of an X gene start codon, which, in some cases, is introduced to restrict or prevent translation initiation. In some embodiments, the variant of the start codon contains one or more nucleotide differences compared to an unmodified X gene start codon, such as a wild-type hepatitis virus X gene start codon. For example, with reference to SEQ ID NO:1 or SEQ ID NO:125, the modified PRE can additionally contain a variant start codon containing one or more nucleotide differences compared to the start codon corresponding to nucleotide positions 411-413 of SEQ ID NO:1 or SEQ ID NO:125. It is readily within the level of a skilled artisan to identify a residue in another hepatitis PRE, such another WPRE, that corresponds to start codon positions 411-413 of SEQ ID NO:1 or SEQ ID NO:125 (see e.g. FIG. 2A-2E). The nucleotide differences can result in restricted or prevented translation initiation.
In some embodiments, the variant of the start codon can be the variant codon found at the position corresponding to an X gene start codon within the variant X gene reading frame of the modified WPRE sequence set forth in SEQ ID NO:119 or SEQ ID NO:216. For example, the variant of the start codon can be a codon corresponding to nucleotide residues 411-413 (TTG) of the sequence of nucleotides set forth in SEQ ID NO:119 or SEQ ID NO:216. In some embodiments, the variant of a start codon can be a codon of the X gene reading frame in the modified WPRE set forth in SEQ ID NO:120. For example, the variant of a start codon can be a codon corresponding to nucleotide residues 411-413 (GGG) of the sequence of nucleotides set forth in SEQ ID NO:120.
Tables 3A and 3B sets forth exemplary variant X genes and modified PRE polynucleotides containing a variant X gene, respectively, in which is contained at least one introduced stop codon and a variant start codon not present in a wild-type or unmodified X gene or PRE. The Table sets forth exemplary positions for introduction of the at least one stop codon in the variant X gene or modified PRE. The corresponding SEQ ID NO for sequences of the exemplary polynucleotides also are set forth. In some embodiments, the variant X gene comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 74-88 or 171-185. In some embodiments, the modified PRE comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 59-73 or 156-170. In some embodiments, provided are polynucleotides containing such PREs operably linked to nucleic acids encoding recombinant molecules, such as recombinant antigen receptors, CARs, TCRs, chimeric receptors, immunomodulators, immunostimulatory molecules, and/or transduction or expression markers, and expression cassettes and vectors containing the same.
In another embodiment, the modified PRE can additionally contain a variant promoter operably linked to the variant X gene therein. The variant promoter can contain one or more nucleotide differences compared to an unmodified hepatitis virus X gene promoter, such as a wild-type hepatitis virus X gene promoter. For example, with reference to SEQ ID NO:1 or SEQ ID NO:125, the variant X gene can contain one or more nucleotide differences compared to the WPRE X gene promoter set forth in SEQ ID NO:11 or set forth as nucleotides 7-20 of SEQ ID NO:11. In some embodiments, the nucleotide differences can result in one or more differences that result in restricted or prevention of transcription from the promoter.
In some embodiments, the modified PRE can contain a variant promoter that has the sequence of the variant promoter operably linked to the modified X gene within the modified PRE sequence set forth as SEQ ID NO:119 or SEQ ID NO:216. For example, the variant promoter can be a variant promoter comprising nucleotide residues corresponding to residues 391-411 (GGGGAAATCATCGTCCTTTCC; SEQ ID NO:217) or nucleotide residues 397-410 (ATCATCGTCCTTTC; SEQ ID NO:218), each of the sequence of nucleotides set forth in SEQ ID NO:119 or SEQ ID NO:216. In some embodiments, the modified PRE can contain a variant promoter that has the sequence of the variant promoter operably linked to the modified X gene within the modified PRE set forth in SEQ ID NO:120. For example, the variant promoter can be a variant promoter comprising nucleotide residues corresponding to residues 391-411 (GGGGAAGGTCTGCTGAGACTC; SEQ ID NO:219) or nucleotide residues 397-410 (GGTCTGCTGAGACT; SEQ ID NO:220), each of the sequence of nucleotides set forth in SEQ ID NO:120.
In some embodiments, the variant promoter can be a variant promoter that is a hepatitis X protein promoter known in the art, such as any described in Sugata et al. (1994) Virology, 205:314-320. In some embodiments, the variant X promoter can be a variant of a promoter set forth in SEQ ID NO:11 or a promoter that comprises nucleotides 6-22 of SEQ ID NO:10 that includes a mutation of the threonine (T) at a position corresponding to position 9 of SEQ ID NO:10 or a mutation of the glycine (G) at a position corresponding to position 10 of SEQ ID NO:10.
Table 3A and 3B sets forth exemplary variant X genes and modified PRE polynucleotides containing a variant X gene, respectively, in which is contained at least one introduced stop codon, a variant promoter and, optionally, a variant start codon not present in a wild-type or unmodified PRE. The Table sets forth exemplary positions for introduction of the at least one stop codon in a variant X gene or modified PRE. The corresponding SEQ ID NO for sequences of the exemplary polynucleotides also are set forth. In some embodiments, the modified PRE comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 89-118 or 186-215. In some embodiments, provided are polynucleotides containing such PREs operably linked to nucleic acids encoding recombinant molecules, such as recombinant antigen receptors, CARs, TCRs, chimeric receptors, immunomodulators, immunostimulatory molecules, and/or transduction or expression markers, and expression cassettes and vectors containing the same.
TABLE 3A
Exemplary Variant X gene with at least one introduced
stop codon and a modified X gene start codon
5′ Position of
Introduced Stop Codon(s)
Relative to
3′ Position
SEQ ID
of X Gene
X gene
NOs
Start Codon
Start Codon
74, 171
9, 13, 17, 21
Modified
75, 172
9, 13, 17
Modified
76, 173
9, 13, 21
Modified
77, 174
9, 17, 21
Modified
78, 175
13, 17, 21
Modified
79, 176
9, 13
Modified
80, 177
9, 17
Modified
81, 178
9, 21
Modified
82, 179
13, 17
Modified
83, 180
13, 21
Modified
84, 181
17, 21
Modified
85, 182
9
Modified
86, 183
13
Modified
87, 184
17
Modified
88, 185
21
Modified
TABLE 3B
Exemplary Variant X gene with at least one introduced stop
codon and a modified X gene start codon and/or promoter
5′ Position of
Introduced Stop Codon(s)
Relative to
Residue
3′ Position
corresponding
SEQ ID
of X Gene
to SEQ ID
X gene
Promoter
NOs
Start Codon
NOs: 1 or 125
Start Codon
Sequence
59, 156
9, 13, 17, 21
420, 424, 428,
Modified
Unmodified
432
60, 157
9, 13, 17
420, 424, 428
Modified
Unmodified
61, 158
9, 13, 21
420, 424, 432
Modified
Unmodified
62, 159
9, 17, 21
420, 428, 432
Modified
Unmodified
63, 160
13, 17, 21
424, 428, 432
Modified
Unmodified
64, 161
9, 13
420, 424
Modified
Unmodified
65, 162
9, 17
420, 428
Modified
Unmodified
66, 163
9, 21
420, 432
Modified
Unmodified
67, 164
13, 17
424, 428
Modified
Unmodified
68, 165
13, 21
424, 432
Modified
Unmodified
69, 166
17, 21
428, 432
Modified
Unmodified
70, 167
9
420
Modified
Unmodified
71, 168
13
424
Modified
Unmodified
72, 169
17
428
Modified
Unmodified
73, 170
21
432
Modified
Unmodified
89, 186
9, 13, 17, 21
420, 424, 428,
Unmodified
Modified
432
90, 187
9, 13, 17
420, 424, 428
Unmodified
Modified
91, 188
9, 13, 21
420, 424, 432
Unmodified
Modified
92, 189
9, 17, 21
420, 428, 432
Unmodified
Modified
93, 190
13, 17, 21
424, 428, 432
Unmodified
Modified
94, 191
9, 13
420, 424
Unmodified
Modified
95, 192
9, 17
420, 428
Unmodified
Modified
96, 193
9, 21
420, 432
Unmodified
Modified
97, 194
13, 17
424, 428
Unmodified
Modified
98, 195
13, 21
424, 432
Unmodified
Modified
99, 196
17, 21
428, 432
Unmodified
Modified
100, 197
9
420
Unmodified
Modified
101, 198
13
424
Unmodified
Modified
102, 199
17
428
Unmodified
Modified
103, 200
21
432
Unmodified
Modified
104, 201
9, 13, 17, 21
420, 424, 428,
Modified
Modified
432
105, 202
9, 13, 17
420, 424, 428
Modified
Modified
106, 203
9, 13, 21
420, 424, 432
Modified
Modified
107, 204
9, 17, 21
420, 428, 432
Modified
Modified
108, 205
13, 17, 21
424, 428, 432
Modified
Modified
109, 206
9, 13
420, 424
Modified
Modified
110, 207
9, 17
420, 428
Modified
Modified
111, 208
9, 21
420, 432
Modified
Modified
112, 209
13, 17
424, 428
Modified
Modified
113, 210
13, 21
424, 432
Modified
Modified
114, 211
17, 21
428, 432
Modified
Modified
115, 212
9
420
Modified
Modified
116, 213
13
424
Modified
Modified
117, 214
17
428
Modified
Modified
118, 215
21
432
Modified
Modified
B. Nucleic Acids Encoding Recombinant Molecules
In some embodiments, the modified PREs in the provided polynucleotides are operably linked to one or more nucleic acid encoding one or more molecule of interest, such as one or more recombinant and/or heterologous molecule, e.g., recombinant protein(s), such as heterologous protein(s). Such recombinant and/or heterologous molecules may include soluble proteins, e.g., secreted proteins, and/or cell surface proteins. In some embodiments, the molecule is or includes a recombinant receptor. Such recombinant receptors may include antigen receptors, such as functional non-TCR antigen receptors, including chimeric antigen receptors (CARs), and other antigen-binding receptors such as transgenic T cell receptors (TCRs). The receptors may also include other receptors, such as other chimeric receptors, such as receptors that bind to particular ligands and having transmembrane and/or intracellular signaling domains similar to those present in a CAR.
In some embodiments, the molecule is a soluble molecule, such as an immunomodulatory and/or immunostimulatory molecule, such as a cytokine, e.g., IL-2, IL-12, IL-6, 41BBL, CD40L, and/or soluble ligand or receptor such as a soluble ligand for an immune cell costimulatory molecule, e.g., CD40L, 41BBL, or a soluble antigen-binding molecule such as an scFv. Also among the molecules are expression or transduction markers and any other molecule(s) known for use in expression vectors and/or cassettes.
In some embodiments, the recombinant antigen receptor, e.g., CAR, specifically binds to one or more ligand on a cell or disease to be targeted, such as a cancer, infectious disease, inflammatory or autoimmune disease, or other disease or condition, including those described herein for targeting with the provided methods and compositions. Exemplary antigens are orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, 0EPHa2, ErbB2, 3, or 4, FBP, fetal acetylcholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1), a cyclin, such as cyclin A1 (CCNA1), and/or biotinylated molecules, and/or molecules expressed by and/or characteristic of or specific for HIV, HCV, HBV, HPV, and/or other pathogens and/or oncogenic versions thereof.
1. Chimeric Antigen Receptors
In some embodiments, the recombinant molecule is or includes a chimeric antigen receptor (CAR). The CAR is generally a genetically engineered receptor with an extracellular ligand binding domain linked to one or more intracellular signaling components. Such molecules typically mimic or approximate a signal through a natural antigen receptor and/or signal through such a receptor in combination with a costimulatory receptor.
In some embodiments, CARs are constructed with a specificity for a particular marker, such as a marker expressed in a particular cell type to be targeted by adoptive therapy, e.g., a cancer marker and/or any of the antigens described. Thus, the CAR typically includes one or more antigen-binding fragment, domain, or portion of an antibody, or one or more antibody variable domains, and/or antibody molecules. In some embodiments, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a variable heavy chain (VH) or antigen-binding portion thereof, or a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).
The antigen-specific binding or recognition component is generally linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR includes a transmembrane domain fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154. The transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and/or transmembrane domain(s).
In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
The receptor, e.g., the CAR, generally includes at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the antigen-binding portion is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR or other chimeric receptor includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25 or CD16.
In some embodiments, upon ligation of the CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptors to initiate signal transduction following antigen receptor engagement, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functional capability.
In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.
Primary cytoplasmic signaling sequences can in some aspects regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.
In some embodiments, the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS.
In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 co-stimulatory domains, linked to a CD3 intracellular domain. In some embodiments, a CAR can also include a transduction marker (e.g., tEGFR). In some embodiments, the intracellular signaling domain of the CD8+ cytotoxic T cells is the same as the intracellular signaling domain of the CD4+ helper T cells. In some embodiments, the intracellular signaling domain of the CD8+ cytotoxic T cells is different than the intracellular signaling domain of the CD4+ helper T cells.
In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. One example is a receptor including intracellular components of CD3-zeta, CD28, and 4-1BB.
In some embodiments, the recombinant molecule(s) encoded by nucleic acid(s) within the provided polynucleotides further include one or more marker, e.g., for purposes of confirming transduction or engineering of the cell to express the receptor and/or selection and/or targeting of cells expressing molecule(s) encoded by the polynucleotide. In some aspects, such a marker may be encoded by a different nucleic acid or polynucleotide, which also may be introduced during the genetic engineering process, typically via the same method, e.g., transduction by the same vector or type of vector.
In some aspects, the marker, e.g., transduction marker, is a protein and/or is a cell surface molecule. Exemplary markers are truncated variants of a naturally-occurring, e.g., endogenous markers, such as naturally-occurring cell surface molecules. In some aspects, the variants have reduced immunogenicity, reduced trafficking function, and/or reduced signaling function compared to the natural or endogenous cell surface molecule.
In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor (e.g., tEGFR).
In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.
In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self” by the immune system of the host into which the cells will be adoptively transferred.
In some embodiments, the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive transfer and encounter with ligand.
Antigen receptors, including CARs and recombinant TCRs, and production and introduction thereof, in some embodiments include those described, for example, in international patent application publication numbers WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 Mar. 18(2): 160-75.
2. T Cell Receptors (TCRs)
In some embodiments, the recombinant molecule(s) encoded by the nucleic acid(s) is or include a recombinant T cell receptor (TCR). In some embodiments, the recombinant TCR is specific for an antigen, generally an antigen present on a target cell, such as a tumor-specific antigen, an antigen expressed on a particular cell type associated with an autoimmune or inflammatory disease, or an antigen derived from a viral pathogen or a bacterial pathogen.
In some embodiments, the TCR is one that has been cloned from naturally occurring T cells. In some embodiments, a high-affinity T cell clone for a target antigen (e.g., a cancer antigen) is identified and isolated from a patient. In some embodiments, the TCR clone for a target antigen has been generated in transgenic mice engineered with human immune system genes (e.g., the human leukocyte antigen system, or HLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al. (2009) Clin Cancer Res. 15:169-180 and Cohen et al. (2005) J Immunol. 175:5799-5808. In some embodiments, phage display is used to isolate TCRs against a target antigen (see, e.g., Varela-Rohena et al. (2008) Nat Med. 14:1390-1395 and Li (2005) Nat Biotechnol. 23:349-354.
In some embodiments, after the T-cell clone is obtained, the TCR alpha and beta chains are isolated and cloned into a gene expression vector. In some embodiments, the TCR alpha and beta genes are linked via a picornavirus 2A ribosomal skip peptide so that both chains are coexpression. In some embodiments, the nucleic acid encoding a TCR further includes a marker to confirm transduction or engineering of the cell to express the receptor.
In some embodiments, the recombinant or heterologous molecule(s) encoded by the nucleic acid within the provided polynucleotide is or includes a nucleic acid molecule, such as an RNA, DNA, or artificial nucleic acid sequence, such as one designed for interference with expression or activity of a target mRNA, such as an short-interfering RNA (siRNA), short hairpin RNA (shRNA), or micro-RNA (miRNA). Such molecules may include those designed to interfere with expression or activity of molecules associated with, promoting, or inhibiting the activity of immune cells, such as immunomodulators, immunoinhibitory molecules, and immune checkpoint molecules. In some embodiments, a nucleotide siRNA or miRNA sequence (e.g. 21-25 nucleotides in length) can, for example, be produced from an expression vector by transcription of a short-hairpin RNA (shRNA) sequence, a longer (e.g. 60-80 nucleotide) precursor sequence, which is subsequently processed by the cellular RNAi machinery to produce either a siRNA or miRNA sequence. Alternatively, a nucleotide siRNA or miRNA sequence (e.g. 21-25 nucleotides in length) can, for example, be synthesized chemically. Chemical synthesis of siRNA or miRNA sequences is commercially available from such corporations as Dharmacon, Inc. (Lafayette, Colo.), Qiagen (Valencia, Calif.), and Ambion (Austin, Tex.). The RNA can be 10 to 30 nucleotides long, such as 19-25 or 21-25 nucleotides in length. For example, an siRNA sequence typically binds a unique sequence within a target mRNA with exact complementarity and results in the degradation of the target mRNA molecule. A siRNA sequence can bind anywhere within the mRNA molecule; sequences targeted by the siRNA include genes expressing a polypeptide of interest, or an upstream or downstream modulator of such a gene, e.g. an upstream or downstream modulator of a gene, such as a transcription factor that binds a gene promoter, a kinase or phosphatase that interacts with a polypeptide of interest, and polypeptides involved in regulatory pathways capable of influencing the polypeptide of interest. A miRNA sequence typically binds a unique sequence within a target mRNA with exact or less than exact complementarity and results in the translational repression of the target mRNA molecule. A miRNA sequence can bind anywhere within mRNA sequence, but generally binds within the 3′ untranslated region of the mRNA molecule.
C. Expression Cassettes and Viral Vectors
In some embodiments, the polynucleotide is provided as or within an expression cassette. The polynucleotide or expression cassette can be contained in an expression vector, such as a viral vector, for expression of the recombinant and/or heterologous molecule encoded by the nucleic acid.
1. Expression Cassette
In some embodiments, the expression cassette can contain the heterologous and/or recombinant nucleic acid under the control of a promoter and operably linked to the modified PRE, such as any described above. The expression cassette also can contain one or more other regulatory elements. In addition to the modified PRE, the nucleic acid, may be operably linked to other nucleic acid sequences, including but not limited to, promoters, enhancers, other post-transcriptional regulatory elements, polyadenylation signals, restriction enzyme sites, multiple cloning sites or coding segments.
a. Promoters
In some embodiments, the expression cassette includes a promoter operably linked to the nucleic acid molecule encoding the recombinant or heterologous protein. The promoter can comprise any promoter desired by the user as appropriate for the expression context. In some embodiments, a promoter can comprise a promoter of eukaryotic or prokaryotic origin that can provide high levels of constitutive expression across a variety of cell types and will be sufficient to direct the transcription of nucleic acid encoding the recombinant or heterologous protein in a cell. In some embodiments, the nucleic acid encoding the recombinant or heterologous protein is a distally located sequence, which is a sequence operably linked to the 5′ end of the promoter sequence. The promoter region can also include control elements for the enhancement or repression of transcription and can be modified as desired by the user and depending on the context.
In some embodiments, a promoter comprises a sequence that functions to position the start site for RNA synthesis. In some embodiments, the promoter comprises the TATA box. In some embodiments, the promoter lacks a TATA box, such as, for example, the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes. In such an embodiment, the promoter can contain a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. In some embodiments, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. To bring a coding sequence “under the control of” a promoter, one positions the 5′ end of the transcription initiation site of the transcriptional reading frame “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream” promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.
In some embodiments, the spacing between promoter elements is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In some embodiments in which the promoter is the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements can function either cooperatively or independently to activate transcription. In some embodiments, a promoter may be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
In some embodiments a promoter may be one that is naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as “endogenous.” In some embodiments an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
Alternatively, in some embodiments the coding nucleic acid segment may be positioned under the control of a recombinant and/or heterologous promoter and/or enhancer, which is not normally associated with the coding nucleic acid sequence in the natural setting. Such promoters or enhancers may include promoters or enhancers which in nature are operably linked to other genes within the species from which the nucleic acid is derived, and promoters or enhancers isolated from other species, such as from other prokaryotic or eukaryotic cells, and promoters or enhancers that are not “naturally occurring,” i.e., that contain different elements of different transcriptional regulatory regions, and/or mutations that alter expression compared with those found in any promoter or enhancer in nature. For example, exemplary promoters used in recombinant DNA construction include, but are not limited to, the β-lactamase (penicillinase), lactose, tryptophan (trp), RNA polymerase (pol) III promoters including, the human and murine U6 pol III promoters as well as the human and murine H1 RNA pol III promoters; RNA polymerase (pol) II promoters; cytomegalovirus immediate early promoter (pCMV), elongation factor-1 alpha (EF-1 alpha), and the Rous Sarcoma virus long terminal repeat promoter (pRSV) promoter systems. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions and methods disclosed herein (see U.S. Pat. Nos. 4,683,202 and 5,928,906, each incorporated herein by reference). Furthermore, in some embodiments the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well. Control sequences comprising promoters, enhancers and other locus or transcription controlling/modulating elements are also referred to as “transcriptional cassettes”.
In some embodiments, the promoter and/or enhancer is operably linked to effectively direct the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, (see, for example Sambrook et al., 1989, incorporated herein by reference). The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous for gene therapy or for applications such as the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.
In some embodiments, a T3, T7 or SP6 cytoplasmic expression system can be employed. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
In some embodiments, an inducible promoter can be used. As used herein, an “inducible promoter” refers to a transcriptional control element that can be regulated in response to specific signals. An inducible promoter is transcriptionally active when bound to a transcriptional activator, which in turn is activated under a specific set of conditions, for example, in the presence of a particular combination of chemical signals that affect binding of the transcriptional activator to the inducible promoter and/or affect function of the transcriptional activator itself. Thus, an inducible promoter is a promoter that, either in the absence of an inducer, does not direct expression, or directs low levels of expression, of a nucleic acid sequence to which the inducible promoter is operably linked; or exhibits a low level of expression in the presence of a regulating factor that, when removed, allows high-level expression from the promoter, for example, the tet system. In the presence of an inducer, an inducible promoter directs transcription at an increased level.
In some embodiments, the tetracycline-(tet)-regulatable system, which is based on the inhibitory action of the tet repression (tetr) of Escherichia coli on the tet operator sequence (TECO), can be modified for use in mammalian systems and used as a regulatable element for expression cassettes. These systems are well known to those of ordinary skill in the art. (See, Goshen and Badgered, Proc. Natl. Acad. Sci. USA 89: 5547-51 (1992), Shockett et al., Proc. Natl. Acad. Sci. USA 92:6522-26 (1996), Lindemann et al., Mol. Med. 3:466-76 (1997)).
b. Other Regulatory Elements
In some embodiments, the expression cassette can additionally include an enhancer that is operably linked to the nucleic acid encoding the recombinant protein, e.g. heterologous protein.
In some embodiments, internal ribosome binding sites (IRES) elements are operably linked to expression cassettes to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5′-methylated cap-dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). Non-limiting examples of IRES elements include, but are not limited to, IRES elements of the picornavirus family (polio and encephalomyocarditis) (Pelletier and Sonenberg, 1988) or an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
In some embodiments, the vectors or constructs will comprise at least one termination signal operably linked to the nucleic acid encoding a recombinant protein, such as a heterologous protein. A “termination signal” or “terminator” is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. In some embodiments, a terminator may be used in vivo to achieve desirable message levels.
In some embodiments comprising eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3′ end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in some embodiments involving eukaryotes, the terminator comprises a signal for the cleavage of the RNA. In some embodiments, the terminator signal promotes polyadenylation of the message. The terminator and/or polyadenylation site elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
Terminators include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.
In some embodiments involving eukaryotic gene expression, the expression cassette may be operably linked to a polyadenylation signal to effect proper polyadenylation of the transcript. Any such sequence may be employed. Some examples include the SV40 polyadenylation signal or the bovine growth hormone polyadenylation signal, convenient and known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.
In some embodiments, the expression cassette or vector contains one or more origins of replication sites (often termed “ori”) in order to propagate in a host cell. An origin of replication is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.
2. Viral Vectors
In some embodiments, the modified PREs, nucleic acids, and/or cassettes are provided within viral vectors. The nucleic acid within the vector can be provided as an expression cassette under the control of a promoter. The viral vectors can be used to transfer the nucleic acid molecule into cells for expression of the heterologous or recombinant protein therein.
Exemplary viral vectors include retroviral vectors, such as lentiviral or gammaretroviral vectors, vectors derived from simian virus 40 (SV40), adenoviruses, and adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into cells using retroviral vectors, such as lentiviral vectors or gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 Nov. 29(11): 550-557. Retroviruses are useful as delivery vectors because of their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell lines (Miller, 1992).
In some embodiments, genetic transfer is accomplished via lentiviral vectors. Lentiviruses, in contrast to other retroviruses, in some contexts may be used for transducing certain non-dividing cells.
Non-limiting examples of lentiviral vectors include those derived from a lentivirus, such as Human Immunodeficiency Virus 1 (HIV-1), HIV-2, an Simian Immunodeficiency Virus (SIV), Human T-lymphotropic virus 1 (HTLV-1), HTLV-2 or equine infection anemia virus (E1AV). For example, lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted, making the vector safer for therapeutic purposes. Lentiviral vectors are known in the art, see Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136). In some embodiments, these viral vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection, and for transfer of the nucleic acid into a host cell. Known lentiviruses can be readily obtained from depositories or collections such as the American Type Culture Collection (“ATCC”; 10801 University Blvd., Manassas, Va. 20110-2209), or isolated from known sources using commonly available techniques.
In some embodiments, two components are involved in making a virus-based gene delivery system: first, packaging plasmids, encompassing the structural proteins as well as the enzymes necessary to generate a viral vector particle, and second, the viral vector itself, i.e., the genetic material to be transferred. Biosafety safeguards can be introduced in the design of one or both of these components. In some embodiments, the packaging plasmid can contain all HIV-1 proteins other than envelope proteins (Naldini et al., 1998). In some embodiments, viral vectors can lack additional viral genes, such as those that are associated with virulence, e.g. vpr, vif, vpu and nef, and/or Tat, a primary transactivator of HIV. In some embodiments, packaging systems for lentiviral vectors, such as HIV-based lentiviral vectors, include separate packaging plasmids that together comprise only three genes of the parental virus: gag, pol and rev, which reduces or eliminates the possibility of reconstitution of a wild-type virus through recombination.
In some aspects of the provided viral vectors, the heterologous nucleic acid encoding a recombinant protein, such as provided as part of an expression cassette containing the transgene under the control of a promoter, is contained and/or located between the 5′ LTR and 3′ LTR sequences of the vector genome, including wildtype LTRs or portions or chimeric portions thereof. In some embodiments, the viral vector, such as an HIV viral vector, lacks additional transcriptional units. In some embodiments, the vector genome can contain deletion in the U3 region of the 3′ LTR of the DNA used to produce the viral vector RNA, which can generate a self-inactivating (SIN) vector. This deletion can then be transferred to the 5′ LTR of the proviral DNA during reverse transcription. In some embodiments, the 3′ LTR is deleted for the promoter and the enhancer of U3. In some embodiments enough sequence can be eliminated, including the removal of a TATA box, to abolish the transcriptional activity of the LTR. This can prevent production of full-length vector RNA in transduced cells. Thus, some embodiments include a deletion in the U3 region of the 3′ LTR of the DNA. In some embodiments, this does not affect vector titers or the in vitro or in vivo properties of the vector.
In some embodiments, the viral vector genome may also contain additional genetic elements. The types of elements that can be included in the constructs are not limited in any way and can be chosen by one with skill in the art. In some embodiments, the vector genome contains sequences derived from a viral genome (e.g. lentiviral genome) that are non-coding regions of the genome that facilitate or provide recognition signals for DNA or RNA synthesis and processing. In some embodiments, such sequences can include cis-acting sequences that can be involved in packaging or encapsidation, reverse transcription and transcription and/or gene transfer or integration. In some embodiments, cis-activating sequences provided as part of the viral vector are derived from the same lentivirus or retrovirus-like organism.
In some embodiments, a signal that facilitates nuclear entry of the viral genome in the target cell may be included. An example of such a signal is the Flap sequence (also called a DNA Flap sequence) formed from the cPPT and CTS components that are part of the pol gene of a viral vector genome, such as a lentiviral vector genome. In some embodiments, a Flap sequence includes a portion of viral nucleic acid that contains a cPPT and/or a CTS region, but in which is deleted 5′ and 3′ portions of the pol gene that are not necessary for Flap function. In some cases, the viral vector does not contain a functional Flap region. As discussed below, in some embodiments a viral vector contains viral nucleic acid containing a variant Flap that lacks all or a portion of one or both of the cPPT and CTS region.
In some embodiments, the lentiviral vector genome can contain elements selected among a splice donor site (SD), a splice acceptor site (SA) and/or a Rev-responsive element (RRE). In some embodiments, RRE is provided to allow export of viral messenger RNA from the nucleus to the cytosol after binding of the Rev protein provided as part of a helper plasmid during viral packaging. In some embodiments, the vector genome can contain the psi (w) packaging signal, which, in some cases, can be derived from the N-terminal fragment of the gag ORF. In some embodiments, the psi packaging signal sequence can be modified by frameshift mutation(s) in order to prevent any interference of a possible transcription/translation of gag peptide, with that of the transgene.
In some embodiments, provided is a viral vector, such as a lentiviral vector, that contains a recombinant genome containing in order between the 5′ and 3′ LTR sequences of the vector genome: an RRE; a polynucleotide containing viral nucleic acid comprising a functional DNA Flap containing a cPPT and CTS that is inserted upstream of a promoter controlling expression of a polynucleotide encoding a recombinant protein; a transgene containing a promoter controlling expression of a polynucleotide encoding the recombinant protein, such as any described above and the polynucleotide encoding the recombinant protein, such as an antigen receptor (e.g. a CAR); and a polynucleotide containing a modified PRE, such as any provided herein, operably linked to the nucleic acid encoding the recombinant protein such as any provided herein. In some embodiments, the recombinant genome comprises the sequence 5′ LTR-RRE-cPPT-CTS-transgene(s)-modified PRE-3′ LTR. In some embodiments, the modified PRE in the viral vector, such as lentiviral vector, has the sequence of nucleotides set forth in any of SEQ ID NOS: 29-43, 59-73, 89-118, 126-140, 156-170 or 186-215. In some embodiments, the modified PRE is or comprises the sequence of nucleotides set forth in SEQ ID NO:29 or SEQ ID NO:126. In some embodiments, the cPPT and CTS are inserted as a fragment of a viral nucleic acid sequence containing an unmodified or wild-type DNA Flap, such as that is or comprises the exemplary sequence set forth in SEQ ID NO:121 or a functional fragment thereof that contains the cPPT and CTS sequences. In some embodiments, the lentiviral vector is an HIV-1 derived lentiviral vector.
In some embodiments, among the provided polynucleotides, including viral vectors, are those containing variations in viral Flap sequences (deemed “variant Flap” polynucleotides or sequences). Such polynucleotides include those containing one or more modifications, e.g., deletion(s), within a viral Flap sequence within the polynucleotide. The variations can include complete deletion of a Flap sequence, or sub-part thereof, within a viral sequence of the polynucleotide. Such polynucleotides include viral vectors, such as a lentiviral vector, containing such variant Flap sequences.
In general, a viral Flap sequence is a viral regulatory region containing a polynucleotide sequence including two components: a central polypurine tract (cPPT) and a central termination sequence (CTS). See, e.g. Iglesias et al., Retrovirology 2011, 8:92. A non-limiting, exemplary cPPT sequence is provided in SEQ ID NO:123. A non-limiting, exemplary CTS sequence is provided in SEQ ID NO:124. In an exemplary wild-type HIV-1, the cPPT and CTS can be operably linked by approximately 70-100 nucleotides. It is reported that the cPPT and CTS are positioned at or about the center of the lentiviral vector genome, such that following reverse transcription, DNA encoded by the cPPT and CTS sequences form an approximately 100 nucleotide overlap, or “DNA Flap”, at the center of the genome. When inserted in viral vectors, such as lentiviral derived vectors, the polynucleotide permitting the DNA flap to be produced during retro-transcription, stimulates gene transfer efficiency and complements the level of nuclear import to wild-type levels.
In some embodiments, the variant Flap polynucleotide contains deletions of all or a portion of nucleotides in the CTS, the cPPT or both the CTS and cPPT with reference to a sequence of a wild-type or unmodified Flap sequence. In some embodiments, the sequence of a wild-type or unmodified Flap sequence is a viral nucleic acid sequence containing the cPPT and CTS that is derived from a retrovirus, such as a lentivirus, or from a retrovirus-like organism such as retrotransposon. In some embodiments, the sequence of a wild-type or unmodified Flap is from a human retrovirus or lentivirus, such as an HIV retrovirus (e.g. HIV-1 or HIV-2). In some embodiments, the sequence of a wild-type or unmodified DNA Flap is from an CAEV (Caprine Arthritis Encephalitis Virus) virus, an EIAV (Equine Infectious Anaemia Virus) virus, a VI SNA virus, the SIV (Simian Immunodeficiency Virus) virus or a FIV (Feline Immunodeficiency Virus) virus. In some embodiments, the viral nucleic acid sequence containing the wild-type or unmodified Flap can contain additional flanking sequence present in the viral genome. In some embodiments, the polynucleotide containing a wild-type or unmodified Flap can have a sequence of about 80 to about 200 nucleotides, depending on its origin and preparation. In some embodiments, the wild-type or unmodified Flap sequence can be prepared synthetically (chemical synthesis) or by amplification of polynucleotides containing the Flap from any retrovirus, such as from a lentivirus nucleic acid, for example by polymerase chain reaction (PCR).
In some embodiments, a wild-type or unmodified Flap sequence can be derived from a polynucleotide sequence present in HIV-1, such as corresponding to a 178 base pair fragment from positions 4757 to 4935 or a 179 base pair fragment from positions 4746 to 4935 of the exemplary HIV-1 vector pNL4-3 (GenBank No. AF324493), or a shorter or longer sequence present therein containing the cPPT and CTS that is capable of producing the Flap upon reverse transcription. In some embodiments, an exemplary polynucleotide sequence containing a wild-type or unmodified Flap is or comprises the sequence set forth in SEQ ID NO:121, a sequence that exhibits at least 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:121 or a functional fragment thereof containing the cPPT and CTS for producing the Flap structure upon reverse transcription. In some embodiments, the cPPT is or comprises a sequence corresponding to nucleotides 30-45 of SEQ ID NO:121 (set forth in SEQ ID NO:123) and the CTS is or comprises a sequence corresponding to nucleotides 117-143 of SEQ ID NO:121 (set forth in SEQ ID NO:124).
In some embodiments of the provided polynucleotides or viral vectors comprising a variant, e.g., deleted, Flap sequence, the variant Flap sequence can be one in which all or a portion of the Flap region of the viral nucleic acid(s) within the vector is deleted and/or mutated, including all or a portion of the cPPT and/or CTS. In some embodiments, the variant Flap sequence includes viral sequences completely lacking or substantially lacking the entire Flap sequence or portion thereof. In some embodiments, the variant Flap contains deletion(s) in the cPPT, the CTS, or both the cPPT and CTS. In some embodiments the variant Flap can contain a cPPT with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotide deletions, which can be contiguous nucleotide deletions, compared to a wild-type or unmodified cPPT sequence, such as compared to the exemplary unmodified cPPT set forth in SEQ ID NO:123. In some embodiments, the cPPT can be deleted entirely. In some embodiments, the variant DNA Flap can contain a CTS with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotide deletions, which can be contiguous nucleotide deletions, compared to a wild-type or unmodified CTS sequence, such as compared to the exemplary unmodified CTS set forth in SEQ ID NO:124. In some embodiments, the CTS can be deleted entirely.
In some embodiments, the variant Flap polynucleotide contains deletion of all or a contiguous portion of the cPPT and contains a wild-type CTS. In some embodiments, the variant Flap polynucleotide contains a wild-type cPPT and contains deletion of all or a contiguous portion the CTS.
In some embodiments, the variant Flap contains a deletion of all or a portion of the cPPT and contains deletion of all or a portion of the CTS. In some embodiments, the variant DNA Flap can contain a cPPT with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotide deletions, which can be contiguous nucleotide deletions, compared to a wild-type or unmodified cPPT, such as compared to the exemplary unmodified cPPT set forth in SEQ ID NO:123, or can have the cPPT deleted entirely and contain a CTS with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotide deletions, which can be contiguous nucleotide deletions, compared to a wild-type or unmodified CTS, such as compared to the exemplary unmodified CTS of SEQ ID NO:124, or have the CTS deleted entirely.
In some embodiments, the polynucleotide containing a variant Flap sequence is or comprises a sequence of nucleotides that exhibits at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 78%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 90% or 95% sequence identity to SEQ ID NO:121, but that lacks all or a portion of the cPPT and/or CTS present in SEQ ID NO:121. In some embodiments, the polynucleotide containing a variant Flap sequence or polynucleotide is, comprises or contains a portion having a nucleotide sequence with at least or about at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:122 in which all or a portion of a cPPT and/or CTS is deleted. A non-limiting, exemplary variant Flap polynucleotide is provided as SEQ ID NO:122.
In some embodiments, the deletion of all or a portion of nucleotides in the CTS and/or the cPPT in a provided variant Flap is introduced into a polynucleotide sequence containing an unmodified or wild-type Flap by recombinant DNA techniques. In some embodiments, the variant Flap polynucleotide is produced synthetically, such as by chemical synthesis.
In some embodiments, the polynucleotide, e.g., viral vector, contains a variant Flap sequence and a PRE. In some embodiments, the PRE can be a modified PRE, such as any described above. Thus, in some embodiments, the polynucleotides contain nucleotide sequences containing variant Flap polynucleotides and containing modified PRE polynucleotides. In some embodiments, such a polynucleotide and/or viral vector contains the sequence of SEQ ID NO: 122 or one having at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:122 in which all or a portion of the cPPT and CTS are deleted, and a sequence for the modified PRE set forth in SEQ ID NO:29 or SEQ ID NO:126 or one having at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:29 or SEQ ID NO:126 containing the introduced stop codon modifications.
In some embodiments, provided is a viral vector, such as a lentiviral vector, that contains a recombinant genome containing in order between the 5′ and 3′ LTR sequences of the vector genome: an RRE; a polynucleotide containing viral nucleic acid comprising a variant DNA Flap containing a deletion of all or a portion of the cPPT and CTS, which variant DNA Flap is inserted upstream of a promoter controlling expression of a polynucleotide encoding a recombinant protein; a transgene containing a promoter controlling expression of a polynucleotide encoding the recombinant protein, such as any described above and the polynucleotide encoding the recombinant protein, such as an antigen receptor (e.g. a CAR); and a polynucleotide containing a modified PRE, such as any provided herein, operably linked to the nucleic acid encoding the recombinant protein such as any provided herein. In some embodiments, the recombinant genome comprises the sequence 5′ LTR-RRE-variant DNA Flap-transgene(s)-modified PRE-3′ LTR. In some embodiments, the modified PRE in the viral vector, such as lentiviral vector, has the sequence of nucleotides set forth in any of SEQ ID NOS: 29-43, 59-73, 89-118, 126-140, 156-170 or 186-215. In some embodiments, the modified PRE is or comprises the sequence of nucleotides set forth in SEQ ID NO:29 or SEQ ID NO:126. In some embodiments, the polynucleotide containing viral nucleic acid containing a variant DNA Flap is or comprises the sequence of nucleotides set forth in SEQ ID NO: 122. In some embodiments, the lentiviral vector is an HIV-1 derived lentiviral vector.
In some embodiments, a polynucleotide, such as a viral vector, containing a variant DNA Flap as provided and/or a modified PRE as provided can be used to transfer a nucleic acid molecule encoding a recombinant or heterologous protein contained therein into cells for transduction and expression of such protein. In some embodiments, while still resulting in successful transduction of cells, such transduction by the provided viral vectors, in some cases, can be less than, such as up to or up to about a 1.1-fold, 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold or more less than or decrease of, transduction as compared to transduction using the same or substantially similar amount and concentration of a corresponding viral vector containing substantially the same genome but that contains an unmodified or wild-type DNA Flap sequence in which is present the cPPT and CTS for formation of the DNA Flap structure. In some cases, where higher transduction efficiency is desired, transduction efficiency can be increased by increasing the volume or the concentration of the viral vector used to transduce the cells. In other cases, the number of transduced cells can be enriched or increased by cell sorting and expansion of transduced cells.
In some embodiments, the vector also can contain sequences for propagation in a host cell, such as a prokaryotic host cell. In some embodiments, the nucleic acid of the viral vector contains one or more origins of replication for propagation in a prokaryotic cell, such as a bacterial cell. In some embodiments, vectors that include a prokaryotic origin of replication also may contain a gene whose expression confers a detectable or selectable marker such as drug resistance.
3. Preparation of Viral Vector Particles
In some embodiments, the nucleic acid, e.g., one encoding the desired sequence, such as the polynucleotide or expression cassette, is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components may be constructed. A recombinant plasmid also can be employed containing a polynucleotide, such as expression cassette, containing nucleic acid encoding a recombinant protein under the operable control of a modified PRE. When a recombinant plasmid together with the retroviral LTR and packaging sequences is introduced into a special cell line (e.g., by calcium phosphate precipitation for example), the packaging sequence may permit the RNA transcript of the recombinant plasmid to be packaged into viral particles, which then may be secreted into the culture media. The media containing the recombinant retroviruses in some embodiments is then collected, optionally concentrated, and used for gene transfer.
In some embodiments, a packaging cell line is transfected with one or more plasmid vectors containing the components necessary to generate the particles. The packaging cell line can express or be made to express essential lentiviral (e.g. HIV-1) genes to allow the generation of lentiviral particles. These genes can be expressed by several plasmids. In some embodiments, multiple vectors are utilized to separate the various genetic components that generate the retroviral vector particles. In some such embodiments, providing separate vectors to the packaging cell reduces the chance of recombination events that might otherwise generate replication competent viruses.
In some embodiments, a packaging cell line can be transfected with a lentiviral expression plasmid containing a cis-acting psi (Y) packaging sequence and the transgene gene inserted between the lentiviral LTRs to allow target cell integration; a packaging plasmid or plasmids encoding the pol, gag, rev and/or tat viral genes and, in some cases, containing the rev-response element (RRE) and a pseudotyping plasmid, such as a plasmid encoding an envelope protein, such as the G protein of the Vesicular Stomatitis Virus (VSV-G) envelope gene.
In some embodiments, a packaging cell line is transfected with a plasmid containing the viral vector genome, including the LTRs, the cis-acting packaging sequence and the sequence of interest, i.e. a nucleic acid encoding a recombinant protein, (e.g. an antigen receptor, such as a CAR) along with several helper plasmids encoding the virus enzymatic and/or structural components, such as Env, Gag, pol and/or rev. In some embodiments, a GagPol packaging plasmid containing the gag and pol genes encoding for structural and enzymatic components and a Rev plasmid containing the rev gene encoding for Rev regulatory protein are separately introduced into a packaging cell line. In some embodiments, a single plasmid vector having all of the retroviral components can be used. In some embodiments, an envelope plasmid encoding an env gene also can be introduced, which, in some cases, can result in viral particles pseudotyped with alternative Env proteins. In some embodiments, the retroviral vector particle, such as lentiviral vector particle, is pseudotyped to increase the transduction efficiency of host cells. For example, a retroviral vector particle, such as a lentiviral vector particle, is pseudotyped with a VSV-G glycoprotein, which provides a broad cell host range extending the cell types that can be transduced.
The env gene can be derived from any appropriate virus, such as a retrovirus. In some embodiments, the env is an amphotropic envelope protein which allows transduction of cells of human and other species. Some embodiments use retroviral-derived env genes, including, but not limited to: Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus (HaMuSV or HSV), murine mammary tumor virus (MuMTV or MMTV), gibbon ape leukemia virus (GaLV or GALV), human immunodeficiency virus (HIV) and Rous sarcoma virus (RSV). In some embodiments, other env genes such as Vesicular stomatitis virus (VSV) protein G (VSVG), that of hepatitis viruses, and of influenza also can be used.
In some embodiments, the packaging plasmid providing the viral env nucleic acid sequence is associated operably linked with regulatory sequences, e.g., a promoter or enhancer. The regulatory sequence in some embodiments can be any eukaryotic promoter or enhancer, including for example, EF1α, PGK, the Moloney murine leukemia virus promoter-enhancer element, the human cytomegalovirus enhancer, the vaccinia P7.5 promoter or the like. In some cases, such as the Moloney murine leukemia virus promoter-enhancer element, the promoter-enhancer elements are located within or adjacent to the LTR sequences. In some embodiments, the regulatory sequence is one which is not endogenous to the lentivirus from which the vector is being constructed. Thus, if the vector is being made from SIV, the SIV regulatory sequence found in the SIV LTR may be replaced by a regulatory element which does not originate from SIV.
In some embodiments, the viral vectors and the packaging plasmids are introduced via transfection or infection into the packaging cell line. The packaging cell line produces viral vector particles that contain the viral vector genome. Methods for transfection or infection are well known. Non-limiting examples include calcium phosphate, DEAE-dextran and lipofection methods, electroporation and microinjection. After cotransfection of the packaging plasmids and the transfer vector to the packaging cell line, the viral vector particles are recovered from the culture media and tittered by standard methods used by those of skill in the art. Thus, the packaging plasmids in some embodiments are introduced into human cell lines by these methods, generally together with a dominant selectable marker, such as neomycin, DHFR, Glutamine synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. The selectable marker gene can be linked physically to the packaging genes in the construct.
In some embodiments, viral vector particles can be produced by stable cell lines wherein the packaging functions are configured to be expressed. Suitable packaging cells are known including, for example, U.S. Pat. No. 5,686,279; and Ory et al., (1996). The packaging cells with a lentiviral vector incorporated in them form producer cells. Producer cells are thus cells or cell-lines that can produce or release viral vector particles carrying the gene of interest. In some embodiments, these cells can further be anchorage dependent, which means that these cells will grow, survive, or maintain function optimally when attached to a surface such as glass or plastic. In some embodiments, the producer cells may be neoplastically transformed cells. In some embodiments, host cells for transfection with the lentiviral vector and packaging plasmids include, for example, mammalian primary cells; established mammalian cell lines, such as COS, CHO, HeLa, NIH3T3, 293T and PC12 cells; amphibian cells, such as Xenopus embryos and oocytes; other vertebrate cells; insect cells (for example, Drosophila), yeast cells (for example, S. cerevisiae, S. pombe, or Pichia pastoris) and prokaryotic cells (for example, E. coli).
In some embodiments, lentiviral vectors can be produced in a packaging cell line, such as an exemplary HEK 293T cell line, by introduction of plasmids to allow generation of lentiviral particles. Approximately two days after transfection of cells, e.g. HEK 293T cells, the cell supernatant contains recombinant lentiviral vectors, which can be used to transduce the target cells. Once in the target cells, the viral RNA is reverse-transcribed, imported into the nucleus and stably integrated into the host genome. One or two days after the integration of the viral RNA, the expression of the recombinant protein can be detected.
D. Methods of Production and Methods of Engineering Cells
Also provided are methods of producing the polynucleotides containing the modified PREs, and/or cassettes, vectors, and/or engineered cells containing the same. In some embodiments, the methods include introducing the modification(s) into a wild-type or unmodified PRE sequence or another starting modified PRE sequence, such as one known to have a particular degree of activity. In some embodiments, such modifications are effected by mutating an existing vector.
The methods further include those for engineering cells by introducing into a cell a polynucleotide containing a modified PRE as provided herein, operably linked to a nucleic acid encoding the recombinant molecule, such as the recombinant protein, such as the heterologous protein, e.g. an antigen receptor, such as a TCR or CAR, or other chimeric receptor. The polynucleotide can be introduced into the cells in the context of a viral vector particle containing in its genome the provided polynucleotide, including the nucleic acid encoding the recombinant molecule, such as the heterologous protein. Also provided are cells containing the polynucleotides, cassettes, and/or vectors with the modified PREs and nucleic acids and compositions containing the same and/or produced by the provided methods.
The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). In some embodiments, the cells are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, and re-introducing them into the same subject, before or after cryopreservation.
Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naïve T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MALT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
The genetic engineering generally involves introduction of the polynucleotide(s) into the cell, such as by retroviral transduction, transfection, or transformation, as described above.
In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.
In some contexts, overexpression of a stimulatory factor (for example, a lymphokine or a cytokine) may be toxic to a subject. Thus, in some contexts, the engineered cells include gene segments that cause the cells to be susceptible to negative selection in vivo, such as upon administration in adoptive immunotherapy. For example in some aspects, the cells are engineered so that they can be eliminated as a result of a change in the in vivo condition of the subject to which they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell II:223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).
In some aspects, the cells further are engineered to promote expression of cytokines or other factors. Various methods for the introduction of genetically engineered components, e.g., antigen receptors, e.g., CARs, are well known and may be used with the provided methods and compositions.
In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).
Other approaches and vectors for transfer of the nucleic acids encoding the recombinant products are those described, e.g., in international patent application, Publication No.: WO2014055668, and U.S. Pat. No. 7,446,190.
Among additional nucleic acids, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.
In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for introduction of the nucleic acid encoding the modified PRE may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig.
In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.
In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, a washing step is accomplished in a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++/Mg++ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.
In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques.
For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (markerhigh) on the positively or negatively selected cells, respectively.
In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.
In embodiments, memory T cells are present in both CD62L+ and CD62L− subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L−CD8+ and/or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.
In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.
CD4+ T helper cells are sorted into naïve, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L− and CD45RO−.
In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher© Humana Press Inc., Totowa, N.J.).
In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynabeads or MACS beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.
In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.
The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.
In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.
In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.
In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.
In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Number WO2009/072003, or US 20110003380 A1.
In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.
In some aspects, the separation and/or other steps is carried out using CliniMACS system (Miltenyi Biotic), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.
The CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.
In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system in some aspects is equipped with a cell processing unit that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood may be automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.
In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.
In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are generally then frozen to −80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.
In some embodiments, the provided methods include cultivation, incubation, culture, and/or genetic engineering steps. For example, in some embodiments, provided are methods for incubating and/or engineering the depleted cell populations and culture-initiating compositions.
Thus, in some embodiments, the cell populations are incubated in a culture-initiating composition. The incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.
In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agents include IL-2 and/or IL-15, for example, an IL-2 concentration of at least about 10 units/mL.
In some aspects, incubation is carried out in accordance with techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, the T cells are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.
In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.
In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigen-specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.
II. Compositions, Methods and Uses
A. Pharmaceutical Compositions and Formulations
Also provided are compositions including the polynucleotides, cassettes, vectors, viral particles and/or cells into which such polynucleotides have been introduced or otherwise containing the same. In some embodiments, provided are pharmaceutical compositions containing cells that have been engineered to express a recombinant protein by transduction methods employing any of the provided vectors or viral particles containing the same. Among the compositions are compositions for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.
The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
In some aspects, the choice of carrier is determined in part by the particular cell and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).
Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.
In some embodiments, the composition includes the cells in an amount effective to reduce burden of the disease or condition, and/or in an amount that does not result in CRS or severe CRS in the subject and/or to effect any of the other outcomes of the methods as described herein.
The cells and compositions may be administered using standard administration techniques, formulations, and/or devices. Administration of the cells can be autologous or allogeneic. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. Parenteral infusions may include intramuscular, intravenous, intraarterial, intraperitoneal, intrathoracic, intracranial, and/or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration of the cells. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyoi (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.
Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
B. Therapeutic and Prophylactic Methods and Uses
Provided are methods of treating a disease or condition in a subject using the provided polynucleotides, cassettes, vectors, cells containing the same, proteins and other molecules encoded thereby, and compositions thereof, such as those in which the recombinant or heterologous molecule encoded by the nucleic acid is a recombinant receptor, such as an antigen receptor, e.g. a CAR or a TCR. In some embodiments, the provided methods include administering to a subject having a disease or condition the provided cells or populations of cells, or pharmaceutical compositions thereof, that have been engineered to express a recombinant protein by transduction methods employing any of the provided vectors or viral particles containing the same. Also provided are uses and compositions for use in treating a disease or condition in a subject employing the provided cells or populations of cells, or compositions thereof, engineered to contain a recombinant receptor, such as an antigen receptor, e.g. a CAR or a TCR.
In some embodiments, the disease or condition treated or prevented is a cancer, an autoimmune disorder, and/or an infectious disease, such as a viral disease, and/or is one or more other diseases, conditions, and/or disorders.
In some embodiments, the cells, compositions, and/or polynucleotides, e.g., vectors, are administered to a subject or patient having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, cells and compositions prepared by the provided methods, such as engineered compositions and end-of-production compositions following incubation and/or other processing steps, are administered to a subject, such as a subject having or at risk for the disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptom of, the disease or condition, such as by lessening tumor burden in a cancer expressing an antigen recognized by an engineered T cell.
In the provided methods or uses, the recombinant or heterologous protein may comprise a recombinant receptor, such as an antigen receptor, e.g. a CAR or a TCR, that specifically binds to a ligand expressed by the disease or condition or a cell or tissue thereof. For example, in some embodiments, the receptor is an antigen receptor and the ligand is an antigen specific for and/or associated with the disease or condition.
Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.
In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.
In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.
The subject, e.g., patient, to whom the cells, cell populations, or compositions are administered is a mammal, and typically is a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent. In some examples, the patient or subject is a validated animal model for disease, adoptive cell therapy, and/or for assessing toxic outcomes such as cytokine release syndrome (CRS).
Among the diseases, conditions, and disorders are tumors, including solid tumors, hematologic malignancies, and melanomas, and infectious diseases, such as infection with a virus or other pathogen, e.g., HIV, HCV, HBV, HPV, CMV, and parasitic disease. In some embodiments, the disease or condition is a tumor, cancer, malignancy, neoplasm, or other proliferative disease. Such diseases include but are not limited to leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), ALL, non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B cell lymphoma, B cell malignancies, cancers of the colon, lung, liver, breast, prostate, ovarian, skin (including melanoma), bone, and brain cancer, ovarian cancer, epithelial cancers, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin lymphoma, cervical carcinoma, colorectal cancer, glioblastoma, neuroblastoma, Ewing sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and/or mesothelioma.
In some embodiments, the disease or condition is an infectious disease or condition, such as, but not limited to, viral, retroviral, bacterial, and protozoal infections, immunodeficiency, Cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus. In some embodiments, the disease or condition is an autoimmune or inflammatory disease or condition, such as arthritis, e.g., rheumatoid arthritis (RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Grave's disease, Crohn's disease multiple sclerosis, asthma, and/or a disease or condition associated with transplant.
In some embodiments, the antigen associated with the disease or disorder is selected from the group consisting of orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, 0EPHa2, ErbB2, 3, or 4, FBP, fetal acetylcholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1), a cyclin, such as cyclin A1 (CCNA1), and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens.
As used herein, a “post-transcriptional regulatory element (PRE),” such as a hepatitis PRE, refers to a DNA sequence that, when transcribed creates a tertiary structure capable of exhibiting post-transcriptional activity to enhance or promote expression of an associated gene operably linked thereto.
As used herein, “post-transcriptional activity” refers to activity of a regulatory element to control or regulate gene expression at the RNA level. Post-transcriptional activity can be manifested by activity that promotes RNA export from the nucleus, provides binding sites for cellular proteins, increases the total amount of RNA, e.g. recombinant and/or heterologous RNA, transcripts, increases RNA stability, increases the number of poly-adenylated transcripts and/or augments the size of the poly-adenylated tails in such transcripts.
As used herein “operably linked” refers to the association of components, such as a DNA sequence, e.g. a heterologous nucleic acid) and a regulatory sequence(s), in such a way as to permit gene expression when the appropriate molecules (e.g. transcriptional activator proteins) are bound to the regulatory sequence. Hence, it means that the components described are in a relationship permitting them to function in their intended manner.
As used herein, “unmodified” with reference to a PRE, an X gene or a Flap sequence refers to a starting polynucleotide that is selected for modification as provided. The starting polynucleotide can be a naturally-occurring, wild-type form of a polynucleotide. In some embodiments, the starting polynucleotide can be altered or mutated, such that it differs from a native wild-type form but is nonetheless referred to as a starting unmodified polynucleotide relative to the subsequently modified polynucleotides as provided. An unmodified polynucleotide does not contain a provided modification (e.g. stop codon, degradation sequence or deletion of all or a portion of a cPPT or CTS as provided) in its sequence. In some embodiments, the unmodified nucleic acid, e.g., PRE, is one that is known to possess or exhibit a desired degree of functionality or activity, such as a PRE with a known degree of post-transcriptional activity, e.g., ability to enhance gene expression. Exemplary of an unmodified PRE sequences are those set forth in SEQ ID NOS:1, 12-27, 119, 120, 125 or 216. Exemplary of an unmodified Flap sequence is set forth in SEQ ID NOS: 121.
As used herein, “wild-type” with reference to a PRE or an X gene refers to a sequence derived from a naturally occurring nucleic acid sequence of a PRE or an X gene that is present as such in a virus or virus genome, such as a hepadnavirus, e.g. an orthohepandanvirus, such as a hepadnavirus isolated from a mammal, in nature. Reference to wild-type without reference to a species is intended to encompass a virus sequence that includes an X gene isolated from any species. Exemplary of a wild-type PRE or X gene are any having a nucleic acid sequence obtained from or that has a sequence that is the same as a PRE or X gene sequence present in a hepadnavirus, including any hepadnavirus found or isolated from a mammalian species, such as a human, a primate, or a squirrel, for example a woodchuck. Examples of a wild-type PRE are those set forth in SEQ ID NOS:1, 12-27 or 125.
As used herein, “wild-type” with reference to a Flap sequence refers to a sequence derived from a naturally occurring viral nucleic acid sequence that contains cPPT and CTS regions for forming a DNA Flap present as such in a virus or virus genome, such as a retrovirus, e.g. a lentivirus, such as HIV-1. Exemplary of a wild-type Flap sequence are any having a nucleic acid sequence obtained from or that has a sequence that is the same as a viral nucleic acid sequence containing cPPT and CTS regions present in a retrovirus, including a lentivirus (e.g. HIV-1). Exemplary of viral nucleic acid containing a wild-type DNA Flap sequence is set forth in SEQ ID NOS:121.
As used herein, “percent (%) sequence identity” and “percent identity” when used with respect to a nucleotide sequence (reference nucleotide sequence) is defined as the percentage of nucleotide residues in a candidate sequence (e.g., the subject PRE or X gene, such as a modified PRE or variant X gene) that are identical with the nucleotide residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
As used herein, “at a position corresponding to” or recitation that nucleotides, amino acid positions or regions “correspond to” nucleotides, amino acid positions or regions in a disclosed sequence, such as set forth in the Sequence listing, refers to positions of nucleotides or amino acids or a region or domain containing nucleotides or amino acids identified upon alignment with the disclosed sequence to maximize identity using a standard alignment algorithm, such as the GAP algorithm. Exemplary described corresponding residues or regions can be identified by alignment of a sequence with an exemplary PRE sequence set forth in SEQ ID NO:1 (or SEQ ID NO:125, which is residues 1-589 of SEQ ID NO:1). By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides. In general, to identify corresponding positions, the sequences of amino acids are aligned so that the highest order match is obtained (see, e.g.: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carrillo et al. (1988) SIAM J Applied Math 48: 1073). FIG. 2A to 2E exemplifies exemplary alignments and identification of exemplary corresponding residues.
The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Among the vectors are viral vectors, such as lentiviral vectors.
The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include “consisting” and/or “consisting essentially of” aspects and variations.
Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.
The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.
As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human.
Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.
The section heading used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
III. Exemplary Embodiments
Among the embodiments provided herein are:
1. A polynucleotide, comprising:
a nucleic acid encoding a recombinant protein;
a modified post-transcriptional regulatory element (PRE) operably linked to said nucleic acid, said modified PRE comprising a variant of a wild-type or unmodified hepatitis virus X gene, said variant X gene comprising a stop codon not present in the wild-type or unmodified X gene.
2. The polynucleotide of embodiment 1, wherein the stop codon comprises at least one stop codon selected from among:
a stop codon beginning at a position within 36 or 24 nucleotides in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the wild-type X protein open reading frame; and/or
a stop codon beginning at a position within 36 or 24 nucleotides in the 3′ direction from a position corresponding to residue 411 of WHV post-transcriptional regulatory element (WPRE) sequence set forth in SEQ ID NO: 1 or SEQ ID NO:125 and/or residue 1503 of the WHV sequence set forth as SEQ ID NO: 2.
3. The polynucleotide of embodiment 1 or embodiment 2, wherein said variant X gene does not comprise an open reading frame of greater than at or about 39 nucleotides in length or greater than at or about 27 nucleotides in length.
4. A polynucleotide, comprising a modified post-transcriptional regulatory element (PRE), said modified PRE comprising a variant of a wild-type or unmodified hepatitis virus X gene, said variant X gene comprising a stop codon not present in the wild-type or unmodified X gene, wherein the stop codon comprises at least one stop codon selected from among:
a stop codon beginning at a position within 32 or 30 nucleotides in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the wild-type X protein open reading frame; and/or
a stop codon beginning at a position within 32 or 30 nucleotides in the 3′ direction from a position in the variant X gene corresponding to residue 411 of WHV post-transcriptional regulatory element (WPRE) sequence set forth in SEQ ID NO: 1 or SEQ ID NO:125 and/or residue 1503 of the WHV sequence set forth as SEQ ID NO: 2.
5. The polynucleotide of embodiment 4, wherein said variant X gene comprises an open reading frame of less than or equal to 33 nucleotides in length.
6. The polynucleotide of any of embodiments 1-5, wherein said stop codon comprises a plurality of stop codons.
7. The polynucleotide of embodiment 6, wherein said plurality of stop codons comprises at least one stop codon in each reading frame present in said variant X gene and/or comprises at least two stop codons in the same reading frame.
8. A polynucleotide, comprising a modified post-transcriptional regulatory element (PRE), said modified PRE comprising a variant of a wild-type or unmodified hepatitis virus X gene, said variant X gene comprising a plurality of stop codons not present in the wild-type or unmodified X gene.
9. The polynucleotide of embodiment 8, wherein the plurality of stop codons comprises at least one stop codon selected from among:
a stop codon beginning at a position within 36, 30 or 24 nucleotides in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the wild-type X protein open reading frame; and/or
a stop codon beginning at a position within 36, 30 or 24 nucleotides in the 3′ direction from a position in the variant X gene corresponding to residue 411 of WHV post-transcriptional regulatory element (WPRE) sequence set forth in SEQ ID NO: 1 or SEQ ID NO:125 and/or residue 1503 of the WHV sequence set forth as SEQ ID NO: 2.
10. The polynucleotide of embodiment 9, wherein said variant X gene does not comprise an open reading frame of greater than at or about 39 nucleotides in length, greater than at or about 33 nucleotides in length or greater than at or about 27 nucleotides in length.
11. The polynucleotide of any of embodiments 8-10, wherein the variant X gene comprises at least 2 stop codons, at least 3 stop codons or at least 4 stop codons.
12. The polynucleotide of any of embodiments 8-11, wherein the variant X gene comprises a stop codon in each reading frame present in said variant X gene.
13. The polynucleotide of any of embodiments 4-12, further comprising a nucleic acid encoding a recombinant protein operably linked to the modified PRE.
14. The polynucleotide of any of embodiments 1-13, wherein the stop codon comprises at least one stop codon selected from among:
a stop codon beginning at a position within or within at least 9, 12, 15, or 18 nucleotides in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the wild-type or unmodified X protein open reading frame; and/or
a stop codon beginning at a position within or within at least 9, 12, 15, or 18 nucleotides in the 3′ direction from a position in the variant X gene corresponding to residue 411 of WHV post-transcriptional regulatory element (WPRE) sequence set forth in SEQ ID NO: 1 and/or residue 1503 of the WHV sequence set forth as SEQ ID NO: 2.
15. The polynucleotide of embodiment 14, wherein said variant X gene does not comprise an open reading frame of greater than at or about 21 nucleotides in length, greater than at or about 18 nucleotides in length, greater than at or about 15 nucleotides in length, or greater than at or about 12 nucleotides in length.
16. The polynucleotide of any of embodiments 1-15, wherein:
the modified PRE contains a beta stem loop corresponding to nucleotide residues 448-470 of SEQ ID NO:1 or SEQ ID NO:125; and/or the modified PRE does not contain a nucleotide change in a position within the beta stem loop corresponding to nucleotides 448-470 of SEQ ID NO:1 or SEQ ID NO:125; and/or
said stop codon or stop codons does not comprise a nucleotide in a position within the beta stem loop corresponding to nucleotides 448-470 of SEQ ID NO:1 or SEQ ID NO:125.
17. The polynucleotide of any of embodiments 1-16, wherein said stop codon is selected from an amber (TAG), ochre (TAA), or opal (TGA) stop codons.
18. The polynucleotide of any of embodiments 1-17, wherein said stop codon is introduced by nucleotide substitution, deletion or insertion.
19. The polynucleotide of any of embodiments 1-18, wherein said stop codon is selected from among:
a stop codon beginning at position 9 in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the wild-type or unmodified X protein open reading frame and/or at a nucleotide position corresponding to position 420 in the sequence set forth in SEQ ID NO:1 or SEQ ID NO:125;
a stop codon beginning at position 13 in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the wild-type or unmodified X protein open reading frame and/or at a nucleotide position corresponding to position 424 in the sequence set forth in SEQ ID NO:1 of SEQ ID NO:125;
a stop codon beginning at position 17 in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the wild-type or unmodified X protein open reading frame and/or at a nucleotide position corresponding to position 428 in the sequence set forth in SEQ ID NO:1 of SEQ ID NO:125;
a stop codon beginning at position 21 in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the wild-type or unmodified X protein open reading frame and/or at a nucleotide position corresponding to position 432 in the sequence set forth in SEQ ID NO:1 of SEQ ID NO:125.
20. The polynucleotide of any of embodiments 17-19, wherein said stop codon is an amber (TAG) or opal (TGA) stop codon.
21. The polynucleotide of any of embodiments 1-20, wherein:
said variant X gene is no more than 180 nucleotides in length or is no more than at or about 210 nucleotides in length; and/or
said variant X gene is at least at or about 90 nucleotides in length or at least about 120 nucleotides in length or is at least at or about 180 nucleotides in length.
22. The polynucleotide of any of embodiments 1-21, wherein:
said variant X gene is a variant of a wild-type or unmodified mammalian hepatitis X gene or a truncated portion thereof, which is, optionally, a truncated portion present in a PRE; or
the modified PRE comprises a variant X gene comprising SEQ ID NO:28.
23. The polynucleotide of embodiment 22, wherein said wild-type or unmodified mammalian hepatitis X gene is a wild-type woodchuck hepatitis virus (WHV) X gene or a truncated portion thereof, which is optionally, a truncated portion present in a wild-type or unmodified PRE.
24. The polynucleotide of embodiment 23, wherein the wild-type or unmodified WHV X gene or the truncated portion of a wild-type X gene present in a PRE comprises the sequence of nucleotides set forth as nucleotides 411-592 of any of SEQ ID NOS: 1 and 12-20 or set forth as nucleotides 411-589 of SEQ ID NO:125.
25. The polynucleotide of embodiment 23 or embodiment 24, wherein said wild-type WHV X gene comprises the nucleotide sequence of SEQ ID NO: 9 or is a truncated portion thereof, which is, optionally, set forth in SEQ ID NO:10 or is a truncated portion present in a wild-type or unmodified PRE.
26. The polynucleotide of any of embodiments 1-25, wherein said modified PRE comprises at least two or at least three cis-acting post-transcriptional regulatory subelements of a wild-type or unmodified hepatitis virus PRE or functional variant(s) thereof.
27. The polynucleotide of embodiment 26, wherein said at least two or at least three subelements comprise a wild-type or unmodified PRE alpha subelement or functional variant thereof, a functional variant of a wild-type or unmodified PRE beta subelement, and/or a wild-type or unmodified PRE gamma subelement or functional variant thereof.
28. The polynucleotide of any of embodiments 1-27, wherein said modified PRE comprises an alpha subelement of a wild-type or unmodified hepatitis virus PRE or functional variant thereof and a functional variant of a wild-type or unmodified PRE beta subelement.
29. The polynucleotide of embodiment 27 or embodiment 28, wherein said alpha subelement comprises the sequence of SEQ ID NO: 3 or a variant thereof, said beta subelement comprises a variant of the sequence of SEQ ID NO: 6, and/or said gamma subunit comprises the sequence of SEQ ID NO: 8 or variant thereof.
30. The polynucleotide of any of embodiments 1-29, wherein the modified PRE comprises nucleotide modifications compared to a wild-type or unmodified hepatitis virus PRE that is a wild-type mammalian hepatitis PRE.
31. The polynucleotide of embodiment 30, wherein said wild-type or unmodified mammalian hepatitis virus PRE comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 1, 12-27 and 125.
32. The polynucleotide of embodiment 30 or embodiment 31, wherein said wild-type or unmodified mammalian hepatitis PRE is a wild-type woodchuck hepatitis virus PRE (WPRE).
33. The polynucleotide of any of embodiments 1-32, wherein the wild-type or unmodified hepatitis PRE comprises:
a) the sequence of nucleotides set forth in SEQ ID NO:1 or SEQ ID NO:125 or a sequence of nucleotides that exhibits at least 94% sequence identity to SEQ ID NO:1 or SEQ ID NO:125, that exhibits substantially the same, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the, post-transcriptional activity of SEQ ID NO:1 or SEQ ID NO:125; and
b) a modified PRE comprising a portion of the sequence of nucleotides of a), wherein the portion exhibits or retains the post-transcriptional activity.
34. The polynucleotide of embodiment 33, wherein the wild-type or unmodified hepatitis PRE comprises a sequence of nucleotides that exhibits at least 95%, 96%, 97%, 98%, 99%, sequence identity to SEQ ID NO:1 or SEQ ID NO:125.
35. The polynucleotide of any of embodiments 1-34, wherein said wild-type or unmodified hepatitis PRE comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 1, 12-20 and 125.
36. The polynucleotide of any of embodiments 1-35, wherein said wild-type or unmodified hepatitis PRE comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:125.
37. The polynucleotide of any of embodiments 1-36, wherein said variant X gene comprises a start codon beginning at a position corresponding to position 411 of SEQ ID NO: 1 or SEQ ID NO:125.
38. The polynucleotide of embodiment 37, wherein said start codon is an ATG start codon.
39. The polynucleotide of any of embodiments 1-38, wherein the variant X gene further comprises a promoter operably linked to said variant X gene.
40. The polynucleotide of embodiment 39, wherein said promoter is a wild-type or unmodified X-gene promoter comprising the sequence set forth in SEQ ID NO: 11 or a wild-type WHV X gene promoter sequence.
41. The polynucleotide of any of embodiments 1-40, wherein the modified PRE is selected from among:
a) a modified PRE comprising a sequence of nucleotides that exhibits at least 65% sequence identity to SEQ ID NO:1 or SEQ ID NO:125, said modified PRE containing a variant X gene comprising at least one stop codon not present in SEQ ID NO:1 or SEQ ID NO:125; and
b) a modified PRE comprising a portion of the sequence of nucleotides of a), said portion comprising a variant X gene comprising the at least one stop codon, wherein the portion exhibits post-transcriptional activity.
42. The polynucleotide of embodiment 41, wherein the modified PRE comprises a sequence of nucleotides that exhibits at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO:1 or SEQ ID NO:125.
43. The polynucleotide of embodiment 41 or embodiment 42, wherein the variant X gene comprises at least 2 stop codons, at least 3 stop codons or at least 4 stop codons.
44. The polynucleotide of any of embodiments 41-43, wherein the variant X gene comprises a stop codon in each reading frame present in said variant X gene.
45. The polynucleotide of any of embodiments 1-44, wherein the variant X gene comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 44-58 or SEQ ID NOS: 141-155 and/or the modified PRE comprises the sequence of nucleotides set forth in any of SEQ ID NOS:29-43 or SEQ ID NOS: 126-140.
46. The polynucleotide of any of embodiments 1-45, wherein the modified PRE does not contain any modifications in addition to the at least one stop codon not present in the wild-type or unmodified PRE.
47. The polynucleotide of any of embodiments 1-46, wherein the modified PRE contains additional modifications in addition to the at least one stop codon not present in the wild-type or unmodified PRE.
48. The polynucleotide of any of embodiments 1-47, wherein said variant X gene further comprises a variant of a start codon comprising one or more nucleotide differences compared to a wild-type or unmodified hepatitis virus X gene start codon and/or compared to the start codon corresponding to nucleotide positions 411-413 of SEQ ID NO: 1 or SEQ ID NO:125.
49. The polynucleotide of embodiment 48, wherein said one or more differences results in restricted or prevented translation initiation from said start codon.
50. The polynucleotide of any of embodiments 47-49, wherein said variant X gene comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 74-88 or 171-185 and/or the modified PRE comprises the sequence of nucleotides set forth in any of SEQ ID NOS:59-73 or 156-170.
51. The polynucleotide of any of embodiments 1-50, wherein said variant X gene comprises a variant promoter operably linked to said variant X gene, said variant promoter comprising one or more nucleotide differences compared to a wild-type or unmodified hepatitis virus X gene promoter and/or compared to a promoter of SEQ ID NO: 11.
52. The polynucleotide of embodiment 51, wherein said one or more differences results in restricted or prevention of transcription from said promoter.
53. The polynucleotide of embodiment 51 or 52, wherein said modified PRE comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 89-118 or 186-215.
54. The polynucleotide of any of embodiments 1-53, wherein upon introduction into a eukaryotic cell, no polypeptide of a length greater than 12, 11, 10, 9, or 8 amino acids in length encoded by said variant X gene is produced; and/or said polynucleotide is incapable of producing a polypeptide of a length greater than 12, 11, 10, 9, or 8 amino acids in length encoded by said variant X gene.
55. The polynucleotide of any of embodiments 1-54, wherein said modified PRE encodes an RNA that promotes nuclear RNA export and/or increases mRNA stability.
56. The polynucleotide of any of embodiments 1-55, wherein said modified PRE encodes an RNA polynucleotide that promotes nuclear RNA export and/or increases mRNA stability, wherein said promotion of nuclear RNA export and/or mRNA stability increases expression of the recombinant protein.
57. The polynucleotide of any of embodiments 1-56, wherein the modified PRE retains the post-transcriptional activity of the corresponding wild-type or unmodified hepatitis PRE and/or the PRE set forth in SEQ ID NO:1, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:125 and/or SEQ ID NO:216, which, in some cases, is at least or about at least 65%, 70%, 75%, 80%, 85%, 90% or 95% of the post-transcriptional activity of the wild-type or unmodified hepatitis PRE and/or the PRE set forth in SEQ ID NO:1, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:125 or SEQ ID NO:216.
58. The polynucleotide of any of embodiments 1-57, wherein the variant X gene further comprises a sequence encoding a post-translational modification signal not present in the wild-type or unmodified hepatitis virus X gene.
59. A polynucleotide, comprising a modified post-transcriptional regulatory element (PRE), said modified PRE comprising a variant of a wild-type or unmodified hepatitis virus X gene, said variant X gene comprising a sequence encoding a post-translational modification signal not present in the wild-type or unmodified hepatitis virus X gene.
60. The polynucleotide of embodiment 58 or embodiment 59, wherein said post-translational modification signal comprises a ubiquitination site.
61. The polynucleotide of embodiment 58 or embodiment 59, wherein said post-translational modification signal comprises:
a first codon beginning at a position within or within at least 2, 3, 4, 5 or 6 nucleotides in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the X protein open reading frame, wherein the first codon encodes a glycine, arginine, glutamic acid, phenylalanine, aspartate, cysteine, lysine, asparagine, serine, tyrosine, tryptophan, histidine, or leucine residue according to the N-end rule; and optionally
a second codon beginning at a position within or within at least 2, 3, 4, 5 or 6 nucleotides in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the X protein open reading frame, wherein the second codon encodes a glutamic acid or asparagine residue according to the N-end rule.
62. The polynucleotide of embodiment 58 or embodiment 59, wherein said post-translational modification signal comprises one or more PEST sequences.
63. The polynucleotide of any of embodiments 59-62, further comprising a nucleic acid encoding a recombinant or protein operably linked to the modified PRE.
64. The polynucleotide of any of embodiments 59-63, wherein:
said variant X gene comprising a sequence encoding a post-translational modification signal contains a beta stem loop corresponding to nucleotide residues 448-470 of SEQ ID NO:1; and/or
said variant X gene comprising a sequence encoding a post-translational modification signal does not contain a nucleotide change in a position within the beta stem loop corresponding to nucleotides 448-470 of SEQ ID NO:1.
65. The polynucleotide of any of embodiments 59-64, wherein:
said variant X gene is no more than 180 nucleotides in length or is no more than at or about 210 nucleotides in length; and/or
said variant X gene is at least at or about 90 nucleotides in length or at least about 120 nucleotides in length or is at least at or about 180 nucleotides in length.
66. The polynucleotide of any of embodiments 59-65, wherein said variant X gene is a variant of a wild-type or unmodified mammalian hepatitis X gene or a truncated portion thereof, which is, optionally, a truncated portion present in a PRE.
67. The polynucleotide of embodiment 66, wherein said wild-type or unmodified mammalian hepatitis X gene is a wild-type woodchuck hepatitis virus (WHV) X gene or a truncated portion thereof, which is optionally, a truncated portion present in a wild-type or unmodified PRE.
68. The polynucleotide of embodiment 67, wherein the wild-type or unmodified WHV X gene or the truncated portion of a wild-type X gene present in a PRE comprises the sequence of nucleotides set forth as nucleotides 1503-1928 of SEQ ID NO: 2, the sequence of nucleotides set forth as nucleotides 411-592 of any of SEQ ID NOS: 1 and 12-20 or the sequence of nucleotides set forth as nucleotides 411-589 of SEQ ID NO:125.
69. The polynucleotide of embodiment 67 or embodiment 68, wherein said wild-type WHV X gene comprises the nucleotide sequence of SEQ ID NO: 9 or is a truncated portion thereof, which is, optionally, set forth in SEQ ID NO:10 or is a truncated portion present in a wild-type or unmodified PRE.
70. The polynucleotide of any of embodiments 59-69, wherein said modified PRE comprises at least two or at least three cis-acting post-transcriptional regulatory subelements of a wild-type or unmodified hepatitis virus PRE or functional variant(s) thereof.
71. The polynucleotide of embodiment 70, wherein said at least two or at least three subelements comprise a wild-type or unmodified PRE alpha subelement or functional variant thereof, a functional variant of a wild-type or unmodified PRE beta subelement, and/or a wild-type or unmodified PRE gamma subelement or functional variant thereof.
72. The polynucleotide of any of embodiments 59-71, wherein said modified PRE comprises an alpha subelement of a wild-type or unmodified hepatitis virus PRE or functional variant thereof and a functional variant of a wild-type or unmodified PRE beta subelement.
73. The polynucleotide of embodiment 71 or embodiment 72, wherein said alpha subelement comprises the sequence of SEQ ID NO: 3 or a variant thereof, said beta subelement comprises a variant of the sequence of SEQ ID NO: 6, and/or said gamma subunit comprises the sequence of SEQ ID NO: 8 or variant thereof.
74. The polynucleotide of any of embodiments 1-73, wherein the modified PRE comprises nucleotide modifications compared to a wild-type or unmodified hepatitis virus PRE that is a wild-type mammalian hepatitis PRE.
75. The polynucleotide of embodiment 74, wherein said wild-type or unmodified mammalian hepatitis virus PRE comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 1, 12-27 and 125.
76. The polynucleotide of embodiment 74 or embodiment 75, wherein said wild-type or unmodified mammalian hepatitis PRE is a wild-type woodchuck hepatitis virus PRE (WPRE).
77. The polynucleotide of any of embodiments 1-76, wherein the wild-type or unmodified hepatitis PRE comprises:
a) the sequence of nucleotides set forth in SEQ ID NO:1 or SEQ ID NO:125 or a sequence of nucleotides that exhibits at least 94% sequence identity to SEQ ID NO:1 or SEQ ID NO:125 that exhibits substantially the same, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the, post-transcriptional activity of SEQ ID NO:1 or SEQ ID NO:125; and
b) a modified PRE comprising a portion of the sequence of nucleotides of a), wherein the portion exhibits or retains the post-transcriptional activity.
78. The polynucleotide of embodiment 77, wherein the wild-type or unmodified hepatitis PRE comprises a sequence of nucleotides that exhibits at least 95%, 96%, 97%, 98%, 99%, sequence identity to SEQ ID NO:1 or SEQ ID NO:125.
79. The polynucleotide of any of embodiments 59-78, wherein said wild-type or unmodified hepatitis PRE comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 1, 12-20 and 125.
80. The polynucleotide of any of embodiments 59-79, wherein said wild-type or unmodified hepatitis PRE comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:125.
81. The polynucleotide of any of embodiments 59-80, wherein said variant X gene comprises a start codon beginning at a position corresponding to position 411 of SEQ ID NO: 1 or SEQ ID NO:125.
82. The polynucleotide of embodiment 81, wherein said start codon is an ATG start codon.
83. The polynucleotide of any of embodiments 59-82, wherein the variant X gene further comprises a promoter operably linked to said variant X gene.
84. The polynucleotide of embodiment 83, wherein said promoter is a wild-type or unmodified X-gene promoter comprising the sequence set forth in SEQ ID NO: 11 or a wild-type WHV X gene promoter sequence.
85. The polynucleotide of any of embodiments 59-84, wherein the modified PRE is selected from among:
a) a modified PRE comprising a sequence of nucleotides that exhibits at least 65% sequence identity to SEQ ID NO:1 or SEQ ID NO:125, said modified PRE containing a variant X gene comprising a sequence encoding a post-translational modification signal not present in SEQ ID NO:1 or SEQ ID NO:125; and
b) a modified PRE comprising a portion of the sequence of nucleotides of a), said portion comprising a variant X gene comprising the sequence encoding a post-translational modification, wherein the portion exhibits post-transcriptional activity.
86. The polynucleotide of embodiment 85, wherein the modified PRE comprises a sequence of nucleotides that exhibits at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity to SEQ ID NO:1 or SEQ ID NO:125.
87. The polynucleotide of any of embodiments 1-86, wherein the variant X gene comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotide changes.
88. The polynucleotide of any of embodiments 59-87, wherein the modified PRE does not contain any modifications in addition to the sequence encoding a post-translational modification signal.
89. The polynucleotide of any of embodiments 59-88, wherein the modified PRE contains additional modification(s) in addition to the sequence encoding a post-translational modification signal not present in the wild-type or unmodified hepatitis virus X gene.
90. The polynucleotide of embodiment 89, wherein the additional modification(s) are in the variant X gene.
91. The polynucleotide of embodiment 90, wherein the additional modification(s) results in a variant X gene encoding an inactive X protein and/or a truncated X protein.
92. The polynucleotide of any of embodiments 59-91, wherein said modified PRE encodes an RNA that promotes nuclear RNA export and/or increases mRNA stability.
93. The polynucleotide of any of embodiments 59-92, wherein said modified PRE encodes an RNA polynucleotide that promotes nuclear RNA export and/or increases mRNA stability, wherein said promotion of nuclear RNA export and/or mRNA stability increases expression of the recombinant protein.
94. The polynucleotide of any of embodiments 59-93, wherein the modified PRE retains the post-transcriptional activity of the corresponding wild-type or unmodified hepatitis PRE and/or the PRE set forth in SEQ ID NO:1, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:125 and/or SEQ ID NO:216, which, in some cases, is at least or about at least 65%, 70%, 75%, 80%, 85%, 90% or 95% of the post-transcriptional activity of the wild-type or unmodified hepatitis PRE and/or the PRE set forth in SEQ ID NO:1, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:125 or SEQ ID NO:216.
95. A polynucleotide, comprising a viral nucleic acid comprising a variant Flap, wherein the variant Flap contains a deletion of all or a portion of the nucleotides corresponding to the central polypurine tract (cPPT) and/or the central termination sequence (CTS) regions of a wild-type or unmodified Flap sequence.
96. The polynucleotide of embodiment 95, wherein the variant Flap comprises a deletion of all or a portion, which can be a contiguous portion, of the nucleotides corresponding to the cPPT and the CTS.
97. The variant Flap polynucleotide of embodiment 95 or 96, wherein:
the wild-type or unmodified Flap sequence comprises from or from about 80 to 200 contiguous nucleotides comprising the cPPT or CTS regions of a retrovirus, which optionally is a lentivirus, which optionally is HIV-1; and/or
the wild-type or unmodified Flap sequence comprises a) the sequence of nucleotides set forth in SEQ ID NO:121; b) a sequence of nucleotides comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence of nucleotides set forth in SEQ ID NO:121 that contains the cPPT and/or CTS regions; or c) a contiguous portion of a) or b) that comprises the cPPT and/or CTS regions.
98. The variant Flap polynucleotide of any of embodiments 95-97, wherein:
the variant Flap comprises deletion of all or a portion, which can be a contiguous portion, of nucleotides corresponding to nucleotides of the cPPT region set forth in SEQ ID NO: 123; and/or
the variant Flap comprises deletion of all or a portion, which can be a contiguous portion, of nucleotides corresponding to the CTS region set forth in SEQ ID NO: 124.
99. The variant Flap polynucleotide of any of embodiment 95-98, wherein the variant Flap comprises deletion of all or a portion, which can be a contiguous portion, of the cPPT region set forth in SEQ ID NO:123 and comprises deletion of all or a portion, which can be a contiguous portion, of the CTS region set forth in SEQ ID NO:124.
100. The variant Flap polynucleotide of any of embodiments 95-99, wherein:
the viral nucleic acid comprising the variant Flap comprises a sequence of nucleotides that exhibits at least 65%, 70%, 75%, 80%, 85%, or 90% sequence identity to SEQ ID NO:121, said variant Flap lacking all or a portion of the cPPT and/or CTS regions; and/or
the viral nucleic acid comprises the variant Flap comprising the sequence set forth in SEQ ID NO:122.
101. The polynucleotide of any of embodiments 1-94, further comprises a polynucleotide comprising viral nucleic acid comprising a variant Flap of any of embodiments 95-100.
102. A polynucleotide of any of embodiments 95-101, comprising:
a variant Flap comprising the sequence of SEQ ID NO: 122 or a sequence having at least at or about 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:122; and
the sequence of SEQ ID NO:29 or SEQ ID NO:126 or a sequence having at least at or about 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:29 or SEQ ID NO:126; and
optionally, comprising nucleic acid encoding a recombinant protein.
103. The polynucleotide of any of embodiments 1-94 and 101-102, wherein the recombinant protein comprises a recombinant receptor.
104. The polynucleotide of embodiment 103, wherein the recombinant receptor is an antigen receptor and/or a chimeric receptor.
105. The polynucleotide of embodiment 104, wherein the recombinant receptor is a functional non-TCR antigen receptor or a transgenic TCR.
106. The polynucleotide of any of embodiments 103-105, wherein the recombinant receptor is a chimeric antigen receptor (CAR).
107. An expression cassette comprising the polynucleotide of any of embodiments 1-106 and a promoter operably linked to the nucleic acid encoding the recombinant protein.
108. A vector comprising the polynucleotide of any of embodiments 1-106 or the expression cassette of embodiment 107.
109. The vector of embodiment 108, which is a viral vector.
110. The vector of embodiment 109, wherein the viral vector is a lentiviral vector, which, optionally, is derived from HIV-1.
111. The vector of embodiment 109, wherein the viral vector is a retroviral vector.
112. A cell comprising the expression cassette of embodiment 107 or the vector of any of embodiments 108-111.
113. The cell of embodiment 112, wherein the cell is a T cell, a natural killer (NK) cell, an iPS cell, or an iPS-derived cell.
114. A virus particle, comprising the vector of any of embodiments 108-111.
115. A method, comprising introducing the expression cassette of embodiment 107, the vector of any of embodiments 108-111, or the virus particle of embodiment 114 to a cell, under conditions whereby expression of the recombinant protein is effected in the cell.
116. The method of embodiment 115, wherein:
said introduction is effected by transducing said cell with said vector or viral particle;
said introduction is effected by transfecting said cell with said vector; and/or
said introduction is effected by electroporation of said cell with said vector.
117. The method of embodiment 116, wherein the recombinant protein is expressed at a level that is increased compared to that achieved by introducing a vector that is the same but that does not contain the modified PRE or does not contain a PRE.
118. A cell produced by the method of any of embodiments 115-117.
119. A pharmaceutical composition comprising the cell of embodiments 112, 113, or 118, and a pharmaceutically effective carrier.
120. A method of treatment, the method comprising administering to a subject having a disease or condition the vector of any of embodiments 108-111, the virus particle of embodiment 114, the cell of embodiment 112, 113, or 118, or the composition of embodiment 119.
121. The method of embodiment 120, wherein the recombinant protein comprises a recombinant receptor that specifically binds to a ligand expressed by the disease or condition or a cell or tissue thereof.
122. The method of embodiment 121, wherein the receptor is an antigen receptor and the ligand is an antigen specific for and/or associated with the disease or condition.
123. The method of embodiment 122, wherein the disease or condition is a cancer, and autoimmune disorder, or an infectious disease.
124. Use of a pharmaceutical composition of embodiment 119 for treating a disease or condition.
125. A pharmaceutical composition of embodiment 119 for use in the preparation of a medicament for treating a disease or condition.
126. The use of embodiment 124 or the pharmaceutical composition of embodiment 125, wherein the recombinant protein expressed by the cell comprises a recombinant receptor that specifically binds to a ligand expressed by the disease or condition or a cell or tissue thereof.
127. The use or pharmaceutical composition of any of embodiments 124-126, wherein the recombinant receptor is an antigen receptor and the ligand is an antigen specific for and/or associated with the disease or condition.
128. The use or pharmaceutical composition of any of embodiments 124-127, wherein the disease or condition is a cancer, an autoimmune disorder or an infectious disease.
IV. Examples
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1
Modified Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element
An exemplary modified woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) polynucleotide was generated, which contained modifications at residues corresponding to positions in an exemplary wild-type WPRE sequence (SEQ ID NO: 1). Stop codons were introduced into each reading frame corresponding to a region of SEQ ID NO: 1 encoding a truncated portion of the WHV X protein (X gene fragment). Specifically, within this region, two stop codons were introduced in the open reading frame encoding the truncated X protein (beginning 9 and 21 nucleotides from the site corresponding to the 5′ position of the X gene start codon), and one in each of the other two reading frames (beginning 13 and 17 nucleotides from the site corresponding to the 5′ position of the start codon). The modified WPRE contained the nucleotide sequence set forth in SEQ ID NO: 126. In the following portion of this sequence, the stop codons are set forth in bold, and introduced mutations with reference to SEQ ID NO:1 are underlined: ATGGCTGCTTAGCTGAGTAGCTGA (SEQ ID NO:28). A control functional modified WPRE, containing the sequence set forth in SEQ ID NO: 216, also was generated. This control functional modified WPRE contains mutations in the WHV X gene promoter region and start codon compared to the wild-type WPRE sequence of SEQ ID NO: 1. It has been used in a plurality of viral vectors for transduction, demonstrating activity sufficient to promote expression of recombinant molecules encoded by such vectors (see, e.g., “Cloning vector pLV.MCS.WHVPRE, complete sequence,” GenBank: JN622008.1; U.S. Pat. No. 7,384,738).
The modified WPRE polynucleotide containing the nucleotide sequence set forth in SEQ ID NO: 126 and the control modified WPRE containing the nucleotide sequence set forth in SEQ ID NO: 216 each were separately incorporated into an exemplary HIV-1-derived lentiviral vector containing mutations within the viral Flap region. Specifically, the lentiviral vector contained a variant Flap region in which the regions corresponding to SEQ ID NO:123 and SEQ ID NO:124 were deleted, resulting in a Flap variant vector, having a portion containing the nucleotide sequence set forth in SEQ ID NO:122 (designated Flap −/−).
A lentiviral vector in which the control modified WPRE of SEQ ID NO:119 was incorporated into an exemplary HIV-1 derived lentiviral vector that contained a complete Flap region also was generated. The lentiviral vector containing the complete Flap region (a wild-type Flap polynucleotide) containing the nucleotide sequence set forth in SEQ ID NO:121 (designated Flap +/+).
Sequences contained in the generated lentiviral vectors are summarized in the Table 4 below:
TABLE 4
WPRE
Flap
(SEQ ID NO)
REGION
Control modified WPRE,
216
121
Flap+/+
Control modified WPRE,
216
122
Flap−/−
Modified WPRE,
126
122
Flap−/−
The vector contained polynucleotides encoding chimeric antigen receptor and truncated epidermal growth factor (EGFRt) transduction marker separated by a T2A linker (see U.S. Pat. No. 8,802,374). Pseudotyped lentiviral vector particles were produced by standard procedures by transiently transfecting HEK-293T cells with the resulting vectors, helper plasmids (containing gagpol plasmids and rev plasmid), and a pseudotyping plasmid and used to transduce cells.
Example 2
Transduction of Cells with Viral Vectors Containing Modified WPRE
The lentiviral vector particles described in Example 1 were used to transduce cells from a human fibrosarcoma cell line (HT1080 ATCC® No. CCL-121™) and primary human T cells. Transduction efficiency was assessed by measuring surface expression of the recombinant EGFRt marker encoded by the vector.
2A. HT1080 Cells
In duplicate studies (A and B), HT1080 cells were transduced in 24-well plates with 250 μL or 125 μL volume of virus in the presence of 8 μg/mL of Polybrene with spinoculation (1200×g for 30 minutes). A sample of untransduced cells was used as a negative control.
Following culture of the cells for 48-72 hours, surface expression of the EGFRt marker was detected by flow cytometry with gating set based on the untransduced control sample. EGFRt was detected using a biotin-conjugated anti-EGFR antibody (cetuximab) followed by detection with a secondary V450-conjugated streptavidin. Table 5 lists the percentage of live cells exhibiting surface EGFRt expression for each of the various conditions in duplicates A and B.
As shown, transduction of HT1080 cells using a lentiviral vector having a variant Flap region and a modified WPRE with stop codons in the X gene fragment resulted in comparable EGFRt expression to that using a vector with a variant Flap region and a control modified WPRE. Thus, as compared to modifications in the X gene start codon and promoter region, the presence of stop codons in the X gene fragment did not interfere with the ability of a WPRE to promote the expression of recombinant protein encoded by the vector.
Also, as shown, a lentiviral vector having a variant Flap region successfully transduced HT1080 cells as detected by EGFRt expression.
TABLE 5
Percentage of live cells
Virus
expressing surface EGFRt
WPRE in vector
(vol.)
A
B
Untransduced control
N/A
0.3%
0.1%
Modified WPRE,
250 μL
58.9%
56.3%
Flap−/−
125 μL
26.2%
20.5%
Control modified WPRE,
250 μL
62.1%
41.4%
Flap−/−
125 μL
34.2%
18.7%
Control modified WPRE,
250 μL
81%
56.7%
Flap+/+
125 μL
56.9%
48.4%
2B. Human T Cells
Primary human T cells were enriched via positive selection from peripheral blood mononuclear cells (PBMCs) obtained from human apheresis samples. The cells were activated using anti-CD3/anti-CD28 beads in the presence IL-2 (100 IU/mL). Activated T cells were transduced with one volume of virus in the presence of Polybrene (8 μg/mL) and spinoculation (1200×g for 30 min) in 24-well plates for each of the vectors described in Example 1. A sample of untransduced cells served as a negative control.
Surface expression of EGFRt was detected as described above in Example 2A. The results are shown in Table 6, including results from duplicate studies (A and B). As shown, transduction of primary human T cells using a lentiviral vector having a variant Flap region and a modified WPRE with stop codons in the X gene fragment resulted in comparable EGFRt expression to that using a vector with a variant Flap region and a control modified WPRE. Thus, as compared to modifications in the X gene start codon and promoter region, the presence of stop codons in the X gene fragment did not interfere with the ability of a WPRE to promote the expression of recombinant protein encoded by the vector. These results were consistent across multiple activation reagents, as similar results were obtained when human primary T cells were activated with various types of anti-CD3/anti-CD28 beads.
Also, as shown, a lentiviral vector having a variant Flap region successfully transduced T cells as observed by EGFRt expression.
TABLE 6
Percentage of live cells
expressing surface EGFRt
WPRE in vector
A
B
Untransduced
0.1%
N/D
Modified WPRE,
24.4%
22.5%
Flap−/−
Control modified WPRE,
20.9%
25.8%
Flap−/−
Control modified WPRE,
39.1%
N/D
Flap+/+
Example 3
Transduction of Cells with Viral Vectors Containing a Complete Flap or Variant Flap Region
Lentiviral vector particles were generated substantially as described in Example 1 in which each encoded a chimeric antigen receptor (CAR) containing a different anti-CD19 scFv antigen-binding domain. Each lentiviral vector also contained WPRE polynucleotides of a control functional modified WPRE that was null for the X gene ORF operably linked to the polynucleotides encoding the CAR, and either a wildtype Flap polynucleotide (Flap +/+) (SEQ ID NO:121) or a variant Flap polynucleotide (Flap −/−) (SEQ ID NO:122). For detection of expression of the CAR, the lentiviral vectors also encoded a truncated EGFR (EGFRt) transduction marker separated from the CAR by a self-cleaving T2A linker.
Primary human CD8+ T cells were enriched via positive selection from peripheral blood mononuclear cells (PBMCs) obtained from human apheresis samples. The lentiviral vector particles were used to transduce CD8+ T cells (essentially as described by Yam et al. (2002) Mol. Ther. 5:479; WO2015/095895). A sample of untransduced cells served as a negative control.
After transduction and expansion, staining with anti-EGFR antibody was used to verify expression of the EGFRt transduction marker on the surface of T cells by flow cytometry as described in Example 2A. Gating was set based on the untransduced control sample. Table 7 lists the percentage of live cells exhibiting surface EGFRt expression for each of the various conditions. As shown, the results confirmed that lentiviral vectors with a Flap −/− polynucleotide could successfully transduce CD8+ T cells.
TABLE 7
Percentage of live CD8+ cells
CAR
expressing surface EGFRt
No.
Flap+/+
Flap−/−
1
48%
26%
2
38%
24%
3
51%
21%
4
35%
23%
5
45%
25%
6
47%
17%
7
59%
23%
8
55%
24%
9
41%
21%
10
50%
22%
11
60%
27%
Following transduction, T cells transduced with each CAR construct, including those containing the variant Flap −/− polynucleotide, were successfully selectively enriched and expanded (at or close to 100% EGFRt+ as confirmed by flow cytometry) by stimulation in the presence of irradiated CD19+ cell lines.
The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.
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14994114
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juno therapeutics, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Mar 30th, 2022 06:04PM
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Mar 30th, 2022 06:04PM
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Bristol-Myers Squibb
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:bmy
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Juno Therapeutics
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Jul 20th, 2021 12:00AM
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Nov 1st, 2018 12:00AM
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https://www.uspto.gov?id=US11066475-20210720
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Chimeric antigen receptors specific for B-cell maturation antigen and encoding polynucleotides
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Provided herein are chimeric receptors, such as chimeric antigen receptors (CARs), comprising BCMA-binding molecules, such as anti-BCMA antibodies and antigen-binding fragments thereof, such as heavy chain variable (VH) regions and single-chain antibody fragments, and encoding polynucleotides. In some embodiments, the anti-BCMA chimeric receptors specifically bind to BCMA. Among the anti-BCMA-binding molecules are human antibodies, including those that compete for binding to BCMA with reference antibodies, such as a non-human reference antibody. Also provided are genetically engineered cells expressing the CARs and uses thereof such as in adoptive cell therapy.
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11066475
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1. A polynucleotide encoding a chimeric antigen receptor, the polynucleotide comprising a nucleic acid sequence encoding: (a) an extracellular antigen-binding domain comprising a single-chain variable fragment (scFv) comprising a variable heavy chain (VH) region and a variable light chain (VL) region that specifically binds B cell maturation antigen (BCMA); (b) a spacer encoded by the nucleic acid sequence set forth in SEQ ID NO:622; (c) a transmembrane domain; and (d) an intracellular signaling region.
2. The polynucleotide of claim 1, wherein:
(A) the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 contained within the VH region amino acid sequence set forth in SEQ ID NO:617; and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 contained within the VL region amino acid sequence set forth in SEQ ID NO:618;
(B) the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 contained within the VH region amino acid sequence set forth in SEQ ID NO:609; and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 contained within the VL region amino acid sequence set forth in SEQ ID NO:610;
(C) the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 contained within the VH region amino acid sequence set forth in SEQ ID NO:115; and the VL region comprises a CDR-LL a CDR-L2 and a CDR-L3 contained within the VL region amino acid sequence set forth in SEQ ID NO:536;
(D) the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 contained within the VH region amino acid sequence set forth in SEQ ID NO:519; and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 contained within the VL region amino acid sequence set forth in SEQ ID NO:535; or
(E) the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 contained within the VH region amino acid sequence set forth in SEQ ID NO:256; and the VL region comprises a CDR-L1, a CDR-L2 and a CDR-L3 contained within the VL region amino acid sequence set forth in SEQ ID NO:267.
3. The polynucleotide of claim 1, wherein the VH region comprises a CDR-H1, a CDR-H2 and a CDR-H3 contained within the VH region amino acid sequence set forth in SEQ ID NO:617; and the VL region comprises a CDR-LL a CDR-L2 and a CDR-L3 contained within the VL region amino acid sequence set forth in SEQ ID NO:618.
4. The polynucleotide of claim 1, wherein:
(A) the VH region and the VL region comprise the amino acid sequences set forth in SEQ ID NOs:617 and 618, respectively;
(B) the VH region and the VL region comprise the amino acid sequences set forth in SEQ ID NOs:609 and 610, respectively;
(C) the VH region and the VL region comprise the amino acid sequences set forth in SEQ ID NOs:115 and 536, respectively;
(D) the VH region and the VL region comprise the amino acid sequences set forth in SEQ ID NOs:519 and 535, respectively; or
(E) the VH region and the VL region comprise the amino acid sequences set forth in SEQ ID NOs:256 and 267, respectively.
5. The polynucleotide of claim 1, wherein the VH region and the VL region comprise the amino acid sequences set forth in SEQ ID NOs:617 and 618, respectively.
6. The polynucleotide of claim 1, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO:478, SEQ ID NO:442, SEQ ID NO:560, SEQ ID NO:559, or SEQ ID NO:278.
7. The polynucleotide of claim 1, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO:478.
8. The polynucleotide of claim 1, wherein the transmembrane domain is from a human CD28.
9. The polynucleotide of claim 1, wherein the intracellular signaling region comprises an activating cytoplasmic signaling domain that is or comprises a cytoplasmic signaling domain of a CD3-zeta (CD3ζ) chain.
10. The polynucleotide of claim 9, wherein the intracellular signaling region further comprises an intracellular signaling domain of a CD28, a 4-1BB, or an ICOS.
11. The polynucleotide of claim 9, wherein the intracellular signaling region further comprises an intracellular signaling domain of a 4-1BB.
12. The polynucleotide of claim 1, wherein the transmembrane domain is from a human CD28, and the intracellular signaling region comprises an activating cytoplasmic signaling domain that is or comprises a cytoplasmic signaling domain of a CD3-zeta (CD3ζ) chain and an intracellular signaling domain of a CD28 or a 4-1BB T cell costimulatory molecule.
13. The polynucleotide of claim 1, wherein the transmembrane domain is from a human CD28, and the intracellular signaling region comprises a cytoplasmic signaling domain of a CD3-zeta (CD3ζ) chain and an intracellular signaling domain of a 4-1BB T cell costimulatory molecule.
14. A vector comprising the polynucleotide of claim 1.
15. An engineered cell, comprising the polynucleotide of claim 1.
16. A T cell, comprising the polynucleotide of claim 1.
17. A composition comprising the engineered cell of claim 15.
18. A composition comprising the T cell of claim 16.
19. A chimeric antigen receptor encoded by the polynucleotide of claim 1.
20. An engineered cell, comprising the chimeric antigen receptor of claim 19.
21. A composition comprising the engineered cell of claim 20.
22. A T cell, comprising the chimeric antigen receptor of claim 19.
23. A composition comprising the T cell of claim 22.
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23
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S. provisional application 62/580,439, filed Nov. 1, 2017, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” U.S. provisional application No. 62/580,445, filed Nov. 1, 2017, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” U.S. provisional application No. 62/582,932, filed Nov. 7, 2017, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” U.S. provisional application No. 62/582,938, filed Nov. 7, 2017, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” U.S. provisional application No. 62/596,765, filed Dec. 8, 2017, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” U.S. provisional application No. 62/596,763, filed Dec. 8, 2017, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” U.S. provisional application No. 62/614,960, filed Jan. 8, 2018, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” U.S. provisional application No. 62/614,963, filed Jan. 8, 2018, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” U.S. provisional application No. 62/665,442, filed May 1, 2018, entitled “CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR B-CELL MATURATION ANTIGEN AND ENCODING POLYNUCLEOTIDES,” and U.S. provisional application No. 62/665,447, filed May 1, 2018, entitled “METHOD OF ASSESSING ACTIVITY OF RECOMBINANT ANTIGEN RECEPTORS,” the contents of which are incorporated by reference in their entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042009900SeqList.txt, created Nov. 1, 2018, which is 593 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.
FIELD
The present disclosure relates in some aspects to chimeric antigen receptors (CARs), which contain antibody portions specific to B-cell maturation antigen (BCMA) and polynucleotides that encode CARs specific for BCMA. The disclosure further relates to genetically engineered cells, containing such BCMA-binding receptors, and uses thereof in adoptive cell therapy.
BACKGROUND
B-cell maturation antigen (BCMA) is a transmembrane type III protein expressed on mature B lymphocytes. Following binding of BCMA to its ligands, B cell activator of the TNF family (BAFF) or a proliferation inducing ligand (APRIL), a pro-survival cell signal is delivered to the B cell which has been found to be required for plasma cell survival. The expression of BCMA has been linked to several diseases including cancer, autoimmune disorders and infectious diseases Due to the role of BCMA in various diseases and conditions, including cancer, BCMA is a therapeutic target. Various BCMA-binding chimeric antigen receptors (CARs), and cells expressing such CARs, are available. However, there remains a need for improved BCMA-binding CARs and engineered BCMA-CAR expressing targeting cells, such as for use in adoptive cell therapy. Provided herein are embodiments that meet such needs.
SUMMARY
Provided are polynucleotides encoding a chimeric antigen receptor, containing nucleic acid encoding: (a) an extracellular antigen-binding domain that specifically recognizes an antigen; (b) a spacer of at least 125 amino acids in length; (c) a transmembrane domain; and (d) an intracellular signaling region, wherein following expression of the polynucleotide in a cell, the transcribed RNA, optionally messenger RNA (mRNA), from the polynucleotide, exhibits at least 70%, 75%, 80%, 85%, 90%, or 95% RNA homogeneity. In some cases, the spacer is derived from an immunoglobulin. In some embodiments, the spacer includes a sequence of a hinge region, a CH2 and a CH3 region. In some embodiments, one of more of the hinge, CH2 and CH3 is derived all or in part from IgG4 or IgG2. In some cases, the hinge, CH2 and CH3 is derived from IgG4. In some aspects, one or more of the hinge, CH2 and CH3 is chimeric and contains sequence derived from IgG4 and IgG2. In some examples, the spacer contains an IgG4/2 chimeric hinge, an IgG2/4 CH2, and an IgG4 CH3 region. In some embodiments, the encoded spacer is or contains (i) the sequence set forth in SEQ ID NO: 649; (ii) a functional variant of SEQ ID NO:649 that has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:649; or (iii) a contiguous portion of (i) or (ii) that is at least 125 amino acids in length. In some embodiments, the encoded spacer is or includes the sequence set forth in SEQ ID NO: 649.
In some of any embodiments, the spacer has a length of 125 to 300 amino acids in length, 125 to 250 amino acids in length, 125 to 230 amino acids in length, 125 to 200 amino acids in length, 125 to 180 amino acids in length, 125 to 150 amino acids in length, 150 to 300 amino acids in length, 150 to 250 amino acids in length, 150 to 230 amino acids in length, 150 to 200 amino acids in length, 150 to 180 amino acids in length, 180 to 300 amino acids in length, 180 to 250 amino acids in length, 180 to 230 amino acids in length, 180 to 200 amino acids in length, 200 to 300 amino acids in length, 200 to 250 amino acids in length, 200 to 230 amino acids in length, 230 to 300 amino acids in length, 230 to 250 amino acids in length or 250 to 300 amino acids in length. In some embodiments, the spacer is at least or at least about or is or is about 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 221, 222, 223, 224, 225, 226, 227, 228 or 229 amino acids in length, or a length between any of the foregoing.
In some embodiments of any of the polynucleotides described herein, the nucleic acid encoding the spacer includes at least one modified splice donor and/or splice acceptor site, said modified splice donor and/or acceptor site containing one or more nucleotide modifications corresponding to a reference splice donor site and/or reference splice acceptor site contained in the sequence set forth in SEQ ID NO:621. In some cases, the one or more nucleotide modifications contains an insertion, deletion, substitution or combinations thereof. In some instances, the reference splice acceptor and/or reference splice donor sites are canonical, non-canonical, or cryptic splice sites. In some examples, the reference splice donor and/or reference splice acceptor site(s) has a splice site prediction score of at least or about 0.4, 0.5, 0.6, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.99, or 1.0; and/or the reference splice donor and/or reference splice acceptor site(s) is/are predicted to be involved in a splice event with a probability of at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%.
In some embodiments of any of the polynucleotides described herein, the reference splice donor site includes the sequence aatctaagtacggac (SEQ ID NO: 705), tcaactggtacgtgg (SEQ ID NO:706), acaattagtaaggca (SEQ ID NO:707) and/or accacaggtgtatac (SEQ ID NO:708); and/or the reference splice acceptor site includes the sequence aagtttctttctgtattccaggctgaccgtggataaatctc (SEQ ID NO:742) and/or gggcaacgtgttctcttgcagtgtcatgcacgaagccctgc (SEQ ID NO:743). In some embodiments, the reference splice donor and/or reference splice acceptor site(s) has a splice site prediction score of at least or about 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.99, or 1.0; and/or the reference splice donor and/or reference splice acceptor site(s) is/are predicted to be involved in a splice event with a probability of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. In some embodiments, the reference splice donor site contains the sequence tcaactggtacgtgg (SEQ ID NO:706); and/or the reference splice acceptor site contains the sequence
(SEQ ID NO: 742)
aagtttctttctgtattccaggctgaccgtggataaatctc.
In some embodiments of any of the polynucleotides described herein, at least one of the one or more nucleotide modifications are within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues of the splice site junction of the reference splice acceptor and/or reference splice donor site. In some aspects, the one or more nucleotide modifications is silent and/or results in a degenerate codon compared to SEQ ID NO:621 and/or does not change the amino acid sequence of the encoded spacer. In some embodiments, the modified splice donor site is set forth in agtctaaatacggac (SEQ ID NO:661), tcaactggtatgtgg (SEQ ID NO:662), accatctccaaggcc (SEQ ID NO:663) and/or gccccaggtttacac (SEQ ID NO:664); and/or the modified splice acceptor site is set forth in cagtttcttcctgtatagtagactcaccgtggataaatcaa (SEQ ID NO:672) gggcaacgtgttcagctgcagcgtgatgcacgaggccctgc (SEQ ID NO: 673) and/or aagtttctttctgtattccagactgaccgtggataaatctc (SEQ ID NO:854). In some cases, the modified splice donor site is set forth in tcaactggtatgtgg (SEQ ID NO:662) and/or the modified acceptor site is set forth in cagtttcttcctgtatagtagactcaccgtggataaatcaa (SEQ ID NO:672). In some of any such embodiments, the spacer is encoded by a sequence of nucleotide set forth in SEQ ID NO:622 or a portion thereof.
Provided is a polynucleotide encoding a chimeric antigen receptor, wherein the polynucleotide includes nucleic acid encoding: (a) an extracellular antigen-binding domain that specifically recognizes an antigen; (b) a spacer, wherein the encoding nucleic acid is or includes the sequence set forth in SEQ ID NO:622 or encodes a sequence of amino acids set forth in SEQ ID NO:649; (c) a transmembrane domain; and (d) an intracellular signaling region.
Also provided is a polynucleotide encoding a chimeric antigen receptor, wherein the polynucleotide including nucleic acid encoding: (a) an extracellular antigen-binding domain that specifically recognizes an antigen; (b) a spacer, wherein the encoding nucleic acid includes or mostly includes the sequence set forth in SEQ ID NO:622 or encodes a sequence of amino acids set forth in SEQ ID NO:649; (c) a transmembrane domain; and (d) an intracellular signaling region.
In some of any of the embodiments, following expression of the polynucleotide in a cell, the transcribed RNA, optionally messenger RNA (mRNA), from the polynucleotide, exhibits at least 70%, 75%, 80%, 85%, 90%, or 95% RNA homogeneity. In some embodiments, following expression in a cell, the transcribed RNA, optionally messenger RNA (mRNA), from the polynucleotide exhibits reduced heterogeneity compared to the heterogeneity of the mRNA transcribed from a reference polynucleotide, said reference polynucleotide encoding the same amino acid sequence as the polynucleotide, wherein the reference polynucleotide differs by the presence of one or more splice donor site and/or one or more splice acceptor site in the nucleic acid encoding the spacer and/or includes one or more nucleotide modifications compared to the polynucleotide. In some instances, the RNA heterogeneity is reduced by greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more. In some cases, the transcribed RNA, optionally messenger RNA (mRNA), from the reference polynucleotide exhibits greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more RNA heterogeneity. In some of any such embodiments, the RNA homogeneity and/or heterogeneity is determined by agarose gel electrophoresis, chip-based capillary electrophoresis, analytical ultracentrifugation, field flow fractionation, or liquid chromatography. In some of any such embodiments, the polynucleotide is codon-optimized.
In some embodiments of any of the polynucleotides described herein, the antigen is associated with the disease or condition or expressed in cells of the environment of a lesion associated with the disease or condition. In some cases, the disease or condition is a cancer. In some examples, the disease or condition is a myeloma, leukemia or lymphoma. In some embodiments, the antigen is ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2/neu (receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folate binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kinase insert domain receptor (kdr), kappa light chain, Lewis Y, L1-cell adhesion molecule, (L1-CAM), Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentially expressed antigen of melanoma (PRAME), survivin, TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2D ligands, CD44v6, dual antigen, a cancer-testes antigen, mesothelin, murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, CCL-1, CD138, a pathogen-specific antigen. In some cases, the antigen is B cell maturation antigen (BCMA).
In some of any such embodiments, the antigen-binding domain is an antibody fragment containing a variable heavy chain (VH) and a variable light chain (VL) region. In some aspects, the VH region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the VH region amino acid sequence set forth in any of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609 617, 772-774, or 814-832; and/or the VL region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VL region amino acid sequence set forth in any of SEQ ID NOs:116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849. In some cases, the VH region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the VH region amino acid sequence set forth in any of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609, 617, 772-774, or 814-832; and/or the VL region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VL region amino acid sequence set forth in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610, 618, 775-777, or 833-849.
In some embodiments of any of the polynucleotides described herein, the VH region is or contains a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region amino acid sequence selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-832; and/or the VL region is or includes a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region amino acid sequence selected from any one of SEQ ID NOs:116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849. In some embodiments, the VH region is or contains a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region amino acid sequence selected from any one of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609, 617, 772-774, or 814-832; and/or the VL region is or includes a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region amino acid sequence selected from any one of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610, 618, 775-777, or 833-849. In some embodiments, the VH region is or includes (a) a heavy chain complementarity determining region 1 (CDR-H1) containing the amino acid sequence selected from any one of SEQ ID NOs:1-3, 140-144, 288, 289, 294, 295, 507, 532, 593, 596, 604, 611; and/or (b) a heavy chain complementarity determining region 2 (CDR-H2) containing the amino acid sequence selected from any one of SEQ ID NOs:4-6, 145-148, 290, 291, 296, 297, 372-374, 513, 551, 594, 597, 605, or 612; and (c) a heavy chain complementarity determining region 3 (CDR-H3) containing the amino acid sequence selected from any one of SEQ ID NOs:7-11, 149-157, 279-287, 292, 293, 376-378, 517, 595, 606, 613; and/or the VL region is or includes (a) a light chain complementarity determining region 1 (CDR-L1) containing the amino acid sequence selected from any one of SEQ ID NOs:26-36, 174-178, 302, 303, 380-392, 394-398, 589, 601, 607 or 614; (b) a light chain complementarity determining region 2 (CDR-L2) containing the amino acid sequence selected from any one of SEQ ID NOs:37-46, 179-183, 304, 305, 399-409, 411-414, 590, 602, 608 or 615; and (c) a light chain complementarity determining region 3 (CDR-L3) containing the amino acid sequence selected from any one of SEQ ID NOs:47-58, 184-194, 306, 307, 415-427, 429-433, 591, or 603.
In some embodiments of any of the polynucleotides described herein, the VH region is or contains (a) a heavy chain complementarity determining region 1 (CDR-H1) containing the amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 3, 141, 143, 144, 288, 289, 507, 593, 604, 611; and/or (b) a heavy chain complementarity determining region 2 (CDR-H2) containing the amino acid sequence selected from any one of SEQ ID NOs: 4, 5, 6, 145, 147, 148, 290, 291, 372, 513, 594, 605 or 612; and (c) a heavy chain complementarity determining region 3 (CDR-H3) containing the amino acid sequence selected from any one of SEQ ID NOs: 7, 8, 9, 10, 149, 153, 154, 155, 156, 157, 292, 293, 376, 517, 595, 606 or 613; and/or the VL region is or contains (a) a light chain complementarity determining region 1 (CDR-L1) containing the amino acid sequence selected from any one of SEQ ID NOs: 26, 27, 28, 30, 31, 33, 34, 174, 176, 177, 178, 302, 303, 380, 381, 382, 589, 601, 607 or 614; (b) a light chain complementarity determining region 2 (CDR-L2) containing the amino acid sequence selected from any one of SEQ ID NOs: 37, 38, 39, 41, 43, 44, 179, 181, 182, 183, 304, 305, 399, 400, 401, 402, 590, 602, 608 or 615; and (c) a light chain complementarity determining region 3 (CDR-L3) containing the amino acid sequence selected from any one of SEQ ID NOs: 47, 48, 49, 51, 52, 55, 56, 185, 189, 190, 191, 192, 193, 194, 306, 307, 415, 417, 418, 421, 591, or 603.
In some embodiments of any of the polynucleotides described herein, the VH region contains a CDR-H1, CDR-H2, and CDR-H3, selected from: a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:1, 4, and 7, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 8, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 9, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 10, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 11, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:140, 145, and 149, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:141, 145, and 149, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:141, 145, and 150, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:142, 146, and 151, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 152, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:143, 147, and 153, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:144, 148, and 154, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 156, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 157, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 372, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 377, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 373, and 152, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 378, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 374, and 9, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:507, 513, and 517, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:604, 605, and 606, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:288, 290, and 292, respectively; or a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:289, 291, and 293, respectively.
In some embodiments of any of the polynucleotides described herein, the VH region contains a CDR-H1, CDR-H2, and CDR-H3, selected from: a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:1, 4, and 7, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 8, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 9, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 10, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:141, 145, and 149, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:143, 147, and 153, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:144, 148, and 154, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 156, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 157, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 372, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:507, 513, and 517, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:604, 605, and 606, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:288, 290, and 292, respectively; or a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:289, 291, and 293, respectively;
In some embodiments of any of the polynucleotides described herein, the VH region is or includes the amino acid sequence set forth in any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-832. In some aspects, the VH region is or includes the amino acid sequence set forth in any of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609 or 617. In some embodiments, the VH region contains a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively; or the VH region includes a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively. In some embodiments, the VH region is or includes the amino acid sequence set forth in SEQ ID NO: 617.
In some embodiments of any of the polynucleotides described herein, the VL region includes a CDR-L1, CDR-L2, and CDR-L3 selected from: a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:26, 37, and 47, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:27, 38, and 48, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:28, 39, and 49, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:29, 40, and 50, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 39, and 51, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:31, 41, and 52, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:32, 42, and 53, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 39, and 54, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:33, 43, and 55, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:34, 44, and 56, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:35, 45, and 57, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:36, 46, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 184, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 185, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 186, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 187, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:175, 180, and 188, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 189, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:176, 181, and 190, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:177, 182, and 191, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 192, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:178, 183, and 193, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:178, 183, and 194, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 399, and 415, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:380, 400, and 416, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:33, 43, and 421, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:381, 401, and 417, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:382, 402, and 418, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:383, 403, and 419, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:384, 39, and 54, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:385, 180, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:175, 180, and 188, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:386, 404, and 420, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:387, 405, and 422, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:388, 406, and 423, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:388, 407, and 424, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:389, 408, and 425, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:390, 183, and 193, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:391, 409, and 426, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:392, 40, and 427, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:394, 39, and 429, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:395, 411, and 430, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:396, 412, and 431, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:396, 412, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:397, 413, and 432, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:398, 414, and 433, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:589, 590, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:607, 608, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:302, 304, and 306, respectively; or a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:303, 305, and 307, respectively.
In some embodiments of any of the polynucleotides described herein, the VL region includes a CDR-L1, CDR-L2, and CDR-L3 selected from: a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:26, 37, and 47, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:27, 38, and 48, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:28, 39, and 49, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 39, and 51, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:31, 41, and 52, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:33, 43, and 55, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:34, 44, and 56, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 185, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 189, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:176, 181, and 190, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:177, 182, and 191, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 192, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:178, 183, and 193, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:178, 183, and 194, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 399, and 415, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:380, 400, and 416, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:33, 43, and 421, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:381, 401, and 417, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:382, 402, and 418, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:589, 590, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:607, 608, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:302, 304, and 306, respectively; or a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:303, 305, and 307, respectively.
In some of any such embodiments, the VL region is or includes the amino acid sequence set forth in any of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849. In some aspects, the VL region is or contains the amino acid sequence set forth in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610, 618, 775-777, or 833-849.
In some embodiments of any of the polynucleotides described herein, the VL region contains a CDR-L1, CDR-L2, and CDR-L3 including the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively; or the VL region contains a CDR-L1, CDR-L2, and CDR-L3 including the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively. In some cases, the VL region is or includes the amino acid sequence set forth in SEQ ID NO:618.
In some of any embodiments, the VH region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the VH region sequence of any of SEQ ID NOs:617, 110-115, 247-256, 324, 325, 518-531, 533, 609, 772-774, or 814-832; and the VL region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VL region sequence of any of SEQ ID NOs: 618, 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 775-777, or 833-849.
In some of any embodiments, the VH region is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region amino acid sequence selected from any one of SEQ ID NOs: 617, 110-115, 247-256, 324, 325, 518-531, 533, 609, 772-774, or 814-832; and the VL region is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region amino acid sequence selected from any one of SEQ ID NOs: 618, 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 775-777, or 833-849.
In some of any embodiments, the VH region is or comprises (a) a CDR-H1 comprising the sequence selected from any one of SEQ ID NOs: 593, 611, 1-3, 140-144, 288, 289, 294, 295, 507, 532, 596, or 604; (b) a CDR-H2 comprising the sequence selected from any one of SEQ ID NOs: 594, 612, 4-6, 145-148, 290, 291, 296, 297, 372-374, 513, 551, 597, or 605; and (c) a CDR-H3 comprising the sequence selected from any one of SEQ ID NOs: 595, 613, 7-11, 149-157, 279-287, 292, 293, 376-378, 517, or 606; and the VL region is or comprises (a) a CDR-L1 comprising the sequence selected from any one of SEQ ID NOs: 601, 614, 26-36, 174-178, 302, 303, 380-392, 394-398, 589, or 607; (b) a CDR-L2 comprising the sequence selected from any one of SEQ ID NOs: 602, 615, 37-46, 179-183, 304, 305, 399-409, 411-414, 590, or 608; and (c) a CDR-L3 comprising the sequence selected from any one of SEQ ID NOs: 603, 47-58, 184-194, 306, 307, 415-427, 429-433, or 591.
In some of any such embodiments, the VH region and the VL regions includes the amino acid sequence set forth in SEQ ID NOs:110 and 116, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 116, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:111 and 117, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:111 and 117, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 119, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 119, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 120, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 120, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 121, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 121, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 122, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 122, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 123, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 123, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:112 and 124, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:112 and 124, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:113 and 125, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:113 and 125, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:114 and 126, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:114 and 126, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:115 and 127, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 127, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:247 and 257, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:247 and 257, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:248 and 258, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:248 and 258, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:249 and 259, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:249 and 259, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:250 and 260, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:250 and 260, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:251 and 261, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:251 and 261, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:252 and 262, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:252 and 262, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:253 and 263, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:253 and 263, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:254 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:254 and 264, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:255 and 265, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:255 and 265, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 266, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 266, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 267, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 267, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:518 and 534, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:518 and 534, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 535, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 535, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:115 and 536, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 536, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:520 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:520 and 264, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:521 and 537, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:521 and 537, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:522 and 538, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 538, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:523 and 539, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:523 and 539, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 540, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 540, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:524 and 541, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:524 and 541, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:525 and 261, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:525 and 261, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:526 and 542, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:526 and 542, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:527 and 543, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:527 and 543, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:528 and 544, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:528 and 544, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:529 and 545, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:529 and 545, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:528 and 546, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:528 and 546, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:522 and 547, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 547, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 548, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 548, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:530 and 549, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:530 and 549, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:531 and 550, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:531 and 550, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 552, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 552, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 553, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 553, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:533 and 554, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:533 and 554, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:115 and 555, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 555, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:524 and 556, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:524 and 556, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 557, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 557, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:609 and 610, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:609 and 610, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:617 and 618, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:324 and 326, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:324 and 326, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:325 and 327, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:325 and 327, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:772 and 775, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:772 and 775, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:773 and 776, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:773 and 776, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:774 and 777, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:774 and 777, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:815 and 833, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:815 and 833, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:816 and 834, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:816 and 834, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:817 and 835, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:817 and 835, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:818 and 836, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:818 and 836, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:819 and 837, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:819 and 837, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:820 and 838, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:820 and 838, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:821 and 839, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:821 and 839, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:822 and 840, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:822 and 840, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:823 and 841, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:823 and 841, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:824 and 842, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:824 and 842, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:825 and 843, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:825 and 843, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:826 and 844, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:826 and 844, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:827 and 845, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:827 and 845, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:828 and 846, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:828 and 846, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:829 and 847, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:829 and 847, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:830 and 847, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:830 and 847, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:831 and 848, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:831 and 848, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:832 and 849, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:832 and 849, respectively.
In some embodiments of any of the polynucleotides described herein, the VH region and the VL regions encoded by the polynucleotides include the amino acid sequence set forth in SEQ ID NOs:110 and 116, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 116, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:111 and 117, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:111 and 117, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 120, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 120, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 121, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 121, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:112 and 124, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:112 and 124, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:113 and 125, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:113 and 125, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:248 and 258, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:248 and 258, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:252 and 262, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:252 and 262, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:253 and 263, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:253 and 263, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:254 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:254 and 264, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:255 and 265, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:255 and 265, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 266, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 266, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 267, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 267, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:518 and 534, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:518 and 534, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 535, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 535, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:115 and 536, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 536, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:520 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:520 and 264, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:521 and 537, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:521 and 537, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:522 and 538, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 538, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:609 and 610, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:609 and 610, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:617 and 618, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:324 and 326, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:324 and 326, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:325 and 327, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:325 and 327, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:772 and 775, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:772 and 775, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:773 and 776, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:773 and 776, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:774 and 777, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:774 and 777, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:815 and 833, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:815 and 833, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:816 and 834, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:816 and 834, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:817 and 835, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:817 and 835, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:818 and 836, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:818 and 836, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:819 and 837, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:819 and 837, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:820 and 838, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:820 and 838, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:821 and 839, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:821 and 839, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:822 and 840, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:822 and 840, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:823 and 841, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:823 and 841, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:824 and 842, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:824 and 842, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:825 and 843, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:825 and 843, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:826 and 844, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:826 and 844, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:827 and 845, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:827 and 845, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:828 and 846, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:828 and 846, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:829 and 847, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:829 and 847, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:830 and 847, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:830 and 847, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:831 and 848, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:831 and 848, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:832 and 849, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:832 and 849, respectively.
In some of any embodiments, the VH region is or comprises the sequence of any of SEQ ID NOs: 617, 110-115, 247-256, 324, 325, 518-531, 533, 609, 772-774, or 814-832; and the VL region is or comprises the sequence of any of SEQ ID NOs: 618, 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 775-777, or 833-849.
In some embodiments of any of the polynucleotides described herein, the fragment includes an scFv. In some embodiments, the VH region and the VL region are joined by a flexible linker. In some embodiments, the scFv includes a linker containing the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:361). In some embodiments, the VH region is amino-terminal to the VL region.
In some embodiments of any of the polynucleotides described herein, the antigen-binding domain includes the amino acid sequence selected from any one of SEQ ID NOs:128-139, 268-278, 329, 442, 478, 558-576, 578-583, 585, or 769-771 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence selected from any one of SEQ ID NOs: 128-139, 268-278, 329, 442, 478, 558-576, 578-583, 585, or 769-771. In some embodiments, the antigen-binding domain includes the amino acid sequence selected from any one of SEQ ID NOs:128-130, 132, 133, 136, 137, 269, 273-278, 329, 442, 478, 558-563 or 585 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence selected from any one of SEQ ID NOs: 128-130, 132, 133, 136, 137, 269, 273-278, 329, 442, 478, 558-563 or 585.
In some embodiments of any of the polynucleotides described herein, the nucleic acid encoding the antigen-binding domain includes (a) the sequence of nucleotides set forth in any of SEQ ID NOS: 330-352, 647, 648, 716 or 718; (b) a sequence of nucleotides that has at least 90% sequence identity to any of SEQ ID NOS: 330-352, 647, 648, 716 or 718; or (c) a degenerate sequence of (a) or (b). In some embodiments, the nucleic acid encoding the antigen-binding domain includes (a) the sequence of nucleotides set forth in any of SEQ ID NOS: 352, 647, 648, 716, or 718; (b) a sequence of nucleotides that has at least 90% sequence identity to any of SEQ ID NOS: 352, 647, 648, 716, or 718; or (c) a degenerate sequence of (a) or (b). In some embodiments, the nucleic acid encoding the antigen-binding domain is codon-optimized. In some embodiments, the nucleic acid encoding the antigen-binding domain includes the sequence of nucleotides set forth in any of SEQ ID NO: 440, 460, 715, 717 or 719. In some embodiments, the nucleic acid encoding the antigen-binding domain includes the sequence of nucleotides set forth in SEQ ID NO:460.
In some embodiments of any of the polynucleotides described herein, the VH region is carboxy-terminal to the VL region. In some embodiments, the scFv includes the amino acid sequence set forth in SEQ ID NOs:328 or 586, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:328 or 586.
Provided are chimeric antigen receptors, comprising: (1) an extracellular antigen-binding domain that specifically binds human B cell maturation antigen (BCMA), wherein the extracellular antigen-binding domain comprises: (i) a variable heavy chain (VH) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VH region sequence of SEQ ID NO: 617; and (ii) a variable light chain (VL) region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VL region sequence of any of SEQ ID NO: 618; (2) a spacer set forth in SEQ ID NO: 649 or wherein the nucleic acid encoding the spacer is or comprises the sequence set forth in SEQ ID NO:622; (3) a transmembrane domain, optionally a transmembrane domain from a human CD28; and (4) an intracellular signaling region comprising a cytoplasmic signaling domain of a CD3-zeta (CD3ζ) chain and an intracellular signaling domain of a T cell costimulatory molecule. Also provided are polynucleotides encoding such a chimeric antigen receptor. In some of any embodiments, the VH region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region sequence of SEQ ID NO: 617; and the VL region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region sequence of SEQ ID NO: 618; or the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:593, 594, and 595, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:596, 597, and 595, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:598, 599, and 595, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; or the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:611, 612, and 613, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:614, 615, and 603, respectively.
Provided are chimeric antigen receptors, comprising: (1) an extracellular antigen-binding domain that specifically binds human B cell maturation antigen (BCMA), wherein the extracellular antigen-binding domain comprises: a variable heavy (VH) region comprising a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region sequence of SEQ ID NO: 617; and a variable light (VL) region comprising a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region sequence of SEQ ID NO: 618; or the VH region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region sequence of SEQ ID NO: 617; and the VL region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region sequence of SEQ ID NO: 618; or the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:593, 594, and 595, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:596, 597, and 595, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:598, 599, and 595, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; or the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:611, 612, and 613, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:614, 615, and 603, respectively; (2) a spacer set forth in SEQ ID NO: 649 or wherein the nucleic acid encoding the spacer is or comprises the sequence set forth in SEQ ID NO:622; (3) a transmembrane domain, optionally a transmembrane domain from a human CD28; and (4) an intracellular signaling region comprising a cytoplasmic signaling domain of a human CD3-zeta (CD3ζ) chain and an intracellular signaling domain of a human 4-1BB or a human CD28. Also provided are polynucleotides encoding such a chimeric antigen receptor. In some of any embodiments, the extracellular antigen-binding domain comprises the VH region sequence of SEQ ID NO:617 and the VL region sequence of SEQ ID NO:618.
In some embodiments, the receptor includes an antigen-binding domain that binds to the same or substantially the same epitope on BCMA, or competes for binding to BCMA with, any of the antibodies and fragments, or antibodies having the provided combinations of VH/VL or CDR sequences, described herein including in any of the foregoing embodiments. In some embodiments, the binding domain recognizes an epitope comprising a portion of one or more amino acid sequences within a BCMA polypeptide. In some aspects, such one or more amino acid sequences are or comprise: MLMAG (SEQ ID NO:640), YFDSL (SEQ ID NO:779), and QLRCSSNTPPL (SEQ ID NO:642). In some aspects, such one or more amino acid sequences are or comprise: MLMAG (SEQ ID NO:640), YFDSLL (SEQ ID NO:641), and QLRCSSNTPPL (SEQ ID NO:642). In some aspects, such one or more amino acid sequences are or comprise: MLMAG (SEQ ID NO:640), QNEYFDSLL (SEQ ID NO:780), and QLRCSSNTPPL (SEQ ID NO:642). In some aspects, such one or more amino acid sequences are or comprise: QNEYF (SEQ ID NO:637), CIPCQL (SEQ ID NO:638), and CQRYC (SEQ ID NO:639). In some aspects, such one or more amino acid sequences are or comprise: CSQNEYF (set forth in SEQ ID NO:410) and LLHACIPCQLR (set forth in SEQ ID NO:428).
In some embodiments of any of the polynucleotides described herein, the intracellular signaling region includes an activating cytoplasmic signaling domain. In some embodiments, the activating cytoplasmic signaling domain is capable of inducing a primary activation signal in a T cell, is a T cell receptor (TCR) component and/or includes an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the activating cytoplasmic signaling domain is or includes a cytoplasmic signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain or a functional variant or signaling portion thereof. In some embodiments, the activating cytoplasmic domain is human or is derived from a human protein. In some embodiments, the activating cytoplasmic domain is or includes the sequence set forth in SEQ ID NO:628 or a sequence of amino acids that has at least 90% sequence identity to SEQ ID NO:628.
In some embodiments of any of the polynucleotides described herein, the nucleic acid encoding the activating cytoplasmic domain is or includes the sequence set forth in SEQ ID NO:627 or is a codon-optimized sequence and/or degenerate sequence thereof. In other embodiments, the nucleic acid encoding the activating cytoplasmic signaling domain is or includes the sequence set forth in SEQ ID NO:652.
In some embodiments of any of the polynucleotides described herein, the intracellular signaling region further includes a costimulatory signaling region. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of 4-1BB. In some embodiments, the costimulatory signaling region is human or is derived from a human protein. In other embodiments, the costimulatory signaling region is or includes the sequence set forth in SEQ ID NO:626 or a sequence of amino acids that exhibits at least 90% sequence identity to the sequence set forth in SEQ ID NO: 626.
In some embodiments of any of the polynucleotides described herein, the nucleic acid encoding the costimulatory region is or includes the sequence set forth in SEQ ID NO:625 or is a codon-optimized sequence and/or degenerate sequence thereof. In some embodiments, the nucleic acid encoding the costimulatory signaling region includes the sequence set forth in SEQ ID NO:681. In some embodiments, the costimulatory signaling region is between the transmembrane domain and the intracellular signaling region. In some embodiments, the transmembrane domain is or includes a transmembrane domain derived from CD4, CD28, or CD8. In some embodiments, the transmembrane domain is or includes a transmembrane domain derived from a CD28. In some embodiments, the transmembrane domain is human or is derived from a human protein. In other embodiments, the transmembrane domain is or includes the sequence set forth in SEQ ID NO:624 or a sequence of amino acids that exhibits at least 90% sequence identity to SEQ ID NO:624.
In some embodiments of any of the polynucleotides described herein, the nucleic acid encoding the transmembrane domain is or includes the sequence set forth in SEQ ID NO:623 or is a codon-optimized sequence and/or degenerate sequence thereof. In some embodiments, the nucleic acid encoding the transmembrane domain includes the sequence set forth in SEQ ID NO:688. In some embodiments of any of the polynucleotides described herein, the encoded chimeric antigen receptor includes from its N to C terminus in order: the antigen-binding domain, the spacer, the transmembrane domain and the intracellular signaling domain.
In some of any of the embodiments, the polynucleotide comprises the sequence set forth in any of SEQ ID NOS: 751-756 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any of SEQ ID NOS: 751-756 and the encoded receptor retains the function to bind to BCMA and retains the reduced RNA heterogeneity. In some of any of the embodiments, the polynucleotide comprises the sequence set forth in any of SEQ ID NOS: 755 and 756 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any of SEQ ID NOS: 755 and 756 and the encoded receptor retains the function to bind to BCMA and retains the reduced RNA heterogeneity. In some of any of the embodiments, the polynucleotide comprises the sequence set forth in SEQ ID NOs:755 or a sequences that exhibits at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and the encoded receptor retains the function to bind to BCMA and retains the reduced RNA heterogeneity. In some of any of the embodiments, the polynucleotide comprises the sequence set forth in SEQ ID NOs:755 and the encoded receptor retains the function to bind to BCMA and retains the reduced RNA heterogeneity.
In some embodiments, the polynucleotide further encodes a truncated receptor
Also provided are vectors comprising any of the polynucleotides described herein. In some of any embodiments, the vector is a viral vector. In some of any embodiments, the viral vector is a retroviral vector. In some of any embodiments, the viral vector is a lentiviral vector.
Provided in some aspects are chimeric antigen receptors encoded a polynucleotide of any of the embodiments described herein. In some embodiments, the chimeric antigen receptor includes: (a) an extracellular antigen-binding domain that specifically recognizes B cell maturation antigen (BCMA); (b) a spacer of at least 125 amino acids in length; (c) a transmembrane domain; and (d) an intracellular signaling region.
In some embodiments of any of the chimeric antigen receptors described herein, the spacer is derived from an immunoglobulin. In some embodiments, the spacer includes a sequence of a hinge region, a CH2 and CH3 region. In some embodiments of any of the chimeric antigen receptors described herein, one of more of the hinge, CH2 and CH3 is derived all or in part from IgG4 or IgG2. In some embodiments, the hinge, CH2 and CH3 is derived from IgG4. In some embodiments, one or more of the hinge, CH2 and CH3 is chimeric and includes sequence derived from IgG4 and IgG2. In some embodiments, the spacer includes an IgG4/2 chimeric hinge, an IgG2/4 CH2, and an IgG4 CH3 region.
In some embodiments of any of the chimeric antigen receptors described herein, the spacer is or includes (i) the sequence set forth in SEQ ID NO: 649; (ii) a functional variant of SEQ ID NO:649 that has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:649; or (iii) a contiguous portion of (i) or (ii) that is at least 125 amino acids in length. In some embodiments, the encoded spacer is or includes the sequence set forth in SEQ ID NO: 649.
Provided in other aspects are chimeric antigen receptors that include (a) an extracellular antigen-binding domain that specifically recognizes B cell maturation antigen (BCMA); (b) a spacer set forth in SEQ ID NO:649; (c) a transmembrane domain; and (d) an intracellular signaling region. In some embodiments of any of the chimeric antigen receptors described herein, the antigen-binding domain is an antibody fragment containing a variable heavy chain (VH) and a variable light chain (VL) region.
In some embodiments of any of the chimeric antigen receptors described herein, the VH region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the VH region amino acid sequence set forth in any of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-832; and/or the VL region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VL region amino acid sequence set forth in any of SEQ ID NOs:116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849.
In some embodiments of any of the chimeric antigen receptors described herein, the VH region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the VH region amino acid sequence set forth in any of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609, 617, 772-774, or 814-832; and/or the VL region is or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VL region amino acid sequence set forth in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610, 618, 775-777, or 833-849.
In some embodiments of any of the chimeric antigen receptors described herein, the VH region is or includes a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region amino acid sequence selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-832; and/or the VL region is or includes a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region amino acid sequence selected from any one of SEQ ID NOs:116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849.
In some embodiments of any of the chimeric antigen receptors described herein, the VH region is or includes a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region amino acid sequence selected from any one of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609, 617, 772-774, or 814-832; and/or the VL region is or includes a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region amino acid sequence selected from any one of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610, 618, 775-777, or 833-849.
In some embodiments of any of the chimeric antigen receptors described herein, the VH region is or includes (a) a heavy chain complementarity determining region 1 (CDR-H1) containing the amino acid sequence selected from any one of SEQ ID NOs:1-3, 140-144, 288, 289, 294, 295, 507, 532, 593, 596, 604, 611; and/or (b) a heavy chain complementarity determining region 2 (CDR-H2) containing the amino acid sequence selected from any one of SEQ ID NOs:4-6, 145-148, 290, 291, 296, 297, 372-374, 513, 551, 594, 597, 605, 612; and (c) a heavy chain complementarity determining region 3 (CDR-H3) containing the amino acid sequence selected from any one of SEQ ID NOs:7-11, 149-157, 279-287, 292, 293, 376-378, 517, 595, 606, 613; and/or the VL region is or includes (a) a light chain complementarity determining region 1 (CDR-L1) containing the amino acid sequence selected from any one of SEQ ID NOs:26-36, 174-178, 302, 303, 380-392, 394-398, 589, 601, 607 or 614; (b) a light chain complementarity determining region 2 (CDR-L2) containing the amino acid sequence selected from any one of SEQ ID NOs:37-46, 179-183, 304, 305, 399-409, 411-414, 590, 602, 608 or 615; and (c) a light chain complementarity determining region 3 (CDR-L3) containing the amino acid sequence selected from any one of SEQ ID NOs:47-58, 184-194, 306, 307, 415-427, 429-433, 591, or 603.
In some embodiments of any of the chimeric antigen receptors described herein, the VH region is or includes (a) a heavy chain complementarity determining region 1 (CDR-H1) containing the amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 3, 141, 143, 144, 288, 289, 507, 593, 604, 611; and/or (b) a heavy chain complementarity determining region 2 (CDR-H2) containing the amino acid sequence selected from any one of SEQ ID NOs: 4, 5, 6, 145, 147, 148, 290, 291, 372, 513, 594, 605 or 612; and (c) a heavy chain complementarity determining region 3 (CDR-H3) containing the amino acid sequence selected from any one of SEQ ID NOs: 7, 8, 9, 10, 149, 153, 154, 155, 156, 157, 292, 293, 376, 517, 595, 606 or 613; and/or the VL region is or includes (a) a light chain complementarity determining region 1 (CDR-L1) containing the amino acid sequence selected from any one of SEQ ID NOs: 26, 27, 28, 30, 31, 33, 34, 174, 176, 177, 178, 302, 303, 380, 381, 382, 589, 601, 607 or 614; (b) a light chain complementarity determining region 2 (CDR-L2) containing the amino acid sequence selected from any one of SEQ ID NOs: 37, 38, 39, 41, 43, 44, 179, 181, 182, 183, 304, 305, 399, 400, 401, 402, 590, 602, 608 or 615; and (c) a light chain complementarity determining region 3 (CDR-L3) containing the amino acid sequence selected from any one of SEQ ID NOs: 47, 48, 49, 51, 52, 55, 56, 185, 189, 190, 191, 192, 193, 194, 306, 307, 415, 417, 418, 421, 591, or 603. In some embodiments of any of the chimeric antigen receptors described herein, the VH region includes a CDR-H1, CDR-H2, and CDR-H3, selected from: a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:1, 4, and 7, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 8, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 9, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 10, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 11, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:140, 145, and 149, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:141, 145, and 149, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:141, 145, and 150, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:142, 146, and 151, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 152, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:143, 147, and 153, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:144, 148, and 154, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 156, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 157, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 372, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 377, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 373, and 152, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 378, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 374, and 9, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:507, 513, and 517, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:604, 605, and 606, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:288, 290, and 292, respectively; or a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:289, 291, and 293, respectively;
In some embodiments of any of the chimeric antigen receptors described herein, the VH region includes a CDR-H1, CDR-H2, and CDR-H3, selected from: a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:1, 4, and 7, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 8, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 9, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 10, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:141, 145, and 149, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:143, 147, and 153, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:144, 148, and 154, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 156, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 5, and 157, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:2, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 372, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:3, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:507, 513, and 517, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:604, 605, and 606, respectively; a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:288, 290, and 292, respectively; or a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:289, 291, and 293, respectively;
In some embodiments of any of the chimeric antigen receptors described herein, the VH region is or includes the amino acid sequence set forth in any of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-832. In some embodiments of any of the chimeric antigen receptors described herein, the VH region is or includes the amino acid sequence set forth in any of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609, 617, 772-774, or 814-832. In some embodiments, the VH region includes a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively; or the VH region includes a CDR-H1, CDR-H2, and CDR-H3 containing the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively. In some embodiments, the VH region is or includes the amino acid sequence set forth in SEQ ID NO:617.
In some embodiments of any of the chimeric antigen receptors described herein, the VL region includes a CDR-L1, CDR-L2, and CDR-L3 selected from: a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:26, 37, and 47, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:27, 38, and 48, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:28, 39, and 49, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:29, 40, and 50, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 39, and 51, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:31, 41, and 52, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:32, 42, and 53, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 39, and 54, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:33, 43, and 55, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:34, 44, and 56, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:35, 45, and 57, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:36, 46, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 184, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 185, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 186, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 187, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:175, 180, and 188, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 189, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:176, 181, and 190, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:177, 182, and 191, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 192, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:178, 183, and 193, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:178, 183, and 194, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 399, and 415, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:380, 400, and 416, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:33, 43, and 421, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:381, 401, and 417, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:382, 402, and 418, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:383, 403, and 419, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:384, 39, and 54, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:385, 180, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:175, 180, and 188, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:386, 404, and 420, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:387, 405, and 422, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:388, 406, and 423, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:388, 407, and 424, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:389, 408, and 425, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:390, 183, and 193, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:391, 409, and 426, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:392, 40, and 427, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:394, 39, and 429, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:395, 411, and 430, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:396, 412, and 431, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:396, 412, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:397, 413, and 432, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:398, 414, and 433, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:589, 590, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:607, 608, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 302, 304, and 306, respectively; or a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:303, 305, and 307, respectively.
In some embodiments of any of the chimeric antigen receptors described herein, the VL region includes a CDR-L1, CDR-L2, and CDR-L3 selected from: a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:26, 37, and 47, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:27, 38, and 48, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:28, 39, and 49, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 39, and 51, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:31, 41, and 52, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:33, 43, and 55, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:34, 44, and 56, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 185, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 189, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:176, 181, and 190, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:177, 182, and 191, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:174, 179, and 192, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:178, 183, and 193, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:178, 183, and 194, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:30, 399, and 415, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:380, 400, and 416, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:33, 43, and 421, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:381, 401, and 417, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:382, 402, and 418, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:589, 590, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:607, 608, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs: 302, 304, and 306, respectively; or a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:303, 305, and 307, respectively.
In some embodiments of any of the chimeric antigen receptors described herein, the VL region is or includes the amino acid sequence set forth in any of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849. In some embodiments of any of the chimeric antigen receptors described herein, the VL region is or includes the amino acid sequence set forth in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610, 618, 775-777, or 833-849.
In some embodiments of any of the chimeric antigen receptors described herein, the VL region includes a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively; or the VL region includes a CDR-L1, CDR-L2, and CDR-L3 containing the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively;
In some embodiments of any of the chimeric antigen receptors described herein, the VL region is or includes the amino acid sequence set forth in SEQ ID NO:618. In some embodiments of any of the chimeric antigen receptors described herein, the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 116, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 116, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:111 and 117, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:111 and 117, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 119, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 119, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 120, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 120, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 121, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 121, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 122, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 122, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 123, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 123, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:112 and 124, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:112 and 124, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:113 and 125, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:113 and 125, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:114 and 126, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:114 and 126, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:115 and 127, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 127, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:247 and 257, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:247 and 257, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:248 and 258, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:248 and 258, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:249 and 259, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:249 and 259, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:250 and 260, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:250 and 260, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:251 and 261, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:251 and 261, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:252 and 262, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:252 and 262, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:253 and 263, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:253 and 263, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:254 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:254 and 264, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:255 and 265, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:255 and 265, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 266, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 266, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 267, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 267, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:518 and 534, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:518 and 534, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 535, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 535, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:115 and 536, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 536, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:520 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:520 and 264, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:521 and 537, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:521 and 537, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:522 and 538, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 538, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:523 and 539, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:523 and 539, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 540, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 540, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:524 and 541, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:524 and 541, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:525 and 261, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:525 and 261, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:526 and 542, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:526 and 542, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:527 and 543, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:527 and 543, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:528 and 544, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:528 and 544, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:529 and 545, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:529 and 545, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:528 and 546, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:528 and 546, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:522 and 547, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 547, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 548, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 548, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:530 and 549, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:530 and 549, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:531 and 550, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:531 and 550, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 552, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 552, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 553, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 553, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:533 and 554, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:533 and 554, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:115 and 555, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 555, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:524 and 556, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:524 and 556, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 557, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 557, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:609 and 610, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:609 and 610, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:617 and 618, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:324 and 326, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:324 and 326, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:325 and 327, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:325 and 327, respectively.
In some embodiments of any of the chimeric antigen receptors described herein, the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 116, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 116, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:111 and 117, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:111 and 117, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 120, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 120, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:110 and 121, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 121, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:112 and 124, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:112 and 124, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:113 and 125, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:113 and 125, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:248 and 258, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:248 and 258, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:252 and 262, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:252 and 262, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:253 and 263, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:253 and 263, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:254 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:254 and 264, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:255 and 265, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:255 and 265, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 266, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 266, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:256 and 267, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 267, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:518 and 534, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:518 and 534, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:519 and 535, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 535, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:115 and 536, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 536, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:520 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:520 and 264, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:521 and 537, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:521 and 537, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:522 and 538, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 538, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:609 and 610, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:609 and 610, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:617 and 618, respectively; the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:324 and 326, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:324 and 326, respectively; or the VH region and the VL regions contain the amino acid sequence set forth in SEQ ID NOs:325 and 327, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:325 and 327, respectively.
In some embodiments of any of the chimeric antigen receptors described herein, the fragment includes an scFv. In some embodiments, the VH region and the VL region are joined by a flexible linker. In some embodiments, the scFv includes a linker containing the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 361). In some embodiments, the VH region is amino-terminal to the VL region.
In some embodiments of any of the chimeric antigen receptors described herein, the antigen-binding domain includes the amino acid sequence selected from any one of SEQ ID NOs:128-139, 268-278, 329, 442, 478, 558-576, 578-583, 585, or 769-771 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence selected from any one of SEQ ID NOs: 128-139, 268-278, 329, 442, 478, 558-576, 578-583, 585, or 769-771. In some embodiments, the antigen-binding domain includes the amino acid sequence selected from any one of SEQ ID NOs:128-130, 132, 133, 136, 137, 269, 273-278, 329, 442, 478, 558-563 or 585 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence selected from any one of SEQ ID NOs: 128-130, 132, 133, 136, 137, 269, 273-278, 329, 442, 478, 558-563 or 585.
In some embodiments of any of the chimeric antigen receptors described herein, the VH region is carboxy-terminal to the VL region. In some embodiments, the scFv includes the amino acid sequence set forth in SEQ ID NOs: 328 or 586, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 328 or 586.
In some embodiments of any of the chimeric antigen receptors described herein, the intracellular signaling region includes an activating cytoplasmic signaling domain. In some embodiments of any of the chimeric antigen receptors described herein, the activating cytoplasmic signaling domain is capable of inducing a primary activation signal in a T cell, is a T cell receptor (TCR) component and/or includes an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the activating cytoplasmic signaling domain is or includes a cytoplasmic signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain or a functional variant or signaling portion thereof. In some embodiments, the activating cytoplasmic domain is human or is derived from a human protein. In some embodiments, the activating cytoplasmic domain is or includes the sequence set forth in SEQ ID NO:628 or a sequence of amino acids that has at least 90% sequence identity to SEQ ID NO:628.
In some embodiments of any of the chimeric antigen receptors described herein, the intracellular signaling region further includes a costimulatory signaling region. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of 4-1BB. In some embodiments, the costimulatory signaling region is human or is derived from a human protein. In some embodiments, the costimulatory signaling region is or includes the sequence set forth in SEQ ID NO:626 or a sequence of amino acids that exhibits at least 90% sequence identity to the sequence set forth in SEQ ID NO: 626. In some embodiments, the costimulatory signaling region is between the transmembrane domain and the intracellular signaling region. In some embodiments, the transmembrane domain is or includes a transmembrane domain derived from CD4, CD28, or CD8. In some embodiments, the transmembrane domain is or includes a transmembrane domain derived from a CD28. In some embodiments, the transmembrane domain is human or is derived from a human protein. In some embodiments of any of the chimeric antigen receptors described herein, the transmembrane domain is or includes the sequence set forth in SEQ ID NO:624 or a sequence of amino acids that exhibits at least 90% sequence identity to SEQ ID NO:624.
In some embodiments of any of the chimeric antigen receptors described herein, the encoded chimeric antigen receptor includes from its N to C terminus in order: the antigen-binding domain, the spacer, the transmembrane domain and the intracellular signaling domain.
In some of any of the embodiments, the chimeric antigen receptor is encoded by a polynucleotide sequence comprising the sequence set forth in any of SEQ ID NOS: 751-756 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any of SEQ ID NOS: 751-756. In some of any of the embodiments, the chimeric antigen receptor is encoded by a polynucleotide sequence comprising the sequence set forth in any of SEQ ID NOS: 755 and 756 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any of SEQ ID NOS: 755 and 756. In some of any of the embodiments, the chimeric antigen receptor is encoded by a polynucleotide sequence comprising the sequence set forth in SEQ ID NO: 755 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any of the embodiments, the chimeric antigen receptor is encoded by a polynucleotide sequence comprising the sequence set forth in SEQ ID NO: 755.
Provided in some embodiments are engineered cells that contain a polynucleotide of any of the embodiments described herein. In some embodiments of any of the engineered cells described herein, the engineered cell contains the chimeric antigen receptor of any of the embodiments described herein.
In some embodiments of any of the engineered cells described herein, the cell is an immune cell. In some embodiments, the immune cell is a primary cell obtained from a subject. In some embodiments, the immune cell is an NK cell or a T cell. In some embodiments, the immune cell is a T cell and the T cell is a CD4+ and/or CD8+ T cell.
In some embodiments of any of the engineered cells described herein, the cell contains transcribed RNA encoding the chimeric antigen receptor, optionally messenger RNA (mRNA), that exhibits at least 70%, 75%, 80%, 85%, 90%, or 95% RNA homogeneity. In some embodiments, the cell contains transcribed RNA encoding the chimeric antigen receptor, optionally messenger RNA (mRNA), that exhibits reduced heterogeneity compared to the heterogeneity of transcribed mRNA in a cell encoding a reference chimeric antigen receptor, said reference chimeric antigen receptor containing the same amino acid sequence as the chimeric antigen receptor but encoded by a different polynucleotide sequence containing one or more nucleotide differences in the polynucleotide encoding the CARs and/or in which the reference chimeric antigen receptor is encoded by a polynucleotide containing one or more splice donor site and/or one or more splice acceptor site in the nucleic acid encoding the spacer. In some embodiments, the RNA heterogeneity is reduced by greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more. In some embodiments, the cell encoding the reference CAR includes transcribed RNA encoding the reference CAR, optionally messenger RNA (mRNA), that exhibits greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more RNA heterogeneity. In some embodiments, the RNA homogeneity and/or heterogeneity is determined by agarose gel electrophoresis, chip-based capillary electrophoresis, analytical ultracentrifugation, field flow fractionation, or liquid chromatography.
In some embodiments of any of the engineered cells described herein, among a plurality of the engineered cells, less than or less than about 10%, 9%, 8%, 7%, 5%, 4%, 3%, 2% or 1% of the cells in the plurality contain a chimeric antigen receptor that exhibits tonic signaling and/or antigen independent activity or signaling.
Also provided are compositions comprising any of the engineered cells provided herein. In some of any such embodiments, the composition comprises CD4+ and CD8+ T cells and the ratio of CD4+ to CD8+ T cells is from or from about 1:3 to 3:1.
Also provided herein are compositions containing a polynucleotide of any of the embodiments described herein, a chimeric antigen receptor of any of the embodiments described herein, or a engineered cell of any of the embodiments described herein. In some embodiments, the composition further contains a pharmaceutically acceptable excipient. In some of any of these embodiments, the composition is sterile.
Provided in other aspects are methods of treatment that involve administering the engineered cells of any of the embodiments described herein or the composition of any of the embodiments described herein to a subject having a disease or disorder. In some of any embodiments, the method comprises administering a dose of the engineered cells or a composition comprising a dose of the engineered cells.
Also provided are uses any of the engineered cells or the compositions described herein for the manufacture of a medicament for the treatment of a disease or disorder. Also provided are uses any of the engineered cells or the compositions described herein for treating a disease or disorder. In some of any such embodiments, the engineered cells or the composition are for use in a treatment regimen, wherein the treatment regimen comprises administering a dose of the engineered cells or a composition comprising a dose of the engineered cells.
In some embodiments of any of the methods described herein, the disease or disorder is associated with expression of B cell maturation antigen (BCMA). In some embodiments, the disease or disorder associated with BCMA is a B cell-related disorder. In some embodiments, the disease or disorder associated with BCMA is an autoimmune disease or disorder. In some embodiments, the autoimmune disease or disorder is systemic lupus erythematosus (SLE), lupus nephritis, inflammatory bowel disease, rheumatoid arthritis, ANCA associated vasculitis, idiopathic thrombocytopenia purpura (ITP), thrombotic thrombocytopenia purpura (TTP), autoimmune thrombocytopenia, Chagas' disease, Grave's disease, Wegener's granulomatosis, poly-arteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, vasculitis, diabetes mellitus, Reynaud's syndrome, anti-phospholipid syndrome, Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia, myasthenia gravis, or progressive glomerulonephritis.
In some embodiments of any of the methods described herein, the disease or disorder associated with BCMA is a cancer. In some embodiments, the cancer is a BCMA-expressing cancer. In some embodiments, the cancer is a B cell malignancy. In some embodiments, the cancer is a lymphoma, a leukemia, or a plasma cell malignancy. In some embodiments, the cancer is a lymphoma and the lymphoma is Burkitt's lymphoma, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, Waldenstrom macroglobulinemia, follicular lymphoma, small non-cleaved cell lymphoma, mucosa-associated lymphatic tissue lymphoma (MALT), marginal zone lymphoma, splenic lymphoma, nodal monocytoid B cell lymphoma, immunoblastic lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, pulmonary B cell angiocentric lymphoma, small lymphocytic lymphoma, primary mediastinal B cell lymphoma, lymphoplasmacytic lymphoma (LPL), or mantle cell lymphoma (MCL). In some embodiments, the cancer is a leukemia and the leukemia is chronic lymphocytic leukemia (CLL), plasma cell leukemia or acute lymphocytic leukemia (ALL). In some embodiments, the cancer is a plasma cell malignancy and the plasma cell malignancy is multiple myeloma (MM) or plasmacytoma. In some embodiments, the cancer is multiple myeloma (MM).
In some of any embodiments, the dose of engineered T cells comprises between at or about 1×107 CAR-expressing T cells and at or about 2×109 CAR-expressing T cells. In some of any embodiments, the dose of engineered T cells comprise between at or about 2.5×107 CAR-expressing T cells and at or about 1.2×109 CAR-expressing T cells, between at or about 5.0×107 CAR-expressing T cells and at or about 4.5×108 CAR-expressing T cells, or between at or about 1.5×108 CAR-expressing T cells and at or about 3.0×108 CAR-expressing T cells. In some of any embodiments, the dose of engineered T cells comprise at or about 2.5×107, at or about 5.0×107, at or about 1.5×108, at or about 3.0×108, at or about 4.5×108, at or about 8.0×108 or at or about 1.2×109 CAR-expressing T cells. In some of any embodiments, the dose of engineered T cells comprise at or about 5.0×107, at or about 1.5×108, at or about 3.0×108 or at or about 4.5×108 CAR-expressing T cells. In some of any embodiments, the dose of engineered T cells comprises a combination of CD4+ T cells and CD8+ T cells, at a defined ratio of CD4+ CAR-expressing T cells to CD8+ CAR-expressing T cells and/or of CD4+ T cells to CD8+ T cells, that is or is approximately 1:1 or is between approximately 1:3 and approximately 3:1.
In some of any embodiments, less than about 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the CAR-expressing T cells in the dose of engineered T cells express a marker of apoptosis, optionally Annexin V or active Caspase 3. In some of any embodiments, less than 5%, 4%, 3%, 2% or 1% of the CAR-expressing T cells in the dose of engineered T cells express Annexin V or active Caspase 3.
In some of any embodiments, prior to the administration, the subject has received a lymphodepleting therapy comprising the administration of fludarabine at or about 20-40 mg/m2 body surface area of the subject, optionally at or about 30 mg/m2, daily, for 2-4 days, and/or cyclophosphamide at or about 200-400 mg/m2 body surface area of the subject, optionally at or about 300 mg/m2, daily, for 2-4 days.
In some of any embodiments, the subject has received a lymphodepleting therapy comprising the administration of fludarabine at or about 30 mg/m2 body surface area of the subject, daily, and cyclophosphamide at or about 300 mg/m2 body surface area of the subject, daily, for 3 days.
In some of any embodiments, at or prior to the administration of the dose of cells, the subject has received three or more therapies selected from among: autologous stem cell transplant (ASCT); an immunomodulatory agent; a proteasome inhibitor; and an anti-CD38 antibody.
In some of any embodiments, the immunomodulatory agent is selected from among thalidomide, lenalidomide or pomalidomide. In some of any embodiments, the proteasome inhibitor is selected from among bortezomib, carfilzomib or ixazomib. In some of any embodiments, the anti-CD38 antibody is or comprises daratumumab.
In some of any embodiments, at the administration of the dose of cells, the subject has not had active or history of plasma cell leukemia (PCL).
In some of any embodiments, when administered to subjects, the dose or the composition is capable of achieving objective response (OR), in at least 50%, 60%, 70%, 80%, 90%, or 95% of subjects that were administered. In some of any embodiments, the OR includes subjects who achieve stringent complete response (sCR), complete response (CR), very good partial response (VGPR), partial response (PR) and minimal response (MR). In some of any embodiments, when administered to subjects, the dose or the composition is capable of achieving stringent complete response (sCR), complete response (CR), very good partial response (VGPR) or partial response (PR), in at least 50%, 60%, 70%, 80%, or 85% of subjects that were administered. In some of any embodiments, when administered to subjects, the dose or the composition is capable of achieving stringent complete response (sCR) or complete response (CR) at least 20%, 30%, 40% 50%, 60% or 70% of subjects that were administered.
In some of any embodiments, the dose of engineered T cells comprise at or about 5.0×107, at or about 1.5×108, at or about 3.0×108 or at or about 4.5×108 CAR-expressing T cells. In some of any embodiments, the dose of the engineered T cells comprise at or about 5.0×107 CAR-expressing T cells.
Also provided herein are methods of determining the heterogeneity of a transcribed nucleic acid of a transgene, the method comprising: a) amplifying a transcribed nucleic acid using at least one 5′ and 3′ primer pair, wherein at least one pair comprises a 5′ primer that is complementary to a nucleic acid sequence within the 5′ untranslated region (5′ UTR) of the transcribed nucleic acid and a 3′ primer that is complementary to a nucleic acid sequence within the 3′ untranslated region (3′ UTR) of the transcribed nucleic acid to generate one or more amplified products; and b) detecting the amplified products, wherein the presence of two or more amplified products from at least one 5′ and 3′ primer pair indicates heterogeneity in the amplified products.
In some embodiments of the method, the detected differences in b) are different lengths of the amplified transcripts. In some embodiments, the differences in b) are differences in chromatographic profiles of the amplified transcripts.
In some embodiments, the differences in the amplified products are determined by agarose gel electrophoresis, chip-based capillary electrophoresis, analytical ultracentrifugation, field flow fractionation, or chromatography. In some embodiments, the 5′ primer is specific to sequence transcribed from the promoter region of the transcribed nucleic acid. In some embodiments, the transcribed nucleic acid is amplified using a 3′ primer specific to a sequence within the amino acid-coding sequence of the polynucleotide, and/or the 3′ untranslated region, on of the transcribed pre-mRNA. In some embodiments, the 3 primer is specific to the polyadenylation sequence or enhancer region of the 3′ untranslated region of the transcribed pre-mRNA.
In some embodiments, step a) is effected by a single amplification reaction, using a single 5′ and 3′ primer pair comprising a 5′ primer that is complementary to a nucleic acid sequence within the 5′ untranslated region (5′ UTR) of the transcribed nucleic acid and a 3′ primer that is complementary to a nucleic acid sequence within the 3′ untranslated region (3′ UTR). In some embodiments, step a) is effected by parallel or subsequent amplification reactions using a first 5′ and 3′ primer pair, a second 5′ and 3′primer pair, and optionally additional 5′ and 3′primer pairs, wherein: the first 5′ and 3′primer pair contains a 5′ primer that is complementary to a nucleic acid sequence within the 5′ UTR of the transcribed nucleic acid and a 3′ primer that is complementary to a nucleic acid sequence within the 3′ UTR of the transcribed nucleic acid; the second 5′ and 3′ primer pair contains a 5′ primer whose sequence is complementary to a portion of the translated sequence of the nucleic acid transcript and a 3′ primer whose sequence is complementary to a nucleic acid sequence within the 3′ UTR of the transcript; and the optionally additional 5′ and 3′primer pairs each contain sequences complementary to sequences within the translated region of the transcript. In some embodiments, the parallel or subsequent amplification reactions amplify overlapping portions of the transcript.
In some embodiments, the amplified products are predicted to be about 1.5 kilobases, 2 kilobases, 2.5 kilobases, 3 kilobases, 3.5 kilobases, 4 kilobases, 4.5 kilobases, 5 kilobases, 5.5 kilobases, 6 kilobases, 7 kilobases, or 8 kilobases in length.
In some of any embodiments, a transcribed nucleic acid that is detected as having heterogeneity is identified as a transgene candidate for removal of one or more splice site. In some of any embodiments, the transcribed nucleic acid of the transgene candidate exhibits at least or at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more heterogeneity following expression in a cell.
Also provided are methods of reducing the heterogeneity of an expressed transgene transcript, the method comprising: a) identifying a transgene candidate for the removal of splice sites according to any of the methods for determining the heterogeneity of a transcribed nucleic acid provided herein; b) identifying one or more potential splice donor and/or splice acceptor sites; and c) modifying the nucleic acid sequence at or near the one or more potential splice donor and/or splice acceptor sites identified in b), thereby generating a modified polynucleotide.
In some of any such embodiments, the method also involves d) assessing the transgene candidacy for the removal of splice sites as in step a). In some of any such embodiments, the method also involves e) repeating steps b)-d) until the heterogeneity of the transcript in step d) is reduced compared to the heterogeneity of the transcript as determined in step a).
In some of any such embodiments, the one or more potential splice donor and/or splice acceptor sites exhibit a score about or at least about 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0 of a splice event, and/or is/are predicted to be involved in a splice event with a probability of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%.
In some of any such embodiments, splice donor sites and splice acceptor sites are identified independently. In some of any such embodiments, the splice acceptor and/or donor site(s) is/are canonical, non-canonical, and/or cryptic splice acceptor and/or donor site(s).
In some of any such embodiments, the transgene is a chimeric antigen receptor or a portion of a chimeric antigen receptor. In some of any such embodiments, the CAR polypeptide comprises an antigen-binding domain comprising an antibody fragment, optionally a single chain antibody fragment (scFv), comprising a variable heavy chain (VH) and a variable light chain (VL), a spacer (e.g., a spacer region located between the ligand-binding domain and the transmembrane domain, of the recombinant receptor), a transmembrane region, and an intracellular signaling region.
In some of any such embodiments, the modified polynucleotide is not modified within the coding sequence for the antigen-binding domain of the encoded CAR polypeptide. In some of any such embodiments, the encoded amino acid sequence of the transgene is unchanged following modification of the polynucleotide. In some of any such embodiments, the RNA transcribed from the modified polynucleotide exhibits at least or at least about 70%, 75%, 80%, 85%, 90%, or 95% homogeneity following expression of the unmodified polynucleotide in a cell.
In some of any such embodiments, the cell is a human cell. In some of any such embodiments, the cell is a T-cell.
In some of any such embodiments, the method is a computer implemented method, and wherein one or more steps a)-c) occur at an electronic device comprising one or more processors and memory.
Also provided are computer systems comprising a processor and memory, the memory comprising instructions operable to cause the processor to carry out any one or more of steps of the methods of reducing the heterogeneity of an expressed transgene transcript.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B depict results of an assay assessing RNA heterogeneity as assessed by agarose gel electrophoresis. FIG. 1A depicts the RNA heterogeneity of several anti-BCMA-CARs, containing a long spacer (LS) region, or a shorter CD28 spacer region. FIG. 1B depicts RNA heterogeneity of three different anti-BCMA CAR encoding sequences, containing the long spacer (LS) region, before and after coding sequence optimization and splice site elimination (O/SSE).
FIG. 2 depicts results of an assay assessing levels of BCMA-LS CAR expression on the surface of transduced T cells before (Non-SSE) and after (O/SSE) optimization and splice site elimination of the coding sequence.
FIG. 3 depicts the comparison of transduction efficiency of lentiviral vectors encoding BMCA-LS CAR constructs and lentiviral vectors encoding BCMA-LS CAR constructs that have been codon optimized and modified to eliminate predicted splice sites (O/SSE).
FIG. 4A depicts results of an assay assessing the cytolytic activity of BMCA-LS CAR-expressing T cells against cell lines that express high (K562/BCMA) or low (RPMI 8226) levels of BCMA at several effector:target cell (E:T) ratios. FIG. 4B depicts the cytolytic activity of several BMCA-LS CAR-expressing T cells against RPMI-8226 cells at an E:T ratio of 3:1.
FIG. 4C and FIG. 4D depict the cytolytic activity of non-optimized BCMA-LS CAR-expressing T cells and optimized (O/SSE) BCMA-LS CAR-expressing T cells on various BCMA-expressing cell lines.
FIG. 5A depicts results of an assay assessing IFNγ, IL-2, and TNFα cytokine release of BMCA-LS CAR-expressing T cells in response to incubation with cell lines that express high (K562/BCMA) or low (RPMI 8226) levels of BCMA at several effector:target cell (E:T) ratios (5:1, 2.5:1, 1.25:1 and 0.6:1 indicated as a, b, c and d, respectively, in the figure). FIG. 5B depicts the IFNγ, IL-2, and TNFα cytokine release of non-optimized BMCA-LS CAR-expressing T cells and optimized (O/SSE) BCMA-LS CAR-expressing T cells in response to incubation with BCMA-expressing K562/BCMA and RPMI 8226 cells at different E:T ratios (3:1, 1.5:1, 0.75:1 and 0.375:1 indicated as a, b, c and d, respectively, in the figure).
FIG. 6 depicts results of an assay assessing cytolytic activity following incubation of BCMA-55-LS-O/SSE CAR-expressing T cells, from two donors, with BCMA-expressing cells that express varying levels of BCMA.
FIG. 7 depicts results of an assay assessing IFNγ release following incubation of BCMA-55-LS CAR O/SSE-expressing T cells, from two donors, with BCMA-expressing cells that express varying levels of BCMA.
FIG. 8 depicts results of an assay assessing cytolytic activity of anti-BCMA-expressing CAR T cells that express CARs containing different spacer regions, on OPM2 target cells.
FIGS. 9A and 9B depict results of an assay assessing cytolytic activity of anti-BCMA CAR-expressing T cells following incubation of anti-BCMA CAR-expressing T cells with OPM2 target cells in the presence of soluble BCMA-Fc.
FIG. 10A depicts results of an assay assessing cytolytic activity of optimized (O/SSE) anti-BCMA CAR-expressing T cells in the presence of supernatant from the H929 multiple myeloma cell line. FIG. 10B depicts results of an assay assessing cytolytic activity of optimize (O/SSE) anti-BCMA CAR-expressing T cells in the presence of recombinant B-cell activating factor (BAFF).
FIGS. 11A and 11B depict results of an assay assessing IFNγ, IL-2, and TNFα cytokine release following incubation of anti-BCMA CAR-expressing T cells with OPM2 target cells in the presence of soluble BCMA-Fc (FIG. 11A) or supernatant from a multiple myeloma cell line H929 (FIG. 11B) at different concentrations (0 ng/mL, 111 ng/mL, 333 ng/mL and 1000 ng/mL indicated as a, b, c and d, respectively, in the figures).
FIG. 12A depicts results of an assay assessing tumor growth in an OPM2 human multiple myeloma xenograft mouse model, following a single intravenous injection of CAR T cells expressing optimized (O/SSE) anti-BCMA CARs. FIG. 12B depicts results of an assay assessing survival in an OPM2 human multiple myeloma xenograft mouse model, following a single intravenous injection of CART cells expressing optimized (O/SSE) anti-BCMA CARs.
FIG. 13A depicts results of an assay assessing tumor growth in an RPMI-8226 (subcutaneous) xenograft mouse model, following a single intravenous injection of CART cells expressing optimized (O/SSE) anti-BCMA CARs. FIG. 13B depicts survival in an RPMI-8226 (subcutaneous) xenograft mouse model, following a single intravenous injection of CART cells expressing optimized (O/SSE) anti-BCMA CARs.
FIGS. 14A and 14B depict results of an assay assessing the number of CD4+(FIG. 14A) and CD8+(FIG. 14B) CAR-positive T cells in the blood from RPMI-8226 (subcutaneous) xenograft mice treated with optimized (O/SSE) anti-BCMA CAR T cells derived from a single donor (Donor 2).
FIGS. 15A and 15B depict results of an assay assessing the number of CD4+(FIG. 15A) and CD8+(FIG. 15B) CAR-positive T cells in the blood from RPMI-8226 (subcutaneous) xenograft mice treated with optimized (O/SSE) anti-BCMA CAR T cells derived from a single donor (Donor 1).
FIG. 16A depicts results of an assay assessing expression level of tdTomato and a truncated receptor (surrogate marker for CAR expression), as detected by flow cytometry, in BCMA-55-LS-O/SSE CAR-expressing cells, incubated for 6 hours in 96-well cell culture plates coated overnight with (0.008 μg/mL, 0.04 μg/mL, 0.2 μg/mL, 1 μg/mL and 5 μg/mL) of BCMA-Fc (soluble human BCMA fused at its C-terminus to an Fc region of IgG) fusion polypeptide. A recombinant Fc polypeptide was used as a control (Fc Control).
FIG. 16B depicts results of an assay assessing percentage of tdTomato+ cells among cells expressing the truncated receptor, in reporter cells expressing BCMA-55-LS-O/SSE CAR, BCMA-26-LS-O/SSE CAR, BCMA-23-LS-O/SSE CAR, and BCMA-25-LS-O/SSE CAR, incubated with ten (10) 2-fold serial dilution of BCMA-Fc. Cells expressing a CAR specific for a different antigen (anti-CD19 CAR) was used as control.
FIG. 17 depicts the percentage of tdTomato+ cells among reporter cells expressing BCMA-55-LS-O/SSE CAR or BCMA-55-SS CAR, following co-cultured with human BCMA-expressing K562 target cells (BCMA.K562) target cells at various E:T ratios.
FIG. 18 depicts the expression level of tdTomato and GFP (surrogate marker for CAR expression), as detected by flow cytometry, in reporter cells expressing an anti-CD19 CAR, BCMA-55-LS-O/SSE CAR, BCMA-26-LS-O/SSE CAR, BCMA-23-LS-O/SSE CAR, or BCMA-52-LS-O/SSE CAR, incubated without antigen stimulation to assess the degree of antigen-independent (tonic) signaling for 3 days.
FIGS. 19A and 19B depict the expression level of tdTomato and truncated receptor (surrogate marker for CAR expression), as detected by flow cytometry, in reporter cells expressing an anti-CD19 CAR, BCMA-55-LS-O/SSE CAR, BCMA-26-LS-O/SSE CAR, BCMA-23-LS-O/SSE CAR, or BCMA-52-LS-O/SSE CAR that contain intracellular domains derived from 4-1BB or CD28 incubated without antigen stimulation to assess the degree of antigen-independent (tonic) signaling.
FIG. 20A depicts the percentage of tdTomato+ cells, as assessed by flow cytometry, among the Nur77-tdTomato reporter cells engineered to express BCMA-55-LS-O/SSE CAR, specific for human BCMA, co-cultured with K562 human myelogenous leukemia cells expressing human BCMA (huBCMA), murine BCMA (muBCMA) or cynomolgus monkey BCMA (cynoBCMA), at an E:T ratio of 2:1 or 5:1. FIGS. 20B and 20C depict the percentage (FIG. 20B) and mean fluorescence intensity (MFI; FIG. 20C) of tdTomato+ cells, as assessed by flow cytometry, among reporter cells expressing BCMA-55-LS-O/SSE CAR, incubated with increasing concentrations (0, 0.1, 0.25, 1, 2.5, 10, 25 and 100 μg/mL) of huBCMA and cynoBCMA coated on 96-well flat-bottom plates.
FIG. 21A depicts an exemplary amplification strategy for a transcript and predicted amplified product. FIG. 21B depicts exemplary amplified products resulting from amplification of a transcript known and unknown (cryptic) splice sites. FIG. 21C depicts exemplary sliding window amplification of a transcript using nested primer pairs.
DETAILED DESCRIPTION
Among the provided embodiments are compositions, articles of manufacture, compounds, methods and uses including those targeting or directed to BCMA and BCMA-expressing cells and diseases. It is observed that BCMA is expressed, e.g., heterogeneously expressed, on certain diseases and conditions such as malignancies or tissues or cells thereof, e.g., on malignant plasma cells such as from all relapsed or newly diagnosed myeloma patients, for example, with little expression on normal tissues. Among the provided embodiments are approaches useful in the treatment of such diseases and conditions and/or for targeting such cell types, including nucleic acid molecules that encode BCMA-binding receptors, including chimeric antigen receptors (CARs), and the encoded receptors such as the encoded CARs, and compositions and articles of manufacture comprising the same. The receptors generally can contain antibodies (including antigen-binding antibody fragments, such as heavy chain variable (VH) regions, single domain antibody fragments and single chain fragments, including scFvs) specific for BCMA. Also provided are cells, such as engineered or recombinant cells expressing such BCMA-binding receptors, e.g., anti-BCMA CARs and/or containing nucleic acids encoding such receptors, and compositions and articles of manufacture and therapeutic doses containing such cells. Also provided are methods of evaluating, optimizing, making and using nucleic acid sequence(s), for example, nucleic acid sequences encoding recombinant BCMA-binding receptors. Also provided are methods of making and using (such as in the treatment or amelioration of BCMA-expressing diseases and conditions) cells (e.g., engineered cells) expressing or containing the recombinant BCMA-binding receptors and recombinant BCMA-binding receptor-encoding polynucleotides or compositions containing such cells.
Adoptive cell therapies (including those involving the administration of cells expressing chimeric receptors specific for a disease or disorder of interest, such as chimeric antigen receptors (CARs) and/or other recombinant antigen receptors, as well as other adoptive immune cell and adoptive T cell therapies) can be effective in the treatment of cancer and other diseases and disorders. In certain contexts, available approaches to adoptive cell therapy may not always be entirely satisfactory. In some aspects, the ability of the administered cells to recognize and bind to a target, e.g., target antigen such as BCMA, to traffic, localize to and successfully enter appropriate sites within the subject, tumors, and environments thereof, to become activated, expand, to exert various effector functions, including cytotoxic killing and secretion of various factors such as cytokines, to persist, including long-term, to differentiate, transition or engage in reprogramming into certain phenotypic states to provide effective and robust recall responses following clearance and re-exposure to target ligand or antigen, and avoid or reduce exhaustion, anergy, terminal differentiation, and/or differentiation into a suppressive state.
In some contexts, optimal response to therapy can depend on the ability of the engineered recombinant receptors such as CARs, to be consistently and reliably expressed on the surface of the cells and/or bind the target antigen. For example, in some cases, heterogeneity of the transcribed RNA from an introduced transgene (e.g., encoding the recombinant receptor) can affect the expression and/or activity of the recombinant receptor, in some cases when expressed in a cell, such as a human T cell, used in cell therapy. In some contexts, the length and type of spacer in the recombinant receptor, such as a CAR, can affect the expression, activity and/or function of the receptor.
Also, in some contexts, certain recombinant receptors can exhibit antigen-independent activity or signaling (also known as “tonic signaling”), which could lead to undesirable effects, such as due to increased differentiation and/or exhaustion of T cells that express the recombinant receptor. In some aspects, such activities may limit the T cell's activity, effect or potency. In some cases, during engineering and ex vivo expansion of the cells for recombinant receptor expression, the cells may exhibit phenotypes indicative of exhaustion, due to tonic signaling through the recombinant receptor.
In some contexts, properties of particular target antigens that the recombinant receptors specifically bind, recognize or target, can that affect the activity of the receptor. In some contexts, B-cell maturation antigen (BCMA), is typically expressed on malignant plasma cells and is an attractive therapeutic target for cell therapy. In some cases, BCMA is can be cleaved by gamma secretase, generating a soluble BCMA (sBCMA), or “shed” form of BCMA, reducing the BCMA expressed on the surface of target cells. In some cases, the activity of the BCMA-binding molecules, such as anti-BCMA chimeric antigen receptors, can be blocked or inhibited by the presence of soluble BCMA. Improved strategies are needed for optimal responses to cell therapies, in particular, for recombinant receptors that specifically bind, recognize or target BCMA.
The provided embodiments, in some contexts, are based on the observation that particular spacers and optimization of the nucleic acid sequences can lead to consistent and robust expression of the recombinant receptor. The provided BCMA-binding recombinant receptors offer advantages over available approaches for cell therapies, in particular, BCMA-targeting cell therapy. In some embodiments, provided BCMA-binding recombinant receptors are observed to exhibit reduced antigen-independent, tonic signaling and lack of inhibition by soluble BCMA. In various aspects, the provided BCMA-binding recombinant receptors, polynucleotides encoding such receptors, engineered cells and cell compositions, exhibit certain desired properties that can overcome or counteract certain limitations that can reduce optimal responses to cell therapy, for example, cell therapy with engineered cells expressing a BCMA-binding recombinant receptor. In some aspects, compositions containing engineered cells expressing an exemplary BCMA-binding recombinant receptor provided herein was observed to exhibit consistency of cell health of the engineered cells, and was associated with clinical response. In some contexts, the provided embodiments, including the recombinant receptors, polynucleotides encoding such receptors, engineered cells and cell compositions, can provide various advantages over available therapies targeting BCMA, to improve the activity of the recombinant receptors and response to BCMA-targeting cell therapies.
I. BCMA-BINDING RECEPTORS AND ENCODING POLYNUCLEOTIDES
Provided in some aspects are BCMA-binding agents, such as cell surface proteins, such as recombinant receptors or chimeric antigen receptors that bind or recognize BCMA molecules and polynucleotides encoding BCMA-binding cell surface proteins, such as recombinant receptors (e.g., CARs), and cells expressing such receptors. The BCMA-binding cell surface proteins generally contain antibodies (e.g., antigen-binding antibody fragments), and/or other binding peptides that specifically recognize, such as specifically bind to BCMA, such as to BCMA proteins, such as human BCMA protein. In some aspects, the agents bind to an extracellular portion of BCMA.
In some embodiments, the polynucleotides are optimized, or contain certain features designed for optimization, such as for codon usage, to reduce RNA heterogeneity and/or to modify, e.g., increase or render more consistent among cell product lots, expression, such as surface expression, of the encoded receptor. In some embodiments, polynucleotides, encoding BCMA-binding cell surface proteins, are modified as compared to a reference polynucleotide, such as to remove cryptic or hidden splice sites, to reduce RNA heterogeneity. In some embodiments, polynucleotides, encoding BCMA-binding cell surface proteins, are codon optimized, such as for expression in a mammalian, e.g., human, cell such as in a human T cell. In some aspects, the modified polynucleotides result in in improved, e.g., increased or more uniform or more consistent level of, expression, e.g., surface expression, when expressed in a cell. Such polynucleotides can be utilized in constructs for generation of engineered cells that express the encoded BCMA-binding cell surface protein. Thus, also provided are cells expressing the recombinant receptors encoded by the polynucleotides provided herein and uses thereof in adoptive cell therapy, such as treatment of diseases and disorders associated with BCMA expression.
Among the provided polynucleotides are those that encode recombinant receptors, such as antigen receptors, that specifically recognize, such as specifically bind, BCMA. In some aspects, the encoded receptors, such as those containing BCMA-binding polypeptides, and compositions and articles of manufacture and uses of the same, also are provided.
Among the BCMA-binding polypeptides are antibodies, such as single-chain antibodies (e.g., antigen binding antibody fragments), or portions thereof. In some examples, the recombinant receptors are chimeric antigen receptors, such as those containing anti-BCMA antibodies or antigen-binding fragments thereof. In any of the embodiments, an antibody or antigen binding fragment, in the provided CARs, that specifically recognizes an antigen, e.g. BCMA, specifically binds to the antigen. The provided polynucleotides can be incorporated into constructs, such as deoxyribonucleic acid (DNA) or RNA constructs, such as those that can be introduced into cells for expression of the encoded recombinant BCMA-binding receptors.
In some cases, the polynucleotide encoding the BCMA-binding receptor contains a signal sequence that encodes a signal peptide, in some cases encoded upstream of the nucleic acid sequences encoding the BCMA-binding receptor, or joined at the 5′ terminus of the nucleic acid sequences encoding the antigen-binding domain. In some cases, the polynucleotide containing nucleic acid sequences encoding the BCMA-binding receptor, e.g., chimeric antigen receptor (CAR), contains a signal sequence that encodes a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. In other aspects, the signal sequence may encode a heterologous or non-native signal peptide. In some aspects, non-limiting exemplary signal peptide include a signal peptide of the IgG kappa chain set forth in SEQ ID NO: 620, or encoded by the nucleotide sequence set forth in SEQ ID NO: 619 or 682-685; a GMCSFR alpha chain set forth in SEQ ID NO:851 and encoded by the nucleotide sequence set forth in SEQ ID NO:850; a CD8 alpha signal peptide set forth in SEQ ID NO:852; or a CD33 signal peptide set forth in SEQ ID NO:853. In some cases, the polynucleotide encoding the BCMA-binding receptor can contain nucleic acid sequence encoding additional molecules, such as a surrogate marker or other markers, or can contain additional components, such as promoters, regulatory elements and/or multicistronic elements. In some embodiments, the nucleic acid sequence encoding the BCMA-binding receptor can be operably linked to any of the additional components.
A. Components of Encoded Recombinant BCMA-Binding Receptors
The provided BCMA-binding receptors generally contain an extracellular binding molecule and an intracellular signaling domain. Among the provided binding molecules are polypeptides containing antibodies, including single chain cell surface proteins, e.g., recombinant receptors such as chimeric antigen receptors, containing such antibodies.
Among the provided binding molecules (e.g., BCMA-binding molecules) are single chain cell surface proteins, such as recombinant receptors (e.g., antigen receptors), that include one of the provided antibodies or fragment thereof (e.g., BCMA-binding fragment). The recombinant receptors include antigen receptors that specifically bind to or specifically recognize BCMA, such as antigen receptors containing the provided anti-BCMA antibodies, e.g., antigen-binding fragments. Among the antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Also provided are cells expressing the recombinant receptors and uses thereof in adoptive cell therapy, such as treatment of diseases and disorders associated with BCMA expression.
Exemplary antigen receptors, including CARs, and methods for engineering and introducing such antigen receptors into cells, include those described, for example, in international patent application publication Nos. WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013166321, WO2013071154, WO2013123061 U.S. patent application publication Nos. US2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application No. EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 Mar. 18(2): 160-75. In some aspects, the antigen receptors include a CAR as described in U.S. Pat. No. 7,446,190, and those described in International Patent Application Publication No. WO2014055668. Exemplary CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190, and 8,389,282, and in which the antigen-binding portion, e.g., scFv, is replaced by an antibody or an antigen-binding fragment thereof, as provided herein.
In some embodiments, the provided CAR has an amino acid sequence selected from among SEQ ID NOs: 757-762, or exhibits at least or about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs 757-762. In some embodiments, the provided CAR is encoded by a polynucleotide, such as an polynucleotide with the nucleic acid sequence set forth in any of SEQ ID NOs 751-756, or a sequences that exhibits at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in any of SEQ ID NOs: 751-756.
In some embodiments, the provided CAR is encoded by a polynucleotide, such as an polynucleotide with the nucleic acid sequence set forth in any of SEQ ID NOs:755 and 756, or a sequences that exhibits at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in any of SEQ ID NOs: 755 and 756.
In some embodiments, the provided CAR is encoded by a polynucleotide, such as an polynucleotide with the nucleic acid sequence set forth in SEQ ID NOs:755 or a sequences that exhibits at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the provided CAR is encoded by a polynucleotide, such as an polynucleotide with the nucleic acid sequence set forth in SEQ ID NOs:755.
In some embodiments, the nucleic acid encoding the antigen-binding domain comprises (a) the sequence of nucleotides set forth in any of SEQ ID NOS: 648, 330-352, 647, 716, or 718; (b) a sequence of nucleotides that has at least 90% sequence identity to any of SEQ ID NOS: 648, 330-352, 647, 716, or 718; or (c) a degenerate sequence of (a) or (b).
1. Antigen-Binding Domain
Among the chimeric receptors are chimeric antigen receptors (CARs). The chimeric receptors, such as CARs, generally include an extracellular antigen binding domain that includes, is, or is comprised within or comprises, one of the provided anti-BCMA antibodies. Thus, the chimeric receptors, e.g., CARs, typically include in their extracellular portions one or more BCMA-binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable regions, and/or antibody molecules, such as those described herein.
The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab′)2 fragments, Fab′ fragments, Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific or trispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof also referred to herein as “antigen-binding fragments.” The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
The terms “complementarity determining region,” and “CDR,” synonymous with “hypervariable region” or “HVR,” are known in the art to refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and/or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). “Framework regions” and “FR” are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).
The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (“Contact” numbering scheme); Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272, (“AbM” numbering scheme).
The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between Kabat and Chothia definitions based on that used by Oxford Molecular's AbM antibody modeling software.
Table 1, below, lists exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FR-L1 located before CDR-L1, FR-L2 located between CDR-L1 and CDR-L2, FR-L3 located between CDR-L2 and CDR-L3 and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.
TABLE 1
Boundaries of CDRs according to various numbering schemes.
CDR
Kabat
Chothia
AbM
Contact
CDR-L1
L24--L34
L24--L34
L24--L34
L30--L36
CDR-L2
L50--L56
L50--L56
L50--L56
L46--L55
CDR-L3
L89--L97
L89--L97
L89--L97
L89--L96
CDR-H1
H31--H35B
H26--H32.34
H26--H35B
H30--H35B
(Kabat
Numbering1)
CDR-H1
H31--H35
H26--H32
H26--H35
H30--H35
(Chothia
Numbering2)
CDR-H2
H50--H65
H52--H56
H50--H58
H47--H58
CDR-H3
H95--H102
H95--H102
H95--H102
H93--H101
1Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD
2Al-Lazikani et al., (1997) JMB 273,927-948
Thus, unless otherwise specified, a “CDR” or “complementary determining region,” or individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the aforementioned schemes, or other known schemes. For example, where it is stated that a particular CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given VH or VL region amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the aforementioned schemes, or other known schemes. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of provided antibodies are described using various numbering schemes, although it is understood that a provided antibody can include CDRs as described according to any of the other aforementioned numbering schemes or other numbering schemes known to a skilled artisan.
Likewise, unless otherwise specified, a FR or individual specified FR(s) (e.g., FR-H1, FR-H2, FR-H3, FR-H4), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) framework region as defined by any of the known schemes. In some instances, the scheme for identification of a particular CDR, FR, or FRs or CDRs is specified, such as the CDR as defined by the Kabat, Chothia, AbM or Contact method, or other known schemes. In other cases, the particular amino acid sequence of a CDR or FR is given.
The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable regions of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
Among the antibodies included in the provided CARs are antibody fragments. An “antibody fragment” or “antigen-binding fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; heavy chain variable (VH) regions, single-chain antibody molecules such as scFvs and single-domain antibodies comprising only the VH region; and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen-binding domain in the provided CARs is or comprises an antibody fragment comprising a variable heavy chain (VH) and a variable light chain (VL) region. In particular embodiments, the antibodies are single-chain antibody fragments comprising a heavy chain variable (VH) region and/or a light chain variable (VL) region, such as scFvs.
Single-domain antibodies (sdAbs) are antibody fragments comprising all or a portion of the heavy chain variable region or all or a portion of the light chain variable region of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody.
Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some aspects, the antibody fragments are scFvs.
A “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
Among the anti-BCMA antibodies included in the provided CARs are human antibodies. A “human antibody” is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences, including human antibody libraries. The term excludes humanized forms of non-human antibodies comprising non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human. The term includes antigen-binding fragments of human antibodies.
Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated. Human antibodies also may be derived from human antibody libraries, including phage display and cell-free libraries, containing antibody-encoding sequences derived from a human repertoire.
Among the antibodies included in the provided CARs are those that are monoclonal antibodies, including monoclonal antibody fragments. The term “monoclonal antibody” as used herein refers to an antibody obtained from or within a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible variants containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. The term is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be made by a variety of techniques, including but not limited to generation from a hybridoma, recombinant DNA methods, phage-display and other antibody display methods.
In some embodiments, the CAR includes a BCMA-binding portion or portions of the antibody molecule, such as a heavy chain variable (VH) region and/or light chain variable (VL) region of the antibody, e.g., an scFv antibody fragment. In some embodiments, the provided BCMA-binding CARs contain an antibody, such as an anti-BCMA antibody, or an antigen-binding fragment thereof that confers the BCMA-binding properties of the provided CAR. In some embodiments, the antibody or antigen-binding domain can be any anti-BCMA antibody described or derived from any anti-BCMA antibody described. See, e.g., Carpenter et al., Clin Cancer Res., 2013, 19(8):2048-2060, WO 2016090320, WO2016090327, WO2010104949 and WO2017173256. Any of such anti-BCMA antibodies or antigen-binding fragments can be used in the provided CARs. In some embodiments, the anti-BCMA CAR contains an antigen-binding domain that is an scFv containing a variable heavy (VH) and/or a variable light (VL) region derived from an antibody described in WO 2016090320 or WO2016090327.
In some embodiments, the antibody, e.g., the anti-BCMA antibody or antigen-binding fragment, contains a heavy and/or light chain variable (VH or VL) region sequence as described, or a sufficient antigen-binding portion thereof. In some embodiments, the anti-BCMA antibody, e.g., antigen-binding fragment, contains a VH region sequence or sufficient antigen-binding portion thereof that contains a CDR-H1, CDR-H2 and/or CDR-H3 as described. In some embodiments, the anti-BCMA antibody, e.g., antigen-binding fragment, contains a VL region sequence or sufficient antigen-binding portion that contains a CDR-L1, CDR-L2 and/or CDR-L3 as described. In some embodiments, the anti-BCMA antibody, e.g., antigen-binding fragment, contains a VH region sequence that contains a CDR-H1, CDR-H2 and/or CDR-H3 as described and contains a VL region sequence that contains a CDR-L1, CDR-L2 and/or CDR-L3 as described. Also among the antibodies are those having sequences at least at or about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to such a sequence.
In some embodiments, the antibody, e.g., antigen-binding fragment thereof, in the provided CAR, has a heavy chain variable (VH) region having the amino acid sequence selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, and 772-774, and 814-832, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VH region amino acid selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832, or contains a CDR-H1, CDR-H2, and/or CDR-H3 present in such a VH sequence. In some embodiments, the antibody or antibody fragment, in the provided CAR, has a VH region of any of the antibodies or antibody binding fragments described in WO 2016090327, WO 2016090320, or WO 2017173256.
In some embodiments, the VH region of the anti-BCMA antibody is one that includes a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO:355), wherein X1 is A, D, E, G, L, V or W; X2 is A, D, G, L, P, Q or S; X3 is A, D, G, L or Y; X4 is D, G, P, R, S, V, Y or null; X5 is D, I, P, S, T, Y or null; X6 is A, G, I, S, T, V, Y or null; X7 is A, D, E, F, L, P, S, Y or null; X8 is P, Q, T, Y or null; X9 is D, G, R, Y or null; X10 is A, F, Y or null; Xii is D, F or null; X12 is F or null; X13 is D, T or Y; and X14 is I, L, N, V or Y. In some such embodiments, in said CDR-H3, X1 is V; X2 is D; X3 is G; X4 is D; X5 is Y; X6 is V; X7 is D; X8 is null; X9 is null; X10 is null; Xii is null; X12 is null; X13 is D; and X14 is Y.
In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H3 comprising the amino acid sequence selected from any one of SEQ ID NOs:7-11, 149-157, 279-287, 292, 293, 376-378, 517, 595, according to Kabat numbering. In some embodiments, the VH region of an antibody or antigen-binding fragment thereof contains a CDR-H3 having the amino acid sequence comprising the amino acid sequence selected from any one of SEQ ID NOs:7-11, 149-157, 279-287, 292, 293, 376-378, 517, and 595 according to Chothia numbering or AbM numbering. In some embodiments, the VH region of an antibody or antigen-binding fragment thereof contains a CDR-H3 having the amino acid sequence comprising the amino acid sequence selected from SEQ ID NOs: 606 and 613. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-H3 having the amino acid sequence of SEQ ID NO: 517, 595, 606, or 613. In any of such examples, the antibody or antigen-binding fragment thereof can contain a VH region sequence selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832 in which the corresponding CDR-H3 sequence contained therein (e.g. corresponding to amino acid residues H95 to H102 by Kabat numbering) is replaced by the CDR-H3 sequence selected from any one of SEQ ID NOs:7-11, 149-157, 279-287, 292, 293, 376-378, 517, and 595 according to Kabat numbering, any one of SEQ ID NOs:7-11, 149-157, 279-287, 292, 293, 376-378, 517, and 595 according to Chothia numbering or AbM numbering, or any one of SEQ ID NOs: 606 and 613.
In some embodiments, the VH region of an antibody or antigen-binding fragment thereof comprises a CDR-H3 contained within the VH region amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832.
In some embodiments, the VH region of the antibody or antigen-binding fragment thereof is one that includes a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of X1X2X3MX4 (SEQ ID NO:353) X1 is D or S; X2 is Y or S; X3 is A, G, W, or Y; and X4 is H, Q, or S. In some embodiments, in said CDR-H1, X1 is D; X2 is Y; X3 is Y; and X4 is S.
In some embodiments, the VH region of an antibody or antigen-binding fragment thereof contains a CDR-H1 having the amino acid sequence comprising the amino acid sequence selected from any one of SEQ ID NOs:1-3, 140-144, 288, 289, 507, and 593 according to Kabat numbering. In some embodiments, the VH region of an antibody or antigen-binding fragment thereof contains a CDR-H1 having the amino acid sequence comprising the amino acid sequence selected from any one of SEQ ID NOs:12-15, 158-160, 294, 295, 532, and 596 according to Chothia numbering. In some embodiments, the VH region of an antibody or antigen-binding fragment thereof contains a CDR-H1 having the amino acid sequence comprising the amino acid sequence selected from any one of SEQ ID NOs:19-22, 165-169, 298, 299, 509, 577, and 598 according to AbM numbering. In some embodiments, the VH region of an antibody or antigen-binding fragment thereof contains a CDR-H1 having the amino acid sequence comprising the amino acid sequence selected from any one of SEQ ID NOs 604, and 611. In some embodiments, the VH region of an antibody or antigen-binding fragment thereof contains a CDR-H1 having the amino acid sequence of SEQ ID NO:507, 532, 577, 593, 596, 598, 604, and 611. In any of such examples, the antibody or antigen-binding fragment thereof can contain a VH region sequence selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832 in which the corresponding CDR-H1 sequence contained therein (e.g. corresponding to amino acid residues H31 to H35 by Kabat numbering) is replaced by the CDR-H1 sequence selected from any one of SEQ ID NOs:1-3, 140-144, 288, 289, 507, and 593 according to Kabat numbering, any one of SEQ ID NOs:12-15, 158-160, 294, 295, 532, and 596 according to Chothia numbering, any one of SEQ ID NOs:19-22, 165-169, 509, 298, 299, 509, 577, and 598 according to AbM numbering, or any one of SEQ ID NOs: 604 and 611.
In some embodiments, the VH region of an antibody or antigen-binding fragment thereof contains a CDR-H1 contained within the VH region amino acid sequence selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832.
In some embodiments, the VH region of an antibody or antigen-binding fragment thereof is one that includes a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of X1IX2X3X4X5X6X7X8X9X10X11YX12 X13 X14 X15 X16 X17 (SEQ ID NO:354), wherein X1 is F, G, H, V, W or Y; X2 is N, R, S or V; X3 is P, Q, S, V, W or Y; X4 is K or null; X5 is A or null; X6 is D, G, N, S, or Y; X7 is G or S; X8 is G or S; X9 is E, G, N, T or S; X10 is I, K, or T; X11 is E, G, N or Y; X12 is A or V; X13 is A, D or Q; X14 is K or S; X15 is F or V; X16 is K or Q; and X17 is E or G. In some embodiments in said CDR-H2, X1 is Y; X2 is 5, X3 is S; X4 is null; X5 is null; X6 is S; X7 is G; X8 is S; X9 is T; X10 is I; Xii is Y; X12 is A; X13 is D; X14 is S; X15 is V; X16 is K; and X17 is G.
In some embodiments, the VH region of an antibody or antigen-binding fragment thereof contains a CDR-H2 comprising the amino acid sequence selected from any one of SEQ ID NOs: 4-6, 145-148, 290, 291, 372-374, 513, and 594 according to Kabat numbering. In some embodiments, the VH region of an antibody or antigen-binding fragment thereof contains a CDR-H2 comprising the amino acid sequence selected from any one of SEQ ID NOs:16-18, 161-164, 296, 297, 514-516, 551, 597 according to Chothia numbering. In some embodiments, the VH region of an antibody or antigen-binding fragment thereof contains a CDR-H2 comprising the amino acid sequence selected from any one of SEQ ID NOs: 23-25, 170-173, 300, 301, 510-512, 587, and 599 according to AbM numbering. In some embodiments, the VH region of an antibody or antigen-binding fragment thereof contains a CDR-H2 comprising the amino acid sequence selected from any one of SEQ ID NOs: 605 and 612. In some embodiments, the VH region of an antibody or antigen-binding fragment thereof contains a CDR-H2 having the amino acid sequence of any of SEQ ID NOs: 513, 551, 587, 594, 597, 599, 605, or 612. In any of such examples, the antibody or antigen-binding fragment thereof can contain a VH region sequence selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832 in which the corresponding CDR-H2 sequence contained therein (e.g. corresponding to amino acid residues H50 to H65 by Kabat numbering) is replaced by the CDR-H2 sequence selected from any one of SEQ ID NOs: 4-6, 145-148, 290, 291, 372-374, 513, and 594 according to Kabat numbering, any one of SEQ ID NOs: 16-18, 161-164, 296, 297, 514-516, 551, 597 according to Chothia numbering, any one of SEQ ID NOs: 23-25, 170-173, 300, 301, 510-512, 587, and 599 according to AbM numbering, or any one of SEQ ID NOs 605 or 612.
In some embodiments, the VH region of an antibody or antigen-binding fragment thereof contains a CDR-H2 contained within the VH region amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832.
In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-H1 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs:1-3, 140-144, 288, 289, 507, and 593 according to Kabat numbering; a CDR-H2 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs: 4-6, 145-148, 290, 291, 372-374, 513, and 594 according to Kabat numbering; and a CDR-H3 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs: 7-11, 149-157, 279-287, 292, 293, 376-378, 517, and 595 according to Kabat numbering. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-H1 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs:12-15, 158-160, 294, 295, 532, and 596 according to Chothia numbering; a CDR-H2 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs: 16-18, 161-164, 296, 297, 514-516, 551, 597 according to Chothia numbering; and a CDR-H3 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs: 7-11, 149-157, 279-287, 292, 293, 376-378, 517, and 595 according to Chothia numbering. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-H1 that is or comprises the amino acid sequence selected from any one of SEQ ID NO:19-22, 165-169, 509, 298, 299, 509, 577, and 598 according to AbM numbering; a CDR-H2 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs:23-25, 170-173, 300, 201, 510-512, 587, and 599 according to AbM numbering; and a CDR-H3 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs:7-11, 149-157, 279-287, 292, 293, 376-378, 517, 595, 606, and 613 according to AbM numbering. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-H1 that is or comprises the amino acid sequence selected from any one of SEQ ID NO:604 and 611; a CDR-H2 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs:605 and 612; and a CDR-H3 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs:606 and 613.
In some embodiments, the VH region of an antibody or antigen-binding fragment thereof comprises a CDR-H1, CDR-H2, and/or CDR-H3 according to Kabat numbering. In some embodiments, the VH region of an antibody or antigen-binding fragment thereof comprises a CDR-H1, CDR-H2, and/or CDR-H3 according to Chothia numbering. In some embodiments, the VH region of an antibody or antigen-binding fragment thereof comprises a CDR-H1, CDR-H2, and/or CDR-H3 according to AbM numbering.
In some embodiments, the antibody or antigen-binding fragment thereof comprises an VH region comprising a CDR-H1, CDR-H2, and CDR-H3 selected from the group consisting of: a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:1, 4, and 7, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 8, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 9, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 10, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 11, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:140, 145, and 149, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:141, 145, and 149, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:141, 145, and 150, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:142, 146, and 151, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 152, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:143, 147, and 153, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:144, 148, and 154, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 156, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 157, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 372, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 376, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 377, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 373, and 152, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 378, respectively; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 374, and 9; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:288, 290, and 292; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:289, 291, 293; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:507, 513, and 517; a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively, according to Kabat numbering.
For example, the antibody or antigen-binding fragment thereof provided herein comprises a VH region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence selected from among: SEQ ID NOs:1, 4, and 7; SEQ ID NOs:2, 5, and 8; SEQ ID NOs:2, 5, and 9; SEQ ID NOs:2, 5, and 10; SEQ ID NOs:3, 6, and 11; SEQ ID NOs:140, 145, and 149; SEQ ID NOs:141, 145, and 149; SEQ ID NOs:141, 145, and 150; SEQ ID NOs:142, 146, and 151; SEQ ID NOs:2, 5, and 152; SEQ ID NOs:143, 147, and 153; SEQ ID NOs:144, 148, and 154; SEQ ID NOs:3, 6, and 155; SEQ ID NOs:2, 5, and 156; SEQ ID NOs:2, 5, and 157; SEQ ID NOs:2, 6, and 376; SEQ ID NOs:3, 372, and 376; SEQ ID NOs:3, 6, and 376; SEQ ID NOs:3, 6, and 377; SEQ ID NOs:2, 373, and 152; SEQ ID NOs:2, 5, and 378; SEQ ID NOs:2, 374, and 9, SEQ ID NOs:288, 290, and 292; SEQ ID NOs:289, 291, 293; SEQ ID NOs:507, 513, and 517; and SEQ ID NOs:593, 594, and 595, respectively, according to Kabat numbering.
In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H1, CDR-H2 and CDR-H3, respectively, comprising the amino acid sequence of a CDR-H1, a CDR-H2, and a CDR-H3 contained within the VH region amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832. In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H1, CDR-H2 and CDR-H3, respectively, comprising the amino acid sequence of a CDR-H1, a CDR-H2, and a CDR-H3 contained within the VH region amino acid sequence of SEQ ID NO:609 or SEQ ID NO: 617. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH region that comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS:593, 594, and 595, respectively; SEQ ID NOS: 596, 597, and 595, respectively; SEQ ID NOS: 598, 599, and 595, respectively; or SEQ ID NOS: 611, 612, and 613, respectively.
In some embodiments of the antibody or antigen-binding fragment thereof provided herein, the VH region comprises any of the CDR-H1, CDR-H2 and CDR-H3 as described and comprises a framework region 1 (FR1), a FR2, a FR3 and/or a FR4 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, respectively, to a FR1, a FR2, a FR3 and/or a FR4 contained within the VH region amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832. For example, the anti-BCMA antibody or antigen-binding fragment thereof can comprise a CDR-H1, CDR-H2 and CDR-H3, respectively, contained within the VH region amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832, and a framework region (e.g., a FR1, a FR2, a FR3 and/or a FR4) that contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a framework region (e.g., a FR1, a FR2, a FR3 and/or a FR4) contained within the VH region amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832. In some embodiments, the VH region comprises a FR1, a FR2, a FR3 and/or a FR4 selected from a FR1 comprising the amino acid sequence selected from any one of SEQ ID NOs:59-63, 195-203, 308, 309, and 434-439; a FR2 comprising the amino acid sequence selected from any one of SEQ ID NOs:64-66, 204-209, 310, and 311; a FR3 comprising the amino acid sequence selected from any one of SEQ ID NOs:67-69, 210-216, 312, 313, 441 and 443; and/or a FR4 comprising the amino acid sequence selected from any one of SEQ ID NOs:70-71, 217-220, 314, 315, 444 and 445. In some embodiments, the VH region comprises a FR1 comprising the amino acid sequence of SEQ ID NO:61, a FR2 comprising the amino acid sequence of SEQ ID NO:65, a FR3 comprising the amino acid sequence of SEQ ID NO:69, and/or a FR4 comprising the amino acid of SEQ ID NO:70.
In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH region comprising the amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832.
Also provided are antibodies and antigen-binding fragments thereof having sequences at least at or about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences. For example, provided herein is an antibody or antigen-binding fragment comprising a VH region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VH region amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832.
In some embodiments, the antibody is a single domain antibody (sdAb) comprising only a VH region sequence or a sufficient antigen-binding portion thereof, such as any of the above described VH sequences (e.g., a CDR-H1, a CDR-H2, a CDR-H3 and/or a CDR-H4).
In some embodiments, an antibody provided herein (e.g., an anti-BCMA antibody) or antigen-binding fragment thereof comprising a VH region further comprises a light chain or a sufficient antigen binding portion thereof. For example, in some embodiments, the antibody or antigen-binding fragment thereof contains a VH region and a VL region, or a sufficient antigen-binding portion of a VH and VL region. In such embodiments, a VH region sequence can be any of the above described VH sequence. In some such embodiments, the antibody is an antigen-binding fragment, such as a Fab or an scFv. In some such embodiments, the antibody is a full-length antibody that also contains a constant region.
In some embodiments, the antibody, e.g., antigen-binding fragment thereof, has a light chain variable (VL) region having the amino acid sequence selected from any one of SEQ ID NOs:116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VL region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. In some embodiments, the antibody or antigen-binding fragment has a VL region described in any of WO 2016090327, WO 2016090320, or WO 2017173256
In some embodiments, the VL region of the antibody described herein (e.g., an anti-BCMA antibody) or antigen-binding fragment thereof is one that includes a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12, (SEQ ID NO:358), wherein X1 is A, C, G, H, I, Q or S; X2 is A, Q, S or V; X3 is 5, W or Y; X4 is D, F, G, H or Y; X5 is D, G, M, R, S or T; X6 is A, G, H, L, R, S, T or Y; X7 is L, P, R, S or null; X8 is D, G, N, R, S, T or null; X9 is A, G, H, L, P or null; X10 is F, S or null; X11 is L, P, W or Y; and X12 is S, T or V. In some embodiments, in said CDR-L3, X1 is H; X2 is V; X3 is W; X4 is D; X5 is R; X6 is 5; X7 is R; X8 is D; X9 is H; X10 is null; Xii is Y; and X12 is V.
In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L3 comprising the amino acid sequence selected from any one of SEQ ID NOs:47-58, 184-194, 306, 307, 415-427, 429-433, 591 and 603 according to Kabat numbering, Chothia numbering or AbM numbering. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L3 having the amino acid sequence of SEQ ID NO:591 or 603 according to Kabat numbering, Chothia numbering or AbM numbering. In any of such examples, the antibody or antigen-binding fragment thereof can contain a VL region sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849 in which the corresponding CDR-L3 sequence contained therein (e.g. corresponding to amino acid residues L89 to L97 by Kabat numbering) is replaced by the CDR-L3 sequence selected from any one of SEQ ID NOs: 47-58, 184-194, 306, 307, 415-427, 429-433, 591 and 603 according to Kabat numbering, Chothia numbering or AbM numbering.
In some embodiments, the VL region of an antibody or antigen-binding fragment thereof comprises a CDR-L3 contained within the VL region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. In some embodiments, the VL region of an antibody or antigen-binding fragment thereof comprises a CDR-L3 contained within the VL region amino acid sequence of SEQ ID NO:610 or SEQ ID NO: 618.
In some embodiments, the VL region of the antibody described herein (e.g., an anti-BCMA antibody) or antigen-binding fragment thereof is one that includes a light chain complementarity determining region 1 (CDR-L1) that contains the amino acid sequence: X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17 (SEQ ID NO:356), wherein X1 is G, K, R, S or T; X2 is A, G or S; X3 is G, N, S or T; X4 is G, K, N, Q, R or S; X5 is S or null; X6 is D, N, V or null; X7 is L, V or null; X8 is H, S, Y or null; X9 is S, T or null; X10 is S or null; X11 is D, G, I, N, S or null; X12 is D, E, G, K, I, N or null; X13 is F, G, K, N, R, S, Y or null; X14 is D, K, N, T or null; X15 is A, D, G, L, N, S, T or Y; X16 is L or V; X17 is A, H, N, Q or S. In some embodiments, X1 is G; X2 is A; X3 is N; X4 is N; X5 is null; X6 is null; X7 is null; X8 is null; X9 is null; X10 is null; Xii is I; X12 is G; X13 is S; X14 is K; X15 is S; X16 is V; X17 is H.
In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L1 comprising the amino acid sequence selected from any one of SEQ ID NOs: 26-36, 174-178, 302, 303, 380-392, 394-398, 589 or 601 according to Kabat numbering, Chothia numbering or AbM numbering. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L1 comprising the amino acid sequence selected from any one of SEQ ID NOs: 607 and 614. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L1 having the amino acid sequence of SEQ ID NO:589 or 601 according to Kabat numbering, Chothia numbering or AbM numbering. In any of such examples, the antibody or antigen-binding fragment thereof can contain a VL region sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849 in which the corresponding CDR-L1 sequence contained therein (e.g. corresponding to amino acid residues L24 to L34 by Kabat numbering) is replaced by the CDR-L1 sequence selected from any one of SEQ ID NOs: 26-36, 174-178, 302, 303, 380-392, 394-398, 589 or 601 according to Kabat numbering, Chothia numbering or AbM numbering.
In some embodiments, the VL region of an antibody or antigen-binding fragment thereof comprises a CDR-L1 contained within the VL region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. In some embodiments, the VL region of an antibody or antigen-binding fragment thereof comprises a CDR-L1 contained within the VL region amino acid sequence of SEQ ID NO:589, 601, 607 or 614.
In some embodiments, the VL region of the antibody provided herein (e.g., an anti-BCMA antibody) or antigen-binding fragment thereof is one that includes a light chain complementarity determining region 2 (CDR-L2) that contains the amino acid sequence of X1X2X3X4X5X6X7 (SEQ ID NO:357), wherein X1 is A, D, E, N, S, V or W; X2 is A, D, N, S or V; X3 is A, D, H, I, N or S; X4 is D, K, N, Q, R or T; X5 is L, R or V; X6 is A, E, P or Q; and X7 is A, D, S or T. In some embodiments, X1 is D; X2 is D; X3 is D; X4 is D; X5 is R; X6 is P; and X7 is S.
In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L2 comprising the amino acid sequence selected from any one of SEQ ID NOs:37-46, 179-183, 304, 305, 399-409, 411-414, 590 and 602 according to Kabat numbering, Chothia numbering or AbM numbering. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L2 comprising the amino acid sequence selected from any one of SEQ ID NOs: 608 and 615. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L2 having the amino acid sequence of SEQ ID NO:590 or SEQ ID NO: 602 according to Kabat numbering, Chothia numbering or AbM numbering. In any of such examples, the antibody or antigen-binding fragment thereof can contain a VL region sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849 in which the corresponding CDR-L2 sequence contained therein (e.g. corresponding to amino acid residues L50 to L56 by Kabat numbering) is replaced by the CDR-L2 sequence selected from any one of SEQ ID NOs: 37-46, 179-183, 304, 305, 399-409, 411-414, 590 and 602 according to Kabat numbering, Chothia numbering or AbM numbering, or with any of SEQ ID NOs: 608 and 615.
In some embodiments, the VL region of an antibody or antigen-binding fragment thereof comprises a CDR-L2 contained within the VL region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. In some embodiments, the VL region of an antibody or antigen-binding fragment thereof comprises a CDR-L2 contained within the VL region amino acid sequence of SEQ ID NO: 589, 601, 607 or 614.
In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L1 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs: 26-36, 174-178, 302, 303, 380-392, 394-398, 589 or 601 according to Kabat numbering, Chothia numbering or AbM numbering; a CDR-L2 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs: 37-46, 179-183, 304, 305, 399-409, 411-414, 590 and 602 according to Kabat numbering, Chothia numbering or AbM numbering; and a CDR-L3 that is or comprises the amino acid sequence selected from any one of SEQ ID NOs: 47-58, 184-194, 306, 307, 415-427, 429-433, 591 and 603 according to Kabat numbering, Chothia numbering or AbM numbering.
In some embodiments, the VL region of an antibody or antigen-binding fragment thereof comprises a CDR-L1, CDR-L2, and/or CDR-L3 according to Kabat numbering. In some embodiments, the VL region of an antibody or antigen-binding fragment thereof comprises a CDR-L1, CDR-L2, and/or CDR-L3 according to Chothia numbering. In some embodiments, the VL region of an antibody or antigen-binding fragment thereof comprises a CDR-L1, CDR-L2, and/or CDR-L3 according to AbM numbering.
In some embodiments of the antibody or antigen-binding fragment thereof provided herein, the VL region comprises a CDR-L1, a CDR-L2, and a CDR-L3 selected from among: a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:26, 37, and 47, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:27, 38, and 48, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:28, 39, and 49, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:29, 40, and 50, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:30, 39, and 51, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:31, 41, and 52, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:32, 42, and 53, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:30, 39, and 54, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:33, 43, and 55, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:34, 44, and 56, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:35, 45, and 57, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:36, 46, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 184, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 185, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 186, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 187, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:175, 180, and 188, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 189, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:176, 181, and 190, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:177, 182, and 191, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 192, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:178, 183, and 193, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:178, 183, and 194, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:30, 399, and 415, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:380, 400, and 416, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:33, 43, and 421, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:381, 401, and 417, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:382, 402, and 418, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:383, 403, and 419, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:384, 39, and 54, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:385, 180, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:175, 180, and 188, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:386, 404, and 420, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:387, 405, and 422, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:388, 406, and 423, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:388, 407, and 424, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:389, 408, and 425, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:390, 183, and 193, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:391, 409, and 426, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:392, 40, and 427, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:394, 39, and 429, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:395, 411, and 430, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:396, 412, and 431, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:396, 412, and 58, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:397, 413, and 432, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:398, 414, and 433, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:302, 304, and 306, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:303, 305, and 307, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:589, 590, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:607, 608, and 591, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 601, 602, and 603, respectively; a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively. In some embodiments of the antibody or antigen-binding fragment thereof provided herein, the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs:589, 590, and 591, respectively; SEQ ID NOs:607, 608, and 591, respectively; SEQ ID NOs: 601, 602, and 603, respectively; or SEQ ID NOs:614, 615, and 603, respectively.
For example, the antibody or antigen-binding fragment thereof provided herein comprises an VL region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence selected from among: SEQ ID NOs:26, 37, and 47; SEQ ID NOs:27, 38, and 48; SEQ ID NOs:28, 39, and 49; SEQ ID NOs:29, 40, and 50; SEQ ID NOs:30, 39, and 51; SEQ ID NOs:31, 41, and 52; SEQ ID NOs:32, 42, and 53; SEQ ID NOs:30, 39, and 54; SEQ ID NOs:33, 43, and 55; SEQ ID NOs:34, 44, and 56; SEQ ID NOs:35, 45, and 57; SEQ ID NOs:36, 46, and 58; SEQ ID NOs:174, 179, and 184; SEQ ID NOs:174, 179, and 185; SEQ ID NOs:174, 179, and 186; SEQ ID NOs:174, 179, and 187; SEQ ID NOs:175, 180, and 188; SEQ ID NOs:174, 179, and 189; SEQ ID NOs:176, 181, and 190; SEQ ID NOs:177, 182, and 191; SEQ ID NOs:174, 179, and 192; SEQ ID NOs:178, 183, and 193; SEQ ID NOs:178, 183, and 194; SEQ ID NOs:30, 399, and 415; SEQ ID NOs:380, 400, and 416; SEQ ID NOs:33, 43, and 421; SEQ ID NOs:381, 401, and 417; SEQ ID NOs:382, 402, and 418; SEQ ID NOs:383, 403, and 419; SEQ ID NOs:384, 39, and 54; SEQ ID NOs:385, 180, and 58; SEQ ID NOs:175, 180, and 188; SEQ ID NOs:386, 404, and 420; SEQ ID NOs:387, 405, and 422; SEQ ID NOs:388, 406, and 423; SEQ ID NOs:388, 407, and 424; SEQ ID NOs:389, 408, and 425; SEQ ID NOs:390, 183, and 193; SEQ ID NOs:391, 409, and 426; SEQ ID NOs:392, 40, and 427; SEQ ID NOs:394, 39, and 429; SEQ ID NOs:395, 411, and 430; SEQ ID NOs:396, 412, and 431; SEQ ID NOs:396, 412, and 58; SEQ ID NOs:397, 413, and 432; SEQ ID NOs:398, 414, and 433; SEQ ID NOs:589, 590, and 591; SEQ ID NOs:607, 608, and 591; SEQ ID NOs: 601, 602, and 603; or SEQ ID NOs:614, 615, and 603, respectively.
In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L1, CDR-L2, and CDR-L3, respectively, contained within the VL region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. In some embodiments, the antibody contains a CDR-L1, CDR-L2, and CDR-L3, respectively, contained within the VL region amino acid sequence selected of SEQ ID NO: 610 or SEQ ID NO: 618.
In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL region that comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS:601, 602, and 603, respectively; or SEQ ID NOS: 614, 615, and 603, respectively.
In some embodiments of the antibody or antigen-binding fragment thereof provided herein, the VL region comprises any of the CDR-L1, CDR-L2 and CDR-L3 as described and comprises a framework region 1 (FR1), a FR2, a FR3 and/or a FR4 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, respectively, to a FR1, a FR2, a FR3 and/or a FR4 contained within the VL region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. For example, the anti-BCMA antibody or antigen-binding fragment thereof can comprise a CDR-L1, CDR-L2 and CDR-L3, respectively, contained within the VL region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849, and a framework region (e.g., a FR1, a FR2, a FR3 and/or a FR4) that contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a framework region (e.g., a FR1, a FR2, a FR3 and/or a FR4) contained within the VL region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. In some embodiments, the VL region comprises a FR1, a FR2, a FR3 and/or a FR4 selected from a FR1 comprising the amino acid sequence selected from any one of SEQ ID NOs:72-82, 221-227, 316, 317, 446-459 and 461-466; a FR2 comprising the amino acid sequence selected from any one of SEQ ID NOs:83-92, 228-232, 318, 319, 467-477 and 479-482; a FR3 comprising the amino acid sequence selected from any one of SEQ ID NOs:93-101, 233-242, 320, 321, 483-495 and 497-501; and/or a FR4 comprising the amino acid sequence selected from any one of SEQ ID NOs:102-109, 243-246, 322, 323, 502-506 and 508. In some embodiments, the VL region comprises a FR1 comprising the amino acid sequence of SEQ ID NO:79, a FR2 comprising the amino acid sequence of SEQ ID NO:89, a FR3 comprising the amino acid sequence of SEQ ID NO:98, and/or a FR4 comprising the amino acid sequence of SEQ ID NO:108.
In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL region comprising an amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849. In some embodiments, the antibody or antigen-binding fragment thereof contains a VL region comprises the amino acid sequence of SEQ ID NO: 610 or SEQ ID NO: 618.
Also provided are antibodies having sequences at least at or about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.
In some embodiments, the VH region of the antibody or fragment comprises the amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832 and the VL region of the antibody or fragment comprises the amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849.
Also provided are antibodies and antigen-binding fragments thereof having sequences at least at or about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences. For example, provided herein is an antibody or antigen-binding fragment containing a VL region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VL region amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849 and/or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a VH region amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832. In some embodiments, the antibody or antigen-binding fragment contains a VL region comprising the amino acid sequence selected from any one of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, and 833-849 and a VH region the amino acid sequence selected from any one of SEQ ID NOs: 110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, and 814-832.
In some embodiments, the VH region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the VH region sequence of any of SEQ ID NOs:617, 110-115, 247-256, 324, 325, 518-531, 533, 609, 772-774, or 814-832; and the VL region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VL region sequence of any of SEQ ID NOs: 618, 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 775-777, or 833-849.
In some embodiments, the VH region and the VL regions comprise the sequence of SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:110 and 116, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:111 and 117, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:110 and 119, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:110 and 120, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:110 and 121, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:110 and 122, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:110 and 123, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:112 and 124, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:113 and 125, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:114 and 126, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:115 and 127, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:247 and 257, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:248 and 258, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:249 and 259, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:250 and 260, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:251 and 261, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:252 and 262, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:253 and 263, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:254 and 264, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:255 and 265, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:256 and 266, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:256 and 267, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:518 and 534, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:519 and 535, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:115 and 536, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:520 and 264, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:521 and 537, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:522 and 538, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:523 and 539, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:519 and 540, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:524 and 541, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:525 and 261, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:526 and 542, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:527 and 543, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:528 and 544, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:529 and 545, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:528 and 546, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:522 and 547, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:256 and 548, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:530 and 549, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:531 and 550, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:519 and 552, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:110 and 553, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:533 and 554, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:115 and 555, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:524 and 556, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:519 and 557, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:324 and 326, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:325 and 327, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:609 and 610, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:772 and 775, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:773 and 776, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:774 and 777, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:815 and 833, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:816 and 834, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:817 and 835, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:818 and 836, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:819 and 837, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:820 and 838, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:821 and 839, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:822 and 840, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:823 and 841, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:824 and 842, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:825 and 843, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:826 and 844, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:827 and 845, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:828 and 846, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:829 and 847, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:830 and 847, respectively, or a sequence of amino acids having at least 90% identity thereto; the VH region and the VL regions comprise the sequence of SEQ ID NOs:831 and 848, respectively, or a sequence of amino acids having at least 90% identity thereto; or the VH region and the VL regions comprise the sequence of SEQ ID NOs:832 and 849, respectively, or a sequence of amino acids having at least 90% identity thereto.
In some embodiments, the VH region of the antibody or antigen-binding fragment thereof comprises a CDR-H1, a CDR-H2, a CDR-H3, respectively, comprising the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained within the VH region amino acid sequence selected from any one of SEQ ID NOs: 617, 110-115, 247-256, 324, 325, 518-531, 533, 609, 772-774, and 814-832; and comprises a CDR-L1, a CDR-L2, a CDR-L3, respectively, comprising the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3, respectively contained within the VL region amino acid sequence selected from any one of SEQ ID NOs: 618, 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 775-777, and 833-849.
In some of any embodiments, the VH is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH sequence of SEQ ID NO: 617; and the VL is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL sequence of SEQ ID NO: 618; the VH is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH sequence of SEQ ID NO: 256; and the VL is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL sequence of SEQ ID NO: 267; the VH is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH sequence of SEQ ID NO: 519; and the VL is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL sequence of SEQ ID NO: 535; the VH is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH sequence of SEQ ID NO:115; and the VL is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL sequence of SEQ ID NO: 536; or the VH is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH sequence of SEQ ID NO: 609; and the VL is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL sequence of SEQ ID NO: 610. In some of any embodiments, the VH region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region amino acid sequence set forth in SEQ ID NO: 617; and the VL region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region amino acid sequence set forth in SEQ ID NO: 618; the VH region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region amino acid sequence set forth in SEQ ID NO: 256; and the VL region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region amino acid sequence set forth in SEQ ID NO: 267; the VH region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region amino acid sequence set forth in SEQ ID NO: 519; and the VL region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region amino acid sequence set forth in SEQ ID NO: 535; the VH region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region amino acid sequence set forth in SEQ ID NO:115; and the VL region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region amino acid sequence set forth in SEQ ID NO: 536; or the VH region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region amino acid sequence set forth in SEQ ID NO: 609; and the VL region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region amino acid sequence set forth in SEQ ID NO: 610.
In some embodiments, the VH region is or comprises (a) a CDR-H1 comprising the sequence selected from any one of SEQ ID NOs: 593, 611, 1-3, 140-144, 288, 289, 294, 295, 507, 532, 596, or 604; (b) a CDR-H2 comprising the sequence selected from any one of SEQ ID NOs: 594, 612, 4-6, 145-148, 290, 291, 296, 297, 372-374, 513, 551, 597, or 605; and (c) a CDR-H3 comprising the sequence selected from any one of SEQ ID NOs: 595, 613, 7-11, 149-157, 279-287, 292, 293, 376-378, 517, or 606; and the VL region is or comprises (a) a CDR-L1 comprising the sequence selected from any one of SEQ ID NOs: 601, 614, 26-36, 174-178, 302, 303, 380-392, 394-398, 589, or 607; (b) a CDR-L2 comprising the sequence selected from any one of SEQ ID NOs: 602, 615, 37-46, 179-183, 304, 305, 399-409, 411-414, 590, or 608; and (c) a CDR-L3 comprising the sequence selected from any one of SEQ ID NOs: 603, 47-58, 184-194, 306, 307, 415-427, 429-433, or 591.
In some embodiments, the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:593, 594, and 595, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:26, 37, and 47, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 8, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:27, 38, and 48, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:28, 39, and 49, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:29, 40, and 50, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:30, 39, and 51, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:31, 41, and 52, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:32, 42, and 53, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:30, 39, and 54, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 9, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:33, 43, and 55, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 10, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:34, 44, and 56, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:3, 6, and 11, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:35, 45, and 57, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 10, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:36, 46, and 58, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:140, 145, and 149, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:174, 179, and 184, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:141, 145, and 149, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:174, 179, and 185, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:141, 145, and 150, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:174, 179, and 186, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:142, 146, and 151, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:174, 179, and 187, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 152, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:175, 180, and 188, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:143, 147, and 153, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:174, 179, and 189, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:144, 148, and 154, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:176, 181, and 190, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:3, 6, and 155, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:177, 182, and 191, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 156, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:174, 179, and 192, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 157, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:178, 183, and 193, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 157, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:178, 183, and 194, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 6, and 376, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:30, 399, and 415, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:380, 400, and 416, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 10, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:33, 43, and 421, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:3, 6, and 155, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:177, 182, and 191, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:3, 372, and 376, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:381, 401, and 417, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:3, 6, and 376, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:382, 402, and 418, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:3, 6, and 377, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:383, 403, and 419, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:384, 39, and 54, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 10, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:385, 180, and 58, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 373, and 152, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:175, 180, and 188, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:3, 6, and 11, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:386, 404, and 420, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 378, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:33, 43, and 421, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 9, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:387, 405, and 422, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 9, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:388, 406, and 423, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 9, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:388, 407, and 424, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:3, 6, and 376, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:389, 408, and 425, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 157, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:390, 183, and 193, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 374, and 9, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:391, 409, and 426, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:392, 40, and 427, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:394, 39, and 429, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:395, 411, and 430, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:28, 39, and 49, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 10, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:396, 412, and 431, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 10, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:396, 412, and 58, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:2, 5, and 10, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:397, 413, and 432, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:1, 4, and 7, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:398, 414, and 433, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:288, 290, and 292, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:302, 304, and 306, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:288, 290, and 292, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:302, 304, and 306, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:289, 291, and 293, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:303, 305, and 307, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:289, 291, and 293, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:303, 305, and 307, respectively; or the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:507, 513, and 517, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:589, 590, and 591, respectively.
In some embodiments, the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:596, 597, and 595, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively. In some embodiments, the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:598, 599, and 595, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively. In some embodiments, the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:611, 612, and 613, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:614, 615, and 603, respectively.
In some embodiments, the VH region is or comprises the sequence of any of SEQ ID NOs: 617, 110-115, 247-256, 324, 325, 518-531, 533, 609, 772-774, or 814-832; and the VL region is or comprises the sequence of any of SEQ ID NOs: 618, 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 775-777, or 833-849.
In some embodiments, the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 116, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:111 and 117, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 118, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 119, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 120, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 121, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 122, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 123, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:112 and 124, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:113 and 125, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:114 and 126, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:115 and 127, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:247 and 257, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:248 and 258, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:249 and 259, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:250 and 260, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:251 and 261, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:252 and 262, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:253 and 263, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:254 and 264, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:255 and 265, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:256 and 266, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:256 and 267, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:518 and 534, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:519 and 535, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:115 and 536, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:520 and 264, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:521 and 537, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:522 and 538, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:523 and 539, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:519 and 540, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:524 and 541, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:525 and 261, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:526 and 542, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:527 and 543, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:528 and 544, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:529 and 545, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:528 and 546, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:522 and 547, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:256 and 548, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:530 and 549, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:531 and 550, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:519 and 552, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 553, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:110 and 118, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:533 and 554, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:115 and 555, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:524 and 556, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:519 and 557, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:324 and 326, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:325 and 327, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:609 and 610, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:617 and 618, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:772 and 775, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:773 and 776, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:774 and 777, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:815 and 833, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs:816 and 834, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:817 and 835, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:818 and 836, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:819 and 837, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:820 and 838, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:821 and 839, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:822 and 840, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:823 and 841, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:824 and 842, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:825 and 843, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:826 and 844, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:827 and 845, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:828 and 846, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:829 and 847, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:830 and 847, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:831 and 848, respectively; the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NO:832 and 849, respectively, or any antibody or antigen-binding fragment thereof that has at least 90% sequence identity to any of the above VH and VL, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
For example, the VH and VL regions of the antibody or antigen-binding fragment thereof provided therein comprise the amino acid sequences selected from: SEQ ID NOs:110 and 116; SEQ ID NOs:111 and 117; SEQ ID NOs:110 and 118; SEQ ID NOs:110 and 119; SEQ ID NOs:110 and 120; SEQ ID NOs:110 and 121; SEQ ID NOs:110 and 122; SEQ ID NOs:110 and 123; SEQ ID NOs:112 and 124; SEQ ID NOs:113 and 125; SEQ ID NOs:114 and 126; SEQ ID NOs:115 and 127; SEQ ID NOs:247 and 257; SEQ ID NOs:248 and 258; SEQ ID NOs:249 and 259; SEQ ID NOs:250 and 260; SEQ ID NOs:251 and 261; SEQ ID NOs:252 and 262; SEQ ID NOs:253 and 263; SEQ ID NOs:254 and 264; SEQ ID NOs:255 and 265; SEQ ID NOs:256 and 266; SEQ ID NOs:256 and 267; SEQ ID NOs:518 and 534; SEQ ID NOs:519 and 535; SEQ ID NOs:115 and 536; SEQ ID NOs:520 and 264; SEQ ID NOs:521 and 537; SEQ ID NOs:522 and 538; SEQ ID NOs:523 and 539; SEQ ID NOs:519 and 540; SEQ ID NOs:524 and 541; SEQ ID NOs:525 and 261; SEQ ID NOs:526 and 542; SEQ ID NOs:527 and 543; SEQ ID NOs:528 and 544; SEQ ID NOs:529 and 545; SEQ ID NOs:528 and 546; SEQ ID NOs:522 and 547; SEQ ID NOs:256 and 548; SEQ ID NOs:530 and 549; SEQ ID NOs:531 and 550; SEQ ID NOs:519 and 552; SEQ ID NOs:110 and 553; SEQ ID NOs:110 and 118; SEQ ID NOs:533 and 554; SEQ ID NOs:115 and 555; SEQ ID NOs:524 and 556; SEQ ID NOs:519 and 557, SEQ ID NOs:324 and 326, SEQ ID NOs:325 and 327, SEQ ID NOs:609 and 610; SEQ ID NOs:617 and 618; SEQ ID NOs:772 and 775; SEQ ID NOs:773 and 776; SEQ ID NOs:774 and 777; SEQ ID NOs:815 and 833; SEQ ID NOs:816 and 834; SEQ ID NO:817 and 835; SEQ ID NO:818 and 836; SEQ ID NO:819 and 837; SEQ ID NO:820 and 838; SEQ ID NO:821 and 839; NO:822 and 840; SEQ ID NO:823 and 841; SEQ ID NO:824 and 842; SEQ ID NO:825 and 843; SEQ ID NO:826 and 844; SEQ ID NO:827 and 845; SEQ ID NO:828 and 846; SEQ ID NO:829 and 847; SEQ ID NO:830 and 847; SEQ ID NO:831 and 848; and SEQ ID NO:832 and 849, respectively, or any antibody or antigen-binding fragment thereof that has at least 90% sequence identity to any of the above VH and VL, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or any antibody or antigen-binding fragment thereof that comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region and a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region of any of the above VH and VL.
In some embodiments, the VH and VL regions of the antibody or antigen-binding fragment thereof provided therein comprise the amino acid sequences selected from: SEQ ID NOs:617 and 618; SEQ ID NOs:256 and 267; SEQ ID NOs:519 and 535; SEQ ID NOs:115 and 536; or SEQ ID NOs:609 and 610; respectively, or any antibody or antigen-binding fragment thereof that has at least 90% sequence identity to any of the above VH and VL, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or any antibody or antigen-binding fragment thereof that comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region and a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region of any of the above VH and VL.
In some embodiments, the VH and VL regions of the antibody or antigen-binding fragment thereof provided therein comprise the amino acid sequences selected from: SEQ ID NOs:617 and 618, or any antibody or antigen-binding fragment thereof that has at least 90% sequence identity to any of the above VH and VL, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or any antibody or antigen-binding fragment thereof that comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region and a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region of any of the above VH and VL.
In some embodiments, the antibody or antigen-binding fragment thereof is a single-chain antibody fragment, such as a single chain variable fragment (scFv) or a diabody or a single domain antibody (sdAb). In some embodiments, the antibody or antigen-binding fragment is a single domain antibody comprising only the VH region. In some embodiments, the antibody or antigen binding fragment is an scFv comprising a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the single-chain antibody fragment (e.g. scFv) includes one or more linkers joining two antibody domains or regions, such as a heavy chain variable (VH) region and a light chain variable (VL) region. The linker typically is a peptide linker, e.g., a flexible and/or soluble peptide linker. Among the linkers are those rich in glycine and serine and/or in some cases threonine. In some embodiments, the linkers further include charged residues such as lysine and/or glutamate, which can improve solubility. In some embodiments, the linkers further include one or more proline.
Accordingly, the provided anti-BCMA antibodies include single-chain antibody fragments, such as scFvs and diabodies, particularly human single-chain antibody fragments, typically comprising linker(s) joining two antibody domains or regions, such VH and VL regions. The linker typically is a peptide linker, e.g., a flexible and/or soluble peptide linker, such as one rich in glycine and serine.
In some aspects, the linkers rich in glycine and serine (and/or threonine) include at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% such amino acid(s). In some embodiments, they include at least at or about 50%, 55%, 60%, 70%, or 75%, glycine, serine, and/or threonine. In some embodiments, the linker is comprised substantially entirely of glycine, serine, and/or threonine. The linkers generally are between about 5 and about 50 amino acids in length, typically between at or about 10 and at or about 30, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and in some examples between 10 and 25 amino acids in length. Exemplary linkers include linkers having various numbers of repeats of the sequence GGGGS (4GS; SEQ ID NO:359) or GGGS (3GS; SEQ ID NO:360), such as between 2, 3, 4, and 5 repeats of such a sequence. Exemplary linkers include those having or consisting of an sequence set forth in SEQ ID NO:361 (GGGGSGGGGSGGGGS). Exemplary linkers further include those having or consisting of the sequence set forth in SEQ ID NO:362 (GSTSGSGKPGSGEGSTKG). Exemplary linkers further include those having or consisting of the sequence set forth in SEQ ID NO:778 (SRGGGGSGGGGSGGGGSLEMA).
Accordingly, in some embodiments, the provided embodiments include single-chain antibody fragments, e.g., scFvs, comprising one or more of the aforementioned linkers, such as glycine/serine rich linkers, including linkers having repeats of GGGS (SEQ ID NO: 360) or GGGGS (SEQ ID NO: 359), such as the linker set forth in SEQ ID NO:361.
In some embodiments, the linker has an amino acid sequence containing the sequence set forth in SEQ ID NO:361. The fragment, e.g., scFv, may include a VH region or portion thereof, followed by the linker, followed by a VL region or portion thereof. The fragment, e.g., the scFv, may include the VL region or portion thereof, followed by the linker, followed by the VH region or portion thereof.
In some embodiments, the antigen-binding domain comprises the sequence selected from any one of SEQ ID NOs: 478, 128-139, 268-278, 329, 442, 558-576, 578-583, 585, or 769-771 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence selected from any one of SEQ ID NOs: 478, 128-139, 268-278, 329, 442, 558-576, 578-583, 585, or 769-771.
In some aspects, an scFv provided herein comprises the amino acid sequence selected from any one of SEQ ID NOs:128-139, 268-278, 328, 329, 442, 478, 558-576, 578-583, 585, 586, and 769-771, or has an amino acid sequence having at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence selected from any one of SEQ ID NOs: 128-139, 268-278, 328, 329, 442, 478, 558-576, 578-583, 585, 586, and 769-771.
For example, the scFv provided herein comprises the amino acid sequence selected from any of SEQ ID NOS:128, 129, 130, 132, 133, 136, 137, 269, 273, 274, 275, 276, 277, 278, 328, 329, 442, 478, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583 585, 586, 769, 770, 771, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, or 813 or has an amino acid sequence having at least at or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence selected from any one of SEQ ID NOS: 128, 129, 130, 132, 133, 136, 137, 269, 273, 274, 275, 276, 277, 278, 328, 329, 442, 478, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583 585, 586, 769, 770, 771, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, or 813.
Table 2 provides the SEQ ID NOS: of exemplary antigen-binding domains, such as antibodies or antigen-binding fragments, that can be comprised in the provided BCMA-binding receptors, such as anti-BCMA chimeric antigen receptors (CARs). In some embodiments, the BCMA-binding receptor contains a BCMA-binding antibody or fragment thereof, comprising a VH region that comprises the CDR-H1, CDR-H2, and CDR-H3 sequence and a VL region that comprises the CDR-L1, CDR-L2 and CDR-L3 sequence set forth in the SEQ ID NOS: listed in each row of Table 2 below (by Kabat numbering). In some embodiments, the BCMA-binding receptor contains a BCMA-binding antibody or fragment thereof, comprising a VH region sequence and a VL region sequence set forth in the SEQ ID NOS: listed in each row of Table 2 below, or an antibody comprising a VH and VL region amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VH region sequence and the VL region sequence set forth in the SEQ ID NOS: listed in each row of Table 2 below. In some embodiments, the BCMA-binding receptor contains a BCMA-binding antibody or fragment thereof, comprising a VH region sequence and a VL region sequence set forth in the SEQ ID NOS: listed in each row of Table 2 below. In some embodiments, the BCMA-binding receptor contains a BCMA-binding antibody or fragment thereof, comprising an scFv sequence set forth in the SEQ ID NOS: listed in each row of Table 2 below, or an antibody comprising an scFv amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the scFv sequence set forth in the SEQ ID NOS: listed in each row of Table 2 below. In some embodiments, the BCMA-binding receptor contains a BCMA-binding antibody or fragment thereof, comprising an scFv sequence set forth in the SEQ ID NOS: listed in each row of Table 2 below.
TABLE 2
Sequence identifier (SEQ ID NO) for Exemplary Antigen-binding Domains
Antigen-binding
CDR-
CDR-
CDR-
CDR-
CDR-
CDR-
domain
H1
H2
H3
L1
L2
L3
VH
VL
scFv
BCMA-1
1
4
7
26
37
47
110
116
128
BCMA-2
2
5
8
27
38
48
111
117
129
BCMA-3
1
4
7
28
39
49
110
118
130
BCMA-4
1
4
7
29
40
50
110
119
131
BCMA-5
1
4
7
30
39
51
110
120
132
BCMA-6
1
4
7
31
41
52
110
121
133
BCMA-7
1
4
7
32
42
53
110
122
134
BCMA-8
1
4
7
30
39
54
110
123
135
BCMA-9
2
5
9
33
43
55
112
124
136
BCMA-10
2
5
10
34
44
56
113
125
137
BCMA-11
3
6
11
35
45
57
114
126
138
BCMA-12
2
5
10
36
46
58
115
127
139
BCMA-13
140
145
149
174
179
184
247
257
268
BCMA-14
141
145
149
174
179
185
248
258
269
BCMA-15
141
145
150
174
179
186
249
259
270
BCMA-16
142
146
151
174
179
187
250
260
271
BCMA-17
2
5
152
175
180
188
251
261
272
BCMA-18
143
147
153
174
179
189
252
262
273
BCMA-19
144
148
154
176
181
190
253
263
274
BCMA-20
3
6
155
177
182
191
254
264
275
BCMA-21
2
5
156
174
179
192
255
265
276
BCMA-22
2
5
157
178
183
193
256
266
277
BCMA-23
2
5
157
178
183
194
256
267
278
BCMA-24
2
6
376
30
399
415
518
534
558
BCMA-25
1
4
7
380
400
416
519
535
559
BCMA-26
2
5
10
33
43
421
115
536
560
BCMA-27
3
6
155
177
182
191
520
264
561
BCMA-28
3
372
376
381
401
417
521
537
562
BCMA-29
3
6
376
382
402
418
522
538
563
BCMA-30
3
6
377
383
403
419
523
539
564
BCMA-31
1
4
7
384
39
54
519
540
565
BCMA-32
2
5
10
385
180
58
524
541
566
BCMA-33
2
373
152
175
180
188
525
261
567
BCMA-34
3
6
11
386
404
420
526
542
568
BCMA-35
2
5
378
33
43
421
527
543
569
BCMA-36
2
5
9
387
405
422
528
544
570
BCMA-37
2
5
9
388
406
423
529
545
571
BCMA-38
2
5
9
388
407
424
528
546
572
BCMA-39
3
6
376
389
408
425
522
547
573
BCMA-40
2
5
157
390
183
193
256
548
574
BCMA-41
2
374
9
391
409
426
530
549
575
BCMA-42
1
4
7
392
40
427
531
550
576
BCMA-44
1
4
7
394
39
429
519
552
578
BCMA-45
1
4
7
395
411
430
110
553
579
BCMA-46
1
4
7
28
39
49
110
118
130
BCMA-47
2
5
10
396
412
431
533
554
580
BCMA-48
2
5
10
396
412
58
115
555
581
BCMA-49
2
5
10
397
413
432
524
556
582
BCMA-51
1
4
7
398
414
433
519
557
583
BCMA-52
507
513
517
589
590
591
609
610
442
BCMA-55
593
594
595
601
602
603
617
618
478
BCMA-C1, VH-
288
290
292
302
304
306
324
326
585
VL
BCMA-C1, VL-
288
290
292
302
304
306
324
326
328
VH
BCMA-C2, VH-
289
291
293
303
305
307
325
327
329
VL
BCMA-C2, VL,
289
291
293
303
305
307
325
327
586
VH
BCMA-D1
772
775
769
BCMA-D2
773
776
770
BCMA-D3
774
777
771
BCMA-D4
814
BCMA-D5
815
833
781
BCMA-D6
816
834
782
BCMA-D7
816
834
783
BCMA-D8
817
835
784
BCMA-D9
817
835
785
BCMA-D10
818
836
786
BCMA-D11
818
836
787
BCMA-D12
819
837
788
BCMA-D13
819
837
789
BCMA-D14
820
838
790
BCMA-D15
820
838
791
BCMA-D16
821
839
792
BCMA-D17
821
839
793
BCMA-D18
822
840
794
BCMA-D19
822
840
795
BCMA-D20
823
841
796
BCMA-D21
823
841
797
BCMA-D22
824
842
798
BCMA-D23
824
842
799
BCMA-D24
824
842
800
BCMA-D25
825
843
801
BCMA-D26
826
844
802
BCMA-D27
827
845
803
BCMA-D28
828
846
804
BCMA-D29
805
BCMA-D30
829
847
806
BCMA-D31
830
847
807
BCMA-D32
831
848
808
BCMA-D33
832
849
809
BCMA-D34
810
BCMA-D35
832
849
811
BCMA-D36
831
848
812
BCMA-D37
813
Among the antibodies, e.g. antigen-binding fragments, in the provided CARs, are human antibodies. In some embodiments of a provided human anti-BCMA antibody, e.g., antigen-binding fragments, the human antibody contains a VH region that comprises a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain V segment, a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain D segment, and/or a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain J segment; and/or contains a VL region that comprises a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain V segment, and/or a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain J segment. In some embodiments, the portion of the VH region corresponds to the CDR-H1, CDR-H2 and/or CDR-H3. In some embodiments, the portion of the VH region corresponds to the framework region 1 (FR1), FR2, FR2 and/or FR4. In some embodiments, the portion of the VL region corresponds to the CDR-L1, CDR-L2 and/or CDR-L3. In some embodiments, the portion of the VL region corresponds to the FR1, FR2, FR2 and/or FR4.
In some embodiments, the human antibody, e.g., antigen-binding fragment, contains a CDR-H1 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-H1 region within a sequence encoded by a germline nucleotide human heavy chain V segment. For example, the human antibody in some embodiments contains a CDR-H1 having a sequence that is 100% identical or with no more than one, two or three amino acid differences as compared to the corresponding CDR-H1 region within a sequence encoded by a germline nucleotide human heavy chain V segment.
In some embodiments, the human antibody, e.g., antigen-binding fragment, contains a CDR-H2 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-H2 region within a sequence encoded by a germline nucleotide human heavy chain V segment. For example, the human antibody in some embodiments contains a CDR-H2 having a sequence that is 100% identical or with no more than one, two or three amino acid difference as compared to the corresponding CDR-H2 region within a sequence encoded by a germline nucleotide human heavy chain V segment.
In some embodiments, the human antibody, e.g., antigen-binding fragment, contains a CDR-H3 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-H3 region within a sequence encoded by a germline nucleotide human heavy chain V segment, D segment and J segment. For example, the human antibody in some embodiments contains a CDR-H3 having a sequence that is 100% identical or with no more than one, two or three amino acid differences as compared to the corresponding CDR-H3 region within a sequence encoded by a germline nucleotide human heavy chain V segment, D segment and J segment.
In some embodiments, the human antibody, e.g., antigen-binding fragment, contains a CDR-L1 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-L1 region within a sequence encoded by a germline nucleotide human light chain V segment. For example, the human antibody in some embodiments contains a CDR-L1 having a sequence that is 100% identical or with no more than one, two or three amino acid differences as compared to the corresponding CDR-L1 region within a sequence encoded by a germline nucleotide human light chain V segment.
In some embodiments, the human antibody, e.g., antigen-binding fragment, contains a CDR-L2 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-L2 region within a sequence encoded by a germline nucleotide human light chain V segment. For example, the human antibody in some embodiments contains a CDR-L2 having a sequence that is 100% identical or with no more than one, two or three amino acid difference as compared to the corresponding CDR-L2 region within a sequence encoded by a germline nucleotide human light chain V segment.
In some embodiments, the human antibody, e.g., antigen-binding fragment, contains a CDR-L3 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-L3 region within a sequence encoded by a germline nucleotide human light chain V segment and J segment. For example, the human antibody in some embodiments contains a CDR-L3 having a sequence that is 100% identical or with no more than one, two or three amino acid differences as compared to the corresponding CDR-L3 region within a sequence encoded by a germline nucleotide human light chain V segment and J segment.
In some embodiments, the human antibody, e.g., antigen-binding fragment, contains a framework region that contains human germline gene segment sequences. For example, in some embodiments, the human antibody contains a VH region in which the framework region, e.g. FR1, FR2, FR3 and FR4, has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a framework region encoded by a human germline antibody segment, such as a V segment and/or J segment. In some embodiments, the human antibody contains a VL region in which the framework region e.g. FR1, FR2, FR3 and FR4, has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a framework region encoded by a human germline antibody segment, such as a V segment and/or J segment. For example, in some such embodiments, the framework region sequence contained within the VH region and/or VL region differs by no more than 10 amino acids, such as no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid, compared to the framework region sequence encoded by a human germline antibody segment.
In some embodiments, the reference antibody can be a mouse anti-BCMA scFv described in International Patent App. Pub. No. WO 2010/104949.
The antibody, e.g., antigen-binding fragment, may contain at least a portion of an immunoglobulin constant region, such as one or more constant region domain. In some embodiments, the constant regions include a light chain constant region and/or a heavy chain constant region 1 (CH1). In some embodiments, the antibody includes a CH2 and/or CH3 domain, such as an Fc region. In some embodiments, the Fc region is an Fc region of a human IgG, such as an IgG1 or IgG4.
2. Spacer
In some embodiments, the recombinant receptor such as a CAR comprising an antibody (e.g., antigen-binding fragment) provided herein, further includes a spacer or spacer region. The spacer typically is a polypeptide spacer and in general is located within the CAR between the antigen binding domain and the transmembrane domain of the CAR. In some aspects, the spacer may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region of an immunoglobulin, such as an IgG hinge region, e.g., an IgG4 or IgG4-derived hinge region, and/or a CH1/CL and/or Fc region. In some embodiments, the constant region or one or more of the portion(s) thereof is of a human IgG, such as of a human IgG4 or IgG1 or IgG2. In general, the spacer, such as the portion of the constant region, serves as a spacer region between the antigen-recognition component (e.g., scFv) and transmembrane domain. In some embodiments, the length and/or composition of the spacer is designed to optimize or promote certain features of the interaction between the CAR and its target; in some aspects, it is designed to optimize the biophysical synapse distance between the CAR-expressing cell and the cell expressing the target of the CAR during or upon or following binding of the CAR to its target on the target-expressing cell; in some aspects, the target expressing cell is a BCMA-expressing tumor cell. In some embodiments, The CAR is expressed by a T-cell, and the length of the spacer is of a length that is compatible for T-cell activation or to optimize CAR T-cell performance. In some embodiments, the spacer is a spacer region, located between the ligand-binding domain and the transmembrane domain, of the recombinant receptor, e.g., CAR. In some embodiments, the spacer region is a region located between the ligand-binding domain and the transmembrane domain, of the recombinant receptor, e.g., CAR.
In some embodiments, the spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer and/or in the presence of a different spacer, such as one different only in length. In some embodiments, the spacer is at least 100 amino acids in length, such as at least 110, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids in length. In some examples, the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 300 amino acids, about 10 to 200 amino acids, about 50 to 175 amino acids, about 50 to 150 amino acids, about 10 to 125 amino acids, about 50 to 100 amino acids, about 100 to 300 amino acids, about 100 to 250 amino acids, about 125 to 250 amino acids, or about 200 to 250 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer or spacer region is at least about 12 amino acids, at least about 119 amino acids or less, at least about 125 amino acids, at least about 200 amino acids, or at least about 220 amino acids, or at least about 225 amino acids in length.
In some embodiments, the spacer has a length of 125 to 300 amino acids in length, 125 to 250 amino acids in length, 125 to 230 amino acids in length, 125 to 200 amino acids in length, 125 to 180 amino acids in length, 125 to 150 amino acids in length, 150 to 300 amino acids in length, 150 to 250 amino acids in length, 150 to 230 amino acids in length, 150 to 200 amino acids in length, 150 to 180 amino acids in length, 180 to 300 amino acids in length, 180 to 250 amino acids in length, 180 to 230 amino acids in length, 180 to 200 amino acids in length, 200 to 300 amino acids in length, 200 to 250 amino acids in length, 200 to 230 amino acids in length, 230 to 300 amino acids in length, 230 to 250 amino acids in length or 250 to 300 amino acids in length. In some embodiments, the spacer is at least or at least about or is or is about 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 221, 222, 223, 224, 225, 226, 227, 228 or 229 amino acids in length, or a length between any of the foregoing.
Exemplary spacers include those containing portion(s) of an immunoglobulin constant region such as those containing an Ig hinge, such as an IgG hinge domain. In some aspects, the spacer includes an IgG hinge alone, an IgG hinge linked to one or more of a CH2 and CH3 domain, or IgG hinge linked to the CH3 domain. In some embodiments, the IgG hinge, CH2 and/or CH3 can be derived all or in part from IgG4 or IgG2. In some embodiments, the spacer can be a chimeric polypeptide containing one or more of a hinge, CH2 and/or CH3 sequence(s) derived from IgG4, IgG2, and/or IgG2 and IgG4. In some embodiments, the hinge region comprises all or a portion of an IgG4 hinge region and/or of an IgG2 hinge region, wherein the IgG4 hinge region is optionally a human IgG4 hinge region and the IgG2 hinge region is optionally a human IgG2 hinge region; the CH2 region comprises all or a portion of an IgG4 CH2 region and/or of an IgG2 CH2 region, wherein the IgG4 CH2 region is optionally a human IgG4 CH2 region and the IgG2 CH2 region is optionally a human IgG2 CH2 region; and/or the CH3 region comprises all or a portion of an IgG4 CH3 region and/or of an IgG2 CH3 region, wherein the IgG4 CH3 region is optionally a human IgG4 CH3 region and the IgG2 CH3 region is optionally a human IgG2 CH3 region. In some embodiments, the hinge, CH2 and CH3 comprises all or a portion of each of a hinge region, CH2 and CH3 from IgG4. In some embodiments, the hinge region is chimeric and comprises a hinge region from human IgG4 and human IgG2; the CH2 region is chimeric and comprises a CH2 region from human IgG4 and human IgG2; and/or the CH3 region is chimeric and comprises a CH3 region from human IgG4 and human IgG2. In some embodiments, the spacer comprises an IgG4/2 chimeric hinge or a modified IgG4 hinge comprising at least one amino acid replacement compared to human IgG4 hinge region; an human IgG2/4 chimeric CH2 region; and a human IgG4 CH3 region.
In some embodiments, the spacer can be derived all or in part from IgG4 and/or IgG2 and can contain mutations, such as one or more single amino acid mutations in one or more domains. In some examples, the amino acid modification is a substitution of a proline (P) for a serine (S) in the hinge region of an IgG4. In some embodiments, the amino acid modification is a substitution of a glutamine (Q) for an asparagine (N) to reduce glycosylation heterogeneity, such as an N177Q mutation at position 177, in the CH2 region, of the full-length IgG4 Fc sequence set forth in SEQ ID NO: 750 or an N176Q. at position 176, in the CH2 region, of the full-length IgG2 Fc sequence set forth in SEQ ID NO: 749. In some embodiments, the spacer is or comprises an IgG4/2 chimeric hinge or a modified IgG4 hinge; an IgG2/4 chimeric CH2 region; and an IgG4 CH3 region and optionally is about 228 amino acids in length; or a spacer set forth in SEQ ID NO: 649. In some embodiments, the spacer comprises the amino acid sequence
(SEQ ID NO: 649)
ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLIVLHQDWLNGK
EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS
RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
encoded by a polynucleotide that has been optimized for codon expression and/or to eliminate splice sites such as cryptic splice sites. In some embodiments, the coding sequence for the spacer comprises the nucleic acid sequence set forth in SEQ ID NO: 622. In some embodiments, the coding sequence for the spacer comprises the nucleic acid sequence set forth in SEQ ID NO: 855 or 856.
Additional exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, Hudecek et al. (2015) Cancer Immunol. Res., 3(2):125-135, or international patent application publication number WO2014031687. In some embodiments, the nucleotide sequence of the spacer is optimized to reduce RNA heterogeneity following expression. In some embodiments, the nucleotide sequence of the spacer is optimized to reduce cryptic splice sites or reduce the likelihood of a splice event at a splice site.
In some embodiments, the spacer has the amino acid sequence set forth in SEQ ID NO:363, and is encoded by the polynucleotide sequence set forth in SEQ ID NO:364. In some embodiments, the spacer has the amino acid sequence set forth in SEQ ID NO:365. In some embodiments, the spacer has the amino acid sequence set forth in SEQ ID NO:366. In some embodiments, the spacer has the amino acid sequence set forth in SEQ ID NO: 630, and is encoded by the polynucleotide sequence set forth in SEQ ID NO: 629. In some embodiments, the spacer has an amino acid sequence set forth in SEQ ID NO: 649, encoded by the polynucleotide sequence set forth in SEQ ID NO: 621, 622, 855 or 856 or a polynucleotide that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 621, 622, 855 or 856. In some embodiments, the spacer has an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 649, encoded by a polynucleotide that has been optionally optimized for codon usage and/or to reduce RNA heterogeneity.
In some embodiments, the spacer is or comprises an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:622.
3. Transmembrane Domain and Intracellular Signaling Components
The antigen-recognition component generally is linked to one or more intracellular signaling regions containing signaling components, such as signaling components that mimic stimulation and/or activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and/or signal via another cell surface receptor. Thus, in some embodiments, the BCMA-binding molecule (e.g., antibody or antigen binding fragment thereof) is linked to one or more transmembrane domains such as those described herein and intracellular signaling regions or domains comprising one or more intracellular components such as those described herein. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane domains include those derived from (i.e. comprise at least the transmembrane domain(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, and/or CD154. For example, the transmembrane domain can be a CD28 transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 624, encoded by the nucleic acid sequence set forth in SEQ ID NO: 623 or SEQ ID NO:688. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and/or transmembrane domain(s).
Among the intracellular signaling regions or domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the intracellular signaling domain of the CAR.
The receptor, e.g., the CAR, generally includes an intracellular signaling region comprising at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component or signaling domain of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the BCMA-binding antibody is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25 or CD16.
In some embodiments, upon or following ligation of the CAR, the cytoplasmic domain or intracellular signaling domain of the CAR stimulates and/or activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functional capability.
In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.
T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such classes of cytoplasmic signaling sequences.
In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary stimulation and/or activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from TCR or CD3 zeta, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon. In some embodiments, the intracellular signaling region or domain in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta. In some embodiments the CD3 zeta comprises the sequence of amino acids set forth in SEQ ID NO: 628, encoded by the nucleic acid sequence set forth in SEQ ID NO: 627 or SEQ ID NO: 652.
In some embodiments, the CAR includes a signaling domain (e.g., an intracellular or cytoplasmic signaling domain) and/or transmembrane portion of a costimulatory molecule, such as a T cell costimulatory molecule. Exemplary costimulatory molecules include CD28, 4-1BB, OX40, DAP10, and ICOS. For example, a costimulatory molecule can be derived from 4-1BB and can comprise the amino acid sequence set forth in SEQ ID NO: 626, encoded by the nucleotide sequence set forth in SEQ ID NO: 625 or SEQ ID NO: 681. In some aspects, the same CAR includes both the stimulatory or activating components (e.g., cytoplasmic signaling sequence) and costimulatory components.
In some embodiments, the stimulatory or activating components are included within one CAR, whereas the costimulatory component is provided by another CAR recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, and costimulatory CARs, both expressed on the same cell (see WO2014/055668). In some aspects, the BCMA-targeting CAR is the stimulatory or activating CAR; in other aspects, it is the costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than BCMA, whereby a stimulatory or an activating signal delivered through the BCMA-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.
In certain embodiments, the intracellular signaling region comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.
In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and a stimulatory or activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.
In some embodiments, the provided chimeric antigen receptor comprises: (a) an extracellular antigen-binding domain that specifically recognizes B cell maturation antigen (BCMA), such as any antigen-binding domain described herein; (b) a spacer of at least 125 amino acids in length; (c) a transmembrane domain; and (d) an intracellular signaling region. In some embodiments, the antigen-binding domain of such receptor, comprising a VH region and a VL region comprising the amino acid sequence of SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids having at least 90% identity to SEQ ID NOS:617 and 618, respectively. In some embodiments, the antigen-binding domain of such receptor, comprising a VH region that is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region amino acid sequence of SEQ ID NO: 617; and a VL region that is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region amino acid sequence of SEQ ID NO: 618. In some embodiments, the antigen-binding domain of such receptor, comprising a VH region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising SEQ ID NOS:593, 594, and 595, respectively, and a VL region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising SEQ ID NOS:601, 602, and 603, respectively. In some embodiments, the antigen-binding domain of such receptor, comprising a VH region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising SEQ ID NOS:596, 597, and 595, respectively, and a VL region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising SEQ ID NOS:601, 602, and 603, respectively. In some embodiments, the antigen-binding domain of such receptor, comprising a VH region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising SEQ ID NOS: 598, 599, and 595, respectively, and a VL region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising SEQ ID NOS:601, 602, and 603, respectively. In some embodiments, the antigen-binding domain of such receptor, comprising a VH region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising SEQ ID NOS: 611, 612, and 613, respectively, and a VL region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising SEQ ID NOS: 614, 615, and 603, respectively. In some embodiments, the antigen-binding domain of such receptor, comprising a VH region that is or comprises the amino acid sequence of SEQ ID NO: 617; and a VL region that is or comprises the amino acid sequence of SEQ ID NO: 618. In some embodiments, the antigen-binding domain of such receptor, comprising the amino acid sequence of SEQ ID NO: 478. In some embodiments, the intracellular signaling region includes an stimulating cytoplasmic signaling domain. In some embodiments, the stimulating cytoplasmic signaling domain is capable of inducing a primary activation signal in a T cell, is a T cell receptor (TCR) component and/or includes an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the stimulating cytoplasmic signaling domain is or includes a cytoplasmic signaling domain of a CD3-zeta (CD3ζ) chain or a functional variant or signaling portion thereof. In some embodiments, the stimulating cytoplasmic domain is human or is derived from a human protein. In some embodiments, the stimulating cytoplasmic domain is or includes the sequence set forth in SEQ ID NO:628 or a sequence of amino acids that has at least 90% sequence identity to SEQ ID NO:628. In some embodiments, the nucleic acid encoding the stimulating cytoplasmic domain is or includes the sequence set forth in SEQ ID NO:627 or is a codon-optimized sequence and/or degenerate sequence thereof. In other embodiments, the nucleic acid encoding the stimulating cytoplasmic signaling domain is or includes the sequence set forth in SEQ ID NO:652. In some embodiments, the intracellular signaling region further includes a costimulatory signaling region. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof. In some embodiments, the costimulatory signaling region includes an intracellular signaling domain of 4-1BB. In some embodiments, the costimulatory signaling region is human or is derived from a human protein. In other embodiments, the costimulatory signaling region is or includes the sequence set forth in SEQ ID NO:626 or a sequence of amino acids that exhibits at least 90% sequence identity to the sequence set forth in SEQ ID NO: 626. In some embodiments, the nucleic acid encoding the costimulatory region is or includes the sequence set forth in SEQ ID NO:625 or is a codon-optimized sequence and/or degenerate sequence thereof. In some embodiments, the nucleic acid encoding the costimulatory signaling region includes the sequence set forth in SEQ ID NO:681. In some embodiments, the costimulatory signaling region is between the transmembrane domain and the intracellular signaling region. In some embodiments, the transmembrane domain is or includes a transmembrane domain derived from CD4, CD28, or CD8. In some embodiments, the transmembrane domain is or includes a transmembrane domain derived from a CD28. In some embodiments, the transmembrane domain is human or is derived from a human protein. In other embodiments, the transmembrane domain is or includes the sequence set forth in SEQ ID NO:624 or a sequence of amino acids that exhibits at least 90% sequence identity to SEQ ID NO:624.
Provided are chimeric antigen receptors, comprising: (1) an extracellular antigen-binding domain that specifically binds human B cell maturation antigen (BCMA), wherein the extracellular antigen-binding domain comprises: (i) a variable heavy chain (VH) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VH region sequence of SEQ ID NO: 617; and (ii) a variable light chain (VL) region comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VL region sequence of any of SEQ ID NO: 618; (2) a spacer set forth in SEQ ID NO: 649 or wherein the nucleic acid encoding the spacer is or comprises the sequence set forth in SEQ ID NO:622; (3) a transmembrane domain, optionally a transmembrane domain from a human CD28; and (4) an intracellular signaling region comprising a cytoplasmic signaling domain of a CD3-zeta (CD3ζ) chain and an intracellular signaling domain of a T cell costimulatory molecule. Also provided are polynucleotides encoding such a chimeric antigen receptor.
In some embodiments, the VH region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region sequence of SEQ ID NO: 617; and the VL region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region sequence of SEQ ID NO: 618; or the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:593, 594, and 595, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:596, 597, and 595, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:598, 599, and 595, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; or the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:611, 612, and 613, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:614, 615, and 603, respectively.
Provided are chimeric antigen receptors, comprising: (1) an extracellular antigen-binding domain that specifically binds human B cell maturation antigen (BCMA), wherein the extracellular antigen-binding domain comprises: a variable heavy (VH) region comprising a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region sequence of SEQ ID NO: 617; and a variable light (VL) region comprising a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region sequence of SEQ ID NO: 618; or the VH region comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region sequence of SEQ ID NO: 617; and the VL region comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region sequence of SEQ ID NO: 618; or the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:593, 594, and 595, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:596, 597, and 595, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:598, 599, and 595, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:601, 602, and 603, respectively; or the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the sequence of SEQ ID NOS:611, 612, and 613, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the sequence of SEQ ID NOS:614, 615, and 603, respectively; (2) a spacer set forth in SEQ ID NO: 649 or wherein the nucleic acid encoding the spacer is or comprises the sequence set forth in SEQ ID NO:622; (3) a transmembrane domain, optionally a transmembrane domain from a human CD28; and (4) an intracellular signaling region comprising a cytoplasmic signaling domain of a human CD3-zeta (CD3ζ) chain and an intracellular signaling domain of a T cell costimulatory molecule, optionally from a human 4-1BB or a human CD28. Also provided are polynucleotides encoding such a chimeric antigen receptor. In some embodiments, the extracellular antigen-binding domain comprises the VH region sequence of SEQ ID NO:617 and the VL region sequence of SEQ ID NO:618. In some embodiments, the antigen-binding domain of such receptor, comprising the amino acid sequence of SEQ ID NO: 478. In some embodiments, other domains, regions, or components of the chimeric antigen receptor includes any domains, regions, or components described herein.
4. Surrogate Marker
In some embodiments, the CAR further includes a surrogate marker, such as a cell surface marker (e.g., a truncated cell surface marker), which may be used to confirm transduction or engineering of the cell to express the receptor. For example, in some aspects, extrinsic marker genes are utilized in connection with engineered cell therapies to permit detection or selection of cells and, in some cases, also to promote cell suicide by ADCC. Exemplary marker genes include truncated epidermal growth factor receptor (EGFRt), which can be co-expressed with a transgene of interest (e.g., a CAR or TCR) in transduced cells (see, e.g., U.S. Pat. No. 8,802,374). EGFRt contains an epitope recognized by the antibody cetuximab (Erbitux®). For this reason, Erbitux® can be used to identify or select cells that have been engineered with the EGFRt construct, including in cells also co-engineered with another recombinant receptor, such as a chimeric antigen receptor (CAR). Additionally, EGFRt is commonly used as a suicide mechanism in connection with cell therapies. In some aspects, when EGFRt is co-expressed in cells with a transgene of interest (e.g. CAR or TCR), it can be targeted by the cetuximab monoclonal antibody to reduce or deplete the transferred gene-modified cells via ADCC (see U.S. Pat. No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434). Importantly, the suicide killing approach using tEGFR requires availability of the antibody epitope. Another example of such a marker gene is prostate-specific membrane antigen (PSMA) or a modified form thereof. PSMA or modified forms thereof may comprise a sequence of amino acids bound by or recognized by a PSMA-targeting molecule, such as an antibody or an antigen-binding fragment thereof. PSMA-targeting molecules can be used to identify or select cells that have been engineered with a PSMA or modified construct, including in cells also co-engineered with another recombinant receptor, such as a chimeric antigen receptor (CAR) provided herein. In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a nerve growth factor receptor (NGFR), epidermal growth factor receptor (e.g., EGFR), or PSMA.
Exemplary surrogate markers can include truncated forms of cell surface polypeptides, such as truncated forms that are non-functional and to not transduce or are not capable of transducing a signal or a signal ordinarily transduced by the full-length form of the cell surface polypeptide, and/or do not or are not capable of internalizing. Exemplary truncated cell surface polypeptides including truncated forms of growth factors or other receptors such as a truncated human epidermal growth factor receptor 2 (tHER2), a truncated epidermal growth factor receptor (tEGFR, exemplary tEGFR sequence set forth in SEQ ID NO:11 or 76) or a prostate-specific membrane antigen (PSMA) or modified form thereof tEGFR may contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibody or binding molecule, which can be used to identify or select cells that have been engineered with the tEGFR construct and an encoded exogenous protein, and/or to eliminate or separate cells expressing the encoded exogenous protein. See U.S. Pat. No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434). In some aspects, the marker, e.g. surrogate marker, includes all or part (e.g., truncated form) of CD34, a NGFR, a CD19 or a truncated CD19, e.g., a truncated non-human CD19, or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the marker is or comprises a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as super-fold GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), and variants thereof, including species variants, monomeric variants, and codon-optimized and/or enhanced variants of the fluorescent proteins. In some embodiments, the marker is or comprises an enzyme, such as a luciferase, the lacZ gene from E. coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyl transferase (CAT). Exemplary light-emitting reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS) or variants thereof.
In some embodiments, the marker is a selection marker. In some embodiments, the selection marker is or comprises a polypeptide that confers resistance to exogenous agents or drugs. In some embodiments, the selection marker is an antibiotic resistance gene. In some embodiments, the selection marker is an antibiotic resistance gene confers antibiotic resistance to a mammalian cell. In some embodiments, the selection marker is or comprises a Puromycin resistance gene, a Hygromycin resistance gene, a Blasticidin resistance gene, a Neomycin resistance gene, a Geneticin resistance gene or a Zeocin resistance gene or a modified form thereof.
In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. See WO2014031687. In some embodiments, introduction of a construct encoding the CAR and surrogate marker, separated by a T2A ribosome switch, can express two proteins from the same construct, such that the surrogate marker can be used as a marker to detect cells expressing such construct. In some embodiments, the surrogate marker, and optionally a linker sequence, can be any as disclosed in international publication no. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) or PSMA that is, optionally, linked to a linker sequence, such as a 2A cleavable linker sequence (e.g., a T2A, P2A, E2A or F2A cleavable linker, described elsewhere herein). An exemplary polypeptide for a truncated EGFR surrogate marker comprises the sequence of amino acids set forth in SEQ ID NO: 634 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 634. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers.
In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.
In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self” by the immune system of the host into which the cells will be adoptively transferred.
In some embodiments, the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells following adoptive transfer and encounter with ligand.
In some cases, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3-chain induced signal upon or in response to antigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR in some aspects is one that includes multiple costimulatory domains of different costimulatory receptors.
In some embodiments, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment includes an scFv or a single-domain antibody comprising only the VH region and the intracellular signaling domain contains an ITAM. In some aspects, the intracellular signaling domain includes a signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some aspects, the transmembrane domain contains a transmembrane portion of CD28. The extracellular domain and transmembrane can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a co-stimulatory molecule (e.g., T cell costimulatory molecule), such as between the transmembrane domain and intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 4-1BB.
In some embodiments, the transmembrane domain of the receptor (e.g., CAR) is a transmembrane domain of human CD28 or variant thereof, e.g., a 27-amino acid transmembrane domain of a human CD28 (Accession No.: P10747.1). In some embodiments, the intracellular signaling domain comprises an intracellular costimulatory signaling domain of human CD28 or functional variant thereof, such as a 41 amino acid domain thereof and/or such a domain with an LL to GG substitution at positions 186-187 of a native CD28 protein. In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of 4-1BB or functional variant thereof, such as a 42-amino acid cytoplasmic domain of a human 4-1BB (Accession No. Q07011.1). In some embodiments, the intracellular signaling domain comprises a human CD3 zeta stimulatory signaling domain or functional variant thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human CD3 (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. No. 7,446,190.
For example, in some embodiments, the CAR includes a BCMA antibody or fragment, such as any of the human BCMA antibodies, including sdAbs and scFvs, described herein, a spacer such as any of the Ig-hinge containing spacers, a CD28 transmembrane domain, a CD28 intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR includes the BCMA antibody or fragment, such as any of the human BCMA antibodies, including sdAbs and scFvs described herein, a spacer such as any of the Ig-hinge containing spacers, a CD28 transmembrane domain, a 4-1BB intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, such CAR constructs further includes a T2A ribosomal skip element and/or a tEGFR sequence, e.g., downstream of the CAR.
In certain embodiments, multispecific binding molecules, e.g., multispecific chimeric receptors, such as multispecific CARs, can contain any of the multispecific antibodies, including, e.g. bispecific antibodies, multispecific single-chain antibodies, e.g., diabodies, triabodies, and tetrabodies, tandem di-scFvs, and tandem tri-scFvs, such as any described above in Section I.A.
B. Exemplary Features
In some aspects, the antibodies or antigen-binding fragments thereof, in the provided CARs, have one or more specified functional features, such as binding properties, including recognizing or binding to particular epitopes, such as to epitopes that are similar to or overlap with those specifically bound by other antibodies such as reference antibodies, or epitopes that are different from those specifically bound by other antibodies such as reference antibodies, the ability to compete for binding with other antibodies such as reference antibodies, and/or particular binding affinities. In other embodiments, the antibodies or antigen-binding fragments thereof, in the provided CARs, recognize, such as specifically recognize, or bind, e.g., specifically bind, to epitopes that are different from, or do not overlap with those specifically bound by other antibodies such as reference antibodies. For example, the epitopes specifically bound by the antibodies, in the provided CARs, are different from those specifically bound by other antibodies such as reference antibodies. In some embodiments, the antibodies and antigen binding fragments thereof do not directly compete for, or compete to a lower degree, with binding with other antibodies such as reference antibodies.
In some embodiments, the antibodies or antigen-binding fragments thereof specifically recognize or specifically bind to BCMA protein. In any of the embodiments, an antibody or antigen binding fragment, in the provided CARs, that specifically recognize BCMA, specifically binds BCMA. In some embodiments provided herein, BCMA protein refers to human BCMA, a mouse BCMA protein, or a non-human primate (e.g., cynomolgus monkey) BCMA protein. In some embodiments of any of the embodiments herein, BCMA protein refers to human BCMA protein. The observation that an antibody or other binding molecule binds to BCMA protein or specifically binds to BCMA protein does not necessarily mean that it binds to a BCMA protein of every species. For example, in some embodiments, features of binding to BCMA protein, such as the ability to specifically bind thereto and/or to compete for binding thereto with a reference antibody, and/or to bind with a particular affinity or compete to a particular degree, in some embodiments, refers to the ability with respect to a human BCMA protein and the antibody may not have this feature with respect to a BCMA protein of another species, such as mouse.
In some embodiments, the antibody or antigen-binding fragment binds to a mammalian BCMA protein, including to naturally occurring variants of BCMA, such as certain splice variants or allelic variants.
In some embodiments, the antibodies specifically bind to human BCMA protein, such as to an epitope or region of human BCMA protein, such as the human BCMA protein comprising the amino acid sequence of SEQ ID NO:367 (GenBank No. BAB60895.1), or SEQ ID NO:368 (NCBI No. NP_001183.2) or an allelic variant or splice variant thereof. In one embodiment, the human BCMA protein is encoded by a transcript variant or is an isoform that has the sequence of amino acids forth in SEQ ID NO:369. In some embodiments, the antibodies bind to cynomolgus monkey BCMA protein, such as the cynomolgus monkey BCMA protein set forth in SEQ ID NO:371 (GenBank No. EHH60172.1). In some embodiments, the antibodies bind to human BCMA but do not bind to or bind in a lower level or degree or affinity to cynomolgus monkey BCMA protein, such as the cynomolgus monkey BCMA protein set forth in SEQ ID NO:371 (GenBank No. EHH60172.1). In some embodiments, the antibodies do not bind to or bind in a lower level or degree or affinity to mouse BCMA protein, such as the mouse BCMA protein set forth in SEQ ID NO:370 (NCBI No. NP_035738.1). In some embodiments, the antibodies bind to mouse BCMA protein, such as the mouse BCMA protein set forth in SEQ ID NO:370 (NCBI No. NP_035738.1). In some embodiments, the antibodies bind to mouse BCMA protein, with lower affinity than its binding to a human BCMA protein and/or a cynomolgus monkey BCMA protein. In some embodiments, the antibodies bind to mouse BCMA protein and/or a cynomolgus monkey BCMA protein with lower affinity than its binding to a human BCMA protein. In some embodiments, the antibodies bind to mouse BCMA protein and/or a cynomolgus monkey BCMA protein with similar binding affinity compared to its binding to a human BCMA protein.
In some embodiments, the provided antigen-binding domain or CAR exhibits preferential binding to membrane-bound BCMA as compared to soluble BCMA. In some embodiments, the provided antigen-binding domain or CAR exhibits greater binding affinity for, membrane-bound BCMA compared to soluble BCMA.
In one embodiment, the extent of binding of an anti-BCMA antibody or antigen-binding domain or CAR to an unrelated, non-BCMA protein, such as a non-human BCMA protein or other non-BCMA protein, is less than at or about 10% of the binding of the antibody or antigen-binding domain or CAR to human BCMA protein or human membrane-bound BCMA as measured, e.g., by a radioimmunoassay (RIA). In some embodiments, among the antibodies or antigen-binding domains in the provided CARs, are antibodies or antigen-binding domains or CARs in which binding to mouse BCMA protein is less than or at or about 10% of the binding of the antibody to human BCMA protein. In some embodiments, among the antibodies or antigen-binding domains in the provided CARs, are antibodies in which binding to cynomolgus monkey BCMA protein is less than or at or about 10% of the binding of the antibody to human BCMA protein. In some embodiments, among the antibodies or antigen-binding domains in the provided CARs, are antibodies in which binding to cynomolgus monkey BCMA protein and/or a mouse BCMA protein is similar to or about the same as the binding of the antibody to human BCMA protein. In some embodiments, among the antibodies or antigen-binding domains in the provided CARs, are antibodies or antigen-binding domains or CARs in which binding to soluble BCMA protein is less than or at or about 10% of the binding of the antibody to membrane-bound BCMA protein.
In some embodiments, the antibody specifically binds to, and/or competes for binding thereto with a reference antibody, and/or binds with a particular affinity or competes to a particular degree, to a BCMA protein, e.g., human BCMA, a mouse BCMA protein, or a non-human primate (e.g., cynomolgus monkey) BCMA protein.
In some embodiments, the antibodies, in the provided CARs, are capable of binding BCMA protein, such as human BCMA protein, with at least a certain affinity, as measured by any of a number of known methods. In some embodiments, the affinity is represented by an equilibrium dissociation constant (KD); in some embodiments, the affinity is represented by EC50.
A variety of assays are known for assessing binding affinity and/or determining whether a binding molecule (e.g., an antibody or fragment thereof) specifically binds to a particular ligand (e.g., an antigen, such as a BCMA protein). It is within the level of a skilled artisan to determine the binding affinity of a binding molecule, e.g., an antibody, for an antigen, e.g., BCMA, such as human BCMA or cynomolgus BCMA or mouse BCMA, such as by using any of a number of binding assays that are well known in the art. For example, in some embodiments, a BIAcore® instrument can be used to determine the binding kinetics and constants of a complex between two proteins (e.g., an antibody or fragment thereof, and an antigen, such as a BCMA protein), using surface plasmon resonance (SPR) analysis (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Pat. Nos. 5,283,173, 5,468,614, or the equivalent).
SPR measures changes in the concentration of molecules at a sensor surface as molecules bind to or dissociate from the surface. The change in the SPR signal is directly proportional to the change in mass concentration close to the surface, thereby allowing measurement of binding kinetics between two molecules. The dissociation constant for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), or determination of binding by monitoring the change in the spectroscopic or optical properties of the proteins through fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blot, ELISA, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing and other methods for detection of expressed polynucleotides or binding of proteins.
In some embodiments, the binding molecule, e.g., antibody or fragment thereof or antigen-binding domain of a CAR, binds, such as specifically binds, to an antigen, e.g., a BCMA protein or an epitope therein, with an affinity or KA (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M; equal to the ratio of the on-rate [kon or ka] to the off-rate [koff or kd] for this association reaction, assuming bimolecular interaction) equal to or greater than 105 M−1. In some embodiments, the antibody or fragment thereof or antigen-binding domain of a CAR exhibits a binding affinity for the peptide epitope with a KD (i.e., an equilibrium dissociation constant of a particular binding interaction with units of M; equal to the ratio of the off-rate [koff or kd] to the on-rate [kon or ka] for this association reaction, assuming bimolecular interaction) of equal to or less than 10−5 M. For example, the equilibrium dissociation constant KD ranges from 10−5 M to 10−13M, such as 10−7 M to 10−11 M, 10−8M to 10−10 M, or 10−9 M to 10−10 M. The on-rate (association rate constant; kon or ka; units of 1/Ms) and the off-rate (dissociation rate constant; koff or kd; units of 1/s) can be determined using any of the assay methods known in the art, for example, surface plasmon resonance (SPR).
In some embodiments, the binding affinity (EC50) and/or the dissociation constant of the antibody (e.g. antigen-binding fragment) or antigen-binding domain of a CAR to about BCMA protein, such as human BCMA protein, is from or from about 0.01 nM to about 500 nM, from or from about 0.01 nM to about 400 nM, from or from about 0.01 nM to about 100 nM, from or from about 0.01 nM to about 50 nM, from or from about 0.01 nM to about 10 nM, from or from about 0.01 nM to about 1 nM, from or from about 0.01 nM to about 0.1 nM, is from or from about 0.1 nM to about 500 nM, from or from about 0.1 nM to about 400 nM, from or from about 0.1 nM to about 100 nM, from or from about 0.1 nM to about 50 nM, from or from about 0.1 nM to about 10 nM, from or from about 0.1 nM to about 1 nM, from or from about 0.5 nM to about 200 nM, from or from about 1 nM to about 500 nM, from or from about 1 nM to about 100 nM, from or from about 1 nM to about 50 nM, from or from about 1 nM to about 10 nM, from or from about 2 nM to about 50 nM, from or from about 10 nM to about 500 nM, from or from about 10 nM to about 100 nM, from or from about 10 nM to about 50 nM, from or from about 50 nM to about 500 nM, from or from about 50 nM to about 100 nM or from or from about 100 nM to about 500 nM. In certain embodiments, the binding affinity (EC50) and/or the equilibrium dissociation constant, KD, of the antibody to a BCMA protein, such as human BCMA protein, is at or less than or about 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less. In some embodiments, the antibodies bind to a BCMA protein, such as human BCMA protein, with a sub-nanomolar binding affinity, for example, with a binding affinity less than about 1 nM, such as less than about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM or about 0.1 nM or less.
In some embodiments, the binding affinity may be classified as high affinity or as low affinity. In some cases, the binding molecule (e.g. antibody or fragment thereof) or antigen-binding domain of a CAR that exhibits low to moderate affinity binding exhibits a KA of up to 107M−1, up to 106M−1, up to 105M−1. In some cases, a binding molecule (e.g. antibody or fragment thereof) that exhibits high affinity binding to a particular epitope interacts with such epitope with a KA of at least 107M−1, at least 108 M−1, at least 109M−1, at least 1010 M−1, at least 1011 M−1, at least 1012 M−1, or at least 1013M−1. In some embodiments, the binding affinity (EC50) and/or the equilibrium dissociation constant, KD, of the binding molecule, e.g., anti-BCMA antibody or fragment thereof or antigen-binding domain of a CAR, to a BCMA protein, is from or from about 0.01 nM to about 1 μM, 0.1 nM to 1 μM, 1 nM to 1 μM, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 10 nM, 10 nM to 500 nM, 10 nM to 100 nM, 10 nM to 50 nM, 50 nM to 500 nM, 50 nM to 100 nM or 100 nM to 500 nM. In certain embodiments, the binding affinity (EC50) and/or the dissociation constant of the equilibrium dissociation constant, KD, of the binding molecule, e.g., anti-BCMA antibody or fragment thereof or antigen-binding domain of a CAR, to a BCMA protein, is at or about or less than at or about 1 μM, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less. The degree of affinity of a particular antibody can be compared with the affinity of a known antibody, such as a reference antibody.
In some embodiments, the binding affinity of a binding molecule, such as an anti-BCMA antibody or antigen-binding domain of a CAR, for different antigens, e.g., BCMA proteins from different species can be compared to determine the species cross-reactivity. For example, species cross-reactivity can be classified as high cross reactivity or low cross reactivity. In some embodiments, the equilibrium dissociation constant, KD, for different antigens, e.g., BCMA proteins from different species such as human, cynomolgus monkey or mouse, can be compared to determine species cross-reactivity. In some embodiments, the species cross-reactivity of an anti-BCMA antibody or antigen-binding domain of a CAR can be high, e.g., the anti-BCMA antibody binds to human BCMA and a species variant BCMA to a similar degree, e.g., the ratio of KD for human BCMA and KD for the species variant BCMA is or is about 1. In some embodiments, the species cross-reactivity of an anti-BCMA antibody or antigen-binding domain of a CAR can be low, e.g., the anti-BCMA antibody has a high affinity for human BCMA but a low affinity for a species variant BCMA, or vice versa. For example, the ratio of KD for the species variant BCMA and KD for the human BCMA is more than 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000 or more, and the anti-BCMA antibody has low species cross-reactivity. The degree of species cross-reactivity can be compared with the species cross-reactivity of a known antibody, such as a reference antibody.
In some embodiments, the binding affinity of the anti-BCMA antibody or antigen-binding domain of a CAR, for different form or topological type of antigens, e.g., soluble BCMA protein compared to the binding affinity to a membrane-bound BCMA, to determine the preferential binding or relative affinity for a particular form or topological type. For example, in some aspects, the provided anti-BCMA antibodies or antigen-binding domains can exhibit preferential binding to membrane-bound BCMA as compared to soluble BCMA and/or exhibit greater binding affinity for, membrane-bound BCMA compared to soluble BCMA. In some embodiments, the equilibrium dissociation constant, KD, for different form or topological type of BCMA proteins, can be compared to determine preferential binding or relative binding affinity. In some embodiments, the preferential binding or relative affinity to a membrane-bound BCMA compared to soluble BCMA can be high. For example, in some cases, the ratio of KD for soluble BCMA and the KD for membrane-bound BCMA is more than 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000 or more and the antibody or antigen-binding domain preferentially binds or has higher binding affinity for membrane-bound BCMA. In some cases, the ratio of KA for membrane-bound BCMA and the KA for soluble BCMA is more than 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000 or more and the antibody or antigen-binding domain preferentially binds or has higher binding affinity for membrane-bound BCMA. In some cases, the antibody or antigen-binding domain of CAR binds soluble BCMA and membrane-bound BCMA to a similar degree, e.g., the ratio of KD for soluble BCMA and KD for membrane-bound BCMA is or is about 1. In some cases, the antibody or antigen-binding domain of CAR binds soluble BCMA and membrane-bound BCMA to a similar degree, e.g., the ratio of KA for soluble BCMA and KA for membrane-bound BCMA is or is about 1. The degree of preferential binding or relative affinity for membrane-bound BCMA or soluble BCMA can be compared with that of a known antibody, such as a reference antibody.
In some embodiments, the antibodies or antigen binding fragments thereof, in the provided CARs, bind to a similar degree to a human BCMA protein and a non-human BCMA protein or other non-BCMA proteins. For example, in some embodiments, the antibodies or antigen binding fragments thereof or antigen-binding domain of a CAR bind to a human BCMA protein, such as the human BCMA protein comprising the amino acid sequence of SEQ ID NO:367 (GenBank No. BAB60895.1), or SEQ ID NO:368 (NCBI No. NP_001183.2) or an allelic variant or splice variant thereof, with an equilibrium dissociation constant (KD), and to a non-human BCMA, such as a cynomolgus monkey BCMA, such as the cynomolgus monkey BCMA protein set forth in SEQ ID NO:371 (GenBank No. EHH60172.1), with a KD that is similar, or about the same, or less than 2-fold different, or less than 5-fold different.
In some embodiments, the antibodies or antigen binding fragments thereof, in the provided CARs, bind to a similar degree to a soluble BCMA protein and a membrane-bound BCMA protein, with an equilibrium dissociation constant (KD) that is similar, or about the same, or less than 2-fold different, or less than 5-fold different.
For example, in some embodiments, the antibodies, in the provided CARs, or antigen binding fragments thereof bind to a human BCMA with a KD of about or less than at or about 1 μM, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less, and binds to a cynomolgus monkey BCMA with a KD of about or less than at or about 1 μM, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less. In some embodiments, the antibodies or antigen binding fragments thereof bind to a mouse BCMA protein with a KD of about or less than at or about 1 μM, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less. In some embodiments, the antibodies or antigen binding fragments thereof, in the provided CARs, bind to a human BCMA, a cynomolgus monkey BCMA and a mouse BCMA with high affinity. In some embodiments, the antibodies or antigen binding fragments thereof bind to a human BCMA and cynomolgus monkey BCMA with a high affinity, and to a mouse BCMA with low affinity. In some embodiments, the antibodies or antigen binding fragments thereof bind to a human BCMA and BCMA from other species, or other variants of the BCMA protein, with high affinity.
In some embodiments, the total binding capacity (Rmax), as measured using particular surface plasmon resonance (SPR) conditions, is used to determine the ability or capacity of binding of the antibody or antigen binding fragment thereof, to the antigen, e.g., a BCMA protein, such as a human BCMA protein. For SPR analysis, the “ligand” is the immobilized target molecule on the surface of the sensor, for example, a BCMA protein, and the “analyte” is the tested molecule, e.g., antibody, for binding to the “ligand”. For example, the “analyte” can be any of the antibodies, or antigen binding fragments thereof, that binds to a BCMA protein. For a particular ligand and analyte pair in SPR, the Rmax can be determined assuming a 1:1 binding stoichiometry model, for a particular condition. Binding capacity (Rmax) was determined using the following formula: Rmax (RU)=(analyte molecular weight)/(ligand molecular weight)×immobilized ligand level (RU). For example, in a particular SPR conditions, the Rmax of binding between any of the antibody or antigen binding fragment thereof and a BCMA protein, such as a human BCMA or a cynomolgus BCMA, is at least or at least about 50 resonance units (RU), such as about 25 RU, 20 RU, 15 RU, 10 RU, 5 RU or 1 RU.
In some embodiments, the antibodies, such as the human antibodies, in the provided CAR, specifically bind to a particular epitope or region of BCMA protein, such as generally an extracellular epitope or region. BCMA protein is a type III membrane 184 amino acid protein that contains an extracellular domain, a transmembrane domain, and a cytoplasmic domain. With reference to a human BCMA amino acid sequence set forth in SEQ ID NO:367, the extracellular domain corresponds to amino acids 1-54, amino acids 55-77 correspond to the transmembrane domain, and amino acids 78-184 correspond to the cytoplasmic domain.
Among the provided CARs are CARs that exhibit antigen-dependent activity or signaling, i.e. signaling activity that is measurably absent or at background levels in the absence of antigen, e.g. BCMA. Thus, in some aspects, provided CARs do not exhibit, or exhibit no more than background or a tolerable or low level of, tonic signaling or antigen-independent activity or signaling in the absence of antigen, e.g. BCMA, being present. In some embodiments, the provided anti-BCMA CAR-expressing cells exhibit biological activity or function, including cytotoxic activity, cytokine production, and ability to proliferate.
In some embodiments, biological activity or functional activity of a chimeric receptor, such as cytotoxic activity, can be measured using any of a number of known methods. The activity can be assessed or determined either in vitro or in vivo. In some embodiments, activity can be assessed once the cells are administered to the subject (e.g., human). Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, e.g., in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells also can be measured by assaying expression and/or secretion of certain cytokines, such as interleukin-2 (IL-2), interferon-gamma (IFNγ), interleukin-4 (IL-4), TNF-alpha (TNFα), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), CD107a, and/or TGF-beta (TGFβ). Assays to measure cytokines are well known in the art, and include but are not limited to, ELISA, intracellular cytokine staining, cytometric bead array, RT-PCR, ELISPOT, flow cytometry and bio-assays in which cells responsive to the relevant cytokine are tested for responsiveness (e.g. proliferation) in the presence of a test sample. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
In some aspects, a reporter cell line can be employed to monitor antigen-independent activity and/or tonic signaling through anti-BCMA CAR-expressing cells. In some embodiments, a T cell line, such as a Jurkat cell line, contains a reporter molecule, such as a fluorescent protein or other detectable molecule, such as a red fluorescent protein, expressed under the control of the endogenous Nur77 transcriptional regulatory elements. In some embodiments, the Nur77 reporter expression is cell intrinsic and dependent upon signaling through a recombinant reporter containing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and/or a signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM), such as a CD3ζ chain. Nur77 expression is generally not affected by other signaling pathways such as cytokine signaling or toll-like receptor (TLR) signaling, which may act in a cell extrinsic manner and may not depend on signaling through the recombinant receptor. Thus, only cells that express the exogenous recombinant receptor, e.g. anti-BCMA CAR, containing the appropriate signaling regions is capable of expressing Nur77 upon stimulation (e.g., binding of the specific antigen). In some cases, Nur77 expression also can show a dose-dependent response to the amount of stimulation (e.g., antigen).
In some embodiments, the provided anti-BCMA CARs exhibit improved expression on the surface of cells, such as compared to an alternative CAR that has an identical amino acid sequence but that is encoded by non-splice site eliminated and/or a codon-optimized nucleotide sequence. In some embodiments, the expression of the recombinant receptor on the surface of the cell can be assessed. Approaches for determining expression of the recombinant receptor on the surface of the cell may include use of chimeric antigen receptor (CAR)-specific antibodies (e.g., Brentjens et al., Sci. Transl. Med. 2013 March; 5(177): 177ra38), Protein L (Zheng et al., J. Transl. Med. 2012 February; 10:29), epitope tags, and monoclonal antibodies that specifically bind to a CAR polypeptide (see international patent application Pub. No. WO2014190273). In some embodiments, the expression of the recombinant receptor on the surface of the cell, e.g., primary T cell, can be assessed, for example, by flow cytometry, using binding molecules that can bind to the recombinant receptor or a portion thereof that can be detected. In some embodiments, the binding molecules used for detecting expression of the recombinant receptor an anti-idiotypic antibody, e.g., an anti-idiotypic agonist antibody specific for a binding domain, e.g., scFv, or a portion thereof. In some embodiments, the binding molecule is or comprises an isolated or purified antigen, e.g., recombinantly expressed antigen.
C. Multispecific Antibodies
In certain embodiments, the BCMA-binding molecules, e.g., antibodies or polypeptides, such as chimeric receptors containing the same, are multispecific. Among the multispecific binding molecules are multispecific antibodies, including, e.g. bispecific antibodies. Multispecific binding partners, e.g., antibodies, have binding specificities for at least two different sites, which may be in the same or different antigens. In certain embodiments, one of the binding specificities is for BCMA and the other is for another antigen. In some embodiments, additional binding molecules bind to and/or recognize a third, or more antigens. In certain embodiments, bispecific antibodies may bind to two different epitopes of BCMA. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express BCMA. Bispecific antibodies can be prepared as full length antibodies or antibody fragments. Among the multispecific antibodies are multispecific single-chain antibodies, e.g., diabodies, triabodies, and tetrabodies, tandem di-scFvs, and tandem tri-scFvs. Also provided are multispecific chimeric receptors, such as multispecific CARs, containing the antibodies (e.g., antigen-binding fragments). Also provided are multispecific cells containing the antibodies or polypeptides including the same, such as cells containing a cell surface protein including the anti-BCMA antibody and an additional cell surface protein, such as an additional chimeric receptor, which binds to a different antigen or a different epitope on BCMA.
Exemplary antigens include B cell specific antigens, other tumor-specific antigens, such as antigens expressed specifically on or associated with a leukemia (e.g., B cell leukemia), lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.), or a myeloma, e.g., a multiple myeloma (MM), a plasma cell malignancy (e.g., plasmacytoma). For example, antigens include those expressed specifically on or associated with B cell chronic lymphocytic leukemia (CLL), a diffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), Burkitt's lymphoma (e.g., endemic Burkitt's lymphoma or sporadic Burkitt's lymphoma), mantle cell lymphoma (MCL), non-small cell lung cancer (NSCLC), chronic myeloid (or myelogenous) leukemia (CML), hairy cell leukemia (HCL), small lymphocytic lymphoma (SLL), Marginal zone lymphoma, Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), Anaplastic large cell lymphoma (ALCL), refractory follicular lymphoma, Waldenstrom macroglobulinemia, follicular lymphoma, small non-cleaved cell lymphoma, mucosa-associated lymphatic tissue lymphoma (MALT), marginal zone lymphoma, nodal monocytoid B cell lymphoma, immunoblastic lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, pulmonary B cell angiocentric lymphoma, small lymphocytic lymphoma, primary mediastinal B cell lymphoma, lymphoplasmacytic lymphoma (LPL), neuroblastoma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell cancer, melanoma, myeloma such as multiple myeloma (e.g., non-secretory multiple myeloma, smoldering multiple myeloma), stomach cancer, esophageal cancer, brain cancer, lung cancer (e.g., small-cell lung cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer (e.g., hepatic carcinoma, hepatoma, etc.), bladder cancer, prostate cancer, testicular cancer, thyroid cancer, uterine cancer, spleen cancer (e.g., splenic lymphoma), adrenal cancer and/or head and neck cancer, and antigens expressed on T cells.
In some embodiments, among the second or additional antigens for multi-targeting strategies includes those in which at least one of the antigens is a universal tumor antigen, or a family member thereof. In some embodiments, the second or additional antigen is an antigen expressed on a tumor. In some embodiments, the BCMA-binding molecules provided herein target an antigen on the same tumor type as the second or additional antigen. In some embodiments, the second or additional antigen may also be a universal tumor antigen or may be a tumor antigen specific to a tumor type.
Exemplary second or additional antigens include CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC-1, Ia, HM1.24, HLA-DR, tenascin, an angiogenesis factor, VEGF, PIGF, ED-B fibronectin, an oncogene, an oncogene product, CD66a-d, necrosis antigens, Ii, IL-2, T101, TAC, IL-6, ROR1, TRAIL-R1 (DR4), TRAIL-R2 (DR5), tEGFR, Her2, L1-CAM, mesothelin, CEA, hepatitis B surface antigen, anti-folate receptor, CD24, CD30, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, erbB dimers, EGFR vIII, FBP, FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, G protein-coupled receptor class C group 5 member D (GPRCSD), HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule (L1-CAM), Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentially expressed antigen of melanoma (PRAME), survivin, EGP2, EGP40, TAG72, B7-H6, IL-13 receptor a2 (IL-13Ra2), CA9, CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2D ligands, dual antigen, an antigen associated with a universal tag, a cancer-testes antigen, MUC1, MUC16, NY-ESO-1, MART-1, gp100, oncofetal antigen, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, 0-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, CCL-1, hTERT, MDM2, CYP1B, WT1, livin, AFP, p53, cyclin (D1), CS-1, BAFF-R, TACI, CD56, TIM-3, CD123, L1-cell adhesion molecule, MAGE-A1, MAGE A3, a cyclin, such as cyclin A1 (CCNA1) and/or a pathogen-specific antigen, biotinylated molecules, molecules expressed by HIV, HCV, HBV and/or other pathogens, and/or in some aspects, neoepitopes or neoantigens thereof. In some embodiments, the antigen is associated with or is a universal tag.
In some aspects, the antigen, e.g., the second or additional antigen, such as the disease-specific antigen and/or related antigen, is expressed on multiple myeloma, such as G protein-coupled receptor class C group 5 member D (GPRCSD), CD38 (cyclic ADP ribose hydrolase), CD138 (syndecan-1, syndecan, SYN-1), CS-1 (CS1, CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), BAFF-R, TACI and/or FcRH5. Other exemplary multiple myeloma antigens include CD56, TIM-3, CD33, CD123, CD44, CD20, CD40, CD74, CD200, EGFR, β2-Microglobulin, HM1.24, IGF-1R, IL-6R, TRAIL-R1, and the activin receptor type IIA (ActRIIA). See Benson and Byrd, J. Clin. Oncol. (2012) 30(16): 2013-15; Tao and Anderson, Bone Marrow Research (2011):924058; Chu et al., Leukemia (2013) 28(4):917-27; Garfall et al., Discov Med. (2014) 17(91):37-46. In some embodiments, the antigens include those present on lymphoid tumors, myeloma, AIDS-associated lymphoma, and/or post-transplant lymphoproliferations, such as CD38. Antibodies or antigen-binding fragments directed against such antigens are known and include, for example, those described in U.S. Pat. Nos. 8,153,765; 8,603,477, 8,008,450; U.S. Pub. No. US20120189622 or US20100260748; and/or International PCT Publication Nos. WO2006099875, WO2009080829 or WO2012092612 or WO2014210064. In some embodiments, such antibodies or antigen-binding fragments thereof (e.g. scFv) are contained in multispecific antibodies, multispecific chimeric receptors, such as multispecific CARs, and/or multispecific cells.
II. METHODS OF OPTIMIZING AND PRODUCING POLYNUCLEOTIDES, E.G., POLYNUCLEOTIDES ENCODING BCMA CARS, AND OPTIMIZED POLYNUCLEOTIDES
Provided herein are methods for optimizing polynucleotides for expression and/or therapeutic use, and polynucleotides optimized, e.g., according to the methods. In some embodiments, the provided methods or optimizations reduce heterogeneity and/or increase homogeneity of transcribed RNA, such as messenger RNA (mRNA), for example, when the polynucleotide is expressed in a cell, such as in a particular cell type, such as in a mammalian, e.g., human cell type such as a human T cell such as a primary human T cell or T cell line. In some embodiments, the methods for optimizing polynucleotides include methods to identify and remove or alter the sequence of one or more cryptic splice site, such as one or both of a donor splice site or an acceptor splice site. In some embodiments, the methods can additionally or further include codon optimization. In some embodiments, codon optimization can be performed prior to and/or after methods of reducing heterogeneity of transcribed RNA (e.g., mRNA), such as by removal or elimination of predicted splice sites. In some embodiments, codon optimization is integrated in any one or more steps of the method of reducing heterogeneity of transcribed RNAs. In some embodiments, methods of reducing heterogeneity, such as by removal or elimination of predicted splice sites, can be performed after codon optimization. In some embodiments, provided are methods in which a polynucleotide encoding a transgene, including a polynucleotide encoding any of the provided anti-BCMA CAR polypeptides, can be optimized for expression and/or for therapeutic use. In some embodiments, the polynucleotides are modified to optimize codon usage. In some embodiments, the polynucleotides are codon optimized for expression in a human cell such as a human T cell such as a primary human T cell. In some embodiments, the polynucleotides, such as those encoding any of the antibodies, receptors (such as antigen receptors such as chimeric antigen receptors) and/or BCMA-specific binding proteins provided herein, are or have been modified to reduce heterogeneity or contain one or more nucleic acid sequences observed herein (such as by the optimization methods) to result in improved features of the polypeptides, such as the CARs, as compared to those containing distinct, reference, sequences or that have not been optimized. Among such features include improvements in RNA heterogeneity, such as that resulting from the presence of one or more splice sites, such as one or more cryptic splice sites, and/or improved expression and/or surface expression of the encoded protein, such as increased levels, uniformity, or consistency of expression among cells or different therapeutic cell compositions engineered to express the polypeptides. In some embodiments, the polynucleotides can be codon optimized for expression in human cells.
Genomic nucleic acid sequences generally, in nature, in a mammalian cell, undergo processing co-transcriptionally or immediately following transcription, wherein a nascent precursor messenger ribonucleic acid (pre-mRNA), transcribed from a genomic deoxyribonucleic acid (DNA) sequence, is in some cases edited by way of splicing, to remove introns, followed by ligation of the exons in eukaryotic cells. Consensus sequences for splice sites are known, but in some aspects, specific nucleotide information defining a splice site may be complex and may not be readily apparent based on available methods. Cryptic splice sites are splice sites that are not predicted based on the standard consensus sequences and are variably activated. Hence, variable splicing of pre-mRNA at cryptic splice sites leads to heterogeneity in the transcribed mRNA products following expression in eukaryotic cells.
Polynucleotides generated for the expression of transgenes are typically constructed from nucleic acid sequences, such as complementary DNA (cDNA), or portions thereof, that do not contain introns. Thus, splicing of such sequences is not expected to occur. However, the presence of cryptic splice sites within the cDNA sequence can lead to unintended or undesired splicing reactions and heterogeneity in the transcribed mRNA. Such heterogeneity results in translation of unintended protein products, such as truncated protein products with variable amino acid sequences that exhibit modified expression and/or activity.
Also provided are methods and approaches for determining the heterogeneity of a transcribed nucleic acid such as one encoding or containing a transgene or encoding a recombinant protein. In some embodiments, the methods include determining the heterogeneity of a transcribed nucleic acid sequence that includes all or a portion of the 5′ untranslated region (5′ UTR), and/or all or a portion of the 3′ untranslated region (3′ UTR), of the transcribed nucleic acid. Also provided herein are methods of identifying the presence of splice sites, such as cryptic splice sites, based on the heterogeneity of the transcribed nucleic acid. Also provided are methods of identifying a transgene candidate for the removal of splice sites, such as cryptic splice sites, using the provided methods of determining the heterogeneity of the transcribed nucleic acid of the transgene. Also provided are methods of reducing the heterogeneity of an expressed transgene transcript.
Also provided herein are methods of identifying a transgene or recombinant protein or nucleic acid candidate for the removal or modification of one or more splice sites, such as cryptic splice sites, such as based on the determined heterogeneity of the transcribed nucleic acid, e.g., of the transgene.
Also provided are methods and approaches for reducing the heterogeneity of a transcribed nucleic acid (e.g., transcript) of a transgene (e.g., an expressed transgene transcript) or other nucleic acid. Such methods and approaches can include identifying a transgene candidate for the removal of splice sites (such as cryptic splice sites) according to the provided methods and identifying one or more potential splice donor and/or splice acceptor sites within the transgene. In embodiments of the provided methods the splice donor and/or splice acceptor sites can be in the translated and/or untranslated regions of the transcribed nucleic acid (e.g., transcript).
In some embodiments, eliminating splice sites, such as cryptic splice sites, can improve or optimize expression of a transgene product, such as a polypeptide translated from the transgene, such as an anti-BCMA CAR polypeptide. Splicing at cryptic splice sites of an encoded transgene, such as an encoded BMCA CAR molecule, can lead to reduced protein expression, e.g., expression on cell surfaces, and/or reduced function, e.g., reduced intracellular signaling. Provided herein are polynucleotides, encoding anti-BMCA CAR proteins that have been optimized to reduce or eliminate cryptic splice sites. Also provided herein are polynucleotides encoding anti-BCMA CAR proteins that have been optimized for codon expression and/or in which one or more sequence, such as one identified by the methods or observations herein regarding splice sites, is present, and/or in which an identified splice site, such as any of the identified splice sites herein, is not present. Among the provided polynucleotides are those exhibiting below a certain degree of RNA heterogeneity or splice forms when expressed under certain conditions and/or introduced into a specified cell type, such as a human T cell, such as a primary human T cell, and cells and compositions and articles of manufacture containing such polypeptides and/or exhibiting such properties.
In some embodiments, reducing RNA heterogeneity or removing potential splice site comprises modifying a polynucleotide. In some embodiments, the modification includes one or more nucleotide modifications, such as a replacement or substitution, compared to a reference polynucleotide such as an unmodified polynucleotide that encodes the same polypeptide. In some embodiments, the reference polynucleotide is one in which the transcribed RNA (e.g. mRNA), when expressed in a cell, exhibits greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more RNA heterogeneity. In some embodiments, the provided methods can result in polynucleotides in which RNA heterogeneity of transcribed RNA is reduced by greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more. In some embodiments, the provided methods produce polynucleotides in which RNA homogeneity of transcribed RNA is at least 70%, 75%, 80%, 85%, 90%, or 95% or greater.
A. Methods of Measuring and Reducing RNA Heterogeneity
Provided herein are methods, approaches, and strategies for measuring, evaluating and/or reducing RNA heterogeneity of a nucleic acid, such as of a transcribed RNA, e.g., when expressed in a particular cell type or context, as well as polynucleotides exhibiting reduction in such heterogeneity and/or risk thereof, as compared to a reference polynucleotide. In some embodiments, a reference polynucleotide can be assessed for RNA heterogeneity, such as by methods as described in this Section. In some embodiments, the provided approaches involve identifying RNA (e.g., mRNA) heterogeneity or likelihood thereof, such as in a particular cell or context, such as due to cryptic splice sites. In some aspects, such heterogeneity is identified by amplifying RNA transcripts using a first primer specific to the 5′ untranslated region (5′ UTR), corresponding to a portion of an element located upstream of the transgene in the transcribed RNA, such as a promoter, and a second primer specific to a 3′ untranslated region (3′ UTR), located downstream of the expressed transgene in the transcribed RNA sequence or specific to a sequence within the transgene. In some embodiments, the methods involve amplifying a transcribed nucleic acid using at least one 5′ and 3′ primer pair, wherein at least one pair comprises a 5′ primer that is complementary to a nucleic acid sequence within the 5′ untranslated region (5′ UTR) of the transcribed nucleic acid and a 3′ primer that is complementary to a nucleic acid sequence within the 3′ untranslated region (3′ UTR) of the transcribed nucleic acid to generate one or more amplified products. In some embodiments, the methods involve detecting the amplified products, wherein the presence of two or more amplified products from at least one 5′ and 3′ primer pair indicates heterogeneity in the amplified products. In some embodiments, the detected difference in transcripts are different lengths of the amplified transcript. In some embodiments, the detected difference in transcripts are differences in chromatographic profiles. Exemplary methods for identifying a polynucleotide with RNA heterogeneity are described below. In some embodiments, the methods comprise evaluating RNA heterogeneity for the need of being modified to reduce heterogeneity. In some embodiments, polynucleotides that exhibit RNA heterogeneity greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more are selected for nucleotide modification to remove one or more splice sites, such as one or more cryptic splice sites.
1. Measuring RNA Heterogeneity
RNA heterogeneity can be determined by any of a number of methods provided herein or described or known. In some embodiments, RNA heterogeneity of a transcribed nucleic acid is determined by amplifying the transcribed nucleic acid, such as by reverse transcriptase polymerase chain reaction (RT-PCR) followed by detecting one or more differences, such as differences in size, in the one or more amplified products. In some embodiments, the RNA heterogeneity is determined based on the number of differently sized amplified products, or the proportion of various differently sized amplified products. For example, in some embodiments, RNA heterogeneity is quantified by determining the number, amount or proportion of differently sized amplified product compared to the number or amount of total amplified products. In some cases, all or substantially all of a particular transcript is determined to be equal in size, and in this case, the RNA heterogeneity is low. In some cases, a variety of differently sized transcripts are present, or a large proportion of a particular transcript is of a different size compared to the predicted size of the amplified product without cryptic or undesired splicing events. In some embodiments, RNA heterogeneity can be calculated by dividing the total number or amount of all of amplified products that are of a different size compared to the predicted size of the amplified product by the total number or amount of all amplified products. In some embodiments, the predicted size of the transcript or amplified product is from an RNA that does not contain or is not predicted to contain a cryptic splice site. In some embodiments, the predicted size of the transcript or amplified product takes into account one or more splice sites that are desired or intentionally placed.
In some embodiments, RNA, such as total RNA or cytoplasmic polyadenylated RNA, is harvested from cells, expressing the transgene to be optimized, and amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using a primer specific to the 5′ untranslated region (5′ UTR), in some cases corresponding to a portion of the promoter sequence in the expression vector, located upstream of the transgene in the transcribed RNA, and a primer specific to the 3′ untranslated region (3′ UTR), located downstream of the expressed transgene in the transcribed RNA sequence or a primer specific to a sequence within the transgene. In particular embodiments, at least one primer complementary to a sequence in the 5′ untranslated region (UTR) and at least one primer complementary to a sequence in the 3′ untranslated region (UTR) are employed to amplify the transgene. An exemplary depiction of the amplification of a transcript and resulting product using a forward primer specific to the 5′ UTR and a primer specific to a nucleotide sequence in the 3′ UTR and a predicted amplified product, where no splice events have occurred, is provided in FIG. 21A. An exemplary depiction of exemplary multiple amplified products (i.e., heterogeneity) resulting from amplification of a transcript that has a 5′ UTR, with a transcribed promoter sequence that contains a known splice donor site (P-SD) and a known splice acceptor site (P-SD), a transcribed transgene containing an unknown (cryptic) splice donor site (T-SD) and two unknown (cryptic) splice acceptor sites (T-SA) and a 3′ UTR, using primers specific to regions of the 5′ UTR and 3′ UTR, is shown in FIG. 21B.
Exemplary primers specific for the 5′ untranslated region (UTR) include primers directed to sequences within the promoter of the transgene. In some examples, a primer specific to an EF1a/HTLV promoter. An exemplary forward primer, specific to an EF1a-HTLV promoter is set forth in SEQ ID NO: 763.
Exemplary primers specific for the 3′ untranslated region (UTR) include primers directed to 3′ posttranscriptional regulatory elements located downstream of the transgene. Exemplary 3′ posttranscriptional regulatory elements include the woodchuck hepatitis virus (WHP) posttranscriptional regulatory element (WPRE), set forth in SEQ ID NO: 636. An exemplary forward primer, specific to a WPRE is set forth in SEQ ID NO: 764.
In some embodiments, multiple primer pairs can be used to amplify the transgene, such as for long transgenes. In some embodiments, sequential or nested pairs of forward and reverse primers, to crease a sliding window of amplified products, can be used to gain full and overlapping coverage of the sequence. Typically, the primers are designed to amplify a length of transgene that is approximately 1.5-6 kb, 2-6 kb, or 3-6 kb. An exemplary depiction of the amplification of a transcript using nested primer pairs is provided in FIG. 21C.
The amplified nucleic acid sequence is then analyzed for heterogeneity in terms of amplified transcript lengths. In some examples, heterogeneity is determined by the number and intensity of the bands for the expressed sequence. In some embodiments, RNA sequences having splice events upon expression generate multiple bands with different mobilities. In some embodiments, a major band is detected at the predicted mobility for a sequence not having any unpredicted splice events, and 1 or more additional bands of varying intensities and mobilities indicate the occurrence of one or more cryptic splice events within the transgene sequence.
The skilled artisan can resolve RNA, such as messenger RNA, and analyze the heterogeneity thereof by several methods. Non-limiting, exemplary methods include agarose gel electrophoresis, chip-based capillary electrophoresis, analytical centrifugation, field flow fractionation, and chromatography, such as size exclusion chromatography or liquid chromatography.
One or more steps of the above techniques can be performed under denaturing conditions, partially denaturing conditions, or non-denaturing conditions. The denaturing conditions can include conditions that cause denaturing of the nucleic acid transcript (e.g., mRNA) due to temperature, chaotropic agents (including salts), organic agents, among other mechanisms for denaturing. With thermal denaturing conditions, an elevated temperature can be applied. The elevated temperature can be one that is sufficient to denature intramolecular hydrogen bonds, to cause a change in or loss of secondary or tertiary structure, and so forth. For example, the temperature or thermal denaturing conditions can include a temperature of 25 degrees Celsius to 95 degrees Celsius, 35 to 85 degrees Celsius, 55 to 75 degrees Celsius, or of another range within those ranges. Similarly, higher or lower temperatures can be used as appropriate to cause the desired level of denaturing. The temperature or thermal denaturing conditions can also be dependent on the identity of the nucleic acid transcript, such that different temperatures are used for different nucleic acid transcripts or types of nucleic acid transcripts. The denaturing conditions can also include using chaotropic agents, such as lithium perchlorate and other perchlorate salts, guanidinium chloride and other guanidinium salts, urea, butanol, ethanol, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, or others. The denaturing conditions can further include organic denaturing agents, such as dimethyl sulfoxide (DMSO), acetonitrile, and glyoxal. In addition, the denaturing conditions can include a combination of two or more of these types of denaturing conditions. Any one or more of the steps of the RNA heterogeneity determining techniques can be performed at an elevated temperature or at ambient temperature, with or without chaotropic or organic agents.
a) Gel Electrophoresis
In some embodiments, RNA transcript topology and apparent (hydrodynamic) size can be analyzed by gel electrophoresis, such as agarose gel electrophoresis. In some examples, RNA transcript can be resolved on a 0.05% to 2% agarose gel, such as a 1.2% agarose gel, and visualized by staining or using probes that are specific to a particular sequence. In some embodiments, RNA transcripts can be directly assessed by gel electrophoresis, or can be assessed after amplification, such as quantitative amplification methods. Nucleic acid stains for visualizing nucleic acid on agarose gel are well known. Exemplary stains include BlueView™ Nucleic Acid Stain (Millipore Sigma), SYBR® Gold Nucleic Acid Stain (ThermoFisher), SYBR® Green Nucleic Acid Stain (Millipore Sigma), SYBR® Green II (ThermoFisher), PicoGreen® nucleic acid stain (Invitrogen), and ethidium bromide: 0.5 μg/mL prepared in distilled water, or incorporated into the gel. In some examples, the nucleic acid is stained using Quant-iT™ PicoGreen® binding followed by fluorescence detection and quantitation of the amplified products. The agarose gel method gives a more quantitative, but less resolving, measure of size distribution. In some embodiments, the nucleic acid fragments, resolved by agarose gel electrophoresis can be visualized by Northern blot for RNA or Southern blot for amplified reverse transcriptase-polymerase chain reaction (RT-PCR) products.
b) Chip-Based Capillary Electrophoresis
Chip-based capillary electrophoresis (e.g., with the AGILENT 2100 BIOANALYZER™) can be used a rapid and routine method for monitoring RNA transcript integrity and its size distribution. The separation is based on hydrodynamic size and charge, and is affected by the nucleotide length and folded structure of the RNA transcript. In one embodiment, the method includes delivering the sample into a channel of a chip with an electrolyte medium and applying an electric field to the chip that causes the RNA transcript and the impurities migrate through the channel. The RNA transcript has a different electrophoretic mobility than the impurities such that the RNA transcript migrates through the channel at rate that is different from a rate at which the impurities migrate through the channel. The electrophoretic mobility of the RNA transcript is proportional to an ionic charge the RNA transcript and inversely proportional to frictional forces in the electrolyte medium. The method also includes collecting from the chip the sample comprising the RNA transcript and one or more separate portions of the sample comprising the impurities. In addition, the method includes characterizing an aspect of at least one of the portion of the sample comprising the RNA transcript and the one or more separate portions of the sample comprising the impurities. The characterizing can include, for example, quantifying charge variants.
c) Analytical Ultracentrifugation (AUC)
Analytical ultracentrifugation (AUC) is a solution phase method for measuring molecular weight distribution, without the potential artifacts that could be introduced by matrix (resin or gel) interaction in the SEC, agarose, or other methods. Both equilibrium AUC and sedimentation ultracentrifugation are used, and the latter provides sedimentation coefficients that are related to both size and shape of the RNA transcript. A BECKMAN™ analytical ultracentrifuge equipped with a scanning UV/visible optics is used for analysis of the RNA transcript.
d) Field Flow Fractionation (FFF)
Another solution phase method for assessing hydrodynamic size distribution is field flow fractionation (FFF). FFF is a separation technique where a field is applied to a fluid suspension or solution pumped through a long and narrow channel, perpendicular to the direction of flow, to cause separation of the polynucleotides (RNA transcripts) present in the fluid, under the force exerted by the field. The field can be asymmetrical flow through a semipermeable membrane, gravitational, centrifugal, thermal-gradient, electrical, magnetic etc.
e) Chromatography
Chromatography also can be used to detect heterogeneity of RNA transcript lengths. Methods of size exclusion chromatography and liquid chromatography for determining mRNA heterogeneity are described in WO2014144711 which is incorporated herein by reference.
B. Methods of Optimizing Polynucleotides, e.g., Polynucleotides Encoding BCMA CARs
In some embodiments, the provided methods include optimizing and/or modifying the polynucleotide, for example, to reduce RNA heterogeneity and/or removing or eliminating cryptic or undesired splice sites. In some aspects, provided are methods of reducing the heterogeneity of an expressed transgene transcript that involves identifying a transgene candidate for the removal of splice sites, such as by the methods described above in Section I.A.; identifying one or more potential splice donor and/or splice acceptor sites; and modifying the nucleic acid sequence at or near the one or more identified splice donor sites that were identified, thereby generating a modified polynucleotide. In some aspects, the methods also involve assessing the transgene candidacy for the removal of splice sites. In some embodiments, the methods also include repeating one or more steps above until the heterogeneity of the transcript is reduced compared to the initial heterogeneity of the transcript as determined (such as before modification).
In some embodiments, methods of reducing heterogeneity, such as by removal or elimination of predicted splice sites, can be performed after codon optimization, or on non codon-optimized RNA. In some aspects, the methods involve identifying splice sites, such as one or more potential splice donor and/or acceptor sites, and modifying or change the RNA sequence (e.g., by replacing or substituting one or more nucleotides at or near the splice site. In some embodiments, codon optimization can be performed prior to and/or after methods of reducing heterogeneity of transcribed RNA (e.g., mRNA), such as by removal or elimination of predicted splice sites. In some embodiments, whether a transcript is a candidate for reducing RNA heterogeneity is determined based on the method of measuring RNA heterogeneity, e.g., as described in Section II.A herein. In some aspects, a transcribed nucleic acid that is detected as having heterogeneity is identified as a transgene candidate for removal of one or more splice site. In some embodiments, a transgene sequence can be a candidate for reducing heterogeneity when the transcribed nucleic acid of the transgene candidate exhibits at least or at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more heterogeneity following expression in a cell. In some embodiments, following transcription and processing of the polynucleotide in a human cell, optionally a human T cell, the messenger RNA (mRNA) from the polynucleotide, exhibits at least 70%, 75%, 80%, 85%, 90%, or 95% RNA homogeneity.
1. Methods of Reducing RNA Heterogeneity
Provided are methods of reducing heterogeneity of an expressed transgene transcript. In some embodiments, the methods involve identifying one or more potential splice donor and/or splice acceptor sites and modifying the nucleic acid sequence at or near the one or more of the identified splice donor sites. In some embodiments, the methods also involve assessing the transgene candidacy for removal of splice sites. In some aspects, one or more steps described herein can be repeated, for example, until the potential RNA heterogeneity is reduced compared to the starting or unmodified transcript.
a) Splice Site Identification
In some aspects, the presence of potential cryptic splice sites (splice donor and/or acceptor sites that are present in a transcript, such as a transgene transcript, can result in RNA heterogeneity of the transcript following expression in a cell. In some embodiments, the methods involve identifying one or more potential splice sites that can be present in the transgene transcript, that are not desired and/or that may be created in a transgene transcript from various underlying sequences, following codon optimization of a transcript and/or by mutation or mistake or error in transcription. In some aspects of the provided embodiments, the splice donor sites and splice acceptor sites are identified independently. In some embodiments, the splice acceptor and/or donor site(s) is/are canonical, non-canonical, and/or cryptic splice acceptor and/or donor site(s).
In some embodiments, the provided methods include identifying one or more potential splice site (e.g., canonical, non-canonical, and/or cryptic splice acceptor and/or donor site(s) or branch sites) in a polynucleotide, such as a polynucleotide encoding a transgene, such as a recombinant receptor, that may exhibit RNA heterogeneity or contain undesired. Also provided are polypeptides having reduced numbers of such splice sites as compared to such reference polynucleotides.
In some aspects, identification of the one or more splice sites in a nucleic acid sequence is an iterative process. In some embodiments, splice sites can be identified using a splice site and/or codon optimization prediction tool, such as by submitting the starting or reference sequence encoding the transgene, such as a BCMA-binding receptor, e.g., anti-BCMA CAR, to a database, a gene synthesis vendor or other source able to computationally or algorithmically compare the starting or reference sequence to identify or predict splice sites and/or for codon optimization and/or splice site removal. In some embodiments, after modifying the sequence for codon optimization and/or splice site removal, one or more further assessment of a sequence, such as a revised or modified nucleic acid sequence, is carried out to further evaluate for splice site removal, such as cryptic splice sites, using one or more other or additional splice site prediction tool(s).
In some aspects, RNA heterogeneity can be a result of the activity of the spliceosome present in a eukaryotic cell. In some aspects, splicing is typically carried out in a series of reactions catalyzed by the spliceosome. Consensus sequences for splice sites are known, but in some aspects, specific nucleotide information defining a splice site may be complex and may not be readily apparent based on available methods. Cryptic splice sites are splice sites that are not predicted based on the standard consensus sequences and are variably activated. Hence, variable splicing of pre-mRNA at cryptic splice sites leads to heterogeneity in the transcribed mRNA products following expression in eukaryotic cells. In some cases, within spliceosomal introns, a donor site (usually at the 5′ end of the intron), a branch site (near the 3′ end of the intron) and an acceptor site (3′ end of the intron) are required for a splicing event. The splice donor site can include a GU sequence at the 5′ end of the intron, with a large less highly conserved region. The splice acceptor site at the 3′ end of the intron can terminate with an AG sequence.
In some embodiments, splice sites, including potential cryptic splice sites can be identified by comparing sequences to known splice site sequences, such as those in a sequence database. In some embodiments, splice sites can be identified by computationally by submitting nucleotide sequences for analysis by splice site prediction tools, such as Human Splice Finder (Desmet et al., Nucl. Acids Res. 37(9):e67 (2009)), a neural network splice site prediction tool, NNSplice (Reese et al., J. Comput. Biol., 4(4):311 (1997)), GeneSplicer (Pertea et al., Nucleic Acids Res. 2001 29(5): 1185-1190) or NetUTR (Eden and Brunak, Nucleic Acids Res. 32(3):1131 (2004)), which identify potential splice sites and the probability of a splicing event at such sites. Additional splice prediction tools include RegRNA, ESEfinder, and MIT splice predictor. Splice site prediction tools such as GeneSplicer has been trained and/or tested successfully on databases for different species, such as human, Drosophila melanogaster, Plasmodium falciparum, Arabidopsis thaliana, and rice. In some embodiments, different prediction tools may be adapted for different extents on different database and/or for different species. In some embodiments, the one or more prediction tools are selected based upon their utility in certain database and/or for certain species. See, e.g., Saxonov et al., (2000) Nucleic Acids Res., 28, 185-190.
In some embodiments, one or more splice site prediction tools are selected for use in the determination of potential splice donor and/or acceptor sites. In some embodiments, splice site prediction tools that can be run locally; that can be retrained with a set of data at the user site; that can use databases for particular species (such as human), that can be compiled for multiple platforms, that allow real-time predictions for sequence selections, and/or that is an OSI certified open source software such that particular tools or plugins can be modified, can be employed. Exemplary tools that can be employed include NNSplice, GeneSplicer or both.
In some aspects, the splice site prediction tools be used to identify a list of potential splice donor and/or splice acceptor sites in a sequence such as a polynucleotide sequence containing transgene sequences. In some aspects, the prediction tools also can generate one or more prediction scores for one or more sequences in the polynucleotide, that can indicate the likelihoods of the one or more sequences being a splice donor or acceptor site sequence.
In some embodiments, the method involves comparing the prediction score for a particular splice site with a threshold score or reference score to determine or identify a particular splice sites that are candidate for elimination or removal. For example, in some embodiments, the predicted splice site is identified as a potential splice site when the prediction score is greater or no less than the threshold score or reference score. In some aspects, considerations for eliminating or removing a particular splice site include the prediction score as compared to a reference score or a threshold score; and whether a particular splice site is desired or intentional (for example, when the splicing event is more advantageous or is required for regulation of transcription and/or translation). In some aspects, the likelihood that the resulting splice variant loses the desired function or has compromised function can also be considered when determining particular donor and/or acceptor sites for elimination or removal. In some aspects, the one or more potential splice donor and/or splice acceptor sites exhibit a score about or at least about 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0 (e.g., on a scale with a maximum of 1.0) of a splice event or probability of a splice event, and the site can be a candidate for splice site elimination or removal. In some aspects, the score, e.g., used by GeneSplicer, at the one or more potential splice donor and/or splice site is based on the difference between the log-odds score returned for that sequence by the true Markov model and the score is computed by the false Markov model. In particular embodiments, the splice donor sites and splice acceptor sites are evaluated independently, or individually. In some embodiments, splice donor sites and splice acceptor sites are evaluated as a splice donor/acceptor pair.
b) Splice Site Elimination
In some embodiments, the provided methods involve eliminating or eliminating one or more splice donor and/or splice acceptor site(s), such as the potential splice donor and/or acceptor sites that may be involved in a cryptic splicing event that is not desired or that results in undesired RNA heterogeneity. In some embodiments, eliminating one or more splice sites comprises modifying one or more nucleotides (e.g., by substitution or replacement) in at, containing or near the splice donor and/or acceptor sites that are candidates for removal. In some aspects, a particular nucleotide within a codon that is at, contains or is near the splice site is modified (e.g., substituted or replaced). In some aspects, the modification (such as substitution or replacement) retains or preserves the amino acid encoded by the particular codon at the site, at the same time removing the potential splice donor and/or acceptor sites.
In some embodiments, the codon at or near the splice site for modification comprises one or more codons that involve one or both of the two nucleotides at the potential splice site (in some cases referred to as “splice site codon”). When the potential splicing is predicted to occur between two nucleotides in a codon, the codon is the only splice site codon for this splice site. If the potential splicing is predicted to occur between two adjacent codons, for example, between the last nucleotide of the first codon and the first nucleotide of the next codon, the two codons are splice site codons. For example, for splice sites that are predicted to be at boundaries of two codons, the two adjacent codons can be candidates for nucleotide modification. In some embodiments, the one or more codons comprise one splice site codon. In some embodiments, the one or more codons comprise both splice site codons. In some embodiments, the method involves eliminating potential splice donor site by modifying one or both splice site codons. In some embodiments, the method involves eliminating a potential splice acceptor donor site by modifying one or both splice site codons. In some embodiments, the one or both codons at the splice site is not modified, for example, when there are no synonymous codon for the splice site codon. In some embodiments, if there are no synonymous codons available for the particular splice site codon, one or more nucleotides in a nearby codon can be modified. In some embodiments, one or more codons that are modified include a splice site codon, wherein the modification comprises changing one or both nucleotides at the splice site to a different nucleotide or different nucleotides. In some embodiments, In some embodiments, the method involves eliminating the splice donor site by modifying one or both splice site codons, wherein the modification does not change one or two of the nucleotides of the at the splice site to a different nucleotide, but a nearby nucleotide, e.g., a part of a codon adjacent to the splice site, is modified. In some embodiments, the nearby or adjacent nucleotides that can be modified include modification of a nucleotide that is a part of a nearby or adjacent codon, such as a codon that is within one, two, three, four, five, six, seven, eight, nine or ten codons upstream or downstream of the splice site codon.
In some cases, manual modification of the polynucleotides can be employed, while preserving the encoded amino acid sequence, to reduce the probability of a predicted splice site. In some embodiments, one or more of the predicted splice sites having at least 80%, 85%, 90%, or 95% probability of a splice site are manually modified to reduce the probability of the splicing event. In some embodiments, the one or more modification(s) is/are by nucleotide replacement or substitution of 1, 2, 3, 4, 5, 6 or 7 nucleotides. In some embodiments, the modification(s) is/are at the junction of the splice donor site or are at the junction of the splice acceptor site. In some embodiments, at least one of the one or more nucleotide modifications is within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues of the splice site junction of the splice acceptor and/or splice donor site. In some embodiments, libraries of modified nucleic acid sequences can be generated with reduced probability of cryptic splice sites. In some embodiments, splice donor sites and splice acceptor sites are evaluated as a splice donor/acceptor pair. In particular embodiments, the splice donor sites and splice acceptor sites are evaluated independently, or individually, and not part as a splice donor/acceptor pair. In some embodiments, one or more predicted splice sites are not eliminated. In some embodiments, splice sites, such as known or predicted splice sites, within the promoter region of the transcript are not eliminated.
In some embodiments, the method involves eliminating one or more potential donor splice site by modifying one or two splice site codons or one or more nearby or adjacent codons (for example, if a synonymous codon is not available for the splice site codon). In some embodiments, the method involves eliminating one or more potential acceptor splice site by modifying one or two splice site codons or one or more nearby or adjacent codons (for example, if a synonymous codon is not available for the splice site codon). In some embodiments, the nearby or adjacent codon that is subject to modification include a codon that is within one, two, three, four, five, six, seven, eight, nine or ten codons upstream or downstream of the splice site codon, such as a codon that is within one, two or three codons from the splice site. In some embodiments, the methods can include removal or elimination of a potential branch site for splicing. In some aspects, a nucleotide within the codon at or near the branch site can be modified, e.g., substituted or replaced, to eliminate cryptic splicing and/or reduce RNA heterogeneity. In some embodiments, the modification of the one or more nucleotides can involve a substitution or replacement of one of the nucleotides that may be involved in splicing (such as at the splice donor site, splice acceptor site or splice branch site), such that the amino acid encoded by the codon is preserved, and the nucleotide substitution or replacement does not change the polypeptide sequence that is encoded by the polynucleotide. In some cases, the third position in the codon is more degenerate than the other two positions. Thus, various synonymous codons can encode a particular amino acid (see, e.g., Section II.B.2 below). In some embodiments, the modification includes replacing the codon with a synonymous codon used in the species of the cell into which the polynucleotide is introduced (e.g., human). In some embodiments, the species is human. In some embodiments, the one or more codon is replaced with a corresponding synonymous codons that the most frequently used in the species or synonymous codons that have a similar frequency of usage (e.g., most closest frequency of usage) as the corresponding codon (see, e.g., Section II.B.2 below).
In some embodiments, the methods also involve assessing the transgene candidacy for the removal of splice sites, after initial proposed modification. In some aspects, the proposed modification can be evaluated again, to assess the proposed modification and identify any further potential splice sites after modification and/or codon optimization. In some aspects, after modifying the sequence for codon optimization and/or splice site removal, one or more further assessment of a sequence, such as a revised or modified nucleic acid sequence, is carried out to further evaluate for splice site removal, such as cryptic splice sites, using the same or one or more other or additional splice site prediction tool(s). In some aspects, proposed modifications are considered for subsequent steps, and iterative optimization can be used. In some aspects, the methods also include repeating any of the identification and/or modification step, for example, until heterogeneity of the transcript is reduced compared to the heterogeneity of the transcript as initially determined. In some embodiments, a further or a different modification, such as with a different nucleotide replacement at the same codon or a modification at a different position or codon, can be done after an interactive evaluation and assessment. In some embodiments, corresponding different synonymous codon can be used, such as the second most frequently used in the particular species or a codon that has a similar frequency of usage (e.g., the next closest frequency of usage) as the corresponding codon (see, e.g., Section II.B.2 below).
In some aspects, a proposed modification can be further evaluated, for example, to assess whether the modification generates an undesired or additional restriction site in the polynucleotide. In some aspects, an additional restriction site may not be desired, and a further or a different modification (e.g., with a different nucleotide replacement at the same codon or a modification at a different position or codon) can be considered. In some aspects, particular restriction site, such as a designated restriction site, is avoided. In some aspects, if the modification does not substantially reduce or, the splice site prediction score, an additional or alternative modification can be proposed. In some embodiments, the splice site prediction score can be is reduced or lowered by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%, after one or more iteration of the methods.
In some embodiments of any of the methods provided herein, a computer system can be used to execute one or more steps, tools, functions, processes or scripts. In certain embodiments, methods provided herein are computer implemented methods and/or are performed with the aid of a computer. In some embodiments, the splice site prediction, evaluation and modification for elimination or removal of a splice site can be performed by computer implemented methods and/or by methods which include steps that are computer implemented steps. In some embodiments, comparison of the sequences to a known database, calculating a splice site prediction score, determining potential nucleotide modifications, codon optimization and/or any one of the iterative steps can be implemented by a computer or using a computer-implemented steps, tools, functions, processes or scripts. In particular embodiments, a computer system comprising a processor and memory is provided, wherein the memory contains instructions operable to cause the processor to carry out any one or more of steps of the methods provided herein. In some embodiments, the methods include steps, functions, processes or scripts that are performed computationally, e.g., performed using one or more computer programs and/or via the use of computational algorithms.
Exemplary steps, functions, processes or scripts of the provided methods for identifying and/or removing possible splice sites include one or more steps of: selecting sequence, writing FASTA format sequences, loading codon table (e.g., from www.kazusa.or.jp/codon, running GeneSplicer, loading predictions, parsing codons, determining overlaps in prediction, identifying next highest usage synonymous codon, reviewing for restriction site, creating annotations or assessing other codons. Particular steps can assess both forward and reverse strands. In some aspects, previously annotated splice site modifications can also be considered, to allow for iterative optimization. In some embodiments, any one or more of the steps, functions, processes or scripts can be repeated.
In certain embodiments, methods provided herein may be practiced, at least in part, with computer system configurations, including single-processor or multi-processor computer systems, minicomputers, mainframe computers, as well as personal computers, hand-held computing devices, microprocessor-based and/or programmable consumer electronics and the like, each of which may operatively communicate with one or more associated devices. In particular embodiments, the methods provided herein may be practiced, at least in part, in distributed computing environments such that certain tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules may be located in local and/or remote memory storage devices. In particular embodiments, some or all steps of the methods provided herein may be practiced on stand-alone computers.
In particular embodiments, some or all of the steps of the methods provided herein can operate in the general context of computer-executable instructions, such as program modules, plugins and/or scripts executed by one or more components. Generally, program modules include routines, programs, objects, data structures and/or scripts, that perform particular tasks or implement particular abstract data types. Typically, the functionality of the program modules may be combined or distributed as desired. In certain embodiments, instructions operable to cause the processor to carry out any one or more steps of the methods provided herein can be embodied on a computer-readable medium having computer-executable instructions and transmitted as signals manufactured to transmit such instructions as well as the results of performing the instructions, for instance, on a network. In some embodiments, also provided are computer systems, computer readable instructions, software, systems, networks and/or devices for carrying out or performing one or more steps of the methods provided herein.
2. Codon Optimization
In some embodiments the polynucleotides are modified by optimization of the codons for expression in humans. In some aspects, codon optimization can be considered before and/or after the steps for splice site identification and/or splice site elimination, and/or at each of the iterative steps for reducing RNA heterogeneity. Codon optimization generally involves balancing the percentages of codons selected with the abundance, e.g., published abundance, of human transfer RNAs, for example, so that none is overloaded or limiting. In some cases, such balancing is necessary or useful because most amino acids are encoded by more than one codon, and codon usage generally varies from organism to organism. Differences in codon usage between transfected or transduced genes or nucleic acids and host cells can have effects on protein expression from the nucleic acid molecule. Table 3 below sets forth an exemplary human codon usage frequency table. In some embodiments, to generate codon-optimized nucleic acid sequences, codons are chosen to select for those codons that are in balance with human usage frequency. The redundancy of the codons for amino acids is such that different codons code for one amino acid, such as depicted in Table 3. In selecting a codon for replacement, it is desired that the resulting mutation is a silent mutation such that the codon change does not affect the amino acid sequence. Generally, the last nucleotide of the codon (e.g., at the third position) can remain unchanged without affecting the amino acid sequence.
TABLE 3
Human Codon Usage Frequency
Hu-
man
co-
amino
freq./
Human
amino
freq./
don
acid
1000
number
codon
acid
1000
number
TTT
F
17.6
714298
TCT
S
15.2
618711
TTC
F
20.3
824692
TCC
S
17.7
718892
TTA
L
7.7
311881
TCA
S
12.2
496448
TTG
L
12.9
525688
TCG
S
4.4
179419
CTT
L
13.2
536515
CCT
P
17.5
713233
CTC
L
19.6
796638
CCC
P
19.8
804620
CTA
L
7.2
290751
CCA
P
16.9
688038
CTG
L
39.6
1611801
CCG
P
6.9
281570
ATT
I
16
650473
ACT
T
13.1
533609
ATC
I
20.8
846466
ACC
T
18.9
768147
ATA
I
7.5
304565
ACA
T
15.1
614523
ATG
M
22
896005
ACG
T
6.1
246105
GTT
V
11
448607
GCT
A
18.4
750096
GTC
V
14.5
588138
GCC
A
27.7
1127679
GTA
V
7.1
287712
GCA
A
15.8
643471
GTG
V
28.1
1143534
GCG
A
7.4
299495
TAT
Y
12.2
495699
TGT
C
10.6
430311
TAC
Y
15.3
622407
TGC
C
12.6
513028
TAA
*
1
40285
TGA
*
1.6
63237
TAG
*
0.8
32109
TGG
W
13.2
535595
CAT
H
10.9
441711
CGT
R
4.5
184609
CAC
H
15.1
613713
CGC
R
10.4
423516
CAA
Q
12.3
501911
CGA
R
6.2
250760
CAG
Q
34.2
1391973
CGG
R
11.4
464485
AAT
N
17
689701
AGT
S
12.1
493429
AAC
N
19.1
776603
AGC
S
19.5
791383
AAA
K
24.4
993621
AGA
R
12.2
494682
AAG
K
31.9
1295568
AGG
R
12
486463
GAT
D
21.8
885429
GGT
G
10.8
437126
GAC
D
25.1
1020595
GGC
G
22.2
903565
GAA
E
29
1177632
GGA
G
16.5
669873
GAG
E
39.6
1609975
GGG
G
16.5
669768
For example, the codons TCT, TCC, TCA, TCG, AGT and AGC all code for Serine (note that T in the DNA equivalent to the U in RNA). From a human codon usage frequency, such as set forth in Table 3 above, the corresponding usage frequencies for these codons are 15.2, 17.7, 12.2, 4.4, 12.1, and 19.5, respectively. Since TCG corresponds to 4.4%, if this codon were commonly used in a gene synthesis, the tRNA for this codon would be limiting. In codon optimization, the goal is to balance the usage of each codon with the normal frequency of usage in the species of animal in which the transgene is intended to be expressed.
C. Optimized Anti-BCMA CAR
In some embodiments, a starting or reference sequence encoding a transgene, such as a BCMA-binding receptor, e.g., anti-BCMA CAR, is assessed for codon optimization and/or splice site removal.
In some embodiments, the methods are carried out on an anti-BCMA CAR, such as a CAR containing an scFv antigen-binding domain specific to BCMA, a spacer, such as a spacer set forth in SEQ ID NO:649, a costimulatory signaling region, such as a costimulatory signaling domain from 4-1BB and a CD3 zeta signaling region. Exemplary identified splice donor sites and splice acceptor sites, and their corresponding scores, are listed in Tables 3 and 4 below for exemplary anti-BCMA CARs.
TABLE 4
Predicted Splice Donor Sites
STARTING SEQUENCE
O/SSE SEQUENCE
Region of
splice
SEQ
Splice
optimized splice
SEQ
Splice
Construct
donor site
ID NO
score
donor site
ID NO
score
promoter
cgtctaggtagttt
689
1
no change
<0.7
scFv-encoding
BCMA-23
gaccaaggtgaccgt
690
N/A
caccaaggtgaccgt
698
0.54
BCMA-26
tgcactggtaccagc
691
0.55
no change
BCMA-52
taaactggtaccagc
692
0.76
tgaactggtatcagc
699
<0.7
BCMA-52
atctcctgtacgggt
693
0.79
atctcttgaaatggt
700
<0.7
BCMA-52
ggtcaaggtactctg
694
0.85
ggccagggcacactg
701
<0.7
BCMA-55
gaggacagtaagcgg
695
0.66
gaggacagcaagagg
702
<0.5
BCMA-55
ggtcaaggtactctg
696
0.85
ggccagggaaccctg
703
<0.5
BCMA-55
tgcctccgtgtctgc
697
<0.50
tgccagcgttagtgc
704
0.60
Spacer-encoding
aatctaagtacggac
705
0.65
agtctaaatacggac
661
<0.7
tcaactggtacgtgg
706
0.96
tcaactggtatgtgg
662
<0.7
tcaattggtacgtgg
616
0.97
tcaactggtatgtgg
662
<0.7
acaattagtaaggca
707
0.43
accatctccaaggcc
663
<0.7
accacaggtgtatac
708
0.42
gccccaggtttacac
664
<0.7
CD3zeta
tttccaggtccgccg
709
0.74
tcagcagatccgccg
665
<0.7
signaling
region-encoding
Truncated receptor surrogate marker-encoding
ctgctctgtgagtta
710
0.56
ctcctgtgtgaactc
666
<0.7
acgcaaagtgtgtaa
711
0.5
tcggaaagtgtgcaa
667
<0.7
caacatggtcagttt
712
0.71
cagcacggccagttt
668
<0.7
aacagaggtgaaaac
713
0.42
aaccggggcgagaac
669
<0.7
ctggagggtgagcca
714
0.82
ctggaaggcgagccc
670
<0.7
tcttcatgtgagcgg
720
0.84
tgttcatgtgagcgg
671
<0.7
Promoter
tggctccgcctttttcccgag
721
0.50
no change
ggtgggggagaaccgtatat
tgaactgcgtccgccgtctag
722
0.71
no change
gtaagtttaaagctcaggtc
ttctgttctgcgccgttacag
723
0.89
no change
atccaagctgtgaccggcgc
scFv-encoding
BCMA-23
ctactacatgagctggatccg
724
N/A
ctactatatgtcctggatcag
735
0.46
ccaggctccagggaaggggc
acaggcacctggcaagggcc
BCMA-23
ggctgattattattgtagctc
725
N/A
ggcagattactattgttctag
736
0.55
atatggaggtagtaggtctt
ctacggcggcagcagatcct
BCMA-25
ctatgccatgtcctggttcag
726
0.95
ctatgccatgtcctggttcaa
737
<0.7
gcaggcaccaggcaagggcc
gcaggcaccaggcaagggcc
BCMA-25
gtccgcctctgtgggcgatag
727
0.50
no change
ggtgaccgtgacatgtcgcg
BCMA-25
gtgggctttatccgctctaaa
728
0.55
no change
gcctacggcggcaccacaga
BCMA-25
gtgacatgtcgcgcctcccag
729
0.67
no change
ggcatctctaactacctggc
BCMA-25
tacagcgcctccaccctgcag
730
0.66
no change
agcggagtgccctcccggtt
BCMA-52
ctggccatcagtggcctccag
731
<0.50
ctggctatttctggactgcag
738
0.62
tctgaggatgaggctgatta
agcgaggacgaggccgacta
BCMA-52
agatacagcccgtccttccaa
732
<0.50
agatacagccctagctttcag
739
0.67
ggccacgtcaccatctcagc
ggccacgtgaccatcagcgc
BCMA-55
cgaggctgattattactgcag
733
0.79
cgaggccgattactactgcag
740
<0.40
ctcaaatacaagaagcagca
cagcaacacccggtccagca
BCMA-55
gccctcaggggtttctaatag
734
<0.50
gcccagcggcgtgtccaatag
741
0.40
cttctctggctccaagtctg
attcagcggcagcaagagcg
Spacer-encoding
cgccttgtcctccttgtccag
765
0.84
cgccttgtcctccttgtccag
766
<0.7
ctcctcctgttgccggacct
ctcctcctgttgccggacct
aagtttctttctgtattccag
742
0.97
cagtttcttcctgtatagtag
672
<0.7
gctgaccgtggataaatctc
actcaccgtggataaatcaa
aagtttctttctgtattccag
742
0.97
aagtttctttctgtattccag
854
gctgaccgtggataaatctc
actgaccgtggataaatctc
gggcaacgtgttctcttgcag
743
0.55
gggcaacgtgttcagctgcag
673
<0.7
tgtcatgcacgaagccctgc
cgtgatgcacgaggccctgc
cagtttcttcctgtatagtag
767
0.74
No change
actcaccgtggataaatcaa
CD28 TM-
aggggtgctggcctgttacag
744
0.4
cggagtgctggcctgttacag
674
0.75
encoding
cctgctggtgacagtcgctt
cctgctggttaccgtggcct
4-1BB/
gctgagagtcaagttttccag
745
0.55
gctgagagtgaagttcagcag
675
<0.7
CD3zeta
gtccgccgacgctccagcct
atccgccgacgctccagcct
signaling
region-encoding
Truncated Receptor Surrogate Marker-encoding
actcctcctctggatccacag
746
0.74
acacctccactggatccccaa
676
<0.7
gaactggatattctgaaaac
gagctggatatcctgaaaac
acagggtttttgctgattcag
747
0.73
accggattcctcctgatccaa
677
<0.7
gcttggcctgaaaacaggac
gcctggccagagaacagaac
accggattcctcctgattcag
768
0.82
accggattcctcctgatccaa
677
<0.7
gcctggccagagaacagaac
gcctggccagagaacagaac
atggtcagttttctcttgcag
748
0.89
acggccagtttagcctggctg
678
<0.7
tcgtcagcctgaacataaca
tggtgtctctgaacatcacc
In some embodiments, the resulting modified nucleic acid sequence(s) is/are then synthesized and used to transduce cells to test for splicing as indicated by RNA heterogeneity. Exemplary methods are as follows and described in the Examples. Briefly, RNA is harvested from the expressing cells, amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and resolved by agarose gel electrophoresis to determine the heterogeneity of the RNA, compared to the starting sequence. In some cases, improved sequences can be resubmitted to the gene synthesis vendor for further codon optimization and splice site removal, followed by further cryptic splice site evaluation, modification, synthesis and testing, until the RNA on the agarose gel exhibits minimal RNA heterogeneity.
In some embodiments, the provided methods for optimizing a coding nucleic acid sequence encoding a transgene, such as an anti-BCMA CAR provided herein, or a construct provided herein, is to both reduce or eliminate cryptic splice sites (see, e.g., SEQ ID NO: 622 for an exemplary codon optimized and splice site eliminated spacer sequence) and optimize human codon usage (see, e.g., SEQ ID NO: 855 for an exemplary codon optimized and spacer sequence). An exemplary optimization strategy is described in the Examples.
In some embodiments, provided are polynucleotides encoding a chimeric antigen receptor, comprising nucleic acid encoding: (a) an extracellular antigen-binding domain that specifically recognizes BCMA, including any of the antigen-binding domains described below; (b) a spacer of at least 125 amino acids in length; (c) a transmembrane domain; and (d) an intracellular signaling region, wherein following expression of the polynucleotide in a cell, the transcribed RNA, optionally messenger RNA (mRNA), from the polynucleotide, exhibits at least 70%, 75%, 80%, 85%, 90%, or 95% RNA homogeneity. In some embodiments the antigen-binding domain comprises a VH region and a VL region comprising the amino acid sequence set forth in SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids having at least 90% identity to SEQ ID NOS:617 and 618, respectively. In some embodiments, the antigen-binding domain comprises a VH region that is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region amino acid sequence selected from SEQ ID NO: 617; and a VL region that is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region amino acid sequence selected from SEQ ID NO: 618. In some embodiments, In some embodiments, the antigen-binding domain comprises a VH region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS:593, 594, and 595, respectively, and a VL region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS:601, 602, and 603, respectively; or a VH region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS:596, 597, and 595, respectively, and a VL region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS:601, 602, and 603, respectively; or a VH region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 598, 599, and 595, respectively, and a VL region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS:601, 602, and 603, respectively; or a VH region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 611, 612, and 613, respectively, and a VL region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS: 614, 615, and 603, respectively; or a VH region that is or comprises the amino acid sequence set forth in SEQ ID NO: 617; and a VL region that is or comprises the amino acid sequence set forth in SEQ ID NO: 618. In some embodiments, exemplary antigen-binding domain in the chimeric antigen receptor encoded by the polynucleotide include those described in each row of Table 2 herein. In any of such embodiments, the transmembrane domain of the CAR is or comprises a transmembrane domain derived from a CD28; the intracellular signaling region comprises a cytoplasmic signaling domain of a CD3-zeta (CD3ζ) chain or a functional variant or signaling portion thereof and a costimulatory signaling region comprises an intracellular signaling domain of 4-1BB.
In some embodiments, provided are polynucleotides encoding a chimeric antigen receptor, comprising nucleic acid encoding: (a) an extracellular antigen-binding domain that specifically recognizes BCMA, including any of the antigen-binding domains described below; (b) (b) a spacer, wherein the encoding nucleic acid is or comprises, or consists or consists essentially of, the sequence set forth in SEQ ID NO:622 or encodes a sequence of amino acids set forth in SEQ ID NO:649; (c) a transmembrane domain; and (d) an intracellular signaling region. In some embodiments the antigen-binding domain comprises a VH region and a VL region comprising the amino acid sequence set forth in SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids having at least 90% identity to SEQ ID NOS:617 and 618, respectively. In some embodiments, the antigen-binding domain comprises a VH region that is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region amino acid sequence selected from SEQ ID NO: 617; and a VL region that is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region amino acid sequence selected from SEQ ID NO: 618. In some embodiments, In some embodiments, the antigen-binding domain comprises a VH region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS:593, 594, and 595, respectively, and a VL region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS:601, 602, and 603, respectively; or a VH region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS:596, 597, and 595, respectively, and a VL region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS:601, 602, and 603, respectively; or a VH region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 598, 599, and 595, respectively, and a VL region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS:601, 602, and 603, respectively; or a VH region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOS: 611, 612, and 613, respectively, and a VL region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOS: 614, 615, and 603, respectively; or a VH region that is or comprises the amino acid sequence set forth in SEQ ID NO: 617; and a VL region that is or comprises the amino acid sequence set forth in SEQ ID NO: 618. In some embodiments, exemplary antigen-binding domain in the chimeric antigen receptor encoded by the polynucleotide include those described in each row of Table 2 herein. In any of such embodiments, the transmembrane domain of the CAR is or comprises a transmembrane domain derived from a CD28; the intracellular signaling region comprises a cytoplasmic signaling domain of a CD3-zeta (CD3ζ) chain or a functional variant or signaling portion thereof and a costimulatory signaling region comprises an intracellular signaling domain of 4-1BB.
Also provided herein are exemplary modified polynucleotides, including polynucleotides that were modified for codon optimization (0) and/or splice site elimination (SSE). Examples of such polynucleotides are set forth in Table 5, wherein exemplary nucleotide (nt) sequences for the components of the exemplary CAR constructs prior to splice site elimination and codon optimization (non-opt), nucleic acid (nt) sequences for the components of the CAR constructs following splice site elimination and optimization (O/SSE), and the corresponding amino acid (aa) sequences encoded by the nucleic acid sequences are provided. The components include the IgG-kappa signaling sequence (ss), the anti-BCMA scFv, spacer region, transmembrane (tm) domain, co-signaling sequence (4-1BB co-sig or CD28 co-sig), CD3-ζ signaling domain (CD3-ζ), T2A ribosomal skip element (T2A) and truncated EGF receptor (EGFRt) sequence. Polynucleotide sequences of exemplary CAR constructs are set forth in SEQ ID NOs: 751-756, encoding the amino acid sequences set forth in SEQ ID NOs: 757-762.
TABLE 5
Exemplary BCMA CAR components (SEQ ID NOs)
Construct
Sequence
ss
scFv
spacer
TM
CD3-ζ
4-1BB
co-stim
BCMA-23-L CAR
non-opt (nt)
619
352
621
623
625
627
BCMA-23-L CAR CO/SSE
O/SSE (nt)
684
715
622 or 856
688
681
652
both
aa
620
278
649
624
626
628
BCMA-25-L CAR
non-opt (nt)
619
716
621
623
625
627
BCMA-25-L CAR CO/SSE
O/SSE (nt)
682
717
622 or 856
688
681
652
both
Aa
620
559
649
624
626
628
BCMA-26-L CAR
non-opt (nt)
619
718
621
623
625
627
BCMA-26-L CAR CO/SSE
O/SSE (nt)
685
719
622 or 856
688
681
652
both
aa
620
560
649
624
626
628
BCMA-52-L CAR
non-opt (nt)
619
647
621
623
625
627
BCMA-52-L CAR CO/SSE
O/SSE (nt)
682
440
622 or 856
688
681
652
both
Aa
620
442
649
624
626
628
BCMA-55-L CAR
non-opt (nt)
619
648
621
623
625
627
BCMA-55-L CAR CO/SSE
O/SSE (nt)
683
460
622 or 856
688
681
652
both
aa
620
478
649
624
626
628
CD28
co-stim
BCMA-55-L-CD28 CAR
non-opt (nt)
619
648
621
623
679
627
BCMA-55-L-CD28 CAR CO/SSE
O/SSE (nt)
683
460
622
688
679
652
both
aa
620
478
649
624
680
628
III. ENGINEERED CELLS
Also provided are cells such as engineered cells that contain a recombinant receptor (e.g., a chimeric antigen receptor) such as one that contains an extracellular domain including an anti-BCMA antibody or fragment as described herein. Also provided are populations of such cells, compositions containing such cells and/or enriched for such cells, such as in which cells expressing the BCMA-binding molecule make up at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or more percent of the total cells in the composition or cells of a certain type such as T cells or CD8+ or CD4+ cells. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.
Thus also provided are genetically engineered cells expressing the recombinant receptors containing the antibodies, e.g., cells containing the CARs. The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, as described herein, and re-introducing them into the same patient, before or after cryopreservation.
Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naïve T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TscM), central memory T (T04), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
In some embodiments, the cells include one or more polynucleotides introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such polynucleotides. In some embodiments, the polynucleotides are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived. In some embodiments, the polynucleotides are not naturally occurring, such as a polynucleotide not found in nature, including one comprising chimeric combinations of polynucleotides encoding various domains from multiple different cell types. In some embodiments, the cells (e.g., engineered cells) comprise a vector (e.g., a viral vector, expression vector, etc.) as described herein such as a vector comprising a nucleic acid encoding a recombinant receptor described herein.
A. Vectors and Methods for Genetic Engineering
Also provided are methods, polynucleotides, compositions, and kits, for expressing the binding molecules (e.g., anti-BCMA binding molecules), including recombinant receptors (e.g., CARs) comprising the binding molecules, and for producing the genetically engineered cells expressing such binding molecules. In some embodiments, one or more binding molecules, including recombinant receptors (e.g., CARs) can be genetically engineered into cells or plurality of cells. The genetic engineering generally involves introduction of a nucleic acid encoding the recombinant or engineered component into the cell, such as by retroviral transduction, transfection, or transformation.
Also provided are polynucleotides encoding the chimeric antigen receptors and/or portions, e.g., chains, thereof. Among the provided polynucleotides are those encoding the anti-BCMA chimeric antigen receptors (e.g., antigen-binding fragment) described herein. Also provided are polynucleotides encoding one or more antibodies and/or portions thereof, e.g., those encoding one or more of the anti-BCMA antibodies (e.g., antigen-binding fragment) described herein and/or other antibodies and/or portions thereof, e.g., antibodies and/or portions thereof that binds other target antigens. The polynucleotides may include those encompassing natural and/or non-naturally occurring nucleotides and bases, e.g., including those with backbone modifications. The terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide” may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide.
Also provided are polynucleotides that have been optimized for codon usage and/or to eliminate splice sites, such as cryptic splice sites. Also provided are methods of optimizing and producing the coding sequences of chimeric antigen receptors, such as any of the chimeric antigen receptors described herein. Such methods are described in Section II herein.
Also provided are vectors containing the polynucleotides, such as any of the polynucleotides described herein, and host cells containing the vectors, e.g., for producing the antibodies or antigen-binding fragments thereof. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, or a lentiviral vector. Also provided are methods for producing the antibodies or antigen-binding fragments thereof. The nucleic acid may encode an amino acid sequence comprising the VL region and/or an amino acid sequence comprising the VH region of the antibody (e.g., the light and/or heavy chains of the antibody). The nucleic acid may encode one or more amino acid sequence comprising the VL region and/or an amino acid sequence comprising the VH region of the antibody (e.g., the light and/or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such polynucleotides are provided. In a further embodiment, a host cell comprising such polynucleotides is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH region of the antibody. In another such embodiment, a host cell comprises (e.g., has been transformed with) (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL region of the antibody and an amino acid sequence comprising the VH region of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL region of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH region of the antibody. In some embodiments, a host cell comprises (e.g., has been transformed with) one or more vectors comprising one or more nucleic acid that encodes one or more an amino acid sequence comprising one or more antibodies and/or portions thereof, e.g., antigen-binding fragments thereof. In some embodiments, one or more such host cells are provided. In some embodiments, a composition containing one or more such host cells are provided. In some embodiments, the one or more host cells can express different antibodies, or the same antibody. In some embodiments, each of the host cells can express more than one antibody.
Also provided are methods of making the anti-BCMA chimeric antigen receptors. For recombinant production of the chimeric receptors, a nucleic acid sequence encoding a chimeric receptor antibody, e.g., as described herein, may be isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid sequences may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). In some embodiments, a method of making the anti-BCMA chimeric antigen receptor is provided, wherein the method comprises culturing a host cell comprising a nucleic acid sequence encoding the antibody, as provided above, under conditions suitable for expression of the receptor.
In some aspects, for production of isolated or secreted polypeptides, in addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been modified to mimic or approximate those in human cells, resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
Exemplary eukaryotic cells that may be used to express polypeptides, including isolated or secreted polypeptides, include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lec13 CHO cells, and FUT8 CHO cells; PER.C6® cells; and NSO cells. In some embodiments, the antibody heavy chains and/or light chains (e.g., VH region and/or VL region) may be expressed in yeast. See, e.g., U.S. Publication No. US 2006/0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and/or light chains (e.g., VH region and/or VL region). For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.
In particular examples immune cells, such as human immune cells are used to express the provided polypeptides encoding chimeric antigen receptors. In some examples, the immune cells are T cells, such as CD4+ and/or CD8+ immune cells, including primary cells, such as primary CD4+ and CD8+ cells.
In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.
In some contexts, overexpression of a stimulatory factor (for example, a lymphokine or a cytokine) may be toxic to a subject. Thus, in some contexts, the engineered cells include gene segments that cause the cells to be susceptible to negative selection in vivo, such as following administration in adoptive immunotherapy. For example in some aspects, the cells are engineered so that they can be eliminated as a result of a change in the in vivo condition of the patient to which they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell 2:223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).
In some aspects, the cells further are engineered to promote expression of cytokines or other factors. Various methods for the introduction of genetically engineered components, e.g., antigen receptors, e.g., CARs, are well known and may be used with the provided methods and compositions. Exemplary methods include those for transfer of polynucleotides encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.
In some embodiments, recombinant polynucleotides are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant polynucleotides are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 Nov. 29(11): 550-557).
In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or human immunodeficiency virus type 1 (HIV-1). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
Methods of lentiviral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.
In some embodiments, recombinant polynucleotides are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant polynucleotides are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).
Other approaches and vectors for transfer of the polynucleotides encoding the recombinant products are those described, e.g., in international patent application, Publication No.: WO2014055668, and U.S. Pat. No. 7,446,190.
Among additional polynucleotides, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.
In some embodiments, one or more binding molecules, including antibodies and/or recombinant receptors (e.g., CARs), can be genetically engineered to be expressed in cells or plurality of cells. In some embodiments, a first recombinant receptor and a second binding molecule, e.g., recombinant receptor, are encoded by the same or separate nucleic acid molecules. In some embodiments, additional binding molecules are engineered to be expressed in cells or a plurality of cells.
In some cases, the polynucleotide containing nucleic acid sequences encoding the BCMA-binding receptor, e.g., chimeric antigen receptor (CAR), contains a signal sequence that encodes a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. In other aspects, the signal sequence may encode a heterologous or non-native signal peptide. In some aspects, non-limiting exemplary signal peptide include a signal peptide of the IgG kappa chain set forth in SEQ ID NO: 620, or encoded by the nucleotide sequence set forth in SEQ ID NO: 619 or 682-685; a GMCSFR alpha chain set forth in SEQ ID NO:851 and encoded by the nucleotide sequence set forth in SEQ ID NO:850; a CD8 alpha signal peptide set forth in SEQ ID NO:852; or a CD33 signal peptide set forth in SEQ ID NO:853.
In some embodiments the vector or construct can contain promoter and/or enhancer or regulatory elements to regulate expression of the encoded recombinant receptor. In some examples the promoter and/or enhancer or regulatory elements can be condition-dependent promoters, enhancers, and/or regulatory elements. In some examples these elements drive expression of the transgene. In some examples, the CAR transgene can be operatively linked to a promoter, such as an EF1alpha promoter with an HTLV1 enhancer (SEQ ID NO: 635). In some examples, the CAR transgene is operatively linked to a Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE; SEQ ID NO: 636), located downstream of the transgene.
In some embodiments, the vector or construct can contain a single promoter that drives the expression of one or more nucleic acid molecules. In some embodiments, such nucleic acid molecules, e.g., transcripts, can be multicistronic (bicistronic or tricistronic, see e.g., U.S. Pat. No. 6,060,273). For example, in some embodiments, transcription units can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows coexpression of gene products (e.g. encoding a first and second chimeric receptor) by a message from a single promoter. Alternatively, in some cases, a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF), two or three genes (e.g. encoding a first and second binding molecules, e.g., antibody recombinant receptor) separated from one another by sequences encoding a self-cleavage peptide (e.g., 2A cleavage sequences) or a protease recognition site (e.g., furin). The ORF thus encodes a single polypeptide, which, either during (in the case of T2A) or after translation, is cleaved into the individual proteins. In some cases, the peptide, such as T2A, can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream (see, for example, de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and deFelipe et al. Traffic 5:616-626 (2004)). Many 2A elements are known. Examples of 2A sequences that can be used in the methods and polynucleotides disclosed herein, without limitation, 2A sequences from the foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 659 or 660), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 657 or 658), Thosea asigna virus (T2A, e.g., SEQ ID NO: 631, 653, or 654), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 655 or 656) as described in U.S. Patent Publication No. 20070116690. In some embodiments, the one or more different or separate promoters drive the expression of one or more nucleic acid molecules encoding the one or more binding molecules, e.g., recombinant receptors.
Any of the binding molecules, e.g., antibodies and/or recombinant receptors provided herein, e.g., BCMA-binding molecules and/or the additional recombinant receptors, can be encoded by polynucleotides containing one or more nucleic acid molecules encoding the receptors, in any combinations or arrangements. For example, one, two, three or more polynucleotides can encode one, two, three or more different receptors or domains. In some embodiments, one vector or construct contains nucleic acid molecules encoding one or more binding molecules, e.g., antibody and/or recombinant receptor, and a separate vector or construct contains nucleic acid molecules encoding an additional binding molecule, e.g., antibody and/or recombinant receptor. Each of the nucleic acid molecules can also encode one or more marker(s), such as a surface marker, e.g., truncated EGFR (tEGFR).
Also provided are compositions containing one or more of the nucleic acid molecules, vectors or constructs, such as any described above. In some embodiments, the nucleic acid molecules, vectors, constructs or compositions can be used to engineer cells, such as T cells, to express any of the binding molecules, e.g., antibody or recombinant receptor, and/or the additional binding molecules.
B. Preparation of Cells for Engineering
In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for introduction of the recombinant receptor (e.g., CAR) may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, or pig.
In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contain cells other than red blood cells and platelets.
In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++/Mg++ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.
In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques.
For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander, MACSiBeads™, etc.).
In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (markerhigh) on the positively or negatively selected cells, respectively.
In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, CD4+ and/or CD8+ selection steps are used to separate CD4+ helper and CD8+ cytotoxic T cells from a composition, such as from a PBMC composition such as one obtained via leukapheresis. Such CD4+ and CD8+ populations, in some aspects, can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations. In some embodiments, CD4+ and CD8+ cells are mixed at a desired ratio
In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.
In embodiments, memory T cells are present in both CD62L+ and CD62L− subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+ and/or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.
In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.
CD4+ T helper cells are sorted into naïve, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L− and CD45RO−.
In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In vitro and In vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher® Humana Press Inc., Totowa, N.J.).
In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynabeads® or MACS® beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.
In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084, are other examples.
The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.
In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.
In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.
In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS®) (Miltenyi Biotec, Auburn, Calif.). Magnetic Activated Cell Sorting (MACS®) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS® operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.
In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Number WO2009/072003, or US 20110003380 A1.
In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.
In some aspects, the separation and/or other steps is carried out using CliniMACS® system (Miltenyi Biotec), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.
The CliniMACS® system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.
In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy® system (Miltenyi Biotec). The CliniMACS Prodigy® system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy® system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood may be automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy® system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.
In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.
In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to −80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.
In some embodiments, the provided methods include cultivation, incubation, culture, and/or genetic engineering steps. For example, in some embodiments, provided are methods for incubating and/or engineering the depleted cell populations and culture-initiating compositions.
Thus, in some embodiments, the cell populations are incubated in a culture-initiating composition. The incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.
In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of stimulating or activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR, e.g. anti-CD3. In some embodiments, the stimulating conditions include one or more agent, e.g. ligand, which is capable of stimulating a costimulatory receptor, e.g., anti-CD28. In some embodiments, such agents and/or ligands may be, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agents include IL-2, IL-15 and/or IL-7. In some aspects, the IL-2 concentration is at least about 10 units/mL.
In some aspects, incubation is carried out in accordance with techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, the T cells are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.
In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.
In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigen-specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.
C. Engineered Cells, Vectors and Compositions for Multi-Targeting
Also provided are cells such as engineered cells that can bind to and/or target multiple antigens. In some embodiments, improved selectivity and specificity is achieved through strategies targeting multiple antigens. Such strategies generally involve multiple antigen-binding domains, which typically are present on distinct genetically engineered antigen receptors and specifically bind to distinct antigens. In some embodiments, the cells are engineered with the ability to bind more than one antigen. For example, in some embodiments, the cells are engineered to express multispecific binding molecules. In some embodiments, the cells express multiple binding molecules, e.g., recombinant receptors, each of which can target one antigen or multiple antigens, e.g., one receptor that targets BCMA, such as any described herein, and another receptor that targets another antigen, e.g., tumor antigen. In some aspects, a plurality of genetically engineered antigen receptors are introduced into the cell, which specifically bind to different antigens, each expressed in or on the disease or condition to be targeted with the cells or tissues or cells thereof. Such features can in some aspects address or reduce the likelihood of off-target effects or increase efficacy. For example, where a single antigen expressed in a disease or condition is also expressed on or in non-diseased or normal cells, such multi-targeting approaches can provide selectivity for desired cell types by requiring binding via multiple antigen receptors in order to activate the cell or induce a particular effector function. In some embodiments, a plurality of cells can be engineered to express one or more different binding molecules, e.g., recombinant receptors, each of which can target one antigen or multiple antigens.
Also provided are multispecific cells containing any of the binding molecules described herein, such as cells containing a cell surface protein including the anti-BCMA antibody and an additional cell surface protein, such as an additional chimeric receptor, which binds to a different antigen or a different epitope on BCMA. In some embodiments, provided are compositions of cells that express recombinant receptors, wherein one or more of the binding molecules, multispecific binding molecules and/or recombinant receptors bind and/or target BCMA. In some embodiments, the multispecific binding molecules and/or recombinant receptors target one or more different epitopes on BCMA.
In some embodiments, provided are composition of cells, wherein each type of cell expresses one or more binding molecules, e.g., recombinant receptors. In some embodiments, the cell comprises (e.g., has been transformed with) one or more vectors comprising one or more nucleic acid that encodes one or more an amino acid sequence comprising one or more antibodies and/or portions thereof, e.g., antigen-binding fragments thereof. In some embodiments, one or more such cells are provided. In some embodiments, a composition containing one or more such cells is provided. In some embodiments, the one or more cells can express different antibodies, or the same antibody. In some embodiments, each of the cells expresses one or more antibodies, such as more than one antibody. In some embodiments, each of the cells expresses a multispecific binding molecule, e.g., a multispecific receptor, e.g., CAR.
In some embodiments, the cells include multi-targeting strategies that target BCMA and a second or additional antigen associated with a particular disease or condition. In some embodiments, the second or additional antigen is targeted by a multispecific binding molecule and/or multiple binding molecules and/or a plurality of cells, e.g., one or more cells, each engineered to express one or more recombinant receptors. In some embodiments, a recombinant receptor targeting a second or additional antigen is expressed on the same cell as a BCMA binding molecule, or on a different cell.
In some embodiments, among the second or additional antigens for multi-targeting strategies includes those in which at least one of the antigens is a universal tumor antigen, or a family member thereof. In some embodiments, the second or additional antigen is an antigen expressed on a tumor. In some embodiments, the BCMA-binding molecules provided herein target an antigen on the same tumor type as the second or additional antigen. In some embodiments, the second or additional antigen may also be a universal tumor antigen or may be a tumor antigen specific to a tumor type. In some embodiments, the cell further comprises an additional genetically engineered antigen receptor that recognizes a second or additional antigen expressed on a disease or condition to be treated and induces a stimulatory or activating signal.
Exemplary antigens include CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC-1, Ia, HM1.24, HLA-DR, tenascin, an angiogenesis factor, VEGF, PIGF, ED-B fibronectin, an oncogene, an oncogene product, CD66a-d, necrosis antigens, Ii, IL-2, T101, TAC, IL-6, ROR1, TRAIL-R1 (DR4), TRAIL-R2 (DR5), B cell maturation antigen (BCMA), tEGFR, Her2, L1-CAM, mesothelin, CEA, hepatitis B surface antigen, anti-folate receptor, CD24, CD30, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, erbB dimers, EGFR vIII, FBP, FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, G protein-coupled receptor class C group 5 member D (GPRCSD), HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule (L1-CAM), Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentially expressed antigen of melanoma (PRAME), survivin, EGP2, EGP40, TAG72, B7-H6, IL-13 receptor a2 (IL-13Ra2), CA9, CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2D ligands, dual antigen, an antigen associated with a universal tag, a cancer-testes antigen, MUC1, MUC16, NY-ESO-1, MART-1, gp100, oncofetal antigen, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, CCL-1, hTERT, MDM2, CYP1B, WT1, livin, AFP, p53, cyclin (D1), CS-1, BCMA, BAFF-R, TACI, CD56, TIM-3, CD123, L1-cell adhesion molecule, MAGE-A1, MAGE A3, a cyclin, such as cyclin A1 (CCNA1) and/or a pathogen-specific antigen, biotinylated molecules, molecules expressed by HIV, HCV, HBV and/or other pathogens, and/or in some aspects, neoepitopes or neoantigens thereof. In some embodiments, the antigen is associated with or is a universal tag.
In some embodiments, the plurality of antigens, e.g., the first antigen, e.g., BCMA, and the second or additional antigens, are expressed on the cell, tissue, or disease or condition being targeted, such as on the cancer cell. In some aspects, the cell, tissue, disease or condition is multiple myeloma or a multiple myeloma cell. One or more of the plurality of antigens generally also is expressed on a cell which it is not desired to target with the cell therapy, such as a normal or non-diseased cell or tissue, and/or the engineered cells themselves. In such embodiments, by requiring ligation of multiple receptors to achieve a response of the cell, specificity and/or efficacy is achieved.
In some aspects, the antigen, e.g., the second or additional antigen, such as the disease-specific antigen and/or related antigen, is expressed on multiple myeloma, such as G protein-coupled receptor class C group 5 member D (GPRCSD), CD38 (cyclic ADP ribose hydrolase), CD138 (syndecan-1, syndecan, SYN-1), CS-1 (CS1, CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), BAFF-R, TACI and/or FcRH5. Other exemplary multiple myeloma antigens include CD56, TIM-3, CD33, CD123, CD44, CD20, CD40, CD74, CD200, EGFR, β2-Microglobulin, HM1.24, IGF-1R, IL-6R, TRAIL-R1, and the activin receptor type IIA (ActRIIA). See Benson and Byrd, J. Clin. Oncol. (2012) 30(16): 2013-15; Tao and Anderson, Bone Marrow Research (2011):924058; Chu et al., Leukemia (2013) 28(4):917-27; Garfall et al., Discov Med. (2014) 17(91):37-46. In some embodiments, the antigens include those present on lymphoid tumors, myeloma, AIDS-associated lymphoma, and/or post-transplant lymphoproliferations, such as CD38. Antibodies or antigen-binding fragments directed against such antigens are known and include, for example, those described in U.S. Pat. Nos. 8,153,765; 8,603,477, 8,008,450; U.S. Pub. No. US20120189622 or US20100260748; and/or International PCT Publication Nos. WO2006099875, WO2009080829 or WO2012092612 or WO2014210064. In some embodiments, such antibodies or antigen-binding fragments thereof (e.g. scFv) are contained in multispecific antibodies, multispecific chimeric receptors, such as multispecific CARs, and/or multispecific cells.
In some embodiments, the cells and methods include multi-targeting strategies, such as expression of two or more genetically engineered receptors on the cell, each recognizing a different antigen and typically each including a different intracellular signaling component. Such multi-targeting strategies are described, for example, in International Patent Application, Publication No.: WO 2014055668 A1 (describing combinations of a stimulatory or activating and costimulatory CARs, e.g., targeting two different antigens present individually on off-target, e.g., normal cells, but present together only on cells of the disease or condition to be treated) and Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013) (describing cells expressing a stimulatory or an activating and an inhibitory CAR, such as those in which the stimulatory or activating CAR binds to one antigen expressed on both normal or non-diseased cells and cells of the disease or condition to be treated, and the inhibitory CAR binds to another antigen expressed only on the normal cells or cells which it is not desired to treat).
In some embodiments, a plurality of cells, each engineered to express one or more recombinant receptors, are provided. For example, in some embodiments, one cell is engineered to express a binding molecule that binds and/or targets BCMA, and another cell is engineered to express a binding molecule that binds and/or targets an additional or second antigen. In some embodiments, the cells can each express a multispecific binding molecule, e.g., a multispecific recombinant receptor, where one or more of the target antigen is BCMA. In some of such embodiments, the plurality of cells can be administered together or separately. In some embodiments, the plurality of cells are administered simultaneously or concurrently with the cells, e.g., administered on the same day, and/or sequentially with or intermittently with, in any order, another engineered cell in the plurality. For example, in some embodiments, an engineered cell expressing a BCMA-binding molecule, e.g., CAR, is administered simultaneously with or sequentially with, in any order, another engineered cell expressing a binding molecule that binds a different target antigen or a different epitope on BCMA. In some embodiments, the plurality of cells can be in the same composition. Exemplary compositions of the cells include compositions described in Section II below.
IV. PHARMACEUTICAL COMPOSITIONS
Also provided are compositions including the BCMA-binding molecules, immunoconjugates, recombinant receptors, and engineered cells, including pharmaceutical compositions and formulations. Among such compositions are those that include engineered cells, such as a plurality of engineered cells, expressing the provided anti-BCMA recombinant receptors (e.g. CARs).
Provided are pharmaceutical formulations comprising a BCMA-binding recombinant chimeric antigen receptors or engineered cells expressing said receptors, a plurality of engineered cells expressing said receptors and/or additional agents for combination treatment or therapy. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier(s) or excipient(s). In some embodiments, the composition includes at least one additional therapeutic agent.
The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
In some aspects, the choice of carrier is determined in part by the particular cell, binding molecule, and/or antibody, and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).
Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
Formulations of the antibodies described herein can include lyophilized formulations and aqueous solutions.
The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the binding molecules or cells, preferably those with activities complementary to the binding molecule or cell, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the cells or antibodies are administered in the form of a salt, e.g., a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p-toluenesulphonic acid.
Active ingredients may be entrapped in microcapsules, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. In certain embodiments, the pharmaceutical composition is formulated as an inclusion complex, such as cyclodextrin inclusion complex, or as a liposome. Liposomes can serve to target the host cells (e.g., T-cells or NK cells) to a particular tissue. Many methods are available for preparing liposomes, such as those described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
The pharmaceutical composition in some aspects can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Many types of release delivery systems are available and known. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.
The pharmaceutical composition in some embodiments contains the binding molecules and/or cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
In certain embodiments, in the context of genetically engineered cells containing the binding molecules, e.g., CAR, a subject is administered the range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges, and/or such a number of cells per kilogram of body weight of the subject. In some aspects, in the context of genetically engineered cells expressing the binding molecules, e.g., CAR, a composition can contain at least the number of cells for administration for a dose of cell therapy, such as about or at least a number of cells described herein for administration, e.g., in Section V.A.
The may be administered using standard administration techniques, formulations, and/or devices. Provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. Administration of the cells can be autologous or heterologous. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, intracranial, intrathoracic, and intraperitoneal administration. In some embodiments, the cell populations are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
Sterile injectable solutions can be prepared by incorporating the binding molecule in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.
Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
Also provided are pharmaceutical compositions for combination therapy. Any of the additional agents for combination therapy described herein, such as agents described in Section III.B, can be prepared and administered as one or more pharmaceutical compositions, with the BCMA-binding molecule (e.g., antibody), immunoconjugate, recombinant receptor (e.g., chimeric antigen receptor) and/or engineered cells expressing said molecules (e.g., recombinant receptor) described herein. The combination therapy can be administered in one or more pharmaceutical compositions, e.g., where the binding molecules, recombinant receptors and/or cells are in the same pharmaceutical composition as the additional agent, or in separate pharmaceutical compositions. For example, in some embodiments, the additional agent is an additional engineered cell, e.g., cell engineered to express a different recombinant receptor, and is administered in the same composition or in a separate composition. In some embodiments, each of the pharmaceutical composition is formulated in a suitable formulation according to the particular binding molecule, recombinant receptor, cell, e.g., engineered cell, and/or additional agent, and the particular dosage regimen and/or method of delivery.
V. METHODS AND USES
Also provided methods of using and uses of the BCMA-binding molecules, immunoconjugates, recombinant receptors, engineered cells, and pharmaceutical compositions and formulations thereof, such as in the treatment of diseases, conditions, and disorders in which BCMA is expressed, and/or detection, diagnostic, and prognostic methods. Among such methods, such as methods of treatment, and uses are those that involve administering to a subject engineered cells, such as a plurality of engineered cells, expressing the provided anti-BCMA recombinant receptors (e.g. CARs). Also provided are methods of combination therapy and/or treatment.
A. Therapeutic and Prophylactic Methods and Uses
Also provided are methods of administering and uses, such as therapeutic and prophylactic uses, of the BCMA-binding molecules, including the anti-BCMA recombinant receptors (e.g., CARs), engineered cells expressing the recombinant receptors (e.g., CARs), plurality of engineered cells expressing the receptors, and/or compositions comprising the same. Such methods and uses include therapeutic methods and uses, for example, involving administration of the molecules (e.g., recombinant receptors), cells (e.g., engineered cells), or compositions containing the same, to a subject having a disease, condition, or disorder associated with BCMA such as a disease, condition, or disorder associated with BCMA expression, and/or in which cells or tissues express, e.g., specifically express, BCMA. In some embodiments, the molecule, cell, and/or composition is/are administered in an effective amount to effect treatment of the disease or disorder. Provided herein are uses of the recombinant receptors (e.g., CARs), and cells (e.g., engineered cells) in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods are carried out by administering the binding molecules or cells, or compositions comprising the same, to the subject having, having had, or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject. Also provided herein are of use of any of the compositions, such as pharmaceutical compositions provided herein, for the treatment of a disease or disorder associated with BCMA, such as use in a treatment regimen.
As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.
As used herein, “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or subject being treated. As sufficient or significant delay can, in effect, encompass prevention, in that the subject does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.
“Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided molecules and compositions are used to delay development of a disease or to slow the progression of a disease.
As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, an antibody or composition or cell which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody or composition or cell.
An “effective amount” of an agent, e.g., a pharmaceutical formulation, binding molecule, antibody, cells, or composition, in the context of administration, refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.
A “therapeutically effective amount” of an agent, e.g., a pharmaceutical formulation, binding molecule, antibody, cells, or composition refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered. In some embodiments, the provided methods involve administering the molecules, antibodies, cells, and/or compositions at effective amounts, e.g., therapeutically effective amounts.
A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
As used herein, a “subject” or an “individual” is a mammal. In some embodiments, a “mammal” includes humans, non-human primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, monkeys, etc. In some embodiments, the subject is human.
Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Pat. App. Pub. No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.
Among the diseases to be treated is any disease or disorder associated with BCMA or any disease or disorder in which BCMA is specifically expressed and/or in which BCMA has been targeted for treatment (also referred to herein interchangeably as a “BCMA-associated disease or disorder”). Cancers associated with BCMA expression include hematologic malignancies such as multiple myeloma, Waldenstrom macroglobulinemia, as well as both Hodgkin's and non-Hodgkin's lymphomas. See Coquery et al., Crit Rev Immunol., 2012, 32(4):287-305 for a review of BCMA. Since BCMA has been implicated in mediating tumor cell survival, it is a potential target for cancer therapy. Chimeric antigen receptors containing mouse anti-human BCMA antibodies and cells expressing such chimeric receptors have been previously described. See Carpenter et al., Clin Cancer Res., 2013, 19(8):2048-2060.
In some embodiments, the disease or disorder associated with BCMA is a B cell-related disorder. In some embodiments, the disease or disorder associated with BCMA is one or more diseases or conditions from among glioblastoma, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, an immunoregulatory disorder, heavy-chain disease, primary or immunocyte-associated amyloidosis, or monoclonal gammopathy of undetermined significance.
In some embodiments, the disease or disorder associated with BCMA is an autoimmune disease or disorder. Such autoimmune diseases or disorder include, but are not limited to, systemic lupus erythematosus (SLE), lupus nephritis, inflammatory bowel disease, rheumatoid arthritis (e.g., juvenile rheumatoid arthritis), ANCA associated vasculitis, idiopathic thrombocytopenia purpura (ITP), thrombotic thrombocytopenia purpura (TTP), autoimmune thrombocytopenia, Chagas' disease, Grave's disease, Wegener's granulomatosis, polyarteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, vasculitis, diabetes mellitus, Reynaud's syndrome, anti-phospholipid syndrome, Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia, myasthenia gravis, or progressive glomerulonephritis.
In certain diseases and conditions, BCMA is expressed on malignant cells and cancers. In some embodiments, the cancer (e.g., a BCMA-expressing cancer) is a B cell malignancy. In some embodiments, the cancer (e.g., a BCMA-expressing cancer) is a lymphoma, a leukemia, or a plasma cell malignancy. Lymphomas contemplated herein include, but are not limited to, Burkitt lymphoma (e.g., endemic Burkitt's lymphoma or sporadic Burkitt's lymphoma), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, Waldenstrom macroglobulinemia, follicular lymphoma, small non-cleaved cell lymphoma, mucosa-associated lymphatic tissue lymphoma (MALT), marginal zone lymphoma, splenic lymphoma, nodal monocytoid B cell lymphoma, immunoblastic lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, pulmonary B cell angiocentric lymphoma, small lymphocytic lymphoma, primary mediastinal B cell lymphoma, lymphoplasmacytic lymphoma (LPL), or mantle cell lymphoma (MCL). Leukemias contemplated here, include, but are not limited to, chronic lymphocytic leukemia (CLL), plasma cell leukemia or acute lymphocytic leukemia (ALL). Also contemplated herein are plasma cell malignancies including, but not limited to, multiple myeloma (e.g., non-secretory multiple myeloma, smoldering multiple myeloma) or plasmacytoma. In some embodiments the disease or condition is multiple myeloma (MM), such as relapsed and/or refractory multiple myeloma (R/R MM). Among the diseases, disorders or conditions associated with BCMA (e.g., a BCMA-expressing cancer) that can be treated include, but are not limited to, neuroblastoma, renal cell carcinoma, colon cancer, colorectal cancer, breast cancer, epithelial squamous cell cancer, melanoma, myeloma (e.g., multiple myeloma), stomach cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, uterine cancer, adrenal cancer and head and neck cancer.
In some embodiments, the methods may identify a subject who has, is suspected to have, or is at risk for developing a BCMA-associated disease or disorder. Hence, provided are methods for identifying subjects with diseases or disorders associated with elevated BCMA expression and selecting them for treatment with a provided BCMA-binding e recombinant receptors (e.g., CARs), and/or engineered cells expressing the recombinant receptors.
For example, a subject may be screened for the presence of a disease or disorder associated with elevated BCMA expression, such as a BCMA-expressing cancer. In some embodiments, the methods include screening for or detecting the presence of a BCMA-associated disease, e.g. a tumor. Thus, in some aspects, a sample may be obtained from a patient suspected of having a disease or disorder associated with elevated BCMA expression and assayed for the expression level of BCMA. In some aspects, a subject who tests positive for a BCMA-associated disease or disorder may be selected for treatment by the present methods, and may be administered a therapeutically effective amount of a recombinant receptor (e.g., CAR) comprising a BCMA-binding molecule, cells containing a recombinant receptor or a pharmaceutical composition thereof as described herein.
In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another BCMA-specific antibody and/or cells expressing a BCMA-targeting chimeric receptor and/or other therapy, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT or autologous HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another BCMA-targeted therapy. In some embodiments, the subject has not relapsed but is determined to be at risk for relapse, such as at a high risk of relapse, and thus the compound or composition is administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.
In some embodiments, the subject is one that is eligible for a transplant, such as is eligible for a hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT or autologous HSCT. In some such embodiments, the subject has not previously received a transplant, despite being eligible, prior to administration of the BCMA-binding molecules, including the anti-BCMA recombinant receptors (e.g., CARs), engineered cells expressing the recombinant receptors (e.g., CARs), plurality of engineered cells expressing the receptors, and/or compositions comprising the same, as provided herein.
In some embodiments, the subject is one that is not eligible for a transplant, such as is not eligible for a hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT or autologous HSCT. In some such embodiments, such a subject is administered the BCMA-binding molecules, including the anti-BCMA recombinant receptors (e.g., CARs), engineered cells expressing the recombinant receptors (e.g., CARs), plurality of engineered cells expressing the receptors, and/or compositions comprising the same, according to the provided embodiments herein.
In some embodiments, prior to the initiation of administration of the engineered cells, the subject has received one or more prior therapies. In some embodiments, the subject has received at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more prior therapies. In some embodiments, the subject has received at least 3, 4, 5, 6, 7, 8, 9, 10 or more prior therapies.
In some aspects, the subject has relapsed or has been refractory to the one or more prior therapies. In some aspects, the prior therapies include treatment with autologous stem cell transplant (ASCT); an immunomodulatory agent; a proteasome inhibitor; and an anti-CD38 antibody; unless the subject was not a candidate for or was contraindicated for one or more of the therapies. In some embodiments, the immunomodulatory agent is selected from among thalidomide, lenalidomide or pomalidomide. In some embodiments, the proteasome inhibitor is selected from among bortezomib, carfilzomib or ixazomib. In some embodiments, the anti-CD38 antibody is or comprises daratumumab. In some embodiments, the subject must have undergone at least 2 consecutive cycles of treatment for each regimen unless progressive disease was the best response to the regimen.
In some embodiments, the method can involve including or excluding particular subjects for therapy with the provided anti-BCMA antibodies, recombinant receptors and/or cells comprising such receptors, based on particular criteria, diagnosis or indication. In some embodiments, at the time of administration of the dose of cells or pre-treatment lymphodepleting chemotherapy, the subject has not had active or history of plasma cell leukemia (PCL). In some embodiments, if the subject had active or a history of PCL at the time of administration, the subject can be excluded from being treated according to the provided methods. In some embodiments, if the subject develops a PCL, such as secondary PCL, at the time of administration, the subject can be excluded from being treated according to the provided methods. In some embodiments, the assessment for the criteria, diagnosis or indication can be performed at the time of screening the subjects for eligibility or suitability of treatment according to the provided methods, at various steps of the treatment regimen, at the time of receiving lymphodepleting therapy, and/or at or immediately prior to the initiation of administration of the engineered cells or composition thereof.
In some embodiments, the treatment does not induce an immune response by the subject to the therapy, and/or does not induce such a response to a degree that prevents effective treatment of the disease or condition. In some aspects, the degree of immunogenicity and/or graft versus host response is less than that observed with a different but comparable treatment. For example, in the case of adoptive cell therapy using cells expressing CARs including the provided anti-BCMA antibodies, the degree of immunogenicity in some embodiments is reduced compared to CARs including a different antibody that binds to a similar, e.g., overlapping epitope and/or that competes for binding to BCMA with the antibody, such as a mouse or monkey or rabbit or humanized antibody.
In some embodiments, the methods include adoptive cell therapy, whereby genetically engineered cells expressing the provided recombinant receptors comprising a BCMA-binding molecule (e.g., CARs comprising anti-BCMA antibody or antigen-binding fragment thereof) are administered to subjects. Such administration can promote activation of the cells (e.g., T cell activation) in a BCMA-targeted manner, such that the cells of the disease or disorder are targeted for destruction.
Thus, the provided methods and uses include methods and uses for adoptive cell therapy. In some embodiments, the methods include administration of the cells or a composition containing the cells to a subject, tissue, or cell, such as one having, at risk for, or suspected of having the disease, condition or disorder. In some embodiments, the cells, populations, and compositions are administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells or compositions are administered to the subject, such as a subject having or at risk for the disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptom of the disease or condition, such as by lessening tumor burden in a BCMA-expressing cancer.
Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.
In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.
In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.
In some embodiments, the subject, to whom the cells, cell populations, or compositions are administered, is a primate, such as a human. In some embodiments, the subject, to whom the cells, cell populations, or compositions are administered, is a non-human primate. In some embodiments, the non-human primate is a monkey (e.g., cynomolgus monkey) or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent (e.g., mouse, rat, etc.). In some examples, the patient or subject is a validated animal model for disease, adoptive cell therapy, and/or for assessing toxic outcomes such as cytokine release syndrome (CRS).
The BCMA-binding molecules such as recombinant receptors (e.g., CARs) and cells expressing the same, can be administered by any suitable means, for example, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjunctival injection, subconjunctival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intracranial, intrathoracic, or subcutaneous administration. Dosing and administration may depend in part on whether the administration is brief or chronic. Various dosing schedules include but are not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion.
For the prevention or treatment of disease, the appropriate dosage of the binding molecule, recombinant receptor or cell may depend on the type of disease to be treated, the type of binding molecule or recombinant receptor, the severity and course of the disease, whether the binding molecule or recombinant receptor is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the recombinant receptor or cell, and the discretion of the attending physician. The compositions and molecules and cells are in some embodiments suitably administered to the patient at one time or over a series of treatments.
In some embodiments, the dose and/or frequency of administration is determined based on efficacy and/or response. In some embodiments, efficacy is determined by evaluating disease status. Exemplary methods for assessing disease status include: measurement of M protein in biological fluids, such as blood and/or urine, by electrophoresis and immunofixation; quantification of sFLC (κ and λ) in blood; skeletal survey; and imaging by positron emission tomography (PET)/computed tomography (CT) in subjects with extramedullary disease. In some embodiments, disease status can be evaluated by bone marrow examination. In some examples, dose and/or frequency of administration is determined by the expansion and persistence of the recombinant receptor or cell in the blood and/or bone marrow. In some embodiments, dose and/or frequency of administration is determined based on the antitumor activity of the recombinant receptor or engineered cell. In some embodiments antitumor activity is determined by the overall response rate (ORR) and/or International Myeloma Working Group (IMWG) Uniform Response Criteria (see Kumar et al. (2016) Lancet Oncol 17(8):e328-346). In some embodiments, response is evaluated using minimal residual disease (MRD) assessment. In some embodiments, MRD can be assessed by methods such as flow cytometry and high-throughput sequencing, e.g., deep sequencing. In some embodiments, response is evaluated based on the duration of response following administration of the recombinant receptor or cells. In some examples, dose and/or frequency of administration can be based on toxicity. In some embodiments, dose and/or frequency can be determined based on health-related quality of life (HRQoL) of the subject to which the recombinant receptor and/or cells is/are administered. In some embodiments, dose and/or frequency of administration can be changed, i.e., increased or decreased, based on any of the above criteria.
In some embodiments, the disease or disorder to be treated is multiple myeloma. In some embodiments, measurable disease criteria for multiple myeloma can include (1) serum M-protein 1 g/dL or greater; (2) Urine M-protein 200 mg or greater/24 hour; (3) involved serum free light chain (sFLC) level 10 mg/dL or greater, with abnormal κ to λ, ratio. In some cases, light chain disease is acceptable only for subjects without measurable disease in the serum or urine.
In some embodiments, the Eastern Cooperative Oncology Group (ECOG) performance status indicator can be used to assess or select subjects for treatment, e.g., subjects who have had poor performance from prior therapies (see, e.g., Oken et al. (1982) Am J Clin Oncol. 5:649-655). The ECOG Scale of Performance Status describes a patient's level of functioning in terms of their ability to care for themselves, daily activity, and physical ability (e.g., walking, working, etc.). In some embodiments, an ECOG performance status of 0 indicates that a subject can perform normal activity. In some aspects, subjects with an ECOG performance status of 1 exhibit some restriction in physical activity but the subject is fully ambulatory. In some aspects, patients with an ECOG performance status of 2 is more than 50% ambulatory. In some cases, the subject with an ECOG performance status of 2 may also be capable of selfcare; see e.g., Sørensen et al., (1993) Br J Cancer 67(4) 773-775. In some embodiments, the subject that are to be administered according to the methods or treatment regimen provided herein include those with an ECOG performance status of 0 or 1.
In some embodiments, the administration can treat the subject despite the subject having become resistant to another therapy. In some embodiments, when administered to subjects according to the embodiments described herein, the dose or the composition is capable of achieving objective response (OR), in at least 50%, 60%, 70%, 80%, 90%, or 95% of subjects that were administered. In some embodiments, OR includes subjects who achieve stringent complete response (sCR), complete response (CR), very good partial response (VGPR), partial response (PR) and minimal response (MR). In some embodiments, when administered to subjects according to the embodiments described herein, the dose or the composition is capable of achieving stringent complete response (sCR), complete response (CR), very good partial response (VGPR) or partial response (PR), in at least 50%, 60%, 70%, 80%, or 85% of subjects that were administered. In some embodiments, when administered to subjects according to the embodiments described herein, the dose or the composition is capable of achieving stringent complete response (sCR) or complete response (CR) at least 20%, 30%, 40% 50%, 60% or 70% of subjects that were administered. In some embodiments, exemplary doses include about 5.0×107, 1.5×108, 3.0×108 or 4.5×108 CAR-expressing T cells. In some aspects, particular response to the treatment, e.g., according to the methods provided herein, can be assessed based on the International Myeloma Working Group (IMWG) Uniform Response Criteria (see Kumar et al. (2016) Lancet Oncol 17(8):e328-346). In some embodiments, exemplary doses to achieve particular outcomes, such as OR, includes about 5.0×107 CAR-expressing T cells.
In some embodiments, toxicity and/or side-effects of treatment can be monitored and used to adjust dose and/or frequency of administration of the recombinant receptor, e.g., CAR, cells, and or compositions. For example, adverse events and laboratory abnormalities can be monitored and used to adjust dose and/or frequency of administration. Adverse events include infusion reactions, cytokine release syndrome (CRS), neurotoxicity, macrophage activation syndrome, and tumor lysis syndrome (TLS). Any of such events can establish dose-limiting toxicities and warrant decrease in dose and/or a termination of treatment. Other side effects or adverse events which can be used as a guideline for establishing dose and/or frequency of administration include non-hematologic adverse events, which include but are not limited to fatigue, fever or febrile neutropenia, increase in transaminases for a set duration (e.g., less than or equal to 2 weeks or less than or equal to 7 days), headache, bone pain, hypotension, hypoxia, chills, diarrhea, nausea/vomiting, neurotoxicity (e.g., confusion, aphasia, seizures, convulsions, lethargy, and/or altered mental status), disseminated intravascular coagulation, other asymptomatic non-hematological clinical laboratory abnormalities, such as electrolyte abnormalities. Other side effects or adverse events which can be used as a guideline for establishing dose and/or frequency of administration include hematologic adverse events, which include but are not limited to neutropenia, leukopenia, thrombocytopenia, animal, and/or B-cell aplasia and hypogammaglobinemia.
In some embodiments, treatment according to the provided methods can result in a lower rate and/or lower degree of toxicity, toxic outcome or symptom, toxicity-promoting profile, factor, or property, such as a symptom or outcome associated with or indicative of cytokine release syndrome (CRS) or neurotoxicity, such as severe CRS or severe neurotoxicity, for example, compared to administration of other therapies.
In certain embodiments, in the context of genetically engineered cells containing the binding molecules or recombinant receptors, a subject is administered the range of about one million to about 100 billion cells and/or that amount of cells per kilogram of body weight, such as, e.g., about 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 25 million cells, about 30 million cells, about 40 million cells, about 50 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 150 million cells, about 250 million cells, about 300 million cells, about 350 million cells, about 450 million cells, about 500 million cells, about 600 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 1 billion cells, about 1.2 billion cells, about 3 billion cells, about 30 billion cells, about 45 billion cells, or about 50 billion cells) or any value in between these ranges and/or per kilogram of body weight. Again, dosages may vary depending on attributes particular to the disease or disorder and/or patient and/or other treatments.
In some embodiments, the methods comprises administering a dose of the engineered cells or a composition comprising a dose of the engineered cells. In some embodiments, the engineered cells or compositions containing engineered cells can be used in a treatment regimen, wherein the treatment regimen comprises administering a dose of the engineered cells or a composition comprising a dose of the engineered cells. In some embodiments, the dose can contain, for example, a particular number or range of recombinant receptor-expressing T cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), such as any number of such cells described herein. In some embodiments, a composition containing a dose of the cells can be administered. In some aspects, the number, amount or proportion of CAR-expressing cells in a cell population or a cell composition can be assessed by detection of a surrogate marker, e.g., by flow cytometry or other means, or by detecting binding of a labelled molecule, such as a labelled antigen, that can specifically bind to the binding molecules or receptors provided herein.
In some embodiments, for example, where the subject is a human, the dose includes more than about 1×106 total recombinant receptor (e.g., CAR)-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs) and fewer than about 2×109 total recombinant receptor (e.g., CAR)-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), e.g., in the range of about 2.5×107 to about 1.2×109 such cells, such as 2.5×107, 5×107, 1.5×108, 3×108, 4.5×108, 8×108, or 1.2×109 total such cells, or the range between any two of the foregoing values.
In some embodiments, the dose of genetically engineered cells comprises between at or about 2.5×107 CAR-expressing T cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), and at or about 1.2×109 CAR-expressing T cells, total T cells, or total PBMCs, between at or about 5.0×107 CAR-expressing T cells and at or about 4.5×108 CAR-expressing T cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), between at or about 1.5×108 CAR-expressing T cells and at or about 3.0×108 CAR-expressing T cells, total T cells, or total PBMCs, each inclusive. In some embodiments, the number is with reference to the total number of CD3+ or CD8+, in some cases also CAR-expressing (e.g. CAR+) cells. In some embodiments, the dose comprises a number of cell from or from about 2.5×107 to or to about 1.2×109 CD3+ or CD8+ total T cells or CD3+ or CD8+ CAR-expressing cells, from or from about 5.0×107 to or to about 4.5×108 CD3+ or CD8+ total T cells or CD3+ or CD8+ CAR-expressing cells, or from or from about 1.5×108 to or to about 3.0×108 CD3+ or CD8+ total T cells or CD3+ or CD8+ CAR-expressing cells, each inclusive.
In some embodiments, the T cells of the dose include CD4+ T cells, CD8+ T cells or CD4+ and CD8+ T cells.
In some embodiments, for example, where the subject is human, the CD8+ T cells of the dose, including in a dose including CD4+ and CD8+ T cells, includes between at or about 1×106 and at or about 2×109 total recombinant receptor (e.g., CAR)-expressing CD8+ cells, e.g., in the range of at or about 5×107 to at or about 4.5×108 such cells, such as at or about 2.5×107, at or about 5×107, at or about 1.5×108, at or about 3×108, at or about 4.5×108, at or about 8×108, or at or about 1.2×109 total such cells, or the range between any two of the foregoing values.
In some embodiments, the dose of cells, e.g., recombinant receptor-expressing T cells, is administered to the subject as a single dose or is administered only one time within a period of two weeks, one month, three months, six months, 1 year or more. In some embodiments, the patient is administered multiple doses, and each of the doses or the total dose can be within any of the foregoing values. In some embodiments, the engineered cells for administration or composition of engineered cells for administration, exhibits properties indicative of or consistent with cell health. In some embodiments, at or about or at least at or about 70, 75, 80, 85, or 90% CAR+ cells of such dose exhibit one or more properties or phenotypes indicative of cell health or biologically active CAR cell, such as absence expression of an apoptotic marker.
In particular embodiments, the phenotype is or includes an absence of apoptosis and/or an indication the cell is undergoing the apoptotic process. Apoptosis is a process of programmed cell death that includes a series of stereotyped morphological and biochemical events that lead to characteristic cell changes and death, including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and global mRNA decay. In some aspects, early stages of apoptosis can be indicated by activation of certain caspases, e.g., 2, 8, 9, and 10. In some aspects, middle to late stages of apoptosis are characterized by further loss of membrane integrity, chromatin condensation and DNA fragmentation, include biochemical events such as activation of caspases 3, 6, and 7.
In particular embodiments, the phenotype is negative expression of one or more factors associated with programmed cell death, for example pro-apoptotic factors known to initiate apoptosis, e.g., members of the death receptor pathway, activated members of the mitochondrial (intrinsic) pathway, such as Bcl-2 family members, e.g., Bax, Bad, and Bid, and caspases. In certain embodiments, the phenotype is the absence of an indicator, e.g., an Annexin V molecule or by TUNEL staining, that will preferentially bind to cells undergoing apoptosis when incubated with or contacted to a cell composition. In some embodiments, the phenotype is or includes the expression of one or more markers that are indicative of an apoptotic state in the cell. In some embodiments, the phenotype is lack of expression and/or activation of a caspase, such as caspase 3. In some aspects, activation of caspase-3 is indicative of an increase or revival of apoptosis. In certain embodiments, caspase activation can be detected by known methods. In some embodiments, an antibody that binds specifically to an activated caspase (i.e., binds specifically to the cleaved polypeptide) can be used to detect caspase activation. In particular embodiments, the phenotype is or includes active caspase 3-. In some embodiments, the marker of apoptosis is a reagent that detects a feature in a cell that is associated with apoptosis. In certain embodiments, the reagent is an annexin V molecule.
In some embodiments, the compositions containing the engineered cells for administration contain a certain number or amount of cells that exhibit phenotypes indicative of or consistent with cell health. In some of any embodiments, less than about 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the CAR-expressing T cells in the dose of engineered T cells express a marker of apoptosis, optionally Annexin V or active Caspase 3. In some of any embodiments, less than 5%, 4%, 3%, 2% or 1% of the CAR-expressing T cells in the dose of engineered T cells express Annexin V or active Caspase 3.
In some embodiments the cells administered are immune cells engineered to express the BCMA-binding recombinant receptor, e.g., CAR. In some embodiments the immune cells are T cells. In some embodiments, the administered cells are CD4+ T cells. In some embodiments the administered cells are CD8+ T cells. In some embodiments, the administered cells are a combination of CD4+ and CD8+ T cells, such as a combination of CD4+ CAR T cells and CD8+ CAR T cells, which in some aspects are within the same vessel or cell composition or suspension. In some examples the ratio of CD4+ cells to CD8+ cells (CD4:CD8) administered, such as ratio within the suspension or composition or vessel, is 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1. In some embodiments, the ratio is between 1:3 and 3:1 or is between at or about 1:4 to at or about 4:1, or between at or about 1:3 to at or about 3:1, or between at or about 1:2 to at or about 2:1, or any of such ratios, within a tolerated error rate. In some aspects, among subjects receiving the therapy and/or among subjects from whom samples are taken and processed to produce the cell compositions, the ratio of CD4+ CAR-T cells to CD8+ CAR-T cells or ratio of CD4+ to CD8+ cells is within a desired range, such as between at or about 1:4 to at or about 4:1, or between at or about 1:3 to at or about 3:1, or between at or about 1:2 to at or about 2:1, or is within such desired ratio for a given percentage of such subjects, such as for at least 65%, at least 70%, at least 75% or at least 80% or at least 85% or at least 90% or at least 95%, of such subjects.
In some embodiments, the cells, binding molecules, or recombinant receptors are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as another antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.
The cells, binding molecules and/or recombinant receptors in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells, binding molecules and/or recombinant receptors are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells, binding molecules and/or recombinant receptors are administered after to the one or more additional therapeutic agents.
In some embodiments, the subject may receive a bridging therapy after leukapheresis and before lymphodepleting chemotherapy. A treating physician can determine if bridging therapy is necessary, for example for disease control, during manufacturing of the provided composition or cells. In some embodiments, bridging therapies do not include biological agents, such as antibodies (e.g., Daratumumab). In some embodiments, bridging therapies are discontinued prior to initiation of lymphodepletion. In some embodiments, bridging therapies are discontinued 1 day, 2 days 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, 21 days, 28 days, 45 days, or 60 days before lymphodepletion.
Once the cells are administered to a mammal (e.g., a human), the biological activity of the engineered cell populations and/or antibodies in some aspects is measured by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells also can be measured by assaying expression and/or secretion of certain cytokines, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
In certain embodiments, engineered cells are modified in any number of ways, such that their therapeutic or prophylactic efficacy is increased. For example, the engineered CAR or TCR expressed by the population in some embodiments are conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating compounds, e.g., the CAR or TCR, to targeting moieties is known in the art. See, for instance, Wadwa et al., J. Drug Targeting, 3(2):111 (1995), and U.S. Pat. No. 5,087,616.
B. Combination Therapy
Also provided are methods of combination therapy that includes administering and uses, such as therapeutic and prophylactic uses, of the BCMA-binding recombinant receptors (e.g., CARs), engineered cells expressing the recombinant receptors (e.g., CARs), plurality of engineered cells expressing the receptors, and/or compositions comprising the same.
In some embodiments, the BCMA-binding recombinant receptor (e.g., chimeric antigen receptor) and/or engineered cells expressing said molecules (e.g., recombinant receptor) described herein are administered as part of a combination treatment or combination therapy, such as simultaneously with, sequentially with or intermittently with, in any order, one or more additional therapeutic intervention. In some embodiments, the one or more additional therapeutic intervention includes, for example, an antibody, an engineered cell, a receptor and/or an agent, such as a cell expressing a recombinant receptor, and/or cytotoxic or therapeutic agent, e.g., a chemotherapeutic agent. In some embodiments, the combination therapy includes administration of one or more additional agents, therapies and/or treatments, e.g., any of the additional agents, therapy and/or treatments described herein. In some embodiments, the combination therapy includes administration of one or more additional agents for treatment or therapy, such as an immunomodulatory agent, immune checkpoint inhibitor, adenosine pathway or adenosine receptor antagonist or agonist and kinase inhibitors. In some embodiments, the combination treatment or combination therapy includes an additional treatment, such as a surgical treatment, transplant, and/or radiation therapy. Also provided are methods of combination treatment or combination therapy that includes BCMA-binding recombinant receptors (e.g., CARs), cells and/or compositions described herein and one or more additional therapeutic interventions.
In some embodiments, the additional agent for combination treatment or combination therapy enhances, boosts and/or promotes the efficacy and/or safety of the therapeutic effect of binding molecules, recombinant receptors, cells and/or compositions. In some embodiments, the additional agent enhances or improves the efficacy, survival or persistence of the administered cells, e.g., cells expressing the binding molecule or a recombinant receptor. In some embodiments, the additional agent is selected from among a protein phosphatase inhibitor, a kinase inhibitor, a cytokine, an immunomodulator, or an agent that decreases the level or activity of a regulatory T (Treg) cell. In some embodiments, the additional agent enhances safety, by virtue of reducing or ameliorating adverse effects of the administered binding molecules, recombinant receptors, cells and/or compositions. In some embodiments, the additional agent can treat the same disease, condition or a comorbidity. In some embodiments, the additional agent can ameliorate, reduce or eliminate one or more toxicities, adverse effects or side effects that are associated with administration of the recombinant receptors, cells and/or compositions, e.g., CAR-expressing cells.
In some embodiments, pain management medication such as acetaminophen, or antihistamine, such as diphenhydramine can be administered prior to, during or after administration of the recombinant receptor, cell or composition provided herein, to ameliorate or reduce or eliminate minor side effects associated with treatment. In some examples, red blood cell and platelet transfusions, and/or colony-stimulating factors can be administered reduce or eliminate one or more toxicities, adverse effects or side effects that are associated with administration of the recombinant receptors, cells and/or compositions, e.g., CAR-expressing cells. In some embodiments, prophylactic or empiric anti-infective agents (e.g., trimethoprim/sulfamethoxazole for pneumocystis pneumonia [PCP] prophylaxis, broad spectrum antibiotics, antifungals, or antiviral agents for febrile neutropenia) can be administered to treat side-effects resulting from treatment. In some examples, when necessary, prophylaxis may be provided to treat lymphopenia and/or neutropenia occurring as a result of treatment.
In some embodiments, the additional therapy, treatment or agent includes chemotherapy, radiation therapy, surgery, transplantation, adoptive cell therapy, antibodies, cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitory agents, anti-hormonal agents, kinase inhibitors, anti-angiogenic agents, cardioprotectants, immunostimulatory agents, immunosuppressive agents, immune checkpoint inhibitors, antibiotics, angiogenesis inhibitors, metabolic modulators or other therapeutic agents or any combination thereof. In some embodiments, the additional agent is a protein, a peptide, a nucleic acid, a small molecule agent, a cell, a toxin, a lipid, a carbohydrate or combinations thereof, or any other type of therapeutic agent, e.g. radiation. In some embodiments, the additional therapy, agent or treatment includes surgery, chemotherapy, radiation therapy, transplantation, administration of cells expressing a recombinant receptor, e.g., CAR, kinase inhibitor, immune checkpoint inhibitor, mTOR pathway inhibitor, immunosuppressive agents, immunomodulators, antibodies, immunoablative agents, antibodies and/or antigen binding fragments thereof, antibody conjugates, other antibody therapies, cytotoxins, steroids, cytokines, peptide vaccines, hormone therapy, antimetabolites, metabolic modulators, drugs that inhibit either the calcium dependent phosphatase calcineurin or the p70S6 kinase FK506) or inhibit the p70S6 kinase, alkylating agents, anthracyclines, vinca alkaloids, proteasome inhibitors, GITR agonists, protein tyrosine phosphatase inhibitors, protein kinase inhibitors, an oncolytic virus, and/or other types of immunotherapy. In some embodiments, the additional agent or treatment is bone marrow transplantation, T cell ablative therapy using chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, and/or antibody therapy.
In some embodiments, the cells, BCMA-binding recombinant receptors and/or compositions, e.g., CAR-expressing cells, are administered in combination with other engineered cells, e.g., other CAR-expressing cells. In some embodiments, the additional agent is a kinase inhibitor, e.g., an inhibitor of Bruton's tyrosine kinase (Btk), e.g., ibrutinib. In some embodiments, the additional agent is an adenosine pathway or adenosine receptor antagonist or agonist. In some embodiments, the additional agent is an immunomodulator such as thalidomide or a thalidomide derivative (e.g., lenalidomide). In some embodiments, the additional agent is a gamma secretase inhibitor, such as a gamma secretase inhibitor that inhibits or reduces intramembrane cleavage of a target of a gamma secretase, e.g. BCMA, on a cell (such as a tumor/cancer cell). In some embodiments, the additional therapy, agent or treatment is a cytotoxic or chemotherapy agent, a biologic therapy (e.g., antibody, e.g., monoclonal antibody, or cellular therapy), or an inhibitor (e.g., kinase inhibitor).
In some embodiments, the additional agent is a chemotherapeutic agent. Exemplary chemotherapeutic agents include an anthracycline (e.g., doxorubicin, such as liposomal doxorubicin); a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine); an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide); an immune cell antibody (e.g., alemtuzumab, gemtuzumab, rituximab, tositumomab); an antimetabolite (including, e.g., folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors such as fludarabine); a TNFR glucocorticoid induced TNFR related protein (GITR) agonist; a proteasome inhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib); an immunomodulatory such as thalidomide or a thalidomide derivative (e.g., lenalidomide).
In some embodiments, the additional therapy or treatment is cell therapy, e.g., adoptive cell therapy. In some embodiments, the additional therapy includes administration of engineered cells, e.g., additional CAR-expressing cell. In some embodiments, the additional engineered cell is a CAR-expressing cell that expresses the same or different recombinant receptor as the engineered cells provided herein, e.g., anti-BCMA CAR-expressing cells. In some embodiments, the recombinant receptor, e.g., CAR, expressed on the additional engineered cell, recognizes a different antigen and/or epitope. In some embodiments, the recombinant receptor, e.g., CAR, expressed on the additional engineered cell, recognizes a different epitope of the same antigen as the recombinant receptors described herein, e.g., BCMA. In some embodiments, the recombinant receptor, e.g., CAR, expressed on the additional engineered cell, recognizes a different antigen, e.g., a different tumor antigen or combination of antigens. For example, in some embodiments, the recombinant receptor, e.g., CAR, expressed on the additional engineered cell, targets cancer cells that express early lineage markers, e.g., cancer stem cells, while other CAR-expressing cells target cancer cells that express later lineage markers. In such embodiments, the additional engineered cell is administered prior to, concurrently with, or after administration (e.g., infusion) of the CAR-expressing cells described herein. In some embodiments, the additional engineered cell expresses allogeneic CAR.
In some embodiments, the configurations of one or more of the CAR molecules comprise a primary intracellular signaling domain and two or more, e.g., 2, 3, 4, or 5 or more, costimulatory signaling domains. In some embodiments, the one or more of the CAR molecules may have the same or a different primary intracellular signaling domain, the same or different costimulatory signaling domains, or the same number or a different number of costimulatory signaling domains. In some embodiments, the one or more of the CAR molecules can be configured as a split CAR, in which one of the CAR molecules comprises an antigen binding domain and a costimulatory domain (e.g., 4-1BB), while the other CAR molecule comprises an antigen binding domain and a primary intracellular signaling domain (e.g., CD3 zeta).
In some embodiments, the additional agent is any of the cells engineered to express one or more of the anti-BCMA binding molecules and/or cells engineered to express additional binding molecules, e.g., recombinant receptors, e.g., CAR, that target a different antigen. In some embodiments, the additional agent includes any of the cells or plurality of cells described herein, e.g., in Section I.C. In some embodiments, the additional agent is a cell engineered to express a recombinant receptor, e.g., CAR, targeting a different epitope and/or antigen, e.g., a different antigen associated with a disease or condition. In some embodiments, the additional agent is a cell engineered to express a recombinant receptor, e.g., CAR, targeting a second or additional antigen expressed in multiple myeloma, e.g., CD38, CD138, CS-1, BAFF-R, TACI and/or FcRH5.
In some embodiments, the additional agent is an immunomodulatory agent. In some embodiments, the combination therapy includes an immunomodulatory agent that can stimulate, amplify and/or otherwise enhance an anti-tumor immune response, e.g. anti-tumor immune response from the administered engineered cells, such as by inhibiting immunosuppressive signaling or enhancing immunostimulant signaling. In some embodiments, the immunomodulatory agent is a peptide, protein or is a small molecule. In some embodiments, the protein can be a fusion protein or a recombinant protein. In some embodiments, the immunomodulatory agent binds to an immunologic target, such as a cell surface receptor expressed on immune cells, such a T cells, B cells or antigen-presenting cells. For example, in some embodiments, the immunomodulatory agent is an antibody or antigen-binding antibody fragment, a fusion protein, a small molecule or a polypeptide. In some embodiments, the recombinant receptors, cells and/or compositions are administered in combination with an additional agent that is an antibody or an antigen-binding fragment thereof, such as a monoclonal antibody.
In some embodiments, the immunomodulatory agent blocks, inhibits or counteracts a component of the immune checkpoint pathway. The immune system has multiple inhibitory pathways that are involved in maintaining self-tolerance and for modulating immune responses. Tumors can use certain immune-checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumor antigens (Pardoll (2012) Nature Reviews Cancer 12:252-264), e.g., engineered cells such as CAR-expressing cells. Because many such immune checkpoints are initiated by ligand-receptor interactions, they can be readily blocked by antibodies against the ligands and/or their receptors.
Therefore, therapy with antagonistic molecules blocking an immune checkpoint pathway, such as small molecules, nucleic acid inhibitors (e.g., RNAi) or antibody molecules, are becoming promising avenues of immunotherapy for cancer and other diseases. In contrast to the majority of anti-cancer agents, checkpoint inhibitors do not necessarily target tumor cells directly, but rather target lymphocyte receptors or their ligands in order to enhance the endogenous antitumor activity of the immune system.
As used herein, the term “immune checkpoint inhibitor” refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more checkpoint proteins. Checkpoint proteins regulate T-cell activation or function. These proteins are responsible for co-stimulatory or inhibitory interactions of T-cell responses. Immune checkpoint proteins regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses. In some embodiments, the subject can be administered an additional agent that can enhance or boost the immune response, e.g., immune response effected by the BCMA-binding recombinant receptors, cells and/or compositions provided herein, against a disease or condition, e.g., a cancer, such as any described herein.
Immune checkpoint inhibitors include any agent that blocks or inhibits in a statistically significant manner, the inhibitory pathways of the immune system. Such inhibitors may include small molecule inhibitors or may include antibodies, or antigen binding fragments thereof, that bind to and block or inhibit immune checkpoint receptors, ligands and/or receptor-ligand interaction. In some embodiments, modulation, enhancement and/or stimulation of particular receptors can overcome immune checkpoint pathway components. Illustrative immune checkpoint molecules that may be targeted for blocking, inhibition, modulation, enhancement and/or stimulation include, but are not limited to, PD-1 (CD279), PD-L1 (CD274, B7-H1), PDL2 (CD273, B7-DC), CTLA-4, LAG-3 (CD223), TIM-3, 4-1BB (CD137), 4-1BBL (CD137L), GITR (TNFRSF18, AITR), CD40, OX40 (CD134, TNFRSF4), CXCR2, tumor associated antigens (TAA), B7-H3, B7-H4, BTLA, HVEM, GAL9, B7H3, B7H4, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK, γδ, and memory CD8+(αβ) T cells), CD160 (also referred to as BY55), CGEN-15049, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, WIC class I, MHC class II, GAL9, adenosine, and a transforming growth factor receptor (TGFR; e.g., TGFR beta). Immune checkpoint inhibitors include antibodies, or antigen binding fragments thereof, or other binding proteins, that bind to and block or inhibit and/or enhance or stimulate the activity of one or more of any of the said molecules.
Exemplary immune checkpoint inhibitors include Tremelimumab (CTLA-4 blocking antibody, also known as ticilimumab, CP-675,206), anti-OX40, PD-L1 monoclonal antibody (Anti-B7-H1; MEDI4736), MK-3475 (PD-1 blocker), nivolumab (anti-PD-1 antibody), CT-011 (anti-PD-1 antibody), BY55 monoclonal antibody, AMP224 (anti-PD-L1 antibody), BMS-936559 (anti-PD-L1 antibody), MPLDL3280A (anti-PD-L1 antibody), MSB0010718C (anti-PD-L1 antibody) and ipilimumab (anti-CTLA-4 antibody, also known as Yervoy®, MDX-010 and MDX-101). Exemplary immunomodulatory antibodies include, but are not limited to, Daclizumab (Zenapax), Bevacizumab (Avastin®), Basiliximab, Ipilimumab, Nivolumab, pembrolizumab, MPDL3280A, Pidilizumab (CT-011), MK-3475, BMS-936559, MPDL3280A (Atezolizumab), tremelimumab, IMP321, BMS-986016, LAG525, urelumab, PF-05082566, TRX518, MK-4166, dacetuzumab (SGN-40), lucatumumab (HCD122), SEA-CD40, CP-870, CP-893, MEDI6469, MEDI6383, MOXR0916, AMP-224, MSB0010718C (Avelumab), MEDI4736, PDR001, rHIgM12B7, Ulocuplumab, BKT140, Varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, ARGX-115, Emactuzumab, CC-90002 and 1VINRP1685A or an antibody-binding fragment thereof. Other exemplary immunomodulators include, e.g., afutuzumab (available from Roche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (mixture of human cytokines including interleukin 1, interleukin 2, and interferon gamma, CAS 951209-71-5, available from IRX Therapeutics).
Programmed cell death 1 (PD-1) is an immune checkpoint protein that is expressed in B cells, NK cells, and T cells (Shinohara et al., 1995, Genomics 23:704-6; Blank et al., 2007, Cancer Immunol Immunother 56:739-45; Finger et al., 1997, Gene 197:177-87; Pardoll (2012) Nature Reviews Cancer 12:252-264). The major role of PD-1 is to limit the activity of T cells in peripheral tissues during inflammation in response to infection, as well as to limit autoimmunity. PD-1 expression is induced in activated T cells and binding of PD-1 to one of its endogenous ligands acts to inhibit T-cell activation by inhibiting stimulatory kinases. PD-1 also acts to inhibit the TCR “stop signal”. PD-1 is highly expressed on Treg cells and may increase their proliferation in the presence of ligand (Pardoll (2012) Nature Reviews Cancer 12:252-264). Anti-PD 1 antibodies have been used for treatment of melanoma, non-small-cell lung cancer, bladder cancer, prostate cancer, colorectal cancer, head and neck cancer, triple-negative breast cancer, leukemia, lymphoma and renal cell cancer (Topalian et al., 2012, N Engl J Med 366:2443-54; Lipson et al., 2013, Clin Cancer Res 19:462-8; Berger et al., 2008, Clin Cancer Res 14:3044-51; Gildener-Leapman et al., 2013, Oral Oncol 49:1089-96; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85). Exemplary anti-PD-1 antibodies include nivolumab (Opdivo by BMS), pembrolizumab (Keytruda by Merck), pidilizumab (CT-011 by Cure Tech), lambrolizumab (MK-3475 by Merck), and AMP-224 (Merck), nivolumab (also referred to as Opdivo, BMS-936558 or MDX1106; Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody which specifically blocks PD-1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are described in U.S. Pat. No. 8,008,449 and WO2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are described in WO2009/101611. Pembrolizumab (formerly known as lambrolizumab, and also referred to as Keytruda, MK03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodies are described in U.S. Pat. No. 8,354,509 and WO2009/114335. Other anti-PD-1 antibodies include AMP 514 (Amplimmune), among others, e.g., anti-PD-1 antibodies described in U.S. Pat. No. 8,609,089, US 2010028330, US 20120114649 and/or US 20150210769. AMP-224 (B7-DCIg; Amplimmune; e.g., described in WO2010/027827 and WO2011/066342), is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD-1 and B7-H1.
PD-L1 (also known as CD274 and B7-H1) and PD-L2 (also known as CD273 and B7-DC) are ligands for PD-1, found on activated T cells, B cells, myeloid cells, macrophages, and some types of tumor cells. Anti-tumor therapies have focused on anti-PD-L1 antibodies. The complex of PD-1 and PD-L1 inhibits proliferation of CD8+ T cells and reduces the immune response (Topalian et al., 2012, N Engl J Med 366:2443-54; Brahmer et al., 2012, N Eng J Med 366:2455-65). Anti-PD-L1 antibodies have been used for treatment of non-small cell lung cancer, melanoma, colorectal cancer, renal-cell cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, and hematologic malignancies (Brahmer et al., 2012, N Eng J Med 366:2455-65; Ott et al., 2013, Clin Cancer Res 19:5300-9; Radvanyi et al., 2013, Clin Cancer Res 19:5541; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85; Berger et al., 2008, Clin Cancer Res 14:13044-51). Exemplary anti-PD-L1 antibodies include MDX-1105 (Medarex), MEDI4736 (Medimmune) MPDL3280A (Genentech), BMS-935559 (Bristol-Myers Squibb) and MSB0010718C. MEDI4736 (Medimmune) is a human monoclonal antibody that binds to PD-L1, and inhibits interaction of the ligand with PD-1. MDPL3280A (Genentech/Roche) is a human Fc optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies to PD-L1 are described in U.S. Pat. No. 7,943,743 and U.S. Publication No. 20120039906. Other anti-PD-L1 binding agents include YW243.55.570 (see WO2010/077634) and MDX-1105 (also referred to as BMS-936559, and, e.g., anti-PD-L1 binding agents described in WO2007/005874).
Cytotoxic T-lymphocyte-associated antigen (CTLA-4), also known as CD152, is a co-inhibitory molecule that functions to regulate T-cell activation. CTLA-4 is a member of the immunoglobulin superfamily that is expressed exclusively on T-cells. CTLA-4 acts to inhibit T-cell activation and is reported to inhibit helper T-cell activity and enhance regulatory T-cell immunosuppressive activity. Although the precise mechanism of action of CTLA-4 remains under investigation, it has been suggested that it inhibits T cell activation by outcompeting CD28 in binding to CD80 and CD86, as well as actively delivering inhibitor signals to the T cell (Pardoll (2012) Nature Reviews Cancer 12:252-264). Anti-CTLA-4 antibodies have been used in clinical trials for the treatment of melanoma, prostate cancer, small cell lung cancer, non-small cell lung cancer (Robert & Ghiringhelli, 2009, Oncologist 14:848-61; Ott et al., 2013, Clin Cancer Res 19:5300; Weber, 2007, Oncologist 12:864-72; Wada et al., 2013, J Transl Med 11:89). A significant feature of anti-CTLA-4 is the kinetics of anti-tumor effect, with a lag period of up to 6 months after initial treatment required for physiologic response. In some cases, tumors may actually increase in size after treatment initiation, before a reduction is seen (Pardoll (2012) Nature Reviews Cancer 12:252-264). Exemplary anti-CTLA-4 antibodies include ipilimumab (Bristol-Myers Squibb) and tremelimumab (Pfizer). Ipilimumab has recently received FDA approval for treatment of metastatic melanoma (Wada et al., 2013, J Transl Med 11:89).
Lymphocyte activation gene-3 (LAG-3), also known as CD223, is another immune checkpoint protein. LAG-3 has been associated with the inhibition of lymphocyte activity and in some cases the induction of lymphocyte anergy. LAG-3 is expressed on various cells in the immune system including B cells, NK cells, and dendritic cells. LAG-3 is a natural ligand for the MHC class II receptor, which is substantially expressed on melanoma-infiltrating T cells including those endowed with potent immune-suppressive activity. Exemplary anti-LAG-3 antibodies include BMS-986016 (Bristol-Myers Squib), which is a monoclonal antibody that targets LAG-3. IMP701 (Immutep) is an antagonist LAG-3 antibody and IMP731 (Immutep and GlaxoSmithKline) is a depleting LAG-3 antibody. Other LAG-3 inhibitors include IMP321 (Immutep), which is a recombinant fusion protein of a soluble portion of LAG-3 and Ig that binds to MHC class II molecules and activates antigen presenting cells (APC). Other antibodies are described, e.g., in WO2010/019570 and US 2015/0259420
T-cell immunoglobulin domain and mucin domain-3 (TIM-3), initially identified on activated Th1 cells, has been shown to be a negative regulator of the immune response. Blockade of TIM-3 promotes T-cell mediated anti-tumor immunity and has anti-tumor activity in a range of mouse tumor models. Combinations of TIM-3 blockade with other immunotherapeutic agents such as TSR-042, anti-CD137 antibodies and others, can be additive or synergistic in increasing anti-tumor effects. TIM-3 expression has been associated with a number of different tumor types including melanoma, NSCLC and renal cancer, and additionally, expression of intratumoral TIM-3 has been shown to correlate with poor prognosis across a range of tumor types including NSCLC, cervical, and gastric cancers. Blockade of TIM-3 is also of interest in promoting increased immunity to a number of chronic viral diseases. TIM-3 has also been shown to interact with a number of ligands including galectin-9, phosphatidylserine and HMGB1, although which of these, if any, are relevant in regulation of anti-tumor responses is not clear at present. In some embodiments, antibodies, antibody fragments, small molecules, or peptide inhibitors that target TIM-3 can bind to the IgV domain of TIM-3 to inhibit interaction with its ligands. Exemplary antibodies and peptides that inhibit TIM-3 are described in US 2015/0218274, WO2013/006490 and US 2010/0247521. Other anti-TIM-3 antibodies include humanized versions of RMT3-23 (Ngiow et al., 2011, Cancer Res, 71:3540-3551), and clone 8B.2C12 (Monney et al., 2002, Nature, 415:536-541). Bi-specific antibodies that inhibit TIM-3 and PD-1 are described in US 2013/0156774.
In some embodiments, the additional agent is a CEACAM inhibitor (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5 inhibitor). In some embodiments, the inhibitor of CEACAM is an anti-CEACAM antibody molecule. Exemplary anti-CEACAM-1 antibodies are described in WO 2010/125571, WO 2013/082366 WO 2014/059251 and WO 2014/022332, e.g., a monoclonal antibody 34B1, 26H7, and 5F4; or a recombinant form thereof, as described in, e.g., US 2004/0047858, U.S. Pat. No. 7,132,255 and WO 99/052552. In some embodiments, the anti-CEACAM antibody binds to CEACAM-5 as described in, e.g., Zheng et al. PLoS One. (2011) 6(6): e21146), or cross reacts with CEACAM-1 and CEACAM-5 as described in, e.g., WO 2013/054331 and US 2014/0271618.
4-1BB, also known as CD137, is transmembrane glycoprotein belonging to the TNFR superfamily. 4-1BB receptors are present on activated T cells and B cells and monocytes. An exemplary anti-4-1BB antibody is urelumab (BMS-663513), which has potential immunostimulatory and antineoplastic activities.
Tumor necrosis factor receptor superfamily, member 4 (TNFRSF4), also known as OX40 and CD134, is another member of the TNFR superfamily. OX40 is not constitutively expressed on resting naïve T cells and acts as a secondary co-stimulatory immune checkpoint molecule. Exemplary anti-OX40 antibodies are MEDI6469 and MOXR0916 (RG7888, Genentech).
In some embodiments, the additional agent includes a molecule that decreases the regulatory T cell (Treg) population. Methods that decrease the number of (e.g., deplete) Treg cells are known in the art and include, e.g., CD25 depletion, cyclophosphamide administration, and modulating Glucocorticoid-induced TNFR family related gene (GITR) function. GITR is a member of the TNFR superfamily that is upregulated on activated T cells, which enhances the immune system. Reducing the number of Treg cells in a subject prior to apheresis or prior to administration of engineered cells, e.g., CAR-expressing cells, can reduce the number of unwanted immune cells (e.g., Tregs) in the tumor microenvironment and reduces the subject's risk of relapse. In some embodiments, the additional agent includes a molecule targeting GITR and/or modulating GITR functions, such as a GITR agonist and/or a GITR antibody that depletes regulatory T cells (Tregs). In some embodiments, the additional agent includes cyclophosphamide. In some embodiments, the GITR binding molecule and/or molecule modulating GITR function (e.g., GITR agonist and/or Treg depleting GITR antibodies) is administered prior to the engineered cells, e.g., CAR-expressing cells. For example, in some embodiments, the GITR agonist can be administered prior to apheresis of the cells. In some embodiments, cyclophosphamide is administered to the subject prior to administration (e.g., infusion or re-infusion) of the engineered cells, e.g., CAR-expressing cells or prior to apheresis of the cells. In some embodiments, cyclophosphamide and an anti-GITR antibody are administered to the subject prior to administration (e.g., infusion or re-infusion) of the engineered cells, e.g., CAR-expressing cells or prior to apheresis of the cells.
In some embodiments, the additional agent is a GITR agonist. Exemplary GITR agonists include, e.g., GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such as, e.g., a GITR fusion protein described in U.S. Pat. No. 6,111,090, European Patent No. 090505B 1, U.S. Pat. No. 8,586,023, PCT Publication Nos.: WO 2010/003118 and 2011/090754, or an anti-GITR antibody described, e.g., in U.S. Pat. No. 7,025,962, European Patent No. 1947183B 1, U.S. Pat. Nos. 7,812,135, 8,388,967, 8,591,886, European Patent No. EP 1866339, PCT Publication No. WO 2011/028683, PCT Publication No. WO 2013/039954, PCT Publication No. WO2005/007190, PCT Publication No. WO 2007/133822, PCT Publication No. WO2005/055808, PCT Publication No. WO 99/40196, PCT Publication No. WO 2001/03720, PCT Publication No. WO99/20758, PCT Publication No. WO2006/083289, PCT Publication No. WO 2005/115451, U.S. Pat. No. 7,618,632, and PCT Publication No. WO 2011/051726. An exemplary anti-GITR antibody is TRX518.
In some embodiments, the additional agent enhances tumor infiltration or transmigration of the administered cells, e.g., CAR-expressing cells. For example, in some embodiments, the additional agent stimulates CD40, such as CD40L, e.g., recombinant human CD40L. Cluster of differentiation 40 (CD40) is also a member of the TNFR superfamily. CD40 is a costimulatory protein found on antigen-presenting cells and mediates a broad variety of immune and inflammatory responses. CD40 is also expressed on some malignancies, where it promotes proliferation. Exemplary anti-CD40 antibodies are dacetuzumab (SGN-40), lucatumumab (Novartis, antagonist), SEA-CD40 (Seattle Genetics), and CP-870,893. In some embodiments, the additional agent that enhances tumor infiltration includes tyrosine kinase inhibitor sunitnib, heparanase, and/or chemokine receptors such as CCR2, CCR4, and CCR7.
In some embodiments, the additional agent includes thalidomide drugs or analogs thereof and/or derivatives thereof, such as lenalidomide, pomalidomide or apremilast. See, e.g., Bertilaccio et al., Blood (2013) 122:4171, Otahal et al., Oncoimmunology (2016) 5(4):e1115940; Fecteau et al., Blood (2014) 124(10):1637-1644 and Kuramitsu et al., Cancer Gene Therapy (2015) 22:487-495). Lenalidomide ((RS)-3-(4-Amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione; also known as Revlimid) is a synthetic derivative of thalidomide, and has multiple immunomodulatory effects, including enforcement of immune synapse formation between T cell and antigen presenting cells (APCs). For example, in some cases, lenalidomide modulates T cell responses and results in increased interleukin (IL)-2 production in CD4+ and CD8+ T cells, induces the shift of T helper (Th) responses from Th2 to Th1, inhibits expansion of regulatory subset of T cells (Tregs), and improves functioning of immunological synapses in follicular lymphoma and chronic lymphocytic leukemia (CLL) (Otahal et al., Oncoimmunology (2016) 5(4):e1115940). Lenalidomide also has direct tumoricidal activity in patients with multiple myeloma (MM) and directly and indirectly modulates survival of CLL tumor cells by affecting supportive cells, such as nurse-like cells found in the microenvironment of lymphoid tissues. Lenalidomide also can enhance T-cell proliferation and interferon-γ production in response to activation of T cells via CD3 ligation or dendritic cell-mediated activation. Lenalidomide can also induce malignant B cells to express higher levels of immunostimulatory molecules such as CD80, CD86, HLA-DR, CD95, and CD40 (Fecteau et al., Blood (2014) 124(10):1637-1644). In some embodiments, lenalidomide is administered at a dosage of from about 1 mg to about 20 mg daily, e.g., from about 1 mg to about 10 mg, from about 2.5 mg to about 7.5 mg, from about 5 mg to about 15 mg, such as about 5 mg, 10 mg, 15 mg or 20 mg daily. In some embodiments, lenalidomide is administered at a dose of from about 10 μg/kg to 5 mg/kg, e.g., about 100 μg/kg to about 2 mg/kg, about 200 μg/kg to about 1 mg/kg, about 400 μg/kg to about 600 μg/kg, such as about 500 μg/kg. In some embodiments, rituximab is administered at a dosage of about 350-550 mg/m2 (e.g., 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/m2), e.g., intravenously. In some embodiments, lenalidomide is administered at a low dose.
In some embodiments, the additional agent is a B-cell inhibitor. In some embodiments, the additional agent is one or more B-cell inhibitors selected from among inhibitors of CD10, CD19, CD20, CD22, CD34, CD123, CD79a, CD79b, CD179b, FLT-3, or ROR1, or a combination thereof. In some embodiments, the B-cell inhibitor is an antibody (e.g., a mono- or bispecific antibody) or an antigen binding fragment thereof. In some embodiments, the additional agent is an engineered cell expressing recombinant receptors that target B-cell targets, e.g., CD10, CD19, CD20, CD22, CD34, CD123, CD79a, CD79b, CD179b, FLT-3, or ROR1.
In some embodiments, the additional agent is a CD20 inhibitor, e.g., an anti-CD20 antibody (e.g., an anti-CD20 mono- or bi-specific antibody) or a fragment thereof. Exemplary anti-CD20 antibodies include but are not limited to rituximab, ofatumumab, ocrelizumab (also known as GA101 or R05072759), veltuzumab, obinutuzumab, TRU-015 (Trubion Pharmaceuticals), ocaratuzumab (also known as AME-133v or ocaratuzumab), and Pro131921 (Genentech). See, e.g., Lim et al. Haematologica. (2010) 95(1):135-43. In some embodiments, the anti-CD20 antibody comprises rituximab. Rituximab is a chimeric mouse/human monoclonal antibody IgG1 kappa that binds to CD20 and causes cytolysis of a CD20 expressing cell. In some embodiments, the additional agent includes rituximab. In some embodiments, the CD20 inhibitor is a small molecule.
In some embodiments, the additional agent is a CD22 inhibitor, e.g., an anti-CD22 antibody (e.g., an anti-CD22 mono- or bi-specific antibody) or a fragment thereof. Exemplary anti-CD22 antibodies include epratuzumab and RFB4. In some embodiments, the CD22 inhibitor is a small molecule. In some embodiments, the antibody is a monospecific antibody, optionally conjugated to a second agent such as a chemotherapeutic agent. For instance, in some embodiments, the antibody is an anti-CD22 monoclonal antibody-MMAE conjugate (e.g., DCDT2980S). In some embodiments, the antibody is an scFv of an anti-CD22 antibody, e.g., an scFv of antibody RFB4. In some embodiments, the scFv is fused to all of or a fragment of Pseudomonas exotoxin-A (e.g., BL22). In some embodiments, the scFv is fused to all of or a fragment of (e.g., a 38 kDa fragment of) Pseudomonas exotoxin-A (e.g., moxetumomab pasudotox). In some embodiments, the anti-CD22 antibody is an anti-CD19/CD22 bispecific antibody, optionally conjugated to a toxin. For instance, in some embodiments, the anti-CD22 antibody comprises an anti-CD19/CD22 bispecific portion, (e.g., two scFv ligands, recognizing human CD19 and CD22) optionally linked to all of or a portion of diphtheria toxin (DT), e.g., first 389 amino acids of diphtheria toxin (DT), DT 390, e.g., a ligand-directed toxin such as DT2219ARL). In some embodiments, the bispecific portion (e.g., anti-CD 19/anti-CD22) is linked to a toxin such as deglycosylated ricin A chain (e.g., Combotox).
In some embodiments, the immunomodulatory agent is a cytokine. In some embodiments, the immunomodulatory agent is a cytokine or is an agent that induces increased expression of a cytokine in the tumor microenvironment. Cytokines have important functions related to T cell expansion, differentiation, survival, and homeostasis. Cytokines that can be administered to the subject receiving the BCMA-binding recombinant receptors, cells and/or compositions provided herein include one or more of IL-2, IL-4, IL-7, IL-9, IL-15, IL-18, and IL-21. In some embodiments, the cytokine administered is IL-7, IL-15, or IL-21, or a combination thereof. In some embodiments, administration of the cytokine to the subject that has sub-optimal response to the administration of the engineered cells, e.g., CAR-expressing cells improves efficacy and/or anti-tumor activity of the administered cells, e.g., CAR-expressing cells.
By “cytokine” is meant a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta, and -gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines. For example, the immunomodulatory agent is a cytokine and the cytokine is IL-4, TNF-α, GM-CSF or IL-2.
In some embodiments, the additional agent includes an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha (IL-15Ra) polypeptide, or combination thereof, e.g., hetIL-15 (Admune Therapeutics, LLC). hetIL-15 is a heterodimeric non-covalent complex of IL-15 and IL-15Ra. hetIL-15 is described in, e.g., U.S. Pat. No. 8,124,084, U.S. 2012/0177598, U.S. 2009/0082299, U.S. 2012/0141413, and U.S. 2011/0081311. In some embodiments, the immunomodulatory agent can contain one or more cytokines. For example, the interleukin can include leukocyte interleukin injection (Multikine), which is a combination of natural cytokines. In some embodiments, the immunomodulatory agent is a Toll-like receptor (TLR) agonist, an adjuvant or a cytokine.
In some embodiments, the additional agent is an agent that ameliorates or neutralizes one or more toxicities or side effects associated with the cell therapy. In some embodiments, the additional agent is selected from among a steroid (e.g., corticosteroid), an inhibitor of TNFα, and an inhibitor of IL-6. An example of a TNFα inhibitor is an anti-TNFα antibody molecule such as, infliximab, adalimumab, certolizumab pegol, and golimumab. Another example of a TNFα inhibitor is a fusion protein such as entanercept. Small molecule inhibitors of TNFα include, but are not limited to, xanthine derivatives (e.g. pentoxifylline) and bupropion. An example of an IL-6 inhibitor is an anti-IL-6 antibody molecule such as tocilizumab, sarilumab, elsilimomab, CNTO 328, ALD518/BMS-945429, CNTO 136, CPSI-2364, CDP6038, VX30, ARGX-109, FE301, and FM101. In some embodiments, the anti-IL-6 antibody molecule is tocilizumab. In some embodiments, the additional agent is an IL-1R inhibitor, such as anakinra.
In some embodiments, the additional agent is a modulator of adenosine levels and/or an adenosine pathway component. Adenosine can function as an immunomodulatory agent in the body. For example, adenosine and some adenosine analogs that non-selectively activate adenosine receptor subtypes decrease neutrophil production of inflammatory oxidative products (Cronstein et al., Ann. N.Y. Acad. Sci. 451:291, 1985; Roberts et al., Biochem. J., 227:669, 1985; Schrier et al., J. Immunol. 137:3284, 1986; Cronstein et al., Clinical Immunol. Immunopath. 42:76, 1987). In some cases, concentration of extracellular adenosine or adenosine analogs can increase in specific environments, e.g., tumor microenvironment (TME). In some cases, adenosine or adenosine analog signaling depends on hypoxia or factors involved in hypoxia or its regulation, e.g., hypoxia inducible factor (HIF). In some embodiments, increase in adenosine signaling can increase in intracellular cAMP and cAMP-dependent protein kinase that results in inhibition of proinflammatory cytokine production, and can lead to the synthesis of immunosuppressive molecules and development of Tregs (Sitkovsky et al., Cancer Immunol Res (2014) 2(7):598-605). In some embodiments, the additional agent can reduce or reverse immunosuppressive effects of adenosine, adenosine analogs and/or adenosine signaling. In some embodiments, the additional agent can reduce or reverse hypoxia-driven A2-adenosinergic T cell immunosuppression. In some embodiments, the additional agent is selected from among antagonists of adenosine receptors, extracellular adenosine-degrading agents, inhibitors of adenosine generation by CD39/CD73 ectoenzymes, and inhibitors of hypoxia-HIF-1α signaling. In some embodiments, the additional agent is an adenosine receptor antagonist or agonist.
Inhibition or reduction of extracellular adenosine or the adenosine receptor by virtue of an inhibitor of extracellular adenosine (such as an agent that prevents the formation of, degrades, renders inactive, and/or decreases extracellular adenosine), and/or an adenosine receptor inhibitor (such as an adenosine receptor antagonist) can enhance immune response, such as a macrophage, neutrophil, granulocyte, dendritic cell, T- and/or B cell-mediated response. In addition, inhibitors of the Gs protein mediated cAMP dependent intracellular pathway and inhibitors of the adenosine receptor-triggered Gi protein mediated intracellular pathways, can also increase acute and chronic inflammation.
In some embodiments, the additional agent is an adenosine receptor antagonist or agonist, e.g., an antagonist or agonist of one or more of the adenosine receptors A2a, A2b, A1, and A3. A1 and A3 inhibit, and A2a and A2b stimulate, respectively, adenylate cyclase activity. Certain adenosine receptors, such as A2a, A2b, and A3, can suppress or reduce the immune response during inflammation. Thus, antagonizing immunosuppressive adenosine receptors can augment, boost or enhance immune response, e.g., immune response from administered cells, e.g., CAR-expressing T cells. In some embodiments, the additional agent inhibits the production of extracellular adenosine and adenosine-triggered signaling through adenosine receptors. For example, enhancement of an immune response, local tissue inflammation, and targeted tissue destruction can be enhanced by inhibiting or reducing the adenosine-producing local tissue hypoxia; by degrading (or rendering inactive) accumulated extracellular adenosine; by preventing or decreasing expression of adenosine receptors on immune cells; and/or by inhibiting/antagonizing signaling by adenosine ligands through adenosine receptors.
An antagonist is any substance that tends to nullify the action of another, as an agent that binds to a cell receptor without eliciting a biological response. In some embodiments, the antagonist is a chemical compound that is an antagonist for an adenosine receptor, such as the A2a, A2b, or A3 receptor. In some embodiments, the antagonist is a peptide, or a pepidomimetic, that binds the adenosine receptor but does not trigger a Gi protein dependent intracellular pathway. Exemplary antagonists are described in U.S. Pat. Nos. 5,565,566; 5,545,627, 5,981,524; 5,861,405; 6,066,642; 6,326,390; 5,670,501; 6,117,998; 6,232,297; 5,786,360; 5,424,297; 6,313,131, 5,504,090; and 6,322,771.
In some embodiments, the additional agent is an A2 receptor (A2R) antagonist, such as an A2a antagonist. Exemplary A2R antagonists include KW6002 (istradefyline), SCH58261, caffeine, paraxanthine, 3,7-dimethyl-1-propargylxanthine (DMPX), 8-(m-chlorostyryl) caffeine (CSC), MSX-2, MSX-3, MSX-4, CGS-15943, ZM-241385, SCH-442416, preladenant, vipadenant (BII014), V2006, ST-1535, SYN-115, PSB-1115, ZM241365, FSPTP, and an inhibitory nucleic acid targeting A2R expression, e.g., siRNA or shRNA, or any antibodies or antigen-binding fragment thereof that targets an A2R. In some embodiments, the additional agent is an A2R antagonist described in, e.g., Ohta et al., Proc Natl Acad Sci USA (2006) 103:13132-13137; Jin et al., Cancer Res. (2010) 70(6):2245-2255; Leone et al., Computational and Structural Biotechnology Journal (2015) 13:265-272; Beavis et al., Proc Natl Acad Sci USA (2013) 110:14711-14716; and Pinna, A., Expert Opin Investig Drugs (2009) 18:1619-1631; Sitkovsky et al., Cancer Immunol Res (2014) 2(7):598-605; U.S. Pat. Nos. 8,080,554; 8,716,301; US 20140056922; WO2008/147482; U.S. Pat. No. 8,883,500; US 20140377240; WO02/055083; U.S. Pat. Nos. 7,141,575; 7,405,219; 8,883,500; 8,450,329 and 8,987,279).
In some embodiments, the antagonist is an antisense molecule, inhibitory nucleic acid molecule (e.g., small inhibitory RNA (siRNA)) or catalytic nucleic acid molecule (e.g. a ribozyme) that specifically binds mRNA encoding an adenosine receptor. In some embodiments, the antisense molecule, inhibitory nucleic acid molecule or catalytic nucleic acid molecule binds nucleic acids encoding A2a, A2b, or A3. In some embodiments, an antisense molecule, inhibitory nucleic acid molecule or catalytic nucleic acid targets biochemical pathways downstream of the adenosine receptor. For example, the antisense molecule or catalytic nucleic acid can inhibit an enzyme involved in the Gs protein- or Gi protein-dependent intracellular pathway. In some embodiments, the additional agent includes dominant negative mutant form of an adenosine receptor, such as A2a, A2b, or A3.
In some embodiments, the additional agent that inhibits extracellular adenosine includes agents that render extracellular adenosine non-functional (or decrease such function), such as a substance that modifies the structure of adenosine to inhibit the ability of adenosine to signal through adenosine receptors. In some embodiments, the additional agent is an extracellular adenosine-generating or adenosine-degrading enzyme, a modified form thereof or a modulator thereof. For example, in some embodiments, the additional agent is an enzyme (e.g. adenosine deaminase) or another catalytic molecule that selectively binds and destroys the adenosine, thereby abolishing or significantly decreasing the ability of endogenously formed adenosine to signal through adenosine receptors and terminate inflammation.
In some embodiments, the additional agent is an adenosine deaminase (ADA) or a modified form thereof, e.g., recombinant ADA and/or polyethylene glycol-modified ADA (ADA-PEG), which can inhibit local tissue accumulation of extracellular adenosine. ADA-PEG has been used in treatment of patients with ADA SCID (Hershfield (1995) Hum Mutat. 5:107). In some embodiments, an agent that inhibits extracellular adenosine includes agents that prevent or decrease formation of extracellular adenosine, and/or prevent or decrease the accumulation of extracellular adenosine, thereby abolishing, or substantially decreasing, the immunosuppressive effects of adenosine. In some embodiments, the additional agent specifically inhibits enzymes and proteins that are involved in regulation of synthesis and/or secretion of pro-inflammatory molecules, including modulators of nuclear transcription factors. Suppression of adenosine receptor expression or expression of the Gs protein- or Gi protein-dependent intracellular pathway, or the cAMP dependent intracellular pathway, can result in an increase/enhancement of immune response.
In some embodiments, the additional agent can target ectoenzymes that generate or produce extracellular adenosine. In some embodiments, the additional agent targets CD39 and CD73 ectoenzymes, which function in tandem to generate extracellular adenosine. CD39 (also called ectonucleoside triphosphate diphosphohydrolase) converts extracellular ATP (or ADP) to 5′AMP. Subsequently, CD73 (also called 5′nucleotidase) converts 5′AMP to adenosine. The activity of CD39 is reversible by the actions of NDP kinase and adenylate kinase, whereas the activity of CD73 is irreversible. CD39 and CD73 are expressed on tumor stromal cells, including endothelial cells and Tregs, and also on many cancer cells. For example, the expression of CD39 and CD73 on endothelial cells is increased under the hypoxic conditions of the tumor microenvironment. Tumor hypoxia can result from inadequate blood supply and disorganized tumor vasculature, impairing delivery of oxygen (Carroll and Ashcroft (2005), Expert. Rev. Mol. Med. 7(6):1-16). Hypoxia also inhibits adenylate kinase (AK), which converts adenosine to AMP, leading to very high extracellular adenosine concentration. Thus, adenosine is released at high concentrations in response to hypoxia, which is a condition that frequently occurs the tumor microenvironment (TME), in or around solid tumors. In some embodiments, the additional agent is one or more of anti-CD39 antibody or antigen binding fragment thereof, anti-CD73 antibody or antigen binding fragment thereof, e.g., MEDI9447 or TY/23, α-β-methylene-adenosine diphosphate (ADP), ARL 67156, POM-3, IPH52 (see, e.g., Allard et al. Clin Cancer Res (2013) 19(20):5626-5635; Hausler et al., Am J Transl Res (2014) 6(2):129-139; Zhang, B., Cancer Res. (2010) 70(16):6407-6411).
In some embodiments, the additional agent is an inhibitor of hypoxia inducible factor 1 alpha (HIF-1α) signaling. Exemplary inhibitors of HIF-1α include digoxin, acriflavine, sirtuin-7 and ganetespib.
In some embodiments, the additional agent includes a protein tyrosine phosphatase inhibitor, e.g., a protein tyrosine phosphatase inhibitor described herein. In some embodiments, the protein tyrosine phosphatase inhibitor is an SHP-1 inhibitor, e.g., an SHP-1 inhibitor described herein, such as, e.g., sodium stibogluconate. In some embodiments, the protein tyrosine phosphatase inhibitor is an SHP-2 inhibitor, e.g., an SHP-2 inhibitor described herein.
In some embodiments, the additional agent is a kinase inhibitor. Kinase inhibitors, such as a CDK4 kinase inhibitor, a BTK kinase inhibitor, a MNK kinase inhibitor, or a DGK kinase inhibitor, can regulate the constitutively active survival pathways that exist in tumor cells and/or modulate the function of immune cells. In some embodiments, the kinase inhibitor is a Bruton's tyrosine kinase (BTK) inhibitor, e.g., ibrutinib. In some embodiments, the kinase inhibitor is a phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibitor. In some embodiments, the kinase inhibitor is a CDK4 inhibitor, e.g., a CDK4/6 inhibitor. In some embodiments, the kinase inhibitor is an mTOR inhibitor, such as, e.g., rapamycin, a rapamycin analog, OSI-027. The mTOR inhibitor can be, e.g., an mTORC1 inhibitor and/or an mTORC2 inhibitor, e.g., an mTORC1 inhibitor and/or mTORC2 inhibitor. In some embodiments, the kinase inhibitor is an MNK inhibitor, or a dual PI3K/mTOR inhibitor. In some embodiments, other exemplary kinase inhibitors include the AKT inhibitor perifosine, the mTOR inhibitor temsirolimus, the Src kinase inhibitors dasatinib and fostamatinib, the JAK2 inhibitors pacritinib and ruxolitinib, the PKCβ inhibitors enzastaurin and bryostatin, and the AAK inhibitor alisertib.
In some embodiments, the kinase inhibitor is a BTK inhibitor selected from ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13. In some embodiments, the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin-2-inducible kinase (ITK), and is selected from GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13.
In some embodiments, the kinase inhibitor is a BTK inhibitor, e.g., ibrutinib (1-[(3R)-3-[4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one; also known as PCI-32765). In some embodiments, the kinase inhibitor is a BTK inhibitor, e.g., ibrutinib (PCI-32765), and the ibrutinib is administered at a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (e.g., 250 mg, 420 mg or 560 mg) daily for a period of time, e.g., daily for 21 day cycle, or daily for 28 day cycle. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of ibrutinib are administered. In some embodiments, the BTK inhibitor is a BTK inhibitor described in International Application WO 2015/079417.
In some embodiments, the kinase inhibitor is a PI3K inhibitor. PI3K is central to the PI3K/Akt/mTOR pathway involved in cell cycle regulation and lymphoma survival. Exemplary PI3K inhibitor includes idelalisib (PI3Kδ inhibitor). In some embodiments, the additional agent is idelalisib and rituximab.
In some embodiments, the additional agent is an inhibitor of mammalian target of rapamycin (mTOR). In some embodiments, the kinase inhibitor is an mTOR inhibitor selected from temsirolimus; ridaforolimus (also known as AP23573 and MK8669); everolimus (RAD001); rapamycin (AY22989); simapimod; AZD8055; PF04691502; SF1126; and XL765. In some embodiments, the additional agent is an inhibitor of mitogen-activated protein kinase (MAPK), such as vemurafenib, dabrafenib, and trametinib.
In some embodiments, the additional agent is an agent that regulates pro- or anti-apoptotic proteins. In some embodiments, the additional agent includes a B-cell lymphoma 2 (BCL-2) inhibitor (e.g., venetoclax, also called ABT-199 or GDC-0199; or ABT-737). Venetoclax is a small molecule (4-(4-{[2-(4-Chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide) that inhibits the anti-apoptotic protein, BCL-2. Other agents that modulate pro- or anti-apoptotic protein include BCL-2 inhibitor ABT-737, navitoclax (ABT-263); Mc1-1 siRNA or Mc1-1 inhibitor retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) for maximal efficacy. In some embodiments, the additional agent provides a pro-apoptotic stimuli, such as recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which can activate the apoptosis pathway by binding to TRAIL death receptors DR-4 and DR-5 on tumor cell surface, or TRAIL-R2 agonistic antibodies.
In some embodiments, the additional agent includes an indoleamine 2,3-dioxygenase (IDO) inhibitor. IDO is an enzyme that catalyzes the degradation of the amino acid, L-tryptophan, to kynurenine. Many cancers overexpress IDO, e.g., prostatic, colorectal, pancreatic, cervical, gastric, ovarian, head, and lung cancer. Plasmacytoid dendritic cells (pDCs), macrophages, and dendritic cells (DCs) can express IDO. In some aspects, a decrease in L-tryptophan (e.g., catalyzed by IDO) results in an immunosuppressive milieu by inducing T-cell anergy and apoptosis. Thus, in some aspects, an IDO inhibitor can enhance the efficacy of the BCMA-binding recombinant receptors, cells and/or compositions described herein, e.g., by decreasing the suppression or death of the administered CAR-expressing cell. Exemplary inhibitors of IDO include but are not limited to 1-methyl-tryptophan, indoximod (New Link Genetics) (see, e.g., Clinical Trial Identifier Nos. NCT01191216; NCT01792050), and INCB024360 (Incyte Corp.) (see, e.g., Clinical Trial Identifier Nos. NCT01604889; NCT01685255).
In some embodiments, the additional agent includes a cytotoxic agent, e.g., CPX-351 (Celator Pharmaceuticals), cytarabine, daunorubicin, vosaroxin (Sunesis Pharmaceuticals), sapacitabine (Cyclacel Pharmaceuticals), idarubicin, or mitoxantrone. In some embodiments, the additional agent includes a hypomethylating agent, e.g., a DNA methyltransferase inhibitor, e.g., azacitidine or decitabine.
In another embodiment, the additional therapy is transplantation, e.g., an allogeneic stem cell transplant.
In some embodiments, the additional therapy is a lymphodepleting therapy. Lymphodepleting chemotherapy is thought to improve engraftment and activity of recombinant receptor-expressing cells, such as CAR T cells. In some embodiments, lymphodepleting chemotherapy may enhance adoptively transferred tumor-specific T cells to proliferate in vivo through homeostatic proliferation (Grossman 2004, Stachel 2004). In some embodiments, chemotherapy may reduce or eliminate CD4+CD25+ regulatory T cells, which can suppress the function of tumor-targeted adoptively transferred T cells (Turk 2004). In some embodiments, lymphodepleting chemotherapy prior to adoptive T-cell therapy may enhance the expression of stromal cell-derived factor 1 (SDF-1) in the bone marrow, enhancing the homing of modified T cells to the primary tumor site through binding of SDF-1 with CXCR-4 expressed on the T-cell surface (Pinthus 2004). In some embodiments, lymphodepleting chemotherapy may further reduce the subject's tumor burden and potentially lower the risk and severity of CRS.
In some embodiments, lymphodepletion is performed on a subject, e.g., prior to administering engineered cells, e.g., CAR-expressing cells. In some embodiments, the lymphodepletion comprises administering one or more of melphalan, Cytoxan, cyclophosphamide, and/or fludarabine. In some embodiments, a lymphodepleting chemotherapy is administered to the subject prior to, concurrently with, or after administration (e.g., infusion) of engineered cells, e.g., CAR-expressing cells. In an example, the lymphodepleting chemotherapy is administered to the subject prior to administration of engineered cells, e.g., CAR-expressing cells. In some embodiments the lymphodepleting chemotherapy is administered 1 to 10 days prior to administration of engineered cells, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to the initiation of administration of engineered cells, or at least 2 days prior, such as at least 3, 4, 5, 6, or 7 days prior, to the initiation of administration of engineered cell. In some embodiments, the subject is administered a preconditioning agent no more than 7 days prior, such as no more than 6, 5, 4, 3, or 2 days prior, to the initiation of administration of engineered cell. The number of days after lymphodepleting chemotherapy that the engineered ells are administered can be determined based on clinical or logistical circumstances. In some examples, dose adjustments or other changes to the lymphodepleting chemotherapy regimen can implemented due to a subject's health, such as the subject's underlying organ function, as determined by the treating physician.
In some embodiments, lymphodepleting chemotherapy comprises administration of a lymphodepleting agent, such as cyclophosphamide, fludarabine, or combinations thereof, In some embodiments, the subject is administered cyclophosphamide at a dose between or between about 20 mg/kg and 100 mg/kg body weight of the subject, such as between or between about 40 mg/kg and 80 mg/kg. In some aspects, the subject is administered about 60 mg/kg of cyclophosphamide. In some embodiments, the cyclophosphamide is administered once daily for one or two days. In some embodiments, where the lymphodepleting agent comprises cyclophosphamide, the subject is administered cyclophosphamide at a dose between or between about 100 mg/m2 and 500 mg/m2 body surface area of the subject, such as between or between about 200 mg/m2 and 400 mg/m2, or 250 mg/m2 and 350 mg/m2, inclusive. In some instances, the subject is administered about 300 mg/m2 of cyclophosphamide. In some embodiments, the cyclophosphamide can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some embodiments, cyclophosphamide is administered daily, such as for 1-5 days, for example, for 2 to 4 days. In some instances, the subject is administered about 300 mg/m2 body surface area of the subject, of cyclophosphamide, daily for 3 days, prior to initiation of the cell therapy.
In some embodiments, where the lymphodepleting agent comprises fludarabine, the subject is administered fludarabine at a dose between or between about 1 mg/m2 and 100 mg/m2 body surface area of the subject, such as between or between about 10 mg/m2 and 75 mg/m2, 15 mg/m2 and 50 mg/m2, 20 mg/m2 and 40 mg/m2, or 24 mg/m2 and 35 mg/m2, inclusive. In some instances, the subject is administered about 30 mg/m2 of fludarabine. In some embodiments, the fludarabine can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some embodiments, fludarabine is administered daily, such as for 1-5 days, for example, for 2 to 4 days. In some instances, the subject is administered about 30 mg/m2 body surface area of the subject, of fludarabine, daily for 3 days, prior to initiation of the cell therapy.
In some embodiments, the lymphodepleting agent comprises a combination of agents, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of agents may include cyclophosphamide at any dose or administration schedule, such as those described above, and fludarabine at any dose or administration schedule, such as those described above. For example, in some aspects, the subject is administered fludarabine at or about 30 mg/m2 body surface area of the subject, daily, and cyclophosphamide at or about 300 mg/m2 body surface area of the subject, daily, for 3 days.
In some embodiments, antiemetic therapy, except dexamethasone or other steroids, may be given prior to lymphodepleting chemotherapy. In some embodiments, Mesna may be used for subjects with a history of hemorrhagic cystitis.
In some embodiments, the additional agent is an oncolytic virus. In some embodiments, oncolytic viruses are capable of selectively replicating in and triggering the death of or slowing the growth of a cancer cell. In some cases, oncolytic viruses have no effect or a minimal effect on non-cancer cells. An oncolytic virus includes but is not limited to an oncolytic adenovirus, oncolytic Herpes Simplex Viruses, oncolytic retrovirus, oncolytic parvovirus, oncolytic vaccinia virus, oncolytic Sinbis virus, oncolytic influenza virus, or oncolytic RNA virus (e.g., oncolytic reovirus, oncolytic Newcastle Disease Virus (NDV), oncolytic measles virus, or oncolytic vesicular stomatitis virus (VSV)).
Other exemplary combination therapy, treatment and/or agents include anti-allergenic agents, anti-emetics, analgesics and adjunct therapies. In some embodiments, the additional agent includes cytoprotective agents, such as neuroprotectants, free-radical scavengers, cardioprotectors, anthracycline extravasation neutralizers and nutrients.
In some embodiments, an antibody used as an additional agent is conjugated or otherwise bound to a therapeutic agent, e.g., a chemotherapeutic agent (e.g., Cytoxan, fludarabine, histone deacetylase inhibitor, demethylating agent, peptide vaccine, anti-tumor antibiotic, tyrosine kinase inhibitor, alkylating agent, anti-microtubule or anti-mitotic agent), anti-allergic agent, anti-nausea agent (or anti-emetic), pain reliever, or cytoprotective agent described herein. In some embodiments, the additional agent is an antibody-drug conjugate.
In some embodiments, the additional agent can modulate, inhibit or stimulate particular factors at the DNA, RNA or protein levels, to enhance or boost the efficacy of the BCMA-binding recombinant receptors, cells and/or compositions provided herein. In some embodiments, the additional agent can modulate the factors at the nucleic acid level, e.g., DNA or RNA, within the administered cells, e.g., cells engineered to express recombinant receptors, e.g., CAR. In some embodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, or a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription-activator like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), can be used to inhibit expression of an inhibitory molecule in the engineered cell, e.g., CAR-expressing cell. In some embodiments the inhibitor is an shRNA. In some embodiments, the inhibitory molecule is inhibited within the engineered cell, e.g., CAR-expressing cell. In some embodiments, a nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of the molecule that modulates or regulates, e.g., inhibits, T-cell function is operably linked to a promoter, e.g., a HI- or a U6-derived promoter such that the dsRNA molecule that inhibits expression of the inhibitory molecule is expressed within the engineered cell, e.g., CAR-expressing cell. See, e.g., Brummelkamp T R, et al. (2002) Science 296: 550-553; Miyagishi M, et al. (2002) Nat. Biotechnol. 19: 497-500.
In some embodiments, the additional agent is capable of disrupting the gene encoding an inhibitory molecule, such as any immune checkpoint inhibitors described herein. In some embodiments, disruption is by deletion, e.g., deletion of an entire gene, exon, or region, and/or replacement with an exogenous sequence, and/or by mutation, e.g., frameshift or missense mutation, within the gene, typically within an exon of the gene. In some embodiments, the disruption results in a premature stop codon being incorporated into the gene, such that the inhibitory molecule is not expressed or is not expressed in a form that is capable of being expressed on the cells surface and/or capable of mediating cell signaling. The disruption is generally carried out at the DNA level. The disruption generally is permanent, irreversible, or not transient.
In some aspects, the disruption is carried out by gene editing, such as using a DNA binding protein or DNA-binding nucleic acid, which specifically binds to or hybridizes to the gene at a region targeted for disruption. In some aspects, the protein or nucleic acid is coupled to or complexed with a nuclease, such as in a chimeric or fusion protein. For example, in some embodiments, the disruption is effected using a fusion comprising a DNA-targeting protein and a nuclease, such as a Zinc Finger Nuclease (ZFN) or TAL-effector nuclease (TALEN), or an RNA-guided nuclease such as a clustered regularly interspersed short palindromic nucleic acid (CRISPR)-Cas system, such as CRISPR-Cas9 system, specific for the gene being disrupted. In some embodiments, methods of producing or generating genetically engineered cells, e.g., CAR-expressing cells, include introducing into a population of cells nucleic acid molecules encoding a genetically engineered antigen receptor (e.g. CAR) and nucleic acid molecules encoding an agent targeting an inhibitory molecule that is a gene editing nuclease, such as a fusion of a DNA-targeting protein and a nuclease such as a ZFN or a TALEN, or an RNA-guided nuclease such as of the CRISPR-Cas9 system, specific for an inhibitory molecule.
Any of the additional agents described herein can be prepared and administered as combination therapy with the BCMA-binding recombinant receptor (e.g., chimeric antigen receptor) and/or engineered cells expressing said molecules (e.g., recombinant receptor) described herein, such as in pharmaceutical compositions comprising one or more agents of the combination therapy and a pharmaceutically acceptable carrier, such as any described herein. In some embodiments, the BCMA-binding recombinant receptor (e.g., chimeric antigen receptor), engineered cells expressing said molecules (e.g., recombinant receptor), plurality of engineered cells expressing said molecules (e.g., recombinant receptor) can be administered simultaneously, concurrently or sequentially, in any order with the additional agents, therapy or treatment, wherein such administration provides therapeutically effective levels each of the agents in the body of the subject. In some embodiments, the additional agent can be co-administered with the BCMA-binding recombinant receptors, cells and/or compositions described herein, for example, as part of the same pharmaceutical composition or using the same method of delivery. In some embodiments, the additional agent is administered simultaneously with the BCMA-binding recombinant receptors, cells and/or compositions described herein, but in separate compositions. In some embodiments, the additional agent is an additional engineered cell, e.g., cell engineered to express a different recombinant receptor, and is administered in the same composition or in a separate composition. In some embodiments, the additional agent is incubated with the engineered cell, e.g., CAR-expressing cells, prior to administration of the cells.
In some examples, the one or more additional agents are administered subsequent to or prior to the administration of the BCMA-binding recombinant receptors, cells and/or compositions described herein, separated by a selected time period. In some examples, the time period is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months. In some examples, the one or more additional agents are administered multiple times and/or the BCMA-binding recombinant receptors, cells and/or compositions described herein, is administered multiple times. For example, in some embodiments, the additional agent is administered prior to the BCMA-binding recombinant receptors, cells and/or compositions described herein, e.g., two weeks, 12 days, 10 days, 8 days, one week, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day before the administration. For example, in some embodiments, the additional agent is administered after the BCMA-binding recombinant receptors, cells and/or compositions described herein, e.g., two weeks, 12 days, 10 days, 8 days, one week, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day after the administration.
The dose of the additional agent can be any therapeutically effective amount, e.g., any dose amount described herein, and the appropriate dosage of the additional agent may depend on the type of disease to be treated, the type, dose and/or frequency of the recombinant receptor, cell and/or composition administered, the severity and course of the disease, whether the recombinant receptor, cell and/or composition is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the recombinant receptor, cell and/or composition, and the discretion of the attending physician. The recombinant receptor, cell and/or composition and/or the additional agent and/or therapy can be administered to the patient at one time, repeated or administered over a series of treatments.
VI. ARTICLES OF MANUFACTURE OR KITS
Also provided are articles of manufacture or kit containing the provided recombinant receptors (e.g., CARs), genetically engineered cells, and/or compositions comprising the same. The articles of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, test tubes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container has a sterile access port. Exemplary containers include an intravenous solution bags, vials, including those with stoppers pierceable by a needle for injection. The article of manufacture or kit may further include a package insert indicating that the compositions can be used to treat a particular condition such as a condition described herein (e.g., multiple myeloma). Alternatively, or additionally, the article of manufacture or kit may further include another or the same container comprising a pharmaceutically-acceptable buffer. It may further include other materials such as other buffers, diluents, filters, needles, and/or syringes.
The label or package insert may indicate that the composition is used for treating the BCMA-expressing or BCMA-associated disease, disorder or condition in an individual. The label or a package insert, which is on or associated with the container, may indicate directions for reconstitution and/or use of the formulation. The label or package insert may further indicate that the formulation is useful or intended for subcutaneous, intravenous, or other modes of administration for treating or preventing a BCMA-expressing or BCMA-associated disease, disorder or condition in an individual.
The container in some embodiments holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition. The article of manufacture or kit may include (a) a first container with a composition contained therein (i.e., first medicament), wherein the composition includes the antibody (e.g., anti-BCMA antibody) or antigen-binding fragment thereof or recombinant receptor (e.g., CAR); and (b) a second container with a composition contained therein (i.e., second medicament), wherein the composition includes a further agent, such as a cytotoxic or otherwise therapeutic agent, and which article or kit further comprises instructions on the label or package insert for treating the subject with the second medicament, in an effective amount.
VII. DEFINITIONS
As used herein, reference to a “corresponding form” of an antibody means that when comparing a property or activity of two antibodies, the property is compared using the same form of the antibody. For example, if it is stated that an antibody has greater activity compared to the activity of the corresponding form of a first antibody, that means that a particular form, such as an scFv of that antibody, has greater activity compared to the scFv form of the first antibody.
The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.
The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).
An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
“Isolated nucleic acid encoding an anti-BCMA antibody” refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the antibodies and antibody chains and other peptides, e.g., linkers and BCMA-binding peptides, may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
As used herein, “percent (%) amino acid sequence identity” and “percent identity” and “sequence identity” when used with respect to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence (e.g., the subject antibody or fragment) that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
An amino acid substitution may include replacement of one amino acid in a polypeptide with another amino acid. Amino acid substitutions may be introduced into a binding molecule, e.g., antibody, of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, or decreased immunogenicity.
Amino acids generally can be grouped according to the following common side-chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro;
(6) aromatic: Trp, Tyr, Phe.
Non-conservative amino acid substitutions will involve exchanging a member of one of these classes for another class.
The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects, embodiments, and variations described herein include “comprising,” “consisting,” and/or “consisting essentially of” aspects, embodiments and variations.
Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.
The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.
As used herein, a “composition” refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
As used herein, a statement that a cell or population of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.
As used herein, a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.
Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
VIII. EXEMPLARY EMBODIMENTS
Among the embodiments provided herein are:
1. A polynucleotide encoding a chimeric antigen receptor, comprising nucleic acid encoding: (a) an extracellular antigen-binding domain that specifically recognizes an antigen; (b) a spacer of at least 125 amino acids in length; (c) a transmembrane domain; and (d) an intracellular signaling region, wherein following expression of the polynucleotide in a cell, the transcribed RNA, optionally messenger RNA (mRNA), from the polynucleotide, exhibits at least 70%, 75%, 80%, 85%, 90%, or 95% RNA homogeneity.
2. The polynucleotide of embodiment 1, wherein the spacer is derived from an immunoglobulin.
3. The polynucleotide of embodiment 1 or embodiment 2, wherein the spacer comprises a sequence of a hinge region, a CH2 and CH3 region.
4. The polynucleotide of any of embodiments 1-3, wherein the encoded spacer is or comprises (i) the sequence set forth in SEQ ID NO: 649; (ii) a functional variant of SEQ ID NO:649 that has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:649; or (iii) a contiguous portion of (i) or (ii) that is at least 125 amino acids in length.
5. The polynucleotide of any of embodiments 1-4, wherein the nucleic acid encoding the spacer comprises at least one modified splice donor and/or splice acceptor site, said modified splice donor and/or acceptor site comprising one or more nucleotide modifications corresponding to a reference splice donor site and/or reference splice acceptor site contained in the sequence set forth in SEQ ID NO:621.
6. The polynucleotide of embodiment 5, wherein the one or more nucleotide modifications comprise an insertion, deletion, substitution or combinations thereof.
7. The polynucleotide of embodiment 5 or embodiment 6, wherein the reference splice acceptor and/or reference splice donor sites are canonical, non-canonical, or cryptic splice sites.
8. The polynucleotide of any of embodiment 5-7, wherein:
the reference splice donor and/or reference splice acceptor site(s) has a splice site prediction score of at least or about 0.4, 0.5, 0.6, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.99, or 1.0; and/or
the reference splice donor and/or reference splice acceptor site(s) is/are predicted to be involved in a splice event with a probability of at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%.
9. The polynucleotide of any of embodiments 5-8, wherein:
the reference splice donor site comprises the sequence aatctaagtacggac (SEQ ID NO: 705), tcaactggtacgtgg (SEQ ID NO:706), acaattagtaaggca (SEQ ID NO:707) and/or accacaggtgtatac (SEQ ID NO:708); and/or
the reference splice acceptor site comprises the sequence
(SEQ ID NO: 742)
aagtttctttctgtattccaggctgaccgtggataaatctc
and/or
(SEQ ID NO: 743)
gggcaacgtgttctcttgcagtgtcatgcacgaagccctgc.
10. The polynucleotide of any of embodiment 5-8, wherein:
the reference splice donor and/or reference splice acceptor site(s) has a splice site prediction score of at least or about 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.99, or 1.0; and/or
the reference splice donor and/or reference splice acceptor site(s) is/are predicted to be involved in a splice event with a probability of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%.
11. The polynucleotide of any of embodiments 5-8 and 10, wherein:
the reference splice donor site comprises the sequence tcaactggtacgtgg (SEQ ID NO:706); and/or
the reference splice acceptor site comprises the sequence aagtttctttctgtattccaggctgaccgtggataaatctc (SEQ ID NO:742).
12. The polynucleotide of any of embodiments 5-11, wherein at least one of the one or more nucleotide modifications are within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues of the splice site junction of the reference splice acceptor and/or reference splice donor site.
13. The polynucleotide of any of embodiments 5-12, wherein the one or more nucleotide modifications is silent and/or results in a degenerate codon compared to SEQ ID NO:621 and/or does not change the amino acid sequence of the encoded spacer.
14. The polynucleotide of any of embodiments 5-9 and 12-13, wherein:
the modified splice donor site is set forth in agtctaaatacggac (SEQ ID NO:661), tcaactggtatgtgg (SEQ ID NO:662), accatctccaaggcc (SEQ ID NO:663) and/or gccccaggtttacac (SEQ ID NO:664); and/or
the modified splice acceptor site is set forth in cagtttcttcctgtatagtagactcaccgtggataaatcaa (SEQ ID NO:672), gggcaacgtgttcagctgcagcgtgatgcacgaggccctgc (SEQ ID NO: 673) and/or cgccttgtcctccttgtcccgctcctcctgttgccggacct (SEQ ID NO:766).
15. The polynucleotide of any of embodiments 5-14, wherein the modified splice donor site is set forth in tcaactggtatgtgg (SEQ ID NO:662) and/or the modified acceptor site is set forth in
(SEQ ID NO: 672)
cagtttcttcctgtatagtagactcaccgtggataaatcaa
and/or
(SEQ ID NO: 766)
cgccttgtcctccttgtcccgctcctcctgttgccggacct.
16. The polynucleotide of any of embodiments 1-15, wherein the spacer is encoded by a sequence of nucleotide set forth in SEQ ID NO:622 or a portion thereof.
17. A polynucleotide encoding a chimeric antigen receptor, wherein the polynucleotide comprises nucleic acid encoding: (a) an extracellular antigen-binding domain that specifically recognizes an antigen; (b) a spacer, wherein the encoding nucleic acid is or comprises the sequence set forth in SEQ ID NO:622 or encodes a sequence of amino acids set forth in SEQ ID NO:649; (c) a transmembrane domain; and (d) an intracellular signaling region.
18. A polynucleotide encoding a chimeric antigen receptor, wherein the polynucleotide comprises nucleic acid encoding: (a) an extracellular antigen-binding domain that specifically recognizes an antigen; (b) a spacer, wherein the encoding nucleic acid consists or consists essentially of the sequence set forth in SEQ ID NO:622 or encodes a sequence of amino acids set forth in SEQ ID NO:649; (c) a transmembrane domain; and (d) an intracellular signaling region.
19. The polynucleotide of embodiment 17 or embodiment 18, wherein following expression of the polynucleotide in a cell, the transcribed RNA, optionally messenger RNA (mRNA), from the polynucleotide, exhibits at least 70%, 75%, 80%, 85%, 90%, or 95% RNA homogeneity.
20. The polynucleotide of any of embodiments 1-19, wherein, following expression in a cell, the transcribed RNA, optionally messenger RNA (mRNA), from the polynucleotide exhibits reduced heterogeneity compared to the heterogeneity of the mRNA transcribed from a reference polynucleotide, said reference polynucleotide encoding the same amino acid sequence as the polynucleotide, wherein the reference polynucleotide differs by the presence of one or more splice donor site and/or one or more splice acceptor site in the nucleic acid encoding the spacer and/or comprises one or more nucleotide modifications compared to the polynucleotide.
21. The polynucleotide of embodiment 20, wherein the RNA heterogeneity is reduced by greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more.
22. The polynucleotide of embodiment 20 or embodiment 21, wherein the transcribed RNA, optionally messenger RNA (mRNA), from the reference polynucleotide exhibits greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more RNA heterogeneity.
23. The polynucleotide of any of embodiments 1-22, wherein the RNA homogeneity and/or heterogeneity is determined by agarose gel electrophoresis, chip-based capillary electrophoresis, analytical ultracentrifugation, field flow fractionation, or liquid chromatography.
24. The polynucleotide of any of embodiments 1-23, wherein the polynucleotide is codon-optimized.
25. The polynucleotide of any of embodiments 1-24, wherein the antigen is associated with the disease or condition or expressed in cells of the environment of a lesion associated with the disease or condition.
26. The polynucleotide of any of embodiments 1-25, wherein the disease or condition is a cancer.
27. The polynucleotide of any of embodiments 1-26, wherein the disease or condition is a myeloma, leukemia or lymphoma.
28. The polynucleotide of any of embodiments 1-27, wherein the antigen is ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2/neu (receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folate binding protein (FBP), FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kinase insert domain receptor (kdr), kappa light chain, Lewis Y, L1-cell adhesion molecule, (L1-CAM), Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentially expressed antigen of melanoma (PRAME), survivin, TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2D ligands, CD44v6, dual antigen, a cancer-testes antigen, mesothelin, murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, 0-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, CCL-1, CD138, a pathogen-specific antigen.
29. The polynucleotide of embodiment 28, wherein the antigen is B cell maturation antigen (BCMA).
30. The polynucleotide of any of embodiments 1-29, wherein the antigen-binding domain is an antibody fragment comprising a variable heavy chain (VH) and a variable light chain (VL) region.
31. The polynucleotide of embodiment 30, wherein:
the VH region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the VH region amino acid sequence set forth in any of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-832; and/or
the VL region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VL region amino acid sequence set forth in any of SEQ ID NOs:116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849.
32. The polynucleotide of embodiment 30 or embodiment 31, wherein:
the VH region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the VH region amino acid sequence set forth in any of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609 or 617; and/or the VL region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VL region amino acid sequence set forth in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610 or 618.
33. The polynucleotide of embodiment 30 or embodiment 31, wherein:
the VH region is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region amino acid sequence selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-832; and/or
the VL region is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region amino acid sequence selected from any one of SEQ ID NOs:116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849.
34. The polynucleotide of any of embodiments 30-33, wherein:
the VH region is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region amino acid sequence selected from any one of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609 or 617; and/or the VL region is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region amino acid sequence selected from any one of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610 or 618.
The polynucleotide of any of embodiments 30-34, wherein:
the VH region is or comprises (a) a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence selected from any one of SEQ ID NOs:1-3, 140-144, 288, 289, 294, 295, 507, 532, 593, 596, 604, 611; and/or (b) a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence selected from any one of SEQ ID NOs:4-6, 145-148, 290, 291, 296, 297, 372-374, 513, 551, 594, 597, 605, 612; and (c) a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence selected from any one of SEQ ID NOs:7-11, 149-157, 279-287, 292, 293, 376-378, 517, 595, 606, 613; and/or
the VL region is or comprises (a) a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence selected from any one of SEQ ID NOs:26-36, 174-178, 302, 303, 380-392, 394-398, 589, 601, 607 or 614; (b) a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence selected from any one of SEQ ID NOs:37-46, 179-183, 304, 305, 399-409, 411-414, 590, 602, 608 or 615; and (c) a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence selected from any one of SEQ ID NOs:47-58, 184-194, 306, 307, 415-427, 429-433, 591, or 603.
36. The polynucleotide of any of embodiments 30-35, wherein:
the VH region is or comprises (a) a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 3, 141, 143, 144, 288, 289, 507, 593, 604, 611; and/or (b) a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence selected from any one of SEQ ID NOs: 4, 5, 6, 145, 147, 148, 290, 291, 372, 513, 594, 605 or 612; and (c) a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence selected from any one of SEQ ID NOs: 7, 8, 9, 10, 149, 153, 154, 155, 156, 157, 292, 293, 376, 517, 595, 606 or 613; and/or
the VL region is or comprises (a) a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence selected from any one of SEQ ID NOs: 26, 27, 28, 30, 31, 33, 34, 174, 176, 177, 178, 302, 303, 380, 381, 382, 589, 601, 607 or 614; (b) a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence selected from any one of SEQ ID NOs: 37, 38, 39, 41, 43, 44, 179, 181, 182, 183, 304, 305, 399, 400, 401, 402, 590, 602, 608 or 615; and (c) a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence selected from any one of SEQ ID NOs: 47, 48, 49, 51, 52, 55, 56, 185, 189, 190, 191, 192, 193, 194, 306, 307, 415, 417, 418, 421, 591, or 603.
37. The polynucleotide of any of embodiments 30-36, wherein the VH region comprises a CDR-H1, CDR-H2, and CDR-H3, selected from:
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:1, 4, and 7, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 8, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 9, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 10, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 11, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:140, 145, and 149, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:141, 145, and 149, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:141, 145, and 150, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:142, 146, and 151, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 152, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:143, 147, and 153, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:144, 148, and 154, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 156, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 157, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 6, and 376, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 372, and 376, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 376, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 377, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 373, and 152, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 378, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 374, and 9, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:507, 513, and 517, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:604, 605, and 606, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:288, 290, and 292, respectively; or a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:289, 291, and 293, respectively.
38. The polynucleotide of any of embodiments 30-37, wherein the VH region comprises a CDR-H1, CDR-H2, and CDR-H3, selected from:
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:1, 4, and 7, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 8, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 9, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 10, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:141, 145, and 149, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:143, 147, and 153, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:144, 148, and 154, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 156, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 157, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 6, and 376, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 372, and 376, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 376, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:507, 513, and 517, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:604, 605, and 606, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:288, 290, and 292, respectively; or a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:289, 291, and 293, respectively.
39. The polynucleotide of any of embodiments 30-38, wherein the VH region is or comprises the amino acid sequence set forth in any of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-832.
40. The polynucleotide of any of embodiments 30-39, wherein the VH region is or comprises the amino acid sequence set forth in any of SEQ ID NOs:110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609 or 617.
41. The polynucleotide of any of embodiments 30-40, wherein:
the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively; or
the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively.
42. The polynucleotide of any of embodiments 30-41, wherein the VH region is or comprises the amino acid sequence set forth in SEQ ID NO:617.
43. The polynucleotide of any one of embodiments 30-42, wherein the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 selected from:
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:26, 37, and 47, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:27, 38, and 48, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:28, 39, and 49, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:29, 40, and 50, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:30, 39, and 51, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:31, 41, and 52, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:32, 42, and 53, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:30, 39, and 54, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:33, 43, and 55, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:34, 44, and 56, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:35, 45, and 57, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:36, 46, and 58, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 184, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 185, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 186, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 187, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:175, 180, and 188, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 189, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:176, 181, and 190, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:177, 182, and 191, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 192, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:178, 183, and 193, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:178, 183, and 194, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:30, 399, and 415, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:380, 400, and 416, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:33, 43, and 421, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:381, 401, and 417, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:382, 402, and 418, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:383, 403, and 419, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:384, 39, and 54, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:385, 180, and 58, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:175, 180, and 188, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:386, 404, and 420, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:387, 405, and 422, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:388, 406, and 423, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:388, 407, and 424, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:389, 408, and 425, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:390, 183, and 193, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:391, 409, and 426, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:392, 40, and 427, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:394, 39, and 429, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:395, 411, and 430, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:396, 412, and 431, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:396, 412, and 58, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:397, 413, and 432, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:398, 414, and 433, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:589, 590, and 591, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:607, 608, and 591, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 302, 304, and 306, respectively; or
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:303, 305, and 307, respectively.
44. The polynucleotide of any one of embodiments 30-43, wherein the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 selected from:
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:26, 37, and 47, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:27, 38, and 48, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:28, 39, and 49, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:30, 39, and 51, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:31, 41, and 52, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:33, 43, and 55, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:34, 44, and 56, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 185, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 189, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:176, 181, and 190, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:177, 182, and 191, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 192, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:178, 183, and 193, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:178, 183, and 194, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:30, 399, and 415, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:380, 400, and 416, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:33, 43, and 421, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:381, 401, and 417, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:382, 402, and 418, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:589, 590, and 591, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:607, 608, and 591, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 302, 304, and 306, respectively; or
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:303, 305, and 307, respectively.
45. The polynucleotide of any of embodiments 30-44, wherein the VL region is or comprises the amino acid sequence set forth in any of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849.
46. The polynucleotide of any of embodiments 30-45, wherein the VL region is or comprises the amino acid sequence set forth in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610 or 618.
47. The polynucleotide of any of embodiments 30-46, wherein:
the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively; or
the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively.
48. The polynucleotide of any of embodiments 30-47, wherein the VL region is or comprises the amino acid sequence set forth in SEQ ID NO:618.
49. The polynucleotide of any of embodiments 30-48, wherein:
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 116, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 116, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:111 and 117, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:111 and 117, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 119, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 119, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 120, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 120, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 121, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 121, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 122, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 122, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 123, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 123, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:112 and 124, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:112 and 124, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:113 and 125, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:113 and 125, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:114 and 126, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:114 and 126, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:115 and 127, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 127, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:247 and 257, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:247 and 257, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:248 and 258, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:248 and 258, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:249 and 259, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:249 and 259, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:250 and 260, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:250 and 260, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:251 and 261, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:251 and 261, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:252 and 262, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:252 and 262, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:253 and 263, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:253 and 263, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:254 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:254 and 264, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:255 and 265, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:255 and 265, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:256 and 266, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 266, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:256 and 267, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 267, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:518 and 534, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:518 and 534, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:519 and 535, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 535, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:115 and 536, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 536, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:520 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:520 and 264, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:521 and 537, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:521 and 537, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:522 and 538, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 538, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:523 and 539, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:523 and 539, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:519 and 540, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 540, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:524 and 541, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:524 and 541, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:525 and 261, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:525 and 261, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:526 and 542, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:526 and 542, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:527 and 543, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:527 and 543, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:528 and 544, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:528 and 544, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:529 and 545, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:529 and 545, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:528 and 546, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:528 and 546, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:522 and 547, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 547, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:256 and 548, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 548, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:530 and 549, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:530 and 549, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:531 and 550, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:531 and 550, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:519 and 552, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 552, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 553, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 553, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:533 and 554, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:533 and 554, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:115 and 555, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 555, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:524 and 556, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:524 and 556, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:519 and 557, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 557, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:609 and 610, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:609 and 610, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:617 and 618, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:324 and 326, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:324 and 326, respectively; or
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:325 and 327, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:325 and 327, respectively.
50. A polynucleotide of any of embodiments 30-49, wherein:
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 116, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 116, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:111 and 117, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:111 and 117, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 120, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 120, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 121, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 121, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:112 and 124, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:112 and 124, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:113 and 125, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:113 and 125, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:248 and 258, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:248 and 258, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:252 and 262, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:252 and 262, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:253 and 263, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:253 and 263, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:254 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:254 and 264, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:255 and 265, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:255 and 265, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:256 and 266, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 266, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:256 and 267, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 267, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:518 and 534, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:518 and 534, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:519 and 535, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 535, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:115 and 536, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 536, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:520 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:520 and 264, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:521 and 537, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:521 and 537, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:522 and 538, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 538, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:609 and 610, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:609 and 610, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:617 and 618, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:324 and 326, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:324 and 326, respectively; or
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:325 and 327, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:325 and 327, respectively.
51. The polynucleotide of any of embodiments 30-50, wherein the fragment comprises an scFv.
52. The polynucleotide of any of embodiments 30-51, when the VH region and the VL region are joined by a flexible linker.
53. The polynucleotide of embodiment 52, wherein the scFv comprises a linker comprising the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:361).
54. The polynucleotide of any of embodiments 30-53, wherein the VH region is amino-terminal to the VL region.
55. The polynucleotide of any of embodiments 30-54, wherein the antigen-binding domain comprises the amino acid sequence selected from any one of SEQ ID NOs:128-139, 268-278, 329, 442, 478, 558-576, 578-583, 585, or 769-771 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence selected from any one of SEQ ID NOs: 128-139, 268-278, 329, 442, 478, 558-576, 578-583, 585, or 769-771.
56. The polynucleotide of any of embodiments 30-55, wherein the antigen-binding domain comprises the amino acid sequence selected from any one of SEQ ID NOs:128-130, 132, 133, 136, 137, 269, 273-278, 329, 442, 478, 558-563 or 585 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence selected from any one of SEQ ID NOs: 128-130, 132, 133, 136, 137, 269, 273-278, 329, 442, 478, 558-563 or 585.
57. The polynucleotide of any of embodiments 30-56, wherein the nucleic acid encoding the antigen-binding domain comprises (a) the sequence of nucleotides set forth in any of SEQ ID NOS: 330-352, 647, 648, 716 or 718; (b) a sequence of nucleotides that has at least 90% sequence identity to any of SEQ ID NOS: 330-352, 647, 648, 716 or 718; or (c) a degenerate sequence of (a) or (b).
58. The polynucleotide of any of embodiments 30-57, wherein the nucleic acid encoding the antigen-binding domain comprises (a) the sequence of nucleotides set forth in any of SEQ ID NOS: 352, 647, 648, 716, or 718; (b) a sequence of nucleotides that has at least 90% sequence identity to any of SEQ ID NOS: 352, 647, 648, 716, or 718; or (c) a degenerate sequence of (a) or (b).
59. The polynucleotide of any of embodiments 30-57, wherein the nucleic acid encoding the antigen-binding domain is codon-optimized.
60. The polynucleotide of any of embodiments 30-57, wherein the nucleic acid encoding the antigen-binding domain comprises the sequence of nucleotides set forth in any of SEQ ID NO: 440, 460, 715, 717 or 719.
61. The polynucleotide of any of embodiments 30-60, wherein the nucleic acid encoding the antigen-binding domain comprises the sequence of nucleotides set forth in SEQ ID NO:460.
62. The polynucleotide of any of embodiments 30-53, wherein the VH region is carboxy-terminal to the VL region.
63. The polynucleotide of any of embodiments 51-53 and 62, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NOs:328 or 586, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:328 or 586.
64. The polynucleotide of any of embodiments 1-59, wherein the intracellular signaling region comprises an activating cytoplasmic signaling domain.
65. The polynucleotide of embodiment 60, wherein the activating cytoplasmic signaling domain is capable of inducing a primary activation signal in a T cell, is a T cell receptor (TCR) component and/or comprises an immunoreceptor tyrosine-based activation motif (ITAM).
66. The polynucleotide of embodiment 64 or embodiment 65, wherein the activating cytoplasmic signaling domain is or comprises a cytoplasmic signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain or a functional variant or signaling portion thereof.
67. The polynucleotide of any of embodiments 64-66, wherein the activating cytoplasmic domain is human or is derived from a human protein.
68. The polynucleotide of any of embodiments 64-67, wherein the activating cytoplasmic domain is or comprises the sequence set forth in SEQ ID NO:628 or a sequence of amino acids that has at least 90% sequence identity to SEQ ID NO:628.
69. The polynucleotide of any of embodiments 64-68, wherein the nucleic acid encoding the activating cytoplasmic domain is or comprises the sequence set forth in SEQ ID NO:627 or is a codon-optimized sequence and/or degenerate sequence thereof.
70. The polynucleotide of any of embodiments 64-69, wherein the nucleic acid encoding the activating cytoplasmic signaling domain is or comprises the sequence set forth in SEQ ID NO:652.
71. The polynucleotide of any of embodiments 64-70, wherein the intracellular signaling region further comprises a costimulatory signaling region.
72. The polynucleotide of embodiment 71, wherein the costimulatory signaling region comprises an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof.
73. The polynucleotide of embodiment 71 or embodiment 72, wherein the costimulatory signaling region comprises an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof.
74. The polynucleotide of any of embodiments 71-73, wherein the costimulatory signaling region comprises an intracellular signaling domain of 4-1BB.
75. The polynucleotide of any of embodiments 71-74, wherein the costimulatory signaling region is human or is derived from a human protein.
76. The polynucleotide of any of embodiments 71-75, wherein the costimulatory signaling region is or comprises the sequence set forth in SEQ ID NO:626 or a sequence of amino acids that exhibits at least 90% sequence identity to the sequence set forth in SEQ ID NO: 626.
77. The polynucleotide of any of embodiments 71-76, wherein the nucleic acid encoding the costimulatory region is or comprises the sequence set forth in SEQ ID NO:625 or is a codon-optimized sequence and/or degenerate sequence thereof.
78. The polynucleotide of any of embodiments 71-77, wherein the nucleic acid encoding the costimulatory signaling region comprises the sequence set forth in SEQ ID NO:681.
79. The polynucleotide of any of embodiments 71-78, wherein the costimulatory signaling region is between the transmembrane domain and the intracellular signaling region.
80. The polynucleotide of any of embodiments 1-79, wherein the transmembrane domain is or comprises a transmembrane domain derived from CD4, CD28, or CD8.
81. The polynucleotide of embodiment 80, wherein the transmembrane domain is or comprises a transmembrane domain derived from a CD28.
82. The polynucleotide of any of embodiments 1-81, wherein the transmembrane domain is human or is derived from a human protein.
83. The polynucleotide of any of embodiments 1-82, wherein the transmembrane domain is or comprises the sequence set forth in SEQ ID NO:624 or a sequence of amino acids that exhibits at least 90% sequence identity to SEQ ID NO:624.
84. The polynucleotide of any of embodiments 1-83, wherein the nucleic acid encoding the transmembrane domain is or comprises the sequence set forth in SEQ ID NO:623 or is a codon-optimized sequence and/or degenerate sequence thereof.
85. The polynucleotide of embodiment 35a, wherein the nucleic acid encoding the transmembrane domain comprises the sequence set forth in SEQ ID NO:688.
86. The polynucleotide of any of embodiments 1-85, wherein the encoded chimeric antigen receptor comprises from its N to C terminus in order: the antigen-binding domain, the spacer, the transmembrane domain and the intracellular signaling domain.
87. The polynucleotide of any of embodiments 1-86, wherein the polynucleotide further encodes a truncated receptor
88. A chimeric antigen receptor encoded by the polynucleotide of any of embodiments 1-87.
89. A chimeric antigen receptor comprising: (a) an extracellular antigen-binding domain that specifically recognizes B cell maturation antigen (BCMA); (b) a spacer of at least 125 amino acids in length; (c) a transmembrane domain; and (d) an intracellular signaling region.
90. The chimeric antigen receptor of embodiment 89, wherein the spacer is derived from an immunoglobulin.
91. The chimeric antigen receptor of embodiment 89 or embodiment 90, wherein the spacer comprises a sequence of a hinge region, a CH2 and CH3 region.
92. The chimeric antigen receptor of embodiment 91, wherein one of more of the hinge, CH2 and CH3 is derived all or in part from IgG4 or IgG2, optionally human IgG4 or human IgG2.
93. The chimeric antigen receptor of embodiment 90 or embodiment 91, wherein the hinge, CH2 and CH3 is derived from IgG4.
94. The chimeric antigen receptor of embodiment 90 or embodiment 91, wherein one or more of the hinge, CH2 and CH3 is chimeric and comprises sequence derived from IgG4 and IgG2.
95. The chimeric antigen receptor of embodiment 94, wherein the spacer comprises an IgG4/2 chimeric hinge or a modified IgG4 comprising at least one amino acid replacement compared to human IgG4, an IgG2/4 chimeric CH2, and an IgG4 CH3 region.
96. The chimeric antigen receptor of any of embodiments 89-92 and 94-95, wherein the spacer is or comprises (i) the sequence set forth in SEQ ID NO: 649; (ii) a functional variant of SEQ ID NO:649 that has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:649; or (iii) a contiguous portion of (i) or (ii) that is at least 125 amino acids in length.
97. The chimeric antigen receptor of any of embodiments 89-92 and 94-96, wherein the encoded spacer is or comprises the sequence set forth in SEQ ID NO: 649.
98. A chimeric antigen receptor comprising: (a) an extracellular antigen-binding domain that specifically recognizes B cell maturation antigen (BCMA); (b) a spacer set forth in SEQ ID NO:649; (c) a transmembrane domain; and (d) an intracellular signaling region.
99. The chimeric antigen receptor of any of embodiments 89-98, wherein the antigen-binding domain is an antibody fragment comprising a variable heavy chain (VH) and a variable light chain (VL) region.
100. The chimeric antigen receptor of embodiment 99, wherein:
the VH region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the VH region amino acid sequence set forth in any of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609 or 617; and/or
the VL region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VL region amino acid sequence set forth in any of SEQ ID NOs:116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849.
101. The chimeric antigen receptor of embodiment 99 or embodiment 100, wherein:
the VH region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the VH region amino acid sequence set forth in any of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609 or 617; and/or
the VL region is or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VL region amino acid sequence set forth in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610 or 618.
102. The chimeric antigen receptor of embodiment 99 or embodiment 100, wherein:
the VH region is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region amino acid sequence selected from any one of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-832; and/or
the VL region is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region amino acid sequence selected from any one of SEQ ID NOs:116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849.
103. The chimeric antigen receptor of any of embodiments 99-102, wherein:
the VH region is or comprises a CDR-H1, CDR-H2 and CDR-H3 contained within the VH region amino acid sequence selected from any one of SEQ ID NOs: 110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609 or 617; and/or
the VL region is or comprises a CDR-L1, CDR-L2 and CDR-L3 contained within the VL region amino acid sequence selected from any one of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610 or 618.
104. The chimeric antigen receptor of any of embodiments 99-103, wherein:
the VH region is or comprises (a) a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence selected from any one of SEQ ID NOs:1-3, 140-144, 288, 289, 294, 295, 507, 532, 593, 596, 604, 611; and/or (b) a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence selected from any one of SEQ ID NOs:4-6, 145-148, 290, 291, 296, 297, 372-374, 513, 551, 594, 597, 605, 612; and (c) a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence selected from any one of SEQ ID NOs:7-11, 149-157, 279-287, 292, 293, 376-378, 517, 595, 606, 613; and/or
the VL region is or comprises (a) a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence selected from any one of SEQ ID NOs:26-36, 174-178, 302, 303, 380-392, 394-398, 589, 601, 607 or 614; (b) a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence selected from any one of SEQ ID NOs:37-46, 179-183, 304, 305, 399-409, 411-414, 590, 602, 608 or 615; and (c) a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence selected from any one of SEQ ID NOs:47-58, 184-194, 306, 307, 415-427, 429-433, 591, or 603.
105. The chimeric antigen receptor of any of embodiments 99-104, wherein:
the VH region is or comprises (a) a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 3, 141, 143, 144, 288, 289, 507, 593, 604, 611; and/or (b) a heavy chain complementarity determining region 2 (CDR-H2) comprising the amino acid sequence selected from any one of SEQ ID NOs: 4, 5, 6, 145, 147, 148, 290, 291, 372, 513, 594, 605 or 612; and (c) a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence selected from any one of SEQ ID NOs: 7, 8, 9, 10, 149, 153, 154, 155, 156, 157, 292, 293, 376, 517, 595, 606 or 613; and/or
the VL region is or comprises (a) a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence selected from any one of SEQ ID NOs: 26, 27, 28, 30, 31, 33, 34, 174, 176, 177, 178, 302, 303, 380, 381, 382, 589, 601, 607 or 614; (b) a light chain complementarity determining region 2 (CDR-L2) comprising the amino acid sequence selected from any one of SEQ ID NOs: 37, 38, 39, 41, 43, 44, 179, 181, 182, 183, 304, 305, 399, 400, 401, 402, 590, 602, 608 or 615; and (c) a light chain complementarity determining region 3 (CDR-L3) comprising the amino acid sequence selected from any one of SEQ ID NOs: 47, 48, 49, 51, 52, 55, 56, 185, 189, 190, 191, 192, 193, 194, 306, 307, 415, 417, 418, 421, 591, or 603.
106. The chimeric antigen receptor of any of embodiments 99-105, wherein the VH region comprises a CDR-H1, CDR-H2, and CDR-H3, selected from:
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:1, 4, and 7, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 8, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 9, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 10, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 11, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:140, 145, and 149, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:141, 145, and 149, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:141, 145, and 150, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:142, 146, and 151, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 152, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:143, 147, and 153, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:144, 148, and 154, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 156, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 157, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 6, and 376, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 372, and 376, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 376, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 377, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 373, and 152, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 378, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 374, and 9, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:507, 513, and 517, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:604, 605, and 606, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:288, 290, and 292, respectively; or
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:289, 291, and 293, respectively.
107. The chimeric antigen receptor of any of embodiments 99-106, wherein the VH region comprises a CDR-H1, CDR-H2, and CDR-H3, selected from:
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:1, 4, and 7, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 8, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 9, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 10, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:141, 145, and 149, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:143, 147, and 153, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:144, 148, and 154, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 156, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 5, and 157, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:2, 6, and 376, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 155, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 372, and 376, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:3, 6, and 376, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:507, 513, and 517, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:604, 605, and 606, respectively;
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:288, 290, and 292, respectively; or
a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:289, 291, and 293, respectively. 108. The chimeric antigen receptor of any of embodiments 99-107, wherein the VH region is or comprises the amino acid sequence set forth in any of SEQ ID NOs:110-115, 247-256, 324, 325, 518-531, 533, 609, 617, 772-774, or 814-832.
109. The chimeric antigen receptor of any of embodiments 99-108, wherein the VH region is or comprises the amino acid sequence set forth in any of SEQ ID NOs:110, 111, 112, 113, 115, 248, 252, 253, 254, 255, 256, 324, 325, 518, 519, 520, 521, 522, 609 or 617.
110. The chimeric antigen receptor of any of embodiments 99-109 wherein:
the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:593, 594, and 595, respectively; or
the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID NOs:611, 612, and 613, respectively;
111. The chimeric antigen receptor of any of embodiments 99-110, wherein the VH region is or comprises the amino acid sequence set forth in SEQ ID NO:617.
112. The chimeric antigen receptor of any one of embodiments 99-111, wherein the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 selected from:
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:26, 37, and 47, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:27, 38, and 48, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:28, 39, and 49, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:29, 40, and 50, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:30, 39, and 51, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:31, 41, and 52, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:32, 42, and 53, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:30, 39, and 54, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:33, 43, and 55, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:34, 44, and 56, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:35, 45, and 57, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:36, 46, and 58, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 184, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 185, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 186, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 187, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:175, 180, and 188, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 189, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:176, 181, and 190, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:177, 182, and 191, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 192, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:178, 183, and 193, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:178, 183, and 194, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:30, 399, and 415, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:380, 400, and 416, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:33, 43, and 421, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:381, 401, and 417, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:382, 402, and 418, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:383, 403, and 419, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:384, 39, and 54, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:385, 180, and 58, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:175, 180, and 188, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:386, 404, and 420, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:387, 405, and 422, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:388, 406, and 423, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:388, 407, and 424, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:389, 408, and 425, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:390, 183, and 193, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:391, 409, and 426, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:392, 40, and 427, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:394, 39, and 429, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:395, 411, and 430, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:396, 412, and 431, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:396, 412, and 58, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:397, 413, and 432, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:398, 414, and 433, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:589, 590, and 591, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:607, 608, and 591, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 302, 304, and 306, respectively; or
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:303, 305, and 307, respectively.
113. The chimeric antigen receptor of any one of embodiments 99-112, wherein the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 selected from:
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:26, 37, and 47, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:27, 38, and 48, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:28, 39, and 49, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:30, 39, and 51, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:31, 41, and 52, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:33, 43, and 55, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:34, 44, and 56, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 185, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 189, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:176, 181, and 190, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:177, 182, and 191, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:174, 179, and 192, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:178, 183, and 193, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:178, 183, and 194, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:30, 399, and 415, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:380, 400, and 416, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:33, 43, and 421, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:381, 401, and 417, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:382, 402, and 418, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:589, 590, and 591, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:607, 608, and 591, respectively;
a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs: 302, 304, and 306, respectively; or a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:303, 305, and 307, respectively.
114. The chimeric antigen receptor of any of embodiments 99-113, wherein the VL region is or comprises the amino acid sequence set forth in any of SEQ ID NOs: 116-127, 257-267, 326, 327, 534-550, 552-557, 610, 618, 775-777, or 833-849.
115. The chimeric antigen receptor of any of embodiments 99-114, wherein the VL region is or comprises the amino acid sequence set forth in any of SEQ ID NOs: 116, 117, 118, 120, 121, 124, 125, 258, 262, 263, 264, 265, 266, 267, 326, 327, 534, 535, 536, 537, 538, 610 or 618.
116. The chimeric antigen receptor of any of embodiments 99-115, wherein:
the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:601, 602, and 603, respectively; or
the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID NOs:614, 615, and 603, respectively.
117. The chimeric antigen receptor of any of embodiments 99-116, wherein the VL region is or comprises the amino acid sequence set forth in SEQ ID NO:618.
118. The chimeric antigen receptor of any of embodiments 99-117, wherein:
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 116, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 116, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:111 and 117, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:111 and 117, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 119, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 119, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 120, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 120, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 121, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 121, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 122, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 122, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 123, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 123, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:112 and 124, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:112 and 124, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:113 and 125, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:113 and 125, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:114 and 126, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:114 and 126, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:115 and 127, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 127, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:247 and 257, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:247 and 257, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:248 and 258, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:248 and 258, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:249 and 259, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:249 and 259, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:250 and 260, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:250 and 260, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:251 and 261, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:251 and 261, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:252 and 262, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:252 and 262, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:253 and 263, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:253 and 263, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:254 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:254 and 264, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:255 and 265, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:255 and 265, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:256 and 266, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 266, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:256 and 267, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 267, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:518 and 534, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:518 and 534, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:519 and 535, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 535, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:115 and 536, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 536, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:520 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:520 and 264, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:521 and 537, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:521 and 537, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:522 and 538, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 538, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:523 and 539, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:523 and 539, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:519 and 540, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 540, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:524 and 541, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:524 and 541, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:525 and 261, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:525 and 261, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:526 and 542, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:526 and 542, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:527 and 543, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:527 and 543, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:528 and 544, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:528 and 544, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:529 and 545, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:529 and 545, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:528 and 546, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:528 and 546, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:522 and 547, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 547, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:256 and 548, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 548, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:530 and 549, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:530 and 549, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:531 and 550, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:531 and 550, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:519 and 552, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 552, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 553, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 553, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:533 and 554, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:533 and 554, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:115 and 555, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 555, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:524 and 556, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:524 and 556, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:519 and 557, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 557, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:609 and 610, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:609 and 610, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:617 and 618, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:324 and 326, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:324 and 326, respectively; or
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:325 and 327, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:325 and 327, respectively.
119. A chimeric antigen receptor of any of embodiments 99-118, wherein:
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 116, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 116, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:111 and 117, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:111 and 117, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 118, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 118, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 120, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 120, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:110 and 121, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:110 and 121, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:112 and 124, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:112 and 124, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:113 and 125, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:113 and 125, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:248 and 258, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:248 and 258, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:252 and 262, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:252 and 262, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:253 and 263, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:253 and 263, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:254 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:254 and 264, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:255 and 265, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:255 and 265, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:256 and 266, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 266, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:256 and 267, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:256 and 267, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:518 and 534, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:518 and 534, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:519 and 535, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:519 and 535, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:115 and 536, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:115 and 536, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:520 and 264, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:520 and 264, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:521 and 537, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:521 and 537, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:522 and 538, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:522 and 538, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:609 and 610, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:609 and 610, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:617 and 618, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:617 and 618, respectively;
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:324 and 326, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:324 and 326, respectively; or
the VH region and the VL regions comprise the amino acid sequence set forth in SEQ ID NOs:325 and 327, respectively, or a sequence of amino acids that has at least 90% identity to SEQ ID NO:325 and 327, respectively.
120. The chimeric antigen receptor of any of embodiments 99-119, wherein the fragment comprises an scFv.
121. The chimeric antigen receptor of any of embodiments 99-120, when the VH region and the VL region are joined by a flexible linker.
122. The chimeric antigen receptor of embodiment 121, wherein the scFv comprises a linker comprising the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:361).
123. The chimeric antigen receptor of any of embodiments 99-122, wherein the VH region is amino-terminal to the VL region.
124. The chimeric antigen receptor of any of embodiments 99-123, wherein the antigen-binding domain comprises the amino acid sequence selected from any one of SEQ ID NOs:128-139, 268-278, 329, 442, 478, 558-576, 578-583, 585, or 769-771 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence selected from any one of SEQ ID NOs: 128-139, 268-278, 329, 442, 478, 558-576, 578-583, 585, or 769-771.
125. The chimeric antigen receptor of any of embodiments 99-124, wherein the antigen-binding domain comprises the amino acid sequence selected from any one of SEQ ID NOs:128-130, 132, 133, 136, 137, 269, 273-278, 329, 442, 478, 558-563 or 585 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence selected from any one of SEQ ID NOs: 128-130, 132, 133, 136, 137, 269, 273-278, 329, 442, 478, 558-563 or 585.
126. The chimeric antigen receptor of any of embodiments 99-122, wherein the VH region is carboxy-terminal to the VL region.
127. The chimeric antigen receptor of any of embodiments 99-122 and 126, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NOs:328 or 586, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:328 or 586.
128. The chimeric antigen receptor of any of embodiments 89-127, wherein the intracellular signaling region comprises an activating cytoplasmic signaling domain.
129. The chimeric antigen receptor of embodiment 128, wherein the activating cytoplasmic signaling domain is capable of inducing a primary activation signal in a T cell, is a T cell receptor (TCR) component and/or comprises an immunoreceptor tyrosine-based activation motif (ITAM).
130. The chimeric antigen receptor of embodiment 128 or embodiment 129, wherein the activating cytoplasmic signaling domain is or comprises a cytoplasmic signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain or a functional variant or signaling portion thereof.
131. The chimeric antigen receptor of any of embodiments 128-130, wherein the activating cytoplasmic domain is human or is derived from a human protein.
132. The chimeric antigen receptor of any of embodiments 128-131, wherein the activating cytoplasmic domain is or comprises the sequence set forth in SEQ ID NO:628 or a sequence of amino acids that has at least 90% sequence identity to SEQ ID NO:628.
133. The chimeric antigen receptor of any of embodiments 128-132, wherein the intracellular signaling region further comprises a costimulatory signaling region.
134. The chimeric antigen receptor of embodiment 133, wherein the costimulatory signaling region comprises an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof.
135. The chimeric antigen receptor of embodiment 133 or embodiment 134, wherein the costimulatory signaling region comprises an intracellular signaling domain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof.
136. The chimeric antigen receptor of any of embodiments 133-135, wherein the costimulatory signaling region comprises an intracellular signaling domain of 4-1BB.
137. The chimeric antigen receptor of any of embodiments 133-136, wherein the costimulatory signaling region is human or is derived from a human protein.
138. The chimeric antigen receptor of any of embodiments 133-137, wherein the costimulatory signaling region is or comprises the sequence set forth in SEQ ID NO:626 or a sequence of amino acids that exhibits at least 90% sequence identity to the sequence set forth in SEQ ID NO: 626.
139. The chimeric antigen receptor of any of embodiments 133-139, wherein the costimulatory signaling region is between the transmembrane domain and the intracellular signaling region.
140. The chimeric antigen receptor of any of embodiments 89-139, wherein the transmembrane domain is or comprises a transmembrane domain derived from CD4, CD28, or CD8.
141. The chimeric antigen receptor of embodiment 140, wherein the transmembrane domain is or comprises a transmembrane domain derived from a CD28.
142. The chimeric antigen receptor of any of embodiments 89-141, wherein the transmembrane domain is human or is derived from a human protein.
143. The chimeric antigen receptor of any of embodiments 89-142, wherein the transmembrane domain is or comprises the sequence set forth in SEQ ID NO:624 or a sequence of amino acids that exhibits at least 90% sequence identity to SEQ ID NO:624.
144. The chimeric antigen receptor of any of embodiments 89-143, wherein the encoded chimeric antigen receptor comprises from its N to C terminus in order: the antigen-binding domain, the spacer, the transmembrane domain and the intracellular signaling domain.
145. An engineered cell, comprising the polynucleotide of any of embodiments 1-87 and 173-180.
146. An engineered cell, comprising the chimeric antigen receptor of any of embodiments 88-144 and 181.
147. The engineered cell of embodiment 145 or embodiment 146, wherein the cell is an immune cell.
148. The engineered cell of embodiment 147, wherein the immune cell is a primary cell obtained from a subject.
149. The engineered cell of embodiment 147 or embodiment 148, wherein the immune cell is an NK cell or a T cell.
150. The engineered cell of any of embodiments 147-149, wherein the immune cell is a T cell and the T cell is a CD4+ and/or CD8+ T cell.
151. The engineered cell of any of embodiments 145-150, wherein the cell comprises transcribed RNA encoding the chimeric antigen receptor, optionally messenger RNA (mRNA), that exhibits at least 70%, 75%, 80%, 85%, 90%, or 95% RNA homogeneity.
152. The engineered cell of any of embodiments 145-151, wherein the cell comprises transcribed RNA encoding the chimeric antigen receptor, optionally messenger RNA (mRNA), that exhibits reduced heterogeneity compared to the heterogeneity of transcribed mRNA in a cell encoding a reference chimeric antigen receptor, said reference chimeric antigen receptor comprising the same amino acid sequence as the chimeric antigen receptor but encoded by a different polynucleotide sequence comprising one or more nucleotide differences in the polynucleotide encoding the CARs and/or in which the reference chimeric antigen receptor is encoded by a polynucleotide comprising one or more splice donor site and/or one or more splice acceptor site in the nucleic acid encoding the spacer.
153. The engineered cell of embodiment 152, wherein the RNA heterogeneity is reduced by greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more.
154. The engineered cell of embodiment 152 or embodiment 153, wherein the cell encoding the reference CAR comprises transcribed RNA encoding the reference CAR, optionally messenger RNA (mRNA), that exhibits greater than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more RNA heterogeneity.
155. The engineered cell of any of embodiments 151-154, wherein the RNA homogeneity and/or heterogeneity is determined by agarose gel electrophoresis, chip-based capillary electrophoresis, analytical ultracentrifugation, field flow fractionation, or liquid chromatography.
156. The engineered cell of any of embodiments 145-155, wherein, among a plurality of the engineered cells, less than or less than about 10%, 9%, 8%, 7%, 5%, 4%, 3%, 2% or 1% of the cells in the plurality comprise a chimeric antigen receptor that exhibits tonic signaling and/or antigen independent activity or signaling.
157. A composition comprising the polynucleotide of any of embodiments 1-87 and 173-179, the chimeric antigen receptor of any one of embodiments 88-144 and 180, or the engineered cell of any one of embodiments 144-156.
158. The composition of embodiment 157, further comprising a pharmaceutically acceptable excipient.
159. The composition of embodiment 157 or embodiment 158 that is sterile.
160. A method of treatment, comprising administering the engineered cells of any of embodiments 144-156 or the composition of any of embodiments 157-159 to a subject having a disease or disorder.
161. The method of embodiment 160, wherein the disease or disorder is associated with expression of B cell maturation antigen (BCMA).
162. The method of any embodiment 160 or embodiment 161, wherein the disease or disorder associated with BCMA is a B cell-related disorder.
163. The method of any one of embodiments 160-162, wherein the disease or disorder associated with BCMA is an autoimmune disease or disorder.
164. The method of embodiment 163, wherein the autoimmune disease or disorder is systemic lupus erythematosus (SLE), lupus nephritis, inflammatory bowel disease, rheumatoid arthritis, ANCA associated vasculitis, idiopathic thrombocytopenia purpura (ITP), thrombotic thrombocytopenia purpura (TTP), autoimmune thrombocytopenia, Chagas' disease, Grave's disease, Wegener's granulomatosis, poly-arteritis nodosa, Sjogren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, vasculitis, diabetes mellitus, Reynaud's syndrome, anti-phospholipid syndrome, Goodpasture's disease, Kawasaki disease, autoimmune hemolytic anemia, myasthenia gravis, or progressive glomerulonephritis.
165. The method of any one of embodiments 160-164, wherein the disease or disorder associated with BCMA is a cancer.
166. The method of embodiment 165, wherein the cancer is a BCMA-expressing cancer.
167. The method of embodiment 165 or 166, wherein the cancer is a B cell malignancy.
168. The method of any one of embodiments 165-167, wherein the cancer is a lymphoma, a leukemia, or a plasma cell malignancy.
169. The method of embodiment 168, wherein the cancer is a lymphoma and the lymphoma is Burkitt's lymphoma, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, Waldenstrom macroglobulinemia, follicular lymphoma, small non-cleaved cell lymphoma, mucosa-associated lymphatic tissue lymphoma (MALT), marginal zone lymphoma, splenic lymphoma, nodal monocytoid B cell lymphoma, immunoblastic lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, pulmonary B cell angiocentric lymphoma, small lymphocytic lymphoma, primary mediastinal B cell lymphoma, lymphoplasmacytic lymphoma (LPL), or mantle cell lymphoma (MCL).
170. The method of embodiment 168, wherein the cancer is a leukemia and the leukemia is chronic lymphocytic leukemia (CLL), plasma cell leukemia or acute lymphocytic leukemia (ALL).
171. The method of embodiment 168, wherein the cancer is a plasma cell malignancy and the plasma cell malignancy is multiple myeloma (MM) or plasmacytoma.
172. The method of any of embodiments 165-168 and 171, wherein the cancer is multiple myeloma (MM).
173. The polynucleotide of any of embodiments 1-87, wherein the antigen-binding domain and/or the encoded chimeric antigen receptor exhibits preferential binding to, and/or exhibits greater binding affinity for, membrane bound BCMA compared to soluble BCMA.
174. The polynucleotide of any of embodiments 1-87 and 173, wherein the polynucleotide comprises the sequence set forth in any of SEQ ID NOS: 751-762 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in any of SEQ ID NOS: 751-762 and retains the function to bind to BCMA and retains the reduced RNA heterogeneity.
175. The polynucleotide of any of embodiments 1-87, 173 and 174, wherein one of more of the hinge, CH2 and CH3 is derived all or in part from IgG4 or IgG2, optionally human IgG4 or human IgG2.
176. The polynucleotide of embodiment any of embodiments 1-87, 173-175, wherein the hinge, CH2 and CH3 is derived from IgG4.
177. The polynucleotide of any of embodiments 1-87 and 173-176, wherein one or more of the hinge, CH2 and CH3 is chimeric and comprises sequence derived from IgG4 and IgG2.
178. The polynucleotide of embodiment 177, wherein the spacer comprises an IgG4/2 chimeric hinge or a modified IgG4 comprising at least one amino acid replacement compared to human IgG4, an IgG2/4 chimeric CH2, and an IgG4 CH3 region.
179. The polynucleotide of any of embodiments 1-87 and 173-178, wherein the encoded spacer is or comprises the sequence set forth in SEQ ID NO: 649.
180. The chimeric antigen receptor of any of embodiments 88-144, wherein the antigen-binding domain and or the chimeric antigen receptor exhibits preferential binding to, and/or exhibits greater binding affinity for, membrane bound BCMA compared to soluble BCMA.
181. The chimeric antigen receptor of any of 181. The chimeric antigen receptor of any of embodiments 88-144, wherein the antigen-binding domain and or the chimeric antigen receptor, or a measure indicative of function or activity of the encoded chimeric antigen receptor following exposure to cells expressing surface BCMA, is not reduced or blocked or is not substantially reduced or blocked in the presence of a soluble or shed form of BCMA.
182. The chimeric antigen receptor of embodiment 181, wherein the concentration or amount of the soluble or shed form of the BCMA corresponds to a concentration or amount present in serum or blood or plasma of the subject or of a multiple myeloma patient, or on average in a patient population for the disease or disorder, or at a concentration or amount of the soluble or shed BCMA at which the binding or measure is reduced or blocked, or is substantially reduced or blocked, for cells expressing a reference anti-BCMA recombinant receptor, optionally a reference anti-BCMA CAR, in the same assay.
183. A method of determining the heterogeneity of a transcribed nucleic acid of a transgene, the method comprising:
a) amplifying a transcribed nucleic acid using at least one 5′ and 3′ primer pair, wherein at least one pair comprises a 5′ primer that is complementary to a nucleic acid sequence within the 5′ untranslated region (5′ UTR) of the transcribed nucleic acid and a 3′ primer that is complementary to a nucleic acid sequence within the 3′ untranslated region (3′ UTR) of the transcribed nucleic acid to generate one or more amplified products; and
b) detecting the amplified products, wherein the presence of two or more amplified products from at least one 5′ and 3′ primer pair indicates heterogeneity in the amplified products.
184. The method of embodiment 183 wherein the detected differences in b) are different lengths of the amplified transcripts.
185. The method of embodiment 183 wherein the differences in b) are differences in chromatographic profiles of the amplified transcripts.
186. The method of any of embodiments 183-185, wherein the differences in the amplified products are determined by agarose gel electrophoresis, chip-based capillary electrophoresis, analytical ultracentrifugation, field flow fractionation, or chromatography.
187. The method of any of embodiments 183-186, wherein the 5′ primer is specific to sequence transcribed from the promoter region of the transcribed nucleic acid.
188. The method of any of embodiments 183-187, wherein the transcribed nucleic acid is amplified using a 3′ primer specific to a sequence within the amino acid-coding sequence of the polynucleotide, and/or the 3′ untranslated region of the transcribed pre-mRNA.
189. The method of any of embodiments 183-188, wherein the 3 primer is specific to the polyadenylation sequence or enhancer region of the 3′ untranslated region of the transcribed pre-mRNA.
190. The method of any of embodiments 183-189, wherein step a) is effected by a single amplification reaction, using a single 5′ and 3′ primer pair comprising a 5′ primer that is complementary to a nucleic acid sequence within the 5′ untranslated region (5′ UTR) of the transcribed nucleic acid and a 3′ primer that is complementary to a nucleic acid sequence within the 3′ untranslated region (3′ UTR).
191. The method of any of embodiments 183-190, wherein step a) is effected by parallel or subsequent amplification reactions using a first 5′ and 3′ primer pair, a second 5′ and 3′primer pair, and optionally additional 5′ and 3′primer pairs, wherein:
the first 5′ and 3′primer pair contains a 5′ primer that is complementary to a nucleic acid sequence within the 5′ UTR of the transcribed nucleic acid and a 3′ primer that is complementary to a nucleic acid sequence within the 3′ UTR of the transcribed nucleic acid;
the second 5′ and 3′ primer pair contains a 5′ primer whose sequence is complementary to a portion of the translated sequence of the nucleic acid transcript and a 3′ primer whose sequence is complementary to a nucleic acid sequence within the 3′ UTR of the transcript; and
the optionally additional 5′ and 3′primer pairs each contain sequences complementary to sequences within the translated region of the transcript.
192. The method of embodiment 191, wherein the parallel or subsequent amplification reactions amplify overlapping portions of the transcript.
193. The method of any of embodiments 183-192, wherein the amplified products are predicted to be about 1.5 kilobases, 2 kilobases, 2.5 kilobases, 3 kilobases, 3.5 kilobases, 4 kilobases, 4.5 kilobases, 5 kilobases, 5.5 kilobases, 6 kilobases, 7 kilobases, or 8 kilobases in length.
194. The method of any of embodiments 183-193, wherein a transcribed nucleic acid that is detected as having heterogeneity is identified as a transgene candidate for removal of one or more splice site.
195. The method of embodiment 194, wherein the transcribed nucleic acid of the transgene candidate exhibits at least or at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more heterogeneity following expression in a cell.
196. A method of reducing the heterogeneity of an expressed transgene transcript, the method comprising:
a) identifying a transgene candidate for the removal of splice sites according to the method of embodiment 194 or embodiment 195;
b) identifying one or more potential splice donor and/or splice acceptor sites; and
c) modifying the nucleic acid sequence at or near the one or more identified splice donor sites identified in b), thereby generating a modified polynucleotide.
197. The method of embodiment 196, further comprising: d) assessing the transgene candidacy for the removal of splice sites as in step a).
198. The method of embodiment 197, further comprising e) repeating steps b)-d) until the heterogeneity of the transcript in step d) is reduced compared to the heterogeneity of the transcript as determined in step a).
199. The method of any of embodiments 196-198, wherein the one or more potential splice donor and/or splice acceptor sites exhibit a score about or at least about 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0 of a splice event or probability of a splice event.
200. The method of any of embodiments 196-199, wherein splice donor sites and splice acceptor sites are identified independently.
201. The method of any of embodiments 196-200, wherein the splice acceptor and/or donor site(s) is/are canonical, non-canonical, and/or cryptic splice acceptor and/or donor site(s).
202. The method of any of embodiments 196-201, wherein the transgene is a chimeric antigen receptor or a portion of a chimeric antigen receptor.
203. The method of embodiment 202, wherein the CAR polypeptide comprises an antigen-binding domain comprising an antibody fragment, optionally a single chain antibody fragment (scFv), comprising a variable heavy chain (VH) and a variable light chain (VL), a spacer region, a transmembrane region, and an intracellular signaling region.
204. The method of embodiment 202 or embodiment 203, wherein the modified polynucleotide is not modified within the coding sequence for the antigen-binding domain of the encoded CAR polypeptide.
205. The method of any of embodiments 196-204, wherein the encoded amino acid sequence of the transgene is unchanged following modification of the polynucleotide.
206. The method of any of embodiments 196-205, wherein the RNA transcribed from the modified polynucleotide exhibits at least or at least about 70%, 75%, 80%, 85%, 90%, or 95% homogeneity following expression of the unmodified polynucleotide in a cell.
207. The method of any of embodiments 183-206, wherein the cell is a human cell. 208. The method of any of embodiments 183-207, wherein the cell is a T-cell.
IX. EXAMPLES
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1: Generation and Assessment of Anti-BCMA Antibodies (VH Chain Only)
Exemplary anti-BCMA antibodies containing a heavy chain variable (VH) region that specifically bound to BCMA, even in the absence of a light chain variable (VL) region, were generated and assessed.
A. Library Selection and Antibody Generation
A number of BCMA-binding VH regions were generated through a series of selection steps carried out on members of a dsDNA-encoded His-tagged human normal donor antibody VH library displayed in a cell-free system. Members of the VH library were subjected to multiple rounds of screening to select VH regions that bound specifically to soluble human BCMA fused to an immunoglobulin Fc region (hBMCA-Fc). VH regions from selected hBCMA-Fc pools were screened, by flow cytometry using a fluorochrome-conjugated anti-HIS antibody, for binding to a recombinant HEK293 cell line expressing human BCMA (hBCMA/HEK293 cell line), as compared to the parental HEK293 cell line not expressing BCMA, as well as to for binding to a human myeloma cell line expressing endogenous BCMA (H929 cells). The results identified VH region clones that exhibited specific binding to hBCMA/HEK293 cells and, to a lesser degree, to H929 cells.
Exemplary VH clones exhibiting specific binding to cell lines expressing BCMA but not to BCMA-negative control cells were sequenced and purified for further characterization. Clones were purified and titrated, and their binding affinities (EC50) to hBCMA/HEK293 cells were measured using a flow cytometry-based assay with the fluorochrome-conjugated anti-HIS-antibody. Table E1 lists heavy chain complementarity determining region 3 (CDR-H3) sequences of exemplary clones, containing human VH3-derived framework regions and their respective binding affinities (EC50) observed in this study.
TABLE E1
CDR3 amino acid sequences
for representative VH clones
VH Clone
Heavy Chain CDR3
CDR-H3 Sequence
EC50
Name
sequence (CDR-H3)a
Identifier Number
(nM)b
VH-1
VDGPPSFDI
SEQ ID NO: 10
>100
VH-2
WSAPTDY
SEQ ID NO: 7
25
VH-3
VDGDDAFDI
SEQ ID NO: 279
>100
VH-4
DPLSWDSSGKGPR
SEQ ID NO: 280
100
VH-5
ENYDFWSWRYYYDMDV
SEQ ID NO: 281
>100
VH-6
VDGPPSYDI
SEQ ID NO: 282
>100
VH-7
GDWDDAFDI
SEQ ID NO: 283
>100
VH-8
VDGDYVDDY
SEQ ID NO: 9
ND
VH-9
VDGDYEDY
SEQ ID NO: 284
>100
VH-10
DVPSSGDDAFDI
SEQ ID NO: 285
>100
VH-11
VDGDDVFDI
SEQ ID NO: 286
>100
VH-12
VDGDAFDI
SEQ ID NO: 287
100
aAccording to Kabat numbering.
bND indicates not detected
Example 2: Generation and Assessment of Anti-BCMA Antibodies (scFvs)
Exemplary anti-BCMA antibodies, formatted as single chain antibody fragments (scFvs), were identified and assessed for binding to BCMA.
A. Library Selection and scFv Antibody Generation
Exemplary anti-BCMA scFv antibodies were generated through various selections, carried out on dsDNA-encoded human normal donor antibody libraries displayed in a cell-free system. In one approach, VH region library members enriched from a first round of screening in the approach described in Example 1 were paired by shuffling with members of a human normal donor VL library, to generate an scFv library, in VH-(G4S)3-VL format. The resulting scFv libraries were enriched in subsequent rounds of selection for specific binding to BCMA-expressing HEK293 cells as compared to parental HEK293 cells.
In another approach, de novo selection was carried out by screening a normal donor-derived human scFv library for BCMA-specific binding to hBCMA-Fc in the presence or absence of competitive elution with a mouse anti-BCMA reference scFv antibody (either BCMA-C1, VL-VH scFv antibody, SEQ ID NO:328; or BCMA-C2, VH-VL scFv antibody, SEQ ID NO:329). After at least 2 rounds of selection, scFv binders were recovered.
Specific binding of resulting scFv clones to BCMA-expressing HEK293 cells, as compared to control cells not expressing BCMA, was assessed by flow cytometry either with in vitro translated crude cell lysate or with bacterially-produced supernatant. Certain scFv clones displaying binding preference for BCMA were further analyzed.
The selected scFv clones were sequenced using forward and reverse primers and purified for further characterization. Table E2 lists sequence identifiers (SEQ ID NO) corresponding to amino acid (aa) and nucleotide (nt) sequences of the scFv and amino acid sequences of the corresponding heavy chain (VH) or light chain (VL) variable regions, CDRs and framework regions (FRs). With respect to clone BCMA-22, the first residue of light chain CDR3 (a cysteine), which was observed to have been inherited from the germline framework region was replaced with a serine to generate an additional scFv, designated BCMA-23. Table E2 also sets forth the sequence of exemplary mouse anti-BCMA reference antibodies used as controls and in competition studies as described in subsequent Examples.
TABLE E2
Sequence identifier (SEQ ID NO) for Exemplary Clones
Heavy Chain
Light Chain
VH FR
VL FR
(FR1, 2,
(FR1, 2,
CDR-H1, CDR-H2,
3, 4
CDR-L1, CDR-L2, CDR-
3, 4,
scFv
Clone #
VH
CDR-H3
Kabat)
VL
L3
Kabat)
aa
nt
BCMA-1
110
1, 4, 7 (Kabat)
59, 64,
116
26, 37, 47 (Kabat)
72, 83,
128
330
12, 16, 7 (Chothia)
67, 70
26, 37, 47 (Chothia)
93, 102
19, 23, 7 (AbM)
26, 37, 47 (AbM)
BCMA-2
111
2, 5, 8 (Kabat)
60, 65,
117
27, 38, 48 (Kabat)
73, 84,
129
331
13, 17, 8 (Chothia)
68, 71
27, 38, 48 (Chothia)
94, 103
20, 24, 8 (AbM)
27, 38, 48 (AbM)
BCMA-3
110
1, 4, 7 (Kabat)
59, 64,
118
28, 39, 49 (Kabat)
74, 85,
130
332
12, 16, 7 (Chothia)
67, 70
28, 39, 49 (Chothia)
93, 104
19, 23, 7 (AbM)
28, 39, 49 (AbM)
BCMA-4
110
1, 4, 7 (Kabat)
59, 64,
119
29, 40, 50 (Kabat)
75, 86,
131
333
12, 16, 7 (Chothia)
67, 70
29, 40, 50 (Chothia)
95, 104
19, 23, 7 (AbM)
29, 40, 50 (AbM)
BCMA-5
110
1, 4, 7 (Kabat)
59, 64,
120
30, 39, 51 (Kabat)
76, 85,
132
334
12, 16, 7 (Chothia)
67, 70
30, 39, 51 (Chothia)
93, 105
19, 23, 7 (AbM)
30, 39, 51 (AbM)
BCMA-6
110
1, 4, 7 (Kabat)
59, 64,
121
31, 41, 52 (Kabat)
77, 87,
133
335
12, 16, 7 (Chothia)
67, 70
31, 41, 52 (Chothia)
96, 104
19, 23, 7 (AbM)
31, 41, 52 (AbM)
BCMA-7
110
1, 4, 7 (Kabat)
59, 64,
122
32, 42, 53 (Kabat)
78, 88,
134
336
12, 16, 7 (Chothia)
67, 70
32, 42, 53 (Chothia)
97, 106
19, 23, 7 (AbM)
32, 42, 53 (AbM)
BCMA-8
110
1, 4, 7 (Kabat)
59, 64,
123
30, 39, 54 (Kabat)
76, 85,
135
337
12, 16, 7 (Chothia)
67, 70
30, 39, 54 (Chothia)
93, 107
19, 23, 7 (AbM)
30, 39, 54 (AbM)
BCMA-9
112
2, 5, 9 (Kabat)
61, 65,
124
33, 43, 55 (Kabat)
79, 89,
136
338
13, 17, 9 (Chothia)
69, 70
33, 43, 55 (Chothia)
98, 108
20, 24, 9 (AbM)
33, 43, 55 (AbM)
BCMA-
113
2, 5, 10 (Kabat)
62, 65,
125
34, 44, 56 (Kabat)
80, 90,
137
339
10
14, 17, 10 (Chothia)
68, 71
34, 44, 56 (Chothia)
99, 108
21, 24, 10 (AbM)
34, 44, 56 (AbM)
BCMA-
114
3, 6, 11 (Kabat)
63, 66,
126
35, 45, 57 (Kabat)
81, 91,
138
340
11
15, 18, 11 (Chothia)
69, 71
35, 45, 57 (Chothia)
100, 108
22, 25, 11 (AbM)
35, 45, 57 (AbM)
BCMA-
115
2, 5, 10 (Kabat)
60, 65,
127
36, 46, 58 (Kabat)
82, 92,
139
341
12
13, 17, 10 (Chothia)
68, 71
36, 46, 58 (Chothia)
101, 109
20, 24, 10 (AbM)
36, 46, 58 (AbM)
BCMA-
247
140, 145, 149 (Kabat)
195, 204,
257
174, 179, 184 (Kabat)
221, 228,
268
342
13
158, 161, 149 (Chothia)
210, 217
174, 179, 184 (Chothia)
233, 243
165, 170, 149 (AbM)
174, 179, 184 (AbM)
BCMA-
248
141, 145, 149 (Kabat)
196, 204,
258
174, 179, 185 (Kabat)
221, 228,
269
343
14
158, 161, 149 (Chothia)
211, 218
174, 179, 185 (Chothia)
234, 109
166, 170, 149 (AbM)
174, 179, 185 (AbM)
BCMA-
249
141, 145, 150 (Kabat)
197, 204,
259
174, 179, 186 (Kabat)
222, 228,
270
344
15
158, 161, 150 (Chothia)
212, 70
174, 179, 186 (Chothia)
235, 109
166, 170, 150 (AbM)
174, 179, 186 (AbM)
BCMA-
250
142, 146, 151 (Kabat)
198, 205,
260
174, 179, 187 (Kabat)
223, 228,
271
345
16
159, 162, 151 (Chothia)
213, 70
174, 179, 187 (Chothia)
235, 109
167, 171, 151 (AbM)
174, 179, 187 (AbM)
BCMA-
251
2, 5, 152 (Kabat)
199, 206,
261
175, 180, 188 (Kabat)
224, 229,
272
346
17
13, 17, 152 (Chothia)
69, 219
175, 180, 188 (Chothia)
237, 109
20, 24, 152 (AbM)
175, 180, 188 (AbM)
BCMA-
252
143, 147, 153 (Kabat)
200, 207,
262
174, 179, 189 (Kabat)
222, 228,
273
347
18
158, 163, 153 (Chothia)
214, 70
174, 179, 189 (Chothia)
238, 109
168, 172, 153 (AbM)
174, 179, 189 (AbM)
BCMA-
253
144, 148, 154 (Kabat)
201, 208,
263
176, 181, 190 (Kabat)
225, 230,
274
348
19
160, 164, 54 (Chothia)
215, 220
176, 181, 190 (Chothia)
239, 244
169, 173, 154 (AbM)
176, 181, 190 (AbM)
BCMA-
254
3, 6, 155 (Kabat)
202, 209,
264
177, 182, 191 (Kabat)
226, 231,
275
349
20
15, 18, 155 (Chothia)
216, 70
177, 182, 191 (Chothia)
240, 245
22, 25, 155 (AbM)
177, 182, 191 (AbM)
BCMA-
255
2, 5, 156 (Kabat)
203, 65,
265
174, 179, 192 (Kabat)
222, 228,
276
350
21
13, 17, 156 (Chothia)
68, 70
174, 179, 192 (Chothia)
241, 246
20, 24, 156 (AbM)
174, 179, 192 (AbM)
BCMA-
256
2, 5, 157 (Kabat)
60, 65,
266
178, 183, 193 (Kabat)
227, 232,
277
351
22
13, 17, 157 (Chothia)
68, 70
178, 183, 193 (Chothia)
242, 246
20, 24, 157 (AbM)
178, 183, 193 (AbM)
BCMA-
256
2, 5, 157 (Kabat)
60, 65,
267
178, 183, 194 (Kabat)
227, 232,
278
352
23
13, 17, 157 (Chothia)
68, 70
178, 183, 194 (Chothia)
242, 246
20, 24, 157 (AbM)
178, 183, 194 (AbM)
BCMA-
518
2, 6, 376 (Kabat)
61, 65,
534
30, 399, 415 (Kabat)
76, 85,
558
24
13, 18, 376 (Chothia)
69, 71
30, 399, 415 (Chothia)
483, 508
20, 25, 376 (AbM)
30, 399, 415 (AbM)
BCMA-
519
1, 4, 7 (Kabat)
436, 64,
535
380, 400, 416 (Kabat)
446, 467,
559
716,
25
12, 16, 7 (Chothia)
67, 70
380, 400, 416 (Chothia)
484, 502
717
19, 23, 7 (AbM)
380, 400, 416 (AbM)
BCMA-
115
2, 5, 10 (Kabat)
60, 65,
536
33, 43, 421 (Kabat)
80, 89,
560
718,
26
13, 17, 10 (Chothia)
68, 71
33, 43, 421 (Chothia)
98, 108
719
20, 24, 10 (AbM)
33, 43, 421 (AbM)
BCMA-
520
3, 6, 155 (Kabat)
434, 209,
264
177, 182, 191 (Kabat)
226, 231,
561
27
15, 18, 155 (Chothia)
216, 70
177, 182, 191 (Chothia)
240, 245
22, 25, 155 (AbM)
177, 182, 191 (AbM)
BCMA-
521
3, 372, 376 (Kabat)
63, 209,
537
381, 401, 417 (Kabat)
447, 468,
562
28
15, 514, 376 (Chothia)
69, 444
381, 401, 417 (Chothia)
485, 508
22, 510, 376 (AbM)
381, 401, 417 (AbM)
BCMA-
522
3, 6, 376 (Kabat)
63, 209,
538
382, 402, 418 (Kabat)
448, 469,
563
29
15, 18, 376 (Chothia)
69, 71
382, 402, 418 (Chothia)
486, 503
22, 25, 376 (AbM)
382, 402, 418 (AbM)
BCMA-
523
3, 6, 377 (Kabat)
435, 209,
539
383, 403, 419 (Kabat)
449, 470,
564
30
12, 18, 377 (Chothia)
69, 71
383, 403, 419 (Chothia)
487, 104
509, 25, 377 (AbM)
383, 403, 419 (AbM)
BCMA-
519
1, 4, 7 (Kabat)
436, 64,
540
384, 39, 54 (Kabat)
450, 471,
565
31
12, 16, 7 (Chothia)
67, 70
384, 39, 54 (Chothia)
93, 504
19, 23, 7 (AbM)
384, 39, 54 (AbM)
BCMA-
524
2, 5, 10 (Kabat)
437, 65,
541
385, 180, 58 (Kabat)
451, 472,
566
32
13, 17, 10 (Chothia)
68, 71
385, 180, 58 (Chothia)
488, 109
20, 24, 10 (AbM)
385, 180, 58 (AbM)
BCMA-
525
2, 373, 152 (Kabat)
199, 65,
261
175, 180, 188 (Kabat)
224, 229,
567
33
13, 515, 152 (Chothia)
69, 219
175, 180, 188 (Chothia)
237, 109
20, 511, 152 (AbM)
175, 180, 188 (AbM)
BCMA-
526
3, 6, 11 (Kabat)
438, 209,
542
386, 404, 420 (Kabat)
452, 84,
568
34
15, 18, 11 (Chothia)
69, 71
386, 404, 420 (Chothia)
489, 504
22, 25, 11 (AbM)
386, 404, 420 (AbM)
BCMA-
527
2, 5, 378 (Kabat)
61, 65,
543
33, 43, 421 (Kabat)
453, 89,
569
35
13, 17, 378 (Chothia)
69, 70
33, 43, 421 (Chothia)
98, 505
20, 24, 378 (AbM)
33, 43, 421 (AbM)
BCMA-
528
2, 5, 9 (Kabat)
199, 65,
544
387, 405, 422 (Kabat)
454, 473,
570
36
13, 17, 9 (Chothia)
69, 70
387, 405, 422 (Chothia)
490, 109
20, 24, 9 (AbM)
387, 405, 422 (AbM)
BCMA-
529
2, 5, 9 (Kabat)
61, 65,
545
388, 406, 423 (Kabat)
455, 474,
571
37
13, 17, 9 (Chothia)
441, 70
388, 406, 423 (Chothia)
491, 109
20, 24, 9 (AbM)
388, 406, 423 (AbM)
BCMA-
528
2, 5, 9 (Kabat)
199, 65,
546
388, 407, 424 (Kabat)
456, 474,
572
38
13, 17, 9 (Chothia)
69, 70
388, 407, 424 (Chothia)
492, 109
20, 24, 9 (AbM)
388, 407, 424 (AbM)
BCMA-
522
3, 6, 376 (Kabat)
63, 209,
547
389, 408, 425 (Kabat)
457, 475,
573
39
15, 18, 376 (Chothia)
69, 71
389, 408, 425 (Chothia)
493, 103
22, 25, 376 (AbM)
389, 408, 425 (AbM)
BCMA-
256
2, 5, 157 (Kabat)
60, 65,
548
390, 183, 193 (Kabat)
227, 232,
574
40
13, 17, 157 (Chothia)
68, 70
390, 183, 193 (Chothia)
242, 108
20, 24, 157 (AbM)
390, 183, 193 (AbM)
BCMA-
530
2, 374, 9 (Kabat)
199, 65,
549
391, 409, 426 (Kabat)
458, 476,
575
584
41
13, 516, 9 (Chothia)
68, 70
391, 409, 426 (Chothia)
494, 109
20, 512, 9 (AbM)
391, 409, 426 (AbM)
BCMA-
531
1, 4, 7 (Kabat)
439, 64,
550
392, 40, 427 (Kabat)
459, 477,
576
42
12, 16, 7 (Chothia)
67, 70
392, 40, 427 (Chothia)
495, 506
19, 23, 7 (AbM)
392, 40, 427 (AbM)
BCMA-
519
1, 4, 7 (Kabat)
436, 64,
552
394, 39, 429 (Kabat)
461, 85,
578
44
12, 16, 7 (Chothia)
67, 70
394, 39, 429 (Chothia)
93, 107
19, 23, 7 (AbM)
394, 39, 429 (AbM)
BCMA-
110
1, 4, 7 (Kabat)
59, 64,
553
395, 411, 430 (Kabat)
462, 479,
579
45
12, 16, 7 (Chothia)
67, 70
395, 411, 430 (Chothia)
497, 105
19, 23, 7 (AbM)
395, 411, 430 (AbM)
BCMA-
110
1, 4, 7 (Kabat)
59, 64,
118
28, 39, 49 (Kabat)
74, 85,
130
46
12, 16, 7 (Chothia)
67, 70
28, 39, 49 (Chothia)
93, 104
19, 23, 7 (AbM)
28, 39, 49 (AbM)
BCMA-
533
2, 5, 10 (Kabat)
197, 65,
554
396, 412, 431 (Kabat)
463, 480,
580
47
13, 17, 10 (Chothia)
443, 445
396, 412, 431 (Chothia)
498, 108
20, 24, 10 (AbM)
396, 412, 431 (AbM)
BCMA-
115
2, 5, 10 (Kabat)
60, 65,
555
396, 412, 58 (Kabat)
464, 480,
581
48
13, 17, 10 (Chothia)
68, 71
396, 412, 58 (Chothia)
499, 109
20, 24, 10 (AbM)
396, 412, 58 (AbM)
BCMA-
524
2, 5, 10 (Kabat)
437, 65,
556
397, 413, 432 (Kabat)
465, 481,
582
49
13, 17, 10 (Chothia)
68, 71
397, 413, 432 (Chothia)
500, 109
20, 24, 10 (AbM)
397, 413, 432 (AbM)
BCMA-
519
1, 4, 7 (Kabat)
436, 64,
557
398, 414, 433 (Kabat)
466, 482,
583
51
12, 16, 7 (Chothia)
67, 70
398, 414, 433 (Chothia)
501, 508
19, 23, 7 (AbM)
398, 414, 433 (AbM)
Exemplary clones were purified and titrated, and their binding affinities (EC50) to hBMCA was tested: BCMA-1, BCMA-2, BCMA-3, BCMA-4, BCMA-5, BCMA-6, BCMA-7, BCMA-8, BCMA-9, BCMA-10, BCMA-11, BCMA-12, BCMA-13, BCMA-14, BCMA-14, BCMA-15, BCMA-16, BCMA-17, BCMA-18, BCMA-19, BCMA-20, BCMA-21, BCMA-22, BCMA-23, BCMA-24, BCMA-25, BCMA-26, BCMA-27, BCMA-28 and BCMA-29. Other anti-BCMA scFv antibodies also were assessed (see Table E3), such as scFvs containing VH and VL sequences of antibodies described in WO2016090327, and scFvs containing VH and VL sequences of BCMA antibodies described in WO2010104949.
TABLE E3
Sequence identifier (SEQ ID NO) for Exemplary Anti-BCMA Antibodies
Heavy Chain
Light Chain
VH FR
VL FR
(FR1, 2,
(FR1, 2,
CDR-H1, CDR-H2,
3, 4
CDR-L1, CDR-L2,
3, 4,
scFv
Clone #
VH
CDR-H3
Kabat)
VL
CDR-L3
Kabat)
aa
nt
BCMA-
609
507, 513, 517 (Kabat)
610
589, 590, 591 (Kabat)
442
647,
52
532, 551, 517 (Chothia)
589, 590, 591 (Chothia)
440
577, 587, 517 (AbM)
589, 590, 591 (AbM)
604, 605, 606
607, 608, 591
BCMA-
617
593, 594, 595 (Kabat)
618
601, 602, 603 (Kabat)
478
648,
55
596, 597, 595 (Chothia)
601, 602, 603 (Chothia)
460
598, 599, 595 (AbM)
601, 602, 603 (AbM)
611, 612, 613
614, 615, 603
BCMA-
324
288, 290, 292 (Kabat)
308, 310,
326
302, 304, 306 (Kabat)
316, 318,
585
C1, VH-
294, 296, 292 (Chothia)
312, 314
302, 304, 306 (Chothia)
320, 322
VL
298, 300, 292 (AbM)
302, 304, 306 (AbM)
BCMA-
324
288, 290, 292 (Kabat)
308, 310,
326
302, 304, 306 (Kabat)
316, 318,
328
C1, VL-
294, 296, 292 (Chothia)
312, 314
302, 304, 306 (Chothia)
320, 322
VH
298, 300, 292 (AbM)
302, 304, 306 (AbM)
BCMA-
325
289, 291, 293 (Kabat)
309, 311,
327
303, 305, 307 (Kabat)
317, 319,
329
C2, VH-
295, 297, 293 (Chothia)
313, 315
303, 305, 307 (Chothia)
321, 323
VL
299, 301, 293 (AbM)
303, 305, 307 (AbM)
BCMA-
325
289, 291, 293 (Kabat)
309, 311,
327
303, 305, 307 (Kabat)
317, 319,
586
C2, VL-
295, 297, 293 (Chothia)
313, 315
303, 305, 307 (Chothia)
321, 323
VH
299, 301, 293 (AbM)
303, 305, 307 (AbM)
Example 3: Generation of Chimeric Antigen Receptors (CARs) Against BCMA and Cells Expressing Anti-BCMA CARs
Polynucleotides encoding exemplary chimeric antigen receptors (CARs), each containing a human anti-BCMA scFv antigen-binding domain, were generated. Among the human anti-BCMA scFvs were those described in Example 2. Also among the CARs generated were CARs containing scFvs containing VH and VL sequences of antibodies described in WO2016090327. Also generated were anti-BCMA CARs containing scFvs with VH and VL sequences of BCMA antibodies described in WO2010104949. In some cases of the scFv, the VH was amino-terminal to the VL and in some cases the VL was amino-terminal to the VH. The scFv regions in generated CARs are set forth in Table E4.
Specifically, the exemplary polynucleotide CAR constructs contained nucleic acid encoding a human IgG-kappa signaling sequence (SEQ ID NO: 619, encoding SEQ ID NO: 620), a human anti-BCMA scFv (SEQ ID NOS: 128-130, 132, 133, 136, 137, 269, 273-277, 442, 478 and 558-563), a spacer (such as a spacer containing a modified IgG4-hinge CH2-CH3 (SEQ ID NO:621, encoding SEQ ID NO:649) (which spacer may in some instances be referred to as “LS”) or, in some cases, a shorter spacer (which may be referred to as “SS”), such as one derived from an IgG hinge region, such as an IgG4-derived hinge region or modified form thereof, or derived from a CD28 extracellular domain; a human CD28 transmembrane domain; a human 4-1BB-derived intracellular co-signaling sequence; and a human CD3-zeta derived intracellular signaling domain. Exemplary spacers included those derived from an IgG4 hinge region (such as those encoded by, e.g., SEQ ID NO:364, and/or containing the amino acid sequence of SEQ ID NO: 363) and CD28 ectodomain-derived spacers such as those encoded by, e.g., the sequence of SEQ ID NO: 629 or those having an amino acid sequence of SEQ ID NO: 630.
A polynucleotide encoding another CAR construct also was generated containing nucleic acid encoding a human IgG-kappa signal sequence (SEQ ID NO: 619, encoding SEQ ID NO: 620), a mouse anti-BCMA scFv (SEQ ID NO: 328 (BCMA-C1; VL—VH), 329 (BCMA-C2; VH—VL), 585 (BCMA-C1; VH—VL), or 586 (BCMAC-2; VL—VH)), a spacer (SEQ ID NO:621, encoding SEQ ID NO:649), a human CD28 transmembrane domain, a human 4-1BB-derived intracellular co-signaling sequence, and a CD3-zeta derived intracellular signaling domain.
TABLE E4
Sequences for exemplary VH-VL scFv clones (SEQ ID NO)
Heavy Chain
Light Chain
Variable
Variable
VH-VL or
(VH)
(VL)
VL-VH scFv
Region
Region
Amino
Construct
Amino Acid
Amino Acid
Nucleotide
Acid
BCMA-1
110
116
330
128
BCMA-2
111
117
331
129
BCMA-3
110
118
332
130
BCMA-5
110
120
334
132
BCMA-6
110
121
335
133
BCMA-9
112
124
338
136
BCMA-10
113
125
339
137
BCMA-14
248
258
343
269
BCMA-18
252
262
347
273
BCMA-19
253
263
348
274
BCMA-20
254
264
349
275
BCMA-21
255
265
350
276
BCMA-22
256
266
351
277
BCMA-23
256
267
352
278
BCMA-24
518
534
558
BCMA-25
519
535
559
BCMA-26
115
536
560
BCMA-27
520
264
561
BCMA-28
521
537
562
BCMA-29
522
538
563
BCMA-52
609
610
647
442
BCMA-55
617
618
648
478
BCMA-C1, VH-VL
324
326
585
BCMA-C1, VL-VH
324
326
328
BCMA-C2, VH-VL
325
327
329
BCMA-C2, VL-VH
325
327
586
cDNA clones encoding such CARs, were linked to a downstream ribosomal skip element (such as T2A-encoding sequence SEQ ID NO: 686 or 687, encoding SEQ ID NO: 654) followed by a truncated receptor-encoding sequence, and cloned into a lentiviral expression vector.
To generate anti-BCMA CAR-expressing T cells, T cells were isolated by immunoaffinity-based enrichment from leukapheresis samples from human donor subjects. Isolated T cells were activated and transduced with lentiviral vectors containing the respective polynucleotides encoding the anti-BCMA CARs. After transduction and expansion, CD4+ and CD8+ T cells were stained with an antibody specific for the truncated receptor and with a fluorescently labeled-recombinant human BCMA and analyzed by flow cytometry, confirming transduction of cells and expression of the anti-BCMA CARs.
Example 4: Assessment of Potential RNA Heterogeneity and Modification
RNA from cells transduced with exemplary anti-BCMA CARs as described in Example 3 were analyzed for heterogeneity by agarose gel electrophoresis, following reverse transcriptase polymerase chain reaction (RT-PCR) using primers specific to the promoter and the WPRE downstream in the 5′ UTR and 3′ UTR of the exemplary CAR transcripts. Multiple bands were observed for various anti-BCMA CAR constructs containing an exemplary spacer including a modified IgG CH2-CH3-hinge region (BCMA-LS CAR) (FIG. 1A), indicating RNA heterogeneity. Less RNA heterogeneity was observed for exemplary CARs containing a shorter spacer, such as that including a portion of a human CD28 extracellular region (see, e.g., BCMA-52-SS CAR).
In the nucleotide sequences encoding various BCMA-LS CARs were assessed for potential splice sites and modified in a conservative manner, including removal of potential predicted splice sites. The sequences prior to modification (starting sequence) and those following modification (optimized sequences) were subjected to analysis to assess the presence of potential cryptic splice sites. Splice donor sites and splice acceptor sites were evaluated independently. Exemplary splice donor and splice acceptor sites of the starting sequences of various regions of the construct were identified (e.g. in promoter region and long spacer region). Exemplary splice donor sites and splice acceptor sites were identified within the long spacer region following initial codon optimization that had a splice site score of >0.7 (>70%), e.g. donor sites set forth in SEQ ID NO: 693 (splice site score of 0.96) and 708 (splice site score of 0.97), respectively. Modified constructs were generated containing additional modifications within regions assessed with a splice site score of >0.7 (>70%) following initial codon optimization (see, e.g., SEQ ID NO:855 for an exemplary initial codon-optimized spacer sequence) were made in order to reduce potential for unwanted splice sites. Among such regions further modified after codon optimization/splice site elimination were those within longer spacer region sequences, e.g. final optimized splice site eliminated (O/SSE) sequences of splice donor site and splice acceptor site is set forth in SEQ ID NOS: 662 and 672, respectively.
The modified sequences were constructed and tested for RNA heterogeneity as described above. Electrophoresis confirmed reduction of RNA heterogeneity. Analysis of BCMA-CAR constructs before and after splice site elimination demonstrated reduced RNA heterogeneity (FIG. 1B). Exemplary O/SSE CAR constructs were generated containing the modifications of the long spacer region, e.g. BCMA-23-LS-O/SSE CAR, BCMA-25-LS-O/SSE CAR, BCMA-26-LS-O/SSE CAR, BCMA-52-LS-O/SSE CAR, and BCMA-55-LS-O/SSE CAR.
Example 5: Assessment of CAR Expression and Function in Primary T Cells
Lentiviral constructs containing anti-BCMA CAR-encoding polynucleotides with starting and optimized sequences, respectively, as described in Example 3, were transduced into T cells and transduced cells were analyzed for transduction (based on expression of a surrogate marker) and for CAR expression based on binding to recombinant BCMA-Fc fusion protein by flow cytometry. A greater percentage of CD4+ and CD8+ T cells transduced using the optimized sequences, BCMA-52-LS-O/SSE CAR and BCMA-55-LS-O/SSE CAR, expressed the anti-BCMA CAR on the surface, compared to cells transduced to express the same corresponding CAR via the polynucleotide having the starting (non-SSE) sequence. Representative data are set forth in FIG. 2 and Table E5 below.
TABLE E5
Percentage of CD4+ and CD8+ T cells expressing anti-BCMA CAR
BCMA-25-
BCMA-52-
BCMA-55-
BCMA-25
O/SSE
BCMA-52
O/SSE
BCMA-55
O/SSE
CD4+
17.9
64.9
36.4
69.6
44.1
50.2
T cells
CD8+
17.7
61.7
31.8
62.4
36.5
43.4
T cells
Various volumes of viral preparations containing lentiviral vectors encoding CAR constructs, BCMA-23-LS CAR, BCMA 26-LS CAR, BCMA 55-LS CAR and BCMA 55-LS-O/SSE CAR, were used to transduce 500,000 donor-derived primary human T cells and transduction efficiency was compared. The percent transduction of T-cells was increased following transduction by optimized sequences (FIG. 3, circles) compared to starting sequences (FIG. 3, triangles).
Example 6: Characterization of BCMA-52 and BCMA-55 scFvs
A. Immunohistochemistry Staining of Tissues
Cells and tissues expressing varying levels of BCMA were assessed by immunohistochemistry for binding of exemplary anti-BCMA antibodies. Binding domains (scFvs) of exemplary human-BCMA-targeted CARs, which had been fused to a mouse IgG1 Fc region peptide, were assessed for binding cells and tissues by immunohistochemistry.
B. Assessment of Binding Kinetics
A CAR with a BCMA-55-derived scFv binding domain, a modified IgG-derived CH2-CH3-hinge spacer, a CD28 transmembrane domain, and 41BB and CD3zeta endodomain, was expressed in a Jurkat T cell line. Kinetics of binding by the CAR to recombinant human BCMA-hFc (rhBCMA hFc) was assessed using a kinetics exclusion assay. Affinity of binding of an Fc fusion protein containing the scFv portion of the CAR (scFv-Fc) to recombinant human BCMA fusion protein was also assessed using a Biacore-based assay. In these studies, the KD for binding by the CAR and scFv-Fc fusion, respectively, were observed to be approximately 1 nM and 10 nM.
In a further experiment, Jurkat cells were transduced with a polynucleotide encoding a CAR with a BCMA-55-derived scFv binding domain and were cultured to a density of ˜2×106. The cells were harvested and spun at 1500 g for 15 minutes at 4° C. The cell pellet was washed and cells were resuspended and serially diluted in 20 nM or 1 nM biotinylated rhBCMA hFc (also referred to in this assay as the constant binding partner (CBP)). After equilibration, cells were spun down and supernatants were harvested for KinExa kinetic exclusion analysis. Briefly, supernatants from equilibrated BCMA-55-LS CAR O/SSE-expressing Jurkat cells containing rhBCMA hFc were flowed over a streptavidin bead flow cell to capture free biotinylated rhBCMA hFc. The rhBCMA was then detected using a secondary anti-hBCMA antibody that was fluorescently labelled. The absorbance of the detected rhBCMA hFc was recorded for each sample, and plotted against the number of cells in each dilution (Darling (2004) Assay Drug. Dev., 2:647-657). In this study, the KD for the interaction of the BCMA-55-LS-O/SSE CAR-expressing cells binding to rhBCMA hFc in this assay was determined to be approximately 1.46 nM, and the expression level (EL) was determined to be approximately 146,500 CARs per CAR-expressing Jurkat cell.
C. Selectivity of BCMA-55 scFv-Fc
A membrane proteome array (MPA) assay was used to assess binding specificity of the BCMA-55-derived binding domain, using an scFv-Fc fusion protein. The interactions of BCMA-55-Fc to HEK293 cells expressing over 4400 unique human extracellular proteins, representing over 85% of the human extracellular proteome, and a fluorescent protein were evaluated using the Retrogenix™ platform. Fluorescent protein was detected to verify transfection, and CTLA4-Fc (tested at 0.2 μg/mL), containing a matched Fc, was also used to screen for CD86 as a positive control. An initial screening involved an scFv binding assay for BCMA-55-scFv against the full protein panel. A follow-up confirmation screen was then performed retesting the interaction of BCMA-55-Fc with a subset of potential hits identified in the initial screen. BCMA was identified as the only strong, specific hit in this assay, consistent with a conclusion that this binding domain is highly selective for BCMA over other extracellular proteins. Some low level signal was observed for Cathepsin G (CTSG), but was observed not to confer functional activity (see Example 16).
Example 7: In Vitro Functional Assessment of T Cells Engineered to Express Various Anti-BCMA Chimeric Antigen Receptor (CARs)
Genetically engineered human T cells expressing various exemplary anti-BCMA CARs were assessed in vitro following co-culture with BCMA-expressing target cells. T cells were transduced with BCMA-52-LS CAR, BCMA-55-LS CAR, BCMA-52-LS-O/SSE CAR, or BCMA-55-LS-O/SSE CAR). Responses were compared to reference anti-BCMA CAR-expressing cells as positive control or mock-processed cells as negative control.
A. Cytolytic Activity Against Target Cells
BCMA-expressing target cells were incubated with T cells expressing the BCMA-52-LS CAR, BCMA-55-LS CAR, or a reference anti-BCMA CAR at an effector to target (E:T) ratio of 5:1, 2.5:1, 1.25:1 and 0.65:1. As a control, target cells were incubated with T cells not expressing a CAR (mock control). Specifically, BCMA-transduced K562 cells (K562/BCMA, BCMAhigh) or RPMI 8226 cells (BCMAlow human multiple myeloma cell line) were used as targets for lysis. Target cells were labeled with NucLight Red (NLR) to permit tracking of target cells by microscopy. Cytolytic activity was assessed by measuring the loss of viable target cells over a period of between 24 and 72 hours, as determined by red fluorescent signal (using the IncuCyte® Live Cell Analysis System, Essen Bioscience). Percent lysis (% Lysis) was normalized to the lysis that occurred in target cells incubated with mock-processed T cells. As shown in FIG. 4A, the anti-BCMA CAR-expressing T cells exhibited antigen-specific cytolytic activity against BCMA+ cells. The magnitude of cell lysis differed depending on the particular cell line and CAR.
In a separate experiment, cytolytic activity was tested with RPMI 8226 target cells at a E:T ratio of 3:1. As shown in FIG. 4B, BCMA-52-LS- and BCMA-55-LS-CAR-expressing cells showed approximately 70% lysis, normalized to the lysis by mock-processed cells not expressing a CAR, whereas the cells expressing the CAR containing the reference anti-BCMA antibody binding domain showed approximately 50% lysis. Thus, the results showed that cytolytic activity of cells engineered to express BCMA-52- or BCMA-55-CARs was similar to or higher than that of the reference binding domain-containing CAR.
To compare cytolytic activity of T cells engineered with the same CAR encoded by an unmodified CAR construct or an optimized CAR construct, T cells were engineered to express an anti-BCMA CAR using a viral vector containing either an unmodified polynucleotide construct (BCMA-52-LS CAR and BCMA-55-LS CAR) or an optimized polynucleotide construct (BCMA-52-LS-O/SSE CAR and BCMA-55-LS-O/SSE CAR). Cytolytic activity of the engineered cells was assayed substantially as described above. The CAR-expressing T cells were incubated with target cells, K562-BCMA, RPMI 8226, MM1.S cells (BCMAmed human multiple myeloma cell line) or OPM2 cells (BCMAmed human multiple myeloma cell line) target cells, at an E:T ratio of 3:1. As shown in FIG. 4C and FIG. 4D, CAR-expressing cells transduced with a CO/SSE CAR construct exhibited greater cytolytic activity compared to cells transduced with the corresponding unmodified construct.
B. Cytokine Release
Cytokine release was assessed following incubation of the various anti-BCMA CAR-expressing cells with antigen-expressing target cells.
BCMA-expressing target cells, K562/BCMA or RPMI 8226 cells, were incubated with T cells expressing the BCMA-52-LS CAR, BCMA-55-LS CAR, or a reference binding domain-containing anti-BCMA CAR at an E:T ratio of 5:1, 2.5:1, 1.25:1 or 0.6:1. As a control, target cells were incubated with T cells not expressing a CAR (mock control). The co-cultured cells were incubated for about 24 hours, and then supernatants were collected for measurement of IFN-γ, TNF-α and IL-2, using a multiplex cytokine immunoassay. As shown in FIG. 5A, the tested anti-BCMA CAR-expressing T cells produced cytokines following antigen stimulation.
To assess antigen-dependent cytokine production of T cells engineered with the same CAR encoded by an unmodified CAR construct or an optimized CAR construct, T cells were engineered to express an anti-BCMA CAR using a viral vector containing either an unmodified polynucleotide construct (BCMA-52-LS CAR and BCMA-55-LS CAR) or an optimized polynucleotide construct (BCMA-52-LS-O/SSE CAR and BCMA-55-LS-O/SSE CAR). CAR-expressing T cells were incubated with target cells, either K562/BCMA, RPMI 8226 cells, MIN1S (BCMAmed human multiple myeloma cell line) or OPM2 cells (BCMAmed human multiple myeloma cell line) target cells, at an E:T ratio of 3:1, 1.5:1, 0.75:1 and 0.375:1. Production of cytokines IFN-γ, TNF-α and IL-2 was assessed as described above. As shown in FIG. 5B, CAR-expressing cells transduced using O/SSE optimized constructs were observed to exhibit higher cytokine production compared to cells transduced with the corresponding unmodified (starting) construct.
C. Cytolytic Activity, Cytokine Release and Proliferation in Response to Targets Expressing Different Levels of Antigen on their Surfaces
Cytolytic activity, cytokine release, and proliferation were assessed following incubation of BCMA-55-LS-O/SSE CAR-expressing T cells with BCMA-expressing cells that expressed different levels of BCMA. All activity was evaluated in the presence or absence of soluble BCMA.
A 1:1 ratio of CD4+ and CD8+ primary T cells, harvested from two human donors (D#1 and D#2), were stimulated with CD3/CD28 beads and transduced with a lentiviral vector to stably express BMCA-55 CAR. Transduced cells were cultured in the presence of BCMA-expressing target cells at an E:T ratio of 1:3, 1:1 or 3:1. Mock-processed T cells from the same donors were also mixed with target cells for use as a control. The BCMA+ target cells, Daudi, RPMI-8226, and K562-BCMA cell, exhibited different levels of BCMA antigen-density of the surface (antigen density: Daudi (<1000 BCMA molecules/cell)<RPMI-8226<K562-BCMA) and were stained with carboxyfluorescein succinimidyl ester (CF SE) prior to incubation with the T cells. An equal number of target-negative cells, not expressing BCMA and stained with cell trace violet (CTV), were also included in the cultures with the T cells and BCMA+ target cells. After a 24 hour incubation, the remaining BCMA+ vs BCMA− target cells were measured by flow cytometry, and the degree of target cell lysis, indicative of cytotoxicity, was assessed.
BCMA-55-LS-O/SSE CAR T cells displayed similar cytolytic activity when cultured with target cells, regardless of BCMA expression levels (FIG. 6). Additionally, similar results were observed for target cells (NCI-H929) expressing a greater than 100,000 molecules per cell. Mock-processed T cells did not show activity against any of the BCMA+ target cell lines. Target cells negative for BCMA expression were not lysed by the BCMA-55-LS-O/SSE CAR T cells from any of the donors tested (data not shown).
The supernatants following the incubation were analyzed for accumulated IFN-γ, TNF-α, and IL-2 cytokines. Data were consistent with a conclusion that BCMA-55-LS-O/SSE CAR T cells had released a range of cytokines following engagement with BCMA-expressing target cells; with the level of cytokines released generally corresponding with increasing level of antigen (i.e., Daudi<RPMI 8226<K562-BCMA). Results for IFN-γ are shown in FIG. 7; similar data were observed for TNF-α and IL-2 (data not shown). BCMA-55-LS CAR O/SSE T cells did not release cytokines in response to BCMA-negative targets, nor did they express cytokines without any target cells present, demonstrating specificity for BCMA+ target cells and lack of tonic signaling.
Activity of BCMA-55-LS-O/SSE CAR-expressing T cells in the presence vs. absence of soluble BCMA was assessed. BCMA-55-LS-O/SSE CAR-expressing T cells were co-cultured with RPMI-8226 tumor cells, with recombinant BCMA-Fc, or with cell culture supernatant derived from NCI-H929 multiple myeloma cells (BCMA-secreting cell line, the supernatant containing soluble BCMA). Neither tumor-cell lysis nor cytokine production was observed to be affected by any of the concentrations of NCI-H929-derived soluble BCMA (up to 1000 ng/mL). Both tumor-cell lysis and cytokine production were only minimally decreased at similarly high physiological levels of recombinant BCMA.
Proliferation in response to BCMA was measured in BCMA-55-LS-O/SSE CAR-expressing T cells and mock-processed T cells. Transduced T cells were labeled with cell trace violet (CTV) and cultured in the presence of BCMA-positive target cells, BCMA-negative target cells, or no cells, at an effector to target (E:T) ratio of 1:1, for 72 hours. Proliferation was measured by flow cytometry. Proliferation of T cells (CD4+ and CD8+ T cells) was observed only for BCMA-55-LS-O/SSE CAR-expressing T cells in response to incubation with BCMA-positive target cells.
D. Transduced T cells Harvested from Healthy Donors and a Myeloma Patient
T cells engineered to express BCMA-55-LS-O/SSE CAR harvested from multiple myeloma patients were compared to those derived from healthy human donors following a 24-hour incubation with BCMA+ and BCMA-K562 target cells. T cells not expressing a CAR were also evaluated as a negative control. CAR T cells derived from multiple myeloma patients demonstrated similar expression, expansion and antigen-specific activities as compared to cells expressing the CAR derived from healthy human donors.
Example 8: Anti-BCMA CARs with Different Spacers
Polynucleotide constructs encoding anti-BCMA CARs were generated that contained different spacer regions between the scFv and transmembrane segments of the encoded CAR polypeptide. Specifically, CARs were generated containing: (1) a spacer derived from an IgG hinge region (e.g., e.g., BCMA-5-SS, BCMA-9-SS, BCMA-18-SS, BCMA-23-SS, BCMA-25-SS, BCMA-26-SS, BCMA-52-SS, BCMA-55-SS, and Referenc1 (VH/VL)-SS); or (2) a short spacer derived from the ectodomain of CD28 (e.g. BCMA-52-SCD28 and BCMA-55-SCD28). T cells expressing such spacer-containing CARs were compared to T cells transduced with polynucleotide constructs encoding exemplary CARs containing spacers as described in Example 3 (e.g. BCMA-1-LS, BCMA-5-LS, BCMA-9-LS, BCMA-18-LS, BCMA-23-LS, BCMA-25-LS, BCMA-26-LS, BCMA-27-LS, BCMA-52-LS, BCMA-55-LS, and Reference1 (VH/VL)-LS).
CAR-expressing cells were assessed for cytolytic activity by monitoring the lysis of OPM2 human multiple myeloma target cells cultured with CAR-expressing T cells at an effector to target (E:T) ratio of 1.25:1 and 0.65:1. Cells that did not express a CAR (mock) were used as a negative control. Cytolytic activity was assessed as described in Example 7. For most assessed CAR-expressing cells, target cell lysis was greater for cells engineered to express a CAR containing a CH2-CH3-hinge spacer as compared to cells engineered with a CAR containing a shorter spacer (FIG. 8).
Example 9: Assessment of Agents on Blocking Activity of Anti-BCMA CAR Activity
The function of anti-BCMA CAR-expressing cells was assessed following incubation with BCMA-expressing target cells and soluble BCMA or other proteins. Cytolytic activity and cytokine production was assessed substantially as described in Example 7.
A. Cytolytic Activity
1. Soluble Recombinant BCMA (rBCMA)—OPM2 Target Cells
Anti-BCMA CAR-expressing T cells, BCMA-52-LS CAR, BCMA-55-LS CAR or Reference binding domain-containing CAR, were incubated with OPM2 target cells at an E:T ratio of 5:1 in the presence of soluble BCMA-Fc at 0, 0.3, 3, 30 or 300 ng/mL. As shown in FIG. 9A cytolytic activity of T cells expressing the Reference binding domain-containing CAR or BCMA-52-LS CAR were substantially reduced in the presence of 3 ng/mL or more BCMA-Fc, however the cytolytic activity of cells expressing BCMA-55-LS CAR was not blocked by the presence of up to 300 ng/mL BCMA-Fc.
In another experiment, Anti-BCMA CAR-expressing T cells (BCMA-1-LS CAR, BCMA-9-LS CAR, BCMA-23-LS CAR, BCMA-25-LS CAR, BCMA-26-LS CAR, BCMA-55-LS CAR and Reference1 (VH/VL)-LS CAR) were incubated with OPM2 target cells at an E:T ratio of 5:1 in the presence of soluble BCMA-Fc at concentrations of 0, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 ng/mL. As shown in FIG. 9B the cytolytic activity of cells expressing BCMA-55-CAR was not blocked by the presence of BCMA-Fc at any of the concentrations tested; however, the presence of variable concentrations of BCMA-Fc blocked activity of cells expressing other anti-BCMA CARs to different extents.
2. Multiple Myeloma Cell Line (H929) Supernatant—OPM2 Target Cells
Optimized, splice site eliminated (O/SSE) anti-BCMA CAR-expressing T cells, BCMA-52-LS-O/SSE CAR, BCMA-55-LS-O/SSE CAR or Reference binding domain-containing CAR, were incubated with OPM2 target cells at an E:T ratio of 5:1 in the presence of 0, 111, 333 and 1000 ng/mL culture supernatant from the H929 multiple myeloma cell line. The concentration of soluble BCMA was quantified from the H929 supernatant by ELISA. As shown in FIG. 10A the cytolytic activity of cells expressing BCMA-52-LS-O/SSE CAR, BCMA-55-LS-O/SSE CAR or Reference CAR were not blocked by the presence of H929 supernatant.
3. Soluble Recombinant BCMA (rBCMA) and H929 Supernatant—RPMI-8226 Target Cells
In a further study, optimized, splice site eliminated (O/SSE) BCMA-55-LS-O/SSE CAR-expressing T cells, were incubated with RPMI-8226 tumor target cells at an E:T ratio of 3:1 in the presence of 0, 111, 333 and 1000 ng/mL soluble BCMA from culture supernatant from the H929 multiple myeloma cell line (soluble BCMA quantitated by ELISA) or BCMA-Fc. The cytolytic activity of cells expressing BCMA-52-LS-O/SSE CAR, BCMA-55-LS-O/SSE CAR or Reference CAR was not blocked by the presence of H929 supernatant.
4. B-Cell Activating Factor (BAFF)
Optimized, splice site eliminated (O/SSE) anti-BCMA CAR-expressing T cells, BCMA-52-LS-O/SSE CAR, BCMA-55-LS-O/SSE CAR or Reference CAR, were incubated with OPM2 target cells at an E:T ratio of 5:1 in the presence of 0, 1, 10, 100 and 1000 ng/mL recombinant B-cell activating factor (BAFF), a ligand for BCMA. As shown in FIG. 10B, cytolytic activity of T cells expressing BCMA-52-LS-O/SSE CAR, BCMA-55-LS-O/SSE CAR or Reference CAR were not blocked by the presence of BAFF.
B. Cytokine Release
1. BCMA-Fc
Anti-BCMA CAR-expressing T cells, BCMA-52-LS CAR, BCMA-55-LS CAR or Reference-LS CAR, were incubated with OPM2 target cells at an E:T ratio of 5:1 in the presence of soluble BCMA-Fc at 0, 111, 333 and 1000 ng/mL T cells not expressing a CAR (mock) also were assessed. Cytokine accumulation of IFN-γ, TNF-α and IL-2 in supernatant was assessed. As shown in FIG. 11A, cytokine accumulation in cultures containing T cells expressing the Reference CAR or BCMA-52-CAR were substantially reduced in the presence of 111 ng/mL or more BCMA-Fc, however less reduction in cytokine accumulation was observed in cultures containing T cells expressing BCMA-55-CAR in the presence of soluble BCMA-Fc at all concentrations tested.
2. Multiple Myeloma Cell Line (H929) Supernatant
Anti-BCMA CAR-expressing T cells, BCMA-52-LS CAR, BCMA-55-LS CAR or Reference-LS CAR, were incubated with OPM2 target cells at an E:T ratio of 5:1 in the presence of 0, 111, 333 and 1000 ng/mL culture supernatant from a multiple myeloma cell line H929. Cytokine accumulation in cultures containing T cells expressing BCMA-52-CAR, BCMA-55-CAR or Reference CAR were not blocked by the presence of H929 supernatant (FIG. 11B)
Example 10: Anti-Tumor Effect of Anti-BCMA CAR-Expressing T Cells after Adoptive Transfer In Vivo in an Animal Model
The anti-tumor effects of exemplary engineered anti-BCMA CAR-expressing primary human T cells were assessed by monitoring tumors following adoptive transfer of cells in tumor-bearing animal models, including OPM2 human multiple myeloma xenograft mouse model (orthotopic bone marrow model) and RPMI 8226 human multiple myeloma xenograft mouse model (subcutaneous implant model).
A. OPM2 (Orthotopic/Bone Marrow) Model
NOD.Cg.PrkdcscidIL2rgtm1Wj1/SzJ (NSG) mice were injected intravenously (i.v.) with 2×106 OPM2 (multiple myeloma) cells transfected with firefly luciferase (OPM2-ffluc). On day 14, following tumor engraftment, mice received a single intravenous (i.v.) injection of anti-BCMA CAR T cells expressing optimized, splice site eliminated (O/SSE) BCMA-23-LS-O/SSE CAR, BCMA-26-LS-O/SSE CAR or BCMA-55-LS-O/SSE CAR. The anti-BCMA CAR-expressing T cells were administered at a dose of either 1×106 (low dose, n=8) or 3×106 (high dose, n=8) CAR-expressing T cells per mouse, and each condition repeated for CAR-expressing T cells derived from two different donors. As a control, mice were administered cells not expressing a CAR (mock, n=8) or were untreated (n=3). Survival and tumor burden were assessed over 90 days.
Anti-tumor activity of the adoptively transferred CAR-expressing (CAR-T) cells was monitored by bioluminescence imaging every 3 to 6 days post CAR-T cell administration for the length of the study. For bioluminescence imaging, mice received intraperitoneal (i.p.) injections of luciferin substrate (CaliperLife Sciences, Hopkinton, Mass.) resuspended in PBS (15 μg/g body weight). Mice were anesthetized and imaged essentially as described in WO2015/095895. The total flux (photon/s) was determined at each time point. For the negative control treated mice, animals were sacrificed between 19 and 23 days after CAR-T cell administration, due to high tumor burden. Representative results from one donor-derived CAR-expressing T cells are shown in FIG. 12A.
As shown in FIG. 12A, for all treated mice, the tumor in mice receiving mock-processed T cells or no T cells continued to grow over the course of the study. Compared to the control mice, mice that received an adoptive transfer of T cells engineered to express BCMA-23-LS-O/SSE CAR, BCMA-26-LS-O/SSE CAR, or BCMA-55-LS-O/SSE CAR, were observed to generally have a lower degree of bioluminescence signal, indicating a reduction in tumor growth over time and/or a lower degree of tumor growth in the treated animals. The effect on tumor growth was greater with the higher dose of anti-BCMA CAR expressing cells for the exemplary tested anti-BCMA CARs.
Survival of mice treated as described above were assessed and compared until day 79 post-infusion of CAR-expressing T cells. Representative survival curves, Kaplan-Meier method (GraphPad Prism 7.0, GraphPad Software, La Jolla), from one donor are shown in FIG. 12B. As shown, the tested anti-BCMA CAR-T cells at the low and high dose resulted in greater percent survival of mice compared to mice receiving no treatment or mock-processed T cells. Mice also were assessed for presentation of clinical signs associated with tumor burden, including hind limb paralysis (HLP), greater than 20% body weight loss (>20% BWL), and graft-versus-host disease (GvHD). The number of mice with these clinical signs was reduced compared to mice receiving no treatment or mock T cells.
B. RPMI-8226 (Subcutaneous) Model
NOD.Cg.PrkdcscidIL2rgtm1Wj1/SzJ (NSG) mice were injected subcutaneously with RPMI 8226 (peripheral blood plasmacytoma) cells. On Day 27, the mice were randomized into groups based on a minimum mean tumor volume of approximately 130 mm3. On Day 29, mice received a single intravenous (i.v.) injection of primary human T cells (CD4+ and CD8+) engineered to express optimized, splice site eliminated (O/SSE) BCMA-23-LS-O/SSE CAR, BCMA-26-LS-O/SSE CAR, or BCMA-55-LS-O/SSE CAR at a dose of 1×106 (low dose, n=8) or 3×106 (high dose, n=8) CAR-expressing T cells. Each condition was repeated for CAR-expressing T cells derived from two different donors. Mice that were administered cells that were mock-processed and untreated mice were used as negative controls. Tumor volume was measured by calipers twice weekly up to Day 152 post CAR T-cell transfer and euthanized when moribund, 20% weight loss, or when tumor volume exceeded 1500 mm3. Survival curves were plotted up to Day 108 post CAR T-cell transfer using the Kaplan-Meier method (GraphPad Prism 7.0, GraphPad)
Representative results for tumor growth and survival from CAR-expressing T cells derived from one donor are shown in FIGS. 13A and 13B, respectively. As shown in FIG. 13A, the tumor continued to grow over the course of the study following adoptive transfer of negative control cells or in mice not receiving treatment. Compared to the control mice, mice that received an adoptive transfer of T cells engineered to express BCMA-23-LS-O/SSE CAR, BCMA-26-LS-O/SSE CAR, or BCMA-55-LS-O/SSE CAR showed substantially reduced tumor volume after receiving the low or high dose of CAR-expressing T cells (FIG. 13A). In this model, mice administered both tested doses of anti-BCMA CAR T cells exhibited complete regression of tumor growth by 20 days post CAR T-cell transfer, which continued throughout the duration of the study assessment shown in FIG. 13A.
The percent survival of mice administered anti-BCMA CAR-expressing T cells also was substantially greater than control groups (FIG. 13B). At 108 days post-CAR T cell infusion, two animals had been lost post-tumor elimination in the group treated with the high dose of BCMA-26-LS-O/SSE CAR-expressing T cells, although this was likely due to graft versus host disease (GVHD) symptoms in this model. All other CAR-T cell treated mice remained alive up to 108 days post-CAR T cell administration.
The presence of CAR+ T cells in the blood was monitored to assess pharmacokinetics of CAR-expressing T cells in the mice from treated. The 8 mice of each treatment group were divided into 2 groups of 4 mice. Blood was drawn weekly, by retro-orbital bleeding, alternating between the 2 groups such that each mouse was bled every other week for 4 weeks post CAR-T cell administration (i.e., on days 7, 14, 21 and 28 post CAR-T cell administration). The collected blood was analyzed for the number of CAR-expressing T cells, as determined using an antibody against the surrogate marker or soluble BCMA-Fc, and non-CAR T cells, per μL blood by flow cytometry (FlowJo software, Treestar Inc., Ashland, Oreg.).
The number of CD4+ and CD8+ T cells per μL of blood at days 7, 14, 21 and 28 are shown in FIG. 14A and FIG. 14B, respectively, for one donor and in FIG. 15A and FIG. 15B, respectively, for the second donor. As shown, CAR-T expansion occurred in high and low dose groups in CD4+ and CD8+ T cells, with maximum or peak expansion observed at day 14 post CAR T-cell transfer for both donors. At all assessed times post CAR-T cell transfer, greater numbers of CD8+ CAR+ T cells were observed compared to CD4+ CAR+ T cells for both donors (compare FIG. 14A and FIG. 14B or FIG. 15A and FIG. 15B). T cells engineered to express BCMA-55-LS-O/SSE CAR exhibited greater CAR expression compared to T cells expressing BCMA-23-LS-O/SSE CAR and BCMA-26-LS-O/SSE CAR constructs, which exhibited comparable expression to each other. These results demonstrate BCMA-55-LS CAR expressing T cells can be identified circulating in the blood during tumor clearance.
Example 11: Assessment of Signals Through Anti-BCMA Chimeric Antigen Receptor (CAR) in a Nur77-tdTomato Reporter Signal in Reporter Cell Line
An exemplary stable Jurkat T cell reporter cell line was generated containing a Nur77 knock-in reporter, where the nucleic acid sequences encoding the reporter molecule was knocked-in at the endogenous Nur77 locus via homology dependent repair (HDR). Orphan nuclear hormone receptor Nur77 (also called Nr4a1) is an immediate-early response gene induced by activation of signal from the T cell receptor and/or via molecules containing immunoreceptor tyrosine-based activation motif (ITAM). The Nur77-reporter cell line was used to assess T cell activation in CAR-engineered cells as Nur77 is an immediate early gene product in T lymphocytes; transcription is initiated specifically downstream of CD3 zeta signaling, and is not influenced by cytokine or TLR mediated signals. In a Jurkat T cell clone E6-1 (ATCC® TIB-152™), nucleic acid sequence encoding a red fluorescent protein (RFP; such as the tdTomato fluorescent protein) was targeted for integration in-frame with the endogenous Nr4a1 (Nur77) gene at the final exon, prior to the stop codon, and after a “self-cleaving” T2A element (sequence set forth in SEQ ID NO:686 or 687, encoding polypeptide sequence set forth in SEQ ID NO: 631, 653 or 654), to allow for coexpression of RFP as a reporter of Nur77 expression, by introducing a genetic disruption using gene editing and targeting a transgene for integration at a site near the genetic disruption by homology-dependent repair (HDR). The Nur77-tdTomato reporter cell line was engineered to express various anti-BCMA chimeric antigen receptors, and reporter expression was assessed.
Viral vectors containing polynucleotides encoding the following anti-BCMA chimeric antigen receptors (CARs), described in Example 3, were introduced into the Nur77-tdTomato reporter Jurkat T cell line: BCMA-55-LS-O/SSE CAR, BCMA-26-LS-O/SSE CAR, BCMA-23-LS-O/SSE CAR, and BCMA-25-LS-O/SSE CAR. Anti-BCMA CAR-expressing reporter cells were evaluated for activity of Nur77 signaling in response to increasing amounts of plate-bound recombinant BCMA or in response to exemplary multiple myeloma cell lines after 20 hours of co-culture.
A. Nur77 Signaling in Response to Plate-Bound Recombinant BCMA
Reporter cells transduced with a viral vector encoding BCMA-55-LS-O/SSE CAR were incubated for 6 hours in 96-well cell culture plates that had been coated overnight with varying concentrations (0.008 μg/mL, 0.04 μg/mL, 0.2 μg/mL, 1 μg/mL and 5 μg/mL) of BCMA-Fc (soluble human BCMA fused at its C-terminus to an Fc region of IgG) fusion polypeptide. A recombinant Fc polypeptide was used as a control (Fc Control). As shown in FIG. 16A, a dose-dependent increase in tdTomato expression was observed following stimulation of anti-BCMA CAR-expressing reporter cells with recombinant antigen.
In another study, reporter cells engineered to express BCMA-55-LS-O/SSE CAR, BCMA-26-LS-O/SSE CAR, BCMA-23-LS-O/SSE CAR, and BCMA-25-LS-O/SSE CAR were incubated with ten (10) 2-fold serial dilutions of BCMA-Fc. Reporter cells expressing an anti-CD19 CAR was used as a non-target control. The percentage of tdTomato-expressing cells within the population of cells expressing the CAR (as determined based on expression of the surrogate marker) was determined. As shown in FIG. 16B, a dose-dependent increase in tdTomato expression was observed following stimulation of with recombinant antigen. No response to stimulation with BCMA-Fc was observed by the control reporter cells expressing a CAR against a non-target antigen.
B. Nur77 Signaling in Response to Multiple Myeloma Cell Lines
Reporter cells transduced with a viral vector encoding BCMA-55-LS-O/SSE CAR were incubated for 20 hours with NALM6, Daudi, RPMI-8226, MM1S, OPM2, and H929 cells. Different levels of RFP expression were observed depending on the cell line which conferred stimulation of the anti-BCMA CAR-expressing reporter cells.
To assess the amounts of BCMA expression on the surface of the multiple myeloma cell lines used to stimulate the anti-BCMA CAR-expressing reporter cells, the cells were stained with anti-human BCMA antibody (BioLegend, San Diego, Calif.), flow cytometry events were collected on an LSRFortessa™ flow cytometer (BD Biosciences, San Jose, Calif.) and data were analyzed with FlowJo software (Treestar Inc., Ashland, Oreg.). BCMA antigen density (AD) was determined by using Quantum™ Simply Cellular® anti-Mouse IgG microsphere beads coated with the same anti-human BCMA antibody. Microspheres were labeled and BCMA antibody binding capacity was calculated. The results confirmed the detection of a parameter (detectable levels of the reporter) indicative of specific CAR activity in CAR-expressing reporter cells, when incubated with each of the various different BCMA-expressing cells, exhibiting a range of different antigen densities, and not when incubated with target-negative cells. The degree of the RFP reporter signal generally correlated with levels of surface BCMA expression. When incubated with cells in which lower levels of surface BCMA expression were observed, CAR-expressing reporter cells exhibited lower levels of the reporter indicative of activity. Likewise, CAR-expressing reporter cells incubated with cell lines in which higher levels of surface BCMA expression was observed exhibited higher levels of the reporter indicative of activity. Thus, the density of BCMA expression on the surface of the various multiple myeloma cell lines was observed to correlate with the level of a parameter indicative of antigen-specific activity of reporter cells expressing the BCMA-55-LS-O/SSE CAR, indicating that cells expressing the CAR can exhibit activity over a range of antigen densities, and in some aspects can exhibit increased activity with increased antigen levels.
Example 12: Assessment of Nur77-tdTomato Reporter Signal in Reporter Cell Lines Expressing Anti-BCMA Chimeric Antigen Receptors (CARs) Containing Spacers of Different Length
Expression of the reporter in cells engineered to express anti-BCMA CARs containing the same antigen-binding domain but spacers of different length was determined after co-culture with target cells. The Jurkat Nur77-tdTomato cells, generated as described in Example 11, were engineered to express BCMA-55-LS-O/SSE CAR (containing a longer spacer derived from modified IgG Hinge-CH2-CH3, set forth in SEQ ID NO:649) or BCMA-55-SS CAR (containing a shorter spacer derived from IgG4 hinge, set forth in SEQ ID NO:363). The cells were co-cultured with human BCMA-expressing K562 target cells (BCMA-K562) target cells at various E:T ratios. Reporter cells expressing a CAR targeting a different antigen (anti-CD19 CAR), were used as control. As shown in FIG. 17, the Nur77-tdTomato expression level was observed to be different in the anti-BCMA CARs containing different spacer lengths, and a dose-dependent response to stimulation with target cells expressing BCMA was observed.
Example 13: Assessment of Antigen-independent (Tonic) Signaling from Different Anti-BCMA Chimeric Antigen Receptors (CARs)
The Nur77-tdTomato reporter cells were transduced with a viral vector encoding anti-CD19 CAR (control), BCMA-55-LS-O/SSE CAR, BCMA-26-LS-O/SSE CAR, BCMA-23-LS-O/SSE CAR, or BCMA-25-LS-O/SSE CAR as described in Example 11 above, with the exception that the surrogate marker for transduction was super-fold green fluorescent protein, sfGFP. In this model, tonic signaling was indicated by tdTomato expression in the absence of BCMA antigen stimulation.
A viral vector encoding an anti-BCMA CAR containing a different anti-BCMA scFv, designated as BCMA-52-LS-O/SSE CAR, also was generated and transduced into the reporter cell. The various CAR-expressing cells were incubated without antigen stimulation to assess the degree of antigen-independent (tonic) signaling for 3 days and evaluated for the expression of tdTomato by flow cytometry.
As shown in FIG. 18, various CAR-expressing cell lines exhibited a varying degree of tdTomato expression in the absence of antigen stimulation. The percentage of tdTomato+ cells (indicative of tonic reporter activation) among CAR-expressing cells (indicated by GFP+ cells) varied from 0.23% to 19.3%, in cells expressing different CARs.
Example 14: Assessment of Antigen-independent (Tonic) Signaling from Anti-BCMA Chimeric Antigen Receptors (CARs) Containing Different Intracellular Domains
Antigen-independent (tonic) signaling was assessed in reporter cells expressing various CARs containing different intracellular signaling regions. The Nur77-tdTomato reporter cells were transduced with a viral vector encoding anti-CD19 CAR, BCMA-55-LS-O/SSE CAR, BCMA-26-LS-O/SSE CAR, BCMA-23-LS-O/SSE CAR, or BCMA-52-LS-O/SSE CAR, generated generally as described in Example 11 and 13, with the exception that the CARs contained intracellular domains derived from 4-1BB or CD28, and the surrogate marker for transduction was a truncated receptor. The various CAR-expressing cells were incubated without antigen stimulation to assess the degree of antigen-independent (tonic) signaling and evaluated for the expression of tdTomato by flow cytometry.
As shown in FIG. 19A and FIG. 19B, the 4-1BB- and CD28-derived intracellular domains in various CARs resulted in different levels of tonic signaling, as indicated by the percentage of tdTomato+ cells among the CAR+ cells (as determined based on expression of the surrogate marker).
Example 15: Assessment of Antigen Cross-Reactivity of Anti-BCMA Chimeric Antigen Receptors (CARs) Using Reporter Cell Line
The Nur77-tdTomato cell line engineered to express BCMA-55-LS-O/SSE CAR, specific for human BCMA and generated as generally described in Example 11, was employed to assess species cross reactivity of the antigen-binding domains of CARs. The reporter cell line expressing BCMA-55-LS-O/SSE CAR was co-cultured with K562 human myelogenous leukemia cells expressing human BCMA (huBCMA), murine BCMA (muBCMA) or cynomolgus monkey BCMA (cynoBCMA), at an E:T ratio of 2:1 or 5:1. The percentage of tdTomato+ cells were determined by flow cytometry.
As shown in FIG. 20A, more than 90% of the BCMA-55-LS-O/SSE CAR-expressing cells were observed to be tdTomato+ when cultured with target cells expressing huBCMA, at both E:T ratios tested. In comparison, when cultured with target cells expressing muBCMA, very few cells were tdTomato+, indicating very low cross-reactivity. When cultured with target cells expressing cynoBCMA, approximately 10 to 20% of the cells were tdTomato+, indicating some cross-reactivity by cynoBCMA.
The reporter cell line expressing BCMA-55-LS-O/SSE CAR was incubated with increasing concentrations (0, 0.1, 0.25, 1, 2.5, 10, 25 and 100 μg/mL) of huBCMA and cynoBCMA coated on 96-well flat-bottom plates. The percentage of tdTomato+ cells and the mean fluorescence intensity (MFI) of the tdTomato signal in CAR+ cells were determined.
As shown in FIGS. 20B and 20C, cynoBCMA did not cross-react with BCMA-55-LS-O/SSE CAR at low concentrations, but did at high concentrations.
Example 16: Assessment of Antigen Specificity of Anti-BCMA Chimeric Antigen Receptors (CARs) Using Reporter Cell Line
The antigen specificity for activation of BCMA-55-LS-O/SSE CAR-expressing cells was tested by comparing the activation of Jurkat Nur77 reporter cells in response to BCMA-expressing MM1S target cells, with K562 target cells engineered to express a non-BCMA protein shown to be recognized at low levels by BCMA-55-scFv Fc in Example 5C, Cathepsin G (CTSG). As a negative control, parental K542 cells also were assessed. Briefly, Nur77 reporter cells, transduced with a viral vector encoding BCMA-55-LS-O/SSE CAR, were incubated 24 hours with the target cells listed above, at 5:1, 1:1, and 1:5 effector:target cell ratios, and activation was determined by measuring the percentage of cells expressing RFP (RFP+) by flow cytometry. The results demonstrated that BCMA-55-LS-O/SSE CAR-expressing cells were activated by BCMA-expressing MM1S cells, but not BCMA-negative target cells (parental or cells expressing the non-BCMA antigen, CTSG).
Example 17: Determining the Binding Epitope for BCMA-52 and BCMA-55 scFvs
Epitopes recognized, e.g., specifically bound to, by exemplary anti-BCMA scFv clones (BCMA-1, BCMA-5, BCMA-9, BCMA-23, BCMA-25, BCMA-26, BCMA-52 and BCMA-55 anti-BCMA scFvs), were assessed using full discontinuous epitope mapping by Chemical Linkage of Peptides onto Scaffolds (CLIPS; Pepscan Presto BV, Lelystad, The Netherlands; see, e.g., Timmerman et al., (2007) J. Mol. Recognit. 20: 283-329). Mapping was carried out using anti-BCMA scFv clones, such as those fused with mouse Fc (scFv-mFc).
Linear and conformational peptide libraries of amino acid residues 1-54 of human BCMA (set forth as amino acid residues 1-54 of SEQ ID NO:367) were generated based on a combinatorial matrix design. Linear peptides and structural mimetics including single loop, α-helix, β-turn, combinatorial and linear disulfide bridge mimics, and discontinuous epitope mimics were used, along with positive and negative control peptides, on an amino-functionalized solid support.
Affinities for binding to the peptides in the epitope library were determined using ELISA. The peptide arrays were incubated with a solution containing the scFv overnight at 4° C. Affinity information was used in iterative screens to define the sequence and conformation of epitopes. Heat maps of affinity information for two or more loops were generated.
scFvs assessed were observed to recognized conformational epitopes that included several discontinuous peptide stretches of the BCMA peptide sequence. BCMA-1, BCMA-5, BCMA-23 and BCMA-25 scFv were observed to bind to a peptide of 30SNTPPLTCQR39 (set forth in SEQ ID NO:379), which could be recognized in a linear form. In some aspects, such antibodies recognize a non-linear or linear epitope including residues of such peptide of SEQ ID NO: 379, and in some aspects to recognize a non-linear epitope further including residues of 21CIPCQLR27 (set forth in SEQ ID NO:375), 30SNTPPLTCQR39 and/or 44SVTNSVK50 (set forth in SEQ ID NO:393). The BCMA-26 scFv was observed to recognize an epitope comprising residues present in 8CSQNEYF14 (set forth in SEQ ID NO:410) and 17LLHACIPCQLR27 (set forth in SEQ ID NO:428). BCMA-52-scFv-mFc was observed to bind to an epitope containing residues of the following discontinuous peptides: 10QNEYF14 (SEQ ID NO:637), 21CIPCQL26 (SEQ ID NO:638), and 7CQRYC41 (SEQ ID NO:639). BCMA-55-scFv-mFc was observed to specifically bind to an epitope containing residues present in peptides comprising discontinuous portions of the BCMA polypeptide sequence, individually comprising the following sequences: 1MLMAG6 (SEQ ID NO:640), 13YFDSL17 (SEQ ID NO:779), and 25QLRCSSNTPPL35 (SEQ ID NO:642). In some embodiments, the provided antibody or receptor specifically binds to an epitope comprising residues present within one or more of, e.g., each of discontinuous peptides having the sequences of: MLMAG (SEQ ID NO:640), YFDSL (SEQ ID NO:779), and QLRCSSNTPPL (SEQ ID NO:642). In some aspects, the provided antibody or receptor specifically binds to an epitope comprising residues present within one or more of, e.g., each of, the following discontinuous peptides having the sequences of: MLMAG (SEQ ID NO:640), YFDSLL (SEQ ID NO:641), and QLRCSSNTPPL (SEQ ID NO:642); in some aspects, the provided antibody or receptor specifically binds to an epitope comprising residues present within one or more of, e.g., each of, the following discontinuous peptides having the sequences of: MLMAG (SEQ ID NO:640), QNEYFDSLL (SEQ ID NO:780), and QLRCSSNTPPL (SEQ ID NO:642).
Example 18: Administration of Anti-BCMA CAR-Expressing Cells to Subjects with Relapsed or Refractory Multiple Myeloma (MM)
Chimeric antigen-receptor (CAR)-expressing T cell compositions containing autologous T cells expressing a CAR specific for B-cell maturation antigen (BCMA) were administered to human subjects with relapsed and/or refractory multiple myeloma (MM).
A. Subjects and Treatment
Compositions containing autologous T cells engineered to express an exemplary CAR specific for BCMA were administered to adult human subjects with relapsed or refractory (R/R) multiple myeloma (MM), who have received 3 or more prior treatments (the 3 or more prior treatments including at least a proteasome inhibitor, an immunomodulatory agent and an anti-CD38 monoclonal antibody, in each case unless the subject was not a candidate to receive such treatment such as by way of it being contraindicated).
The administered T cell compositions had been generated by a process including immunoaffinity-based enrichment of CD4+ and CD8+ cell populations from leukapheresis samples from individual subjects with MM, combining cells of such populations, such as at or at approximately a 1:1 ratio, and subjecting the cells to processing steps including for cell transduction and expansion, and cryopreservation, and generation of cells with a range of CD4+ to CD8+ CAR T cell ratios. The CAR contained a BCMA-55-derived scFv binding domain, a modified IgG-derived CH2-CH3-hinge spacer, a CD28 transmembrane domain, and an intracellular signaling region including, in series, a 4-1BB endodomain and a CD3zeta endodomains. The polynucleotide sequence encoding the anti-BCMA CAR did not include identified potential cryptic splice donor and acceptor sites.
Two to seven days prior to CAR+ T cell infusion (and completed at least 48 hours prior to CAR-T infusion) subjects received a lymphodepleting chemotherapy (LDC) with flurdarabine (flu, 30 mg/m2/day) and cyclophosphamide (Cy, 300 mg/m2/day) for 3 days, the LDC completed at least 48 hours prior to CAR-T infusion. The cryopreserved cell compositions were thawed at bedside prior to intravenous administration, with the day of infusion being designated day 1. On day 1, subjects were administered a dose of CAR-expressing T cells as follows: a single dose of dose level 1 (DL1) containing 5×107 total CAR-expressing T cells, or a single dose of dose level 2 (DL2) containing 1.5×108 total CAR-expressing T cells.
At a particular timepoint of analysis, 19 adult subjects had been enrolled in an ongoing clinical study involving such therapy. Of these 19 subjects at this particular timepoint, 13 subjects had been administered the anti-BCMA CAR+ cells, each either at DL1 or DL2. Of these 13 subjects, at this particular timepoint in the ongoing study, 8 subjects were evaluable for attributes indicative of safety (evaluability based on ≥1 mo. follow-up) (n=5 DL1; n=3 DL2). One subject had been unable to receive CAR+ T cells, due to sepsis after LDC, leading to death before CAR+ T cell administration. Three subjects (all DL1) were evaluable at this timepoint for confirmed response (evaluability based on ≥2 mo. follow-up) according to International Myeloma Working Group (IMWG) uniform response criteria (Kumar et al. (2016) Lancet Oncol 17(8):e328-346).
For these 8 subjects assessed at this timepoint, median follow-up was 5 weeks (range 4-13 weeks). Median age was 53 years (range 36-66) with a median time from diagnosis of 4 years (range 2-12). Subjects had received a median of 10 prior regimens (range 4-15) for MM. Of the 8 subjects, 4 (50%) had been refractory (no response or progression within 60 days of last therapy) to bortezomib, carfilzomib, lenalidomide, pomalidomide and an anti-CD38 monoclonal antibody. Seven of the 8 subjects (88%) had had prior autologous stem cell transplant and 4 of 8 (50%) had IMWG high risk cytogenetics.
At the time of the assessment at the timepoint in the ongoing study, no DLTs had been observed in the subjects assessed receiving DL1 or DL2. Cytokine release syndrome (CRS), all grade 1 or 2, had been observed in 6 of the 8 (75%) subjects at the timepoint. Median onset of CRS at the timepoint among the 8 subjects was 9 days (range 4-10) with a median duration of 4.5 days (range 2-19 days). None of the subjects with grade 2 CRS at the timepoint had required vasopressor support and only 1 subject had received tocilizumab. None of the subjects had exhibited CRS of grade 3 or higher. Three of 8 (38%) subjects had experienced neurologic adverse events (AE). Two of the eight subjects at the timepoint had exhibited grade 1 events, and 1 had exhibited a grade 3 event (lethargy), which had resolved within 24 hours after receiving steroids. Onset of neurologic AEs was 9, 11 and 12 days, with a duration of 2, 3 and 1 days, respectively, for the 3 subjects experiencing neurological AE. The subject who had experienced grade 3 neurotoxicity (NT) as-of the analysis at this timepoint had developed secondary plasma cell leukemia (PCL) just prior to receiving LDC.
All 8 subjects at the timepoint were observed to have had evidence of objective response, including the subject with secondary PCL. Three subjects, all administered DL1, were observed to have achieved confirmed responses (1 partial response, PR; 2 stringent complete response, sCR), whereas the remaining subjects remained unconfirmed (1 complete response, CR; 2 very good partial response, VGPR; 1 PR, 1 MR). As of the timepoint for assessment, no subject had been observed to have progressed.
The results showed that at the assessed dose levels, administration of the anti-BCMA CAR cell therapy exhibited favorable safety profiles, with no DLTs reported at this timepoint in an ongoing clinical study. The results were consistent with a conclusion that at this timepoint the incidence of grade 3 or higher NT was low, and no grade 3 or higher CRS had been observed with clinical response.
The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.
SEQUENCES
SEQ
ID
NO
SEQUENCE
Description
1
DYAMS
BCMA-1, -3, -4, -5, -6,
-7, -8, -25, -31, -42,
-44, -45, -46, -51
CDR-H1 (aa) Kabat
numbering
2
DYYMS
BCMA-2, -9, -10, -12,
-17, -21, -22, -23, -24,
-26, -32, -33, -35, -36,
-37, -38, -40, -41, -47,
-48, -49 CDR-H1 (aa)
Kabat numbering
3
DYAMH
BCMA-11, -20, -27, -28,
-29, -30, -34, -39 CDR-H1
(aa) Kabat numbering
4
FIRSKAYGGTTEYAASVKG
BCMA-1, -3, -4, -5, -6,
-7, -8, -25, -31, -42,
-44, -45, -46, -51 CDR-H2
(aa) Kabat numbering
5
YISSSGSTIYYADSVKG
BCMA-2, -9, -10, -12,
-17, -21, -22, -23, -26,
-32, -35, -36, -37, -38,
-40, -47, -48, -49 CDR-H2
(aa) Kabat numbering
6
GISWNSGSIGYADSVKG
BCMA-11, -20, -24, -27,
-29, -30, -34, -39 CDR-H2
(aa) Kabat numbering
7
WSAPTDY
BCMA-1, -3, -4, -5, -6,
-7, -8, -25, -31, -42,
-44, -45, -46, -51 and
VH-2 CDR-H3 (aa)
8
VDGPPSSDI
BCMA-2 CDR-H3 (aa)
9
VDGDYVDDY
BCMA-9, -36, -37, -38,
-41 and VH-8 CDR-H3
(aa)
10
VDGPPSFDI
BCMA-10, -12, -26, -32,
-47, -48, -49 and VH-1
CDR-H3 (aa)
11
DLGPDYDPDAFDI
BCMA-11, -34 CDR-H3
(aa)
12
GFTFGDY
BCMA-1, -3, -4, -5, -6,
-7, -8, -25, -30, -31,
-42, -44, -45, -46, -51
CDR-H1 (aa) Chothia
numbering
13
GFTFSDY
BCMA-2, -9, -12, -17,
-21, -22, -23, -24, -26,
-32, -33, -35, -36, -37,
-38, -40, -41, -47, -48,
-49 CDR-H1 (aa) Chothia
numbering
14
GFPFSDY
BCMA-10 CDR-H1 (aa)
Chothia numbering
15
GFTFDDY
BCMA-11, -20, -27, -28,
-29, -34, -39 CDR-H1 (aa)
Chothia numbering
16
RSKAYGGT
BCMA-1, -3, -4, -5, -6,
-7, -8, -25, -31, -42,
-44, -45, -46, -51 CDR-H2
(aa) Chothia numbering
17
SSSGST
BCMA-2, -9, -10, -12,
-17, -21, -22, -23, -26,
-32, -35, -36, -37, -38,
-40, -47, -48 CDR-H2 (aa)
Chothia numbering
18
SWNSGS
BCMA-11, -20, -24, -27,
-29, -30, -34, -39 CDR-H2
(aa) Chothia numbering
19
GFTFGDYAMS
BCMA-1, -3, -4, -5, -6,
-7, -8, -25, -31, -42,
-44, -45, -46, -51 CDR-H1
(aa) AbM numbering
20
GFTFSDYYMS
BCMA-2, -9, -12, -17,
-21, -22, -23, -24, -26,
-32, -33, -35, -36, -37,
-38, -40, -41, -47, -48,
-49 CDR-H1 (aa) AbM
numbering
21
GFPFSDYYMS
BCMA-10 CDR-H1 (aa)
AbM numbering
22
GFTFDDYAMH
BCMA-11, -20, -27, -28,
-29, -34, -39 CDR-H1 (aa)
AbM numbering
23
FIRSKAYGGTTE
BCMA-1, -3, -4, -5, -6,
-7, -8, -25, -31, -42,
-44, -45, -46, -51 CDR-H2
(aa) AbM numbering
24
YISSSGSTIY
BCMA-2, -9, -10, -12,
-17, -21, -22, -23, -26,
-32, -35, -36, -37, -38,
-40, -47, -48, -49 CDR-H2
(aa) AbM numbering
25
GISWNSGSIG
BCMA-11, -20, -24, -27,
-29, -30, -34, -39 CDR-H2
(aa) AbM numbering
26
KSSQSVLSTSNNKNYLA
BCMA-1 CDR-L1 (aa)
27
RASQSIKTNLA
BCMA-2 CDR-L1 (aa)
28
KSSQSVLHSSNNKNYLA
BCMA-3, -46 CDR-L1
(aa)
29
RASQDIRNSLA
BCMA-4 CDR-L1 (aa)
30
KSSQSVLYSSNNKNYLA
BCMA-5, -8, -24
CDR-L1 (aa)
31
RASQSISNSLA
BCMA-6 CDR-L1 (aa)
32
RASQDIGDYLA
BCMA-7 CDR-L1 (aa)
33
GANNIGSKSVH
BCMA-9, -26, -35
CDR-L1 (aa)
34
GGNNIERKNVH
BCMA-10 CDR-L1 (aa)
35
SGSSSNIGSNAVN
BCMA-11 CDR-L1 (aa)
36
SGSRSNIGNNYVS
BCMA-12 CDR-L1 (aa)
37
WASTREA
BCMA-1 CDR-L2 (aa)
38
AASTRAT
BCMA-2 CDR-L2 (aa)
39
WASTRES
BCMA-3, -5, -8, -31, -44,
-46 CDR-L2 (aa)
40
AASRLES
BCMA-4, -42 CDR-L2
(aa)
41
AASNVED
BCMA-6 CDR-L2 (aa)
42
VASTLQS
BCMA-7 CDR-L2 (aa)
43
DDDDRPS
BCMA-9, -26, -35 CDR-
L2 (aa)
44
DDSDRAS
BCMA-10 CDR-L2 (aa)
45
NSHQRPS
BCMA-11 CDR-L2 (aa)
46
DNAKRPS
BCMA-12 CDR-L2 (aa)
47
QQYFSSPYT
BCMA-1 CDR-L3 (aa)
48
QQYGSSPT
BCMA-2 CDR-L3 (aa)
49
QQYYTTPLT
BCMA-3, -46 CDR-L3
(aa)
50
QQYYSLPLS
BCMA-4 CDR-L3 (aa)
51
QQYYSTPWT
BCMA-5 CDR-L3 (aa)
52
QQSHMYPPT
BCMA-6 CDR-L3 (aa)
53
QQYHSHPWT
BCMA-7 CDR-L3 (aa)
54
QQYYSTPYT
BCMA-8, -31 CDR-L3
(aa)
55
HVWDRSRDHYV
BCMA-9 CDR-L3 (aa)
56
QAWDSSSTLYV
BCMA-10 CDR-L3 (aa)
57
AAWDDSLRGYV
BCMA-11 CDR-L3 (aa)
58
QVWDSSSDHWV
BCMA-12, -32, -48 CDR-
L3 (aa)
59
QVQLVQSGGGLVQPGRSLRLSCTASGFTFG
BCMA-1, -3, -4, -5, -6,
-7, -8, -45, -46 VH FR1
(aa)
60
EVQLVESGGGLVKPGGSLRLSCAASGFTFS
26, -40, -48 VH FR1 (aa)
61
EVQLVQSGGGLVKPGGSLRLSCAASGFTFS
BCMA-9, -24, -35, -37
VH FR1 (aa)
62
EVQLVESGGGLVKPGGSLRLSCAASGFPFS
BCMA-10 VH FR1 (aa)
63
QVQLVQSGGGLVQPGRSLRLSCAASGFTFD
BCMA-11, -28, -29, -39
VH FR1 (aa)
64
WFRQAPGKGLEWVG
BCMA-1, -3, -4, -5, -6,
-7, -8, -25, -31, -42,
-44, -45, -46, -51 VH
FR2 (aa)
65
WIRQAPGKGLEWVS
BCMA-2, -9, -10, -12,
-21, -22, -23, -24, -26,
-32, -33, -35, -36, -37,
-38, -40, -41, -47, -48,
-49 VH FR2 (aa)
66
WVRRAPGKGLEWVS
BCMA-11 VH FR2 (aa)
67
RFTISRDDSKSIAYLQMNSLKTEDTAVYYCAA
BCMA-1, -3, -4, -5, -6,
-7, -8, -25, -31, -42,
-44, -45, -46, -51 VH
FR3 (aa)
68
RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAK
BCMA-2, -10, -12, -21,
-22, -23, -26, -32, -40,
-41, -48, -49 VH FR3 (aa)
69
RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
BCMA-9, -11, -17, -24,
-28, -29, -30, -33, -34,
-35, -36, -38, -39 VH
FR3 (aa)
70
WGQGTLVTVSS
BCMA-1, -3, -4, -5, -6,
-7, -8, -9, -15, -16, -18,
-20, -21, -22, -23, -25,
-27, -31, -35, -36, -37, -38,
-40, -41, -42, -44, -45, -46,
-51 VH FR4 (aa)
71
WGQGTMVTVSS
BCMA-2, -10, -11, -12,
-24, -26, -29, -30, -32, -34,
-39, -48, -49, -50 VH FR4
(aa)
72
DIVMTQSPDSLSVSPGERATISC
BCMA-1 VL FR1 (aa)
73
EIVMTQSPATLSVSPGETATLSC
BCMA-2 VL FR1 (aa)
74
DIVMTQSPDSLVVSLGERATINC
BCMA-3, -46 VL FR1
(aa)
75
AIRMTQSPSSLSASLGDRVTITC
BCMA-4 VL FR1 (aa)
76
DIVMTQSPDSLAVSLGERATINC
BCMA-5, -8, -24 VL FR1
(aa)
77
DIVMTQSPSSLSVSVGERVTITC
BCMA-6 VL FR1 (aa)
78
VIQLTQSPSSLSASVGDRVTITC
BCMA-7 VL FR1 (aa)
79
SYELTQPPSVSVAPGQTARVTC
BCMA-9 VL FR1 (aa)
80
SYVLTQPPSVSVAPGQTARITC
BCMA-10, -26 VL FR1
(aa)
81
QLVLTQPPSASGTPGQRVTISC
BCMA-11 VL FR1 (aa)
82
QSALTQPPSVSAAPGQKVTISC
BCMA-12 VL FR1 (aa)
83
WYQQKPGQPPRLLLY
BCMA-1 VL FR2 (aa)
84
WYQQKPGQAPRLLIY
BCMA-2, -34 VL FR2
(aa)
85
WYQQKPGQPPKLLIY
BCMA-3, -5, -8, -24, -44,
-46 VL FR2 (aa)
86
WYQQRPGKAPKLLLS
BCMA-4 VL FR2 (aa)
87
WYKQRPGEAPKLLIH
BCMA-6 VL FR2 (aa)
88
WFQQRPGKAPKSLIY
BCMA-7 VL FR2 (aa)
89
WYQQKPGQAPMLVVY
BCMA-9, -26, -35 VL
FR2 (aa)
90
WYQQKPGQAPVPVVY
BCMA-10 VL FR2 (aa)
91
WYQQLPGTAPEVLIY
BCMA-11 VL FR2 (aa)
92
WYQQLPGTAPKLLIY
BCMA-12 VL FR2 (aa)
93
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC
BCMA-1, -3, -5, -8, -31,
-44, -46 VL FR3 (aa)
94
GIPDRFSGSGSGTDFTLTITRLEPEDFAVYYC
BCMA-2 VL FR3 (aa)
95
GVPSRFSGTTSGAEYALSISSLQPEDVASYFC
BCMA-4 VL FR3 (aa)
96
GVPSRFSGRGSGTVFTLAISNVQPEDFATYYC
BCMA-6 VL FR3 (aa)
97
GVPSRFSGSGSGTHFTLTINSLQPEDFATYYC
BCMA-7 VL FR3 (aa)
98
GIPERFSGSNSGNTATLTISGVEAGDEADYFC
BCMA-9, -26, -35 VL
FR3 (aa)
99
GIPERFSASNSGNTATLTISGAQATDEAEYYC
BCMA-10 VL FR3 (aa)
100
GVPDRFSGSKSGTSASLAINGLQSEDEADYYC
BCMA-11 VL FR3 (aa)
101
GIPDRFSGSKSGTSATLDIAGLQTGDEADYYC
BCMA-12 VL FR3 (aa)
102
FGHGTKLEIK
BCMA-1 VL FR4 (aa)
103
FGRGTKLEIK
BCMA-2, -39 VL FR4
(aa)
104
FGGGTKVEIK
BCMA-3, -4, -6, -30, -46
VL FR4 (aa)
105
FGQGTKVDIK
BCMA-5, -45 VL FR4
(aa)
106
FGPGTKVDIK
BCMA-7 VL FR4 (aa)
107
FGQGTKLEIK
BCMA-8, -44 VL FR4
(aa)
108
FGTGTKLTVL
BCMA-9, -10, -11, -26,
-40, -47 VL FR4 (aa)
109
FGGGTKLTVL
BCMA-12, -14, -15, -16,
-17, -18, -32, -33, -36,
-37, -38, -41, -48, -49,
-50 VL FR4 (aa)
110
QVQLVQSGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEY
BCMA-1, -3, -4, -5, -6,
AASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAAWSAPTDYWGQGTLVTVSS
-7, -8, -45, -46 VH Chain
(aa)
111
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-2 VH Chain (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGPPSSDIWGQGTMVTVSS
112
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-9 VH Chain (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVDGDYVDDYWGQGTLVTVSS
113
EVQLVESGGGLVKPGGSLRLSCAASGFPFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-10 VH Chain (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGPPSFDIWGQGTMVTVSS
114
QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRRAPGKGLEWVSGISWNSGSIGYAD
BCMA-11 VH Chain (aa)
SVKGRFTISRDNAKNSLYLQMNSSLRAEDTAVYYCARDLGPDYDPDAFDIWGQGTMVTVS
115
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-12, -26, -48 VH
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGPPSFDIWGQGTMVTVSS
Chain (aa)
116
DIVMTQSPDSLSVSPGERATISCKSSQSVLSTSNNKNYLAWYQQKPGQPPRLLLYWASTREA
BCMA-1 VL Chain (aa)
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYFSSPYTFGHGTKLEIK
117
EIVMTQSPATLSVSPGETATLSCRASQSIKTNLAWYQQKPGQAPRLLIYAASTRATGIPDRF
BCMA-2 VL Chain (aa)
SGSGSGTDFTLTITRLEPEDFAVYYCQQYGSSPTFGRGTKLEIK
118
DIVMTQSPDSLVVSLGERATINCKSSQSVLHSSNNKNYLAWYQQKPGQPPKLLIYWASTRES
BCMA-3, -46 VL Chain
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPLTFGGGTKVEIK
(aa)
119
AIRMTQSPSSLSASLGDRVTITCRASQDIRNSLAWYQQRPGKAPKLLLSAASRLESGVPSRF
BCMA-4 VL Chain (aa)
SGTTSGAEYALSISSLQPEDVASYFCQQYYSLPLSFGGGTKVEIK
120
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRES
BCMA-5 VL Chain (aa)
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPWTFGQGTKVDIK
121
DIVMTQSPSSLSVSVGERVTITCRASQSISNSLAWYKQRPGEAPKLLIHAASNVEDGVPSRF
BCMA-6 VL Chain (aa)
SGRGSGTVFTLAISNVQPEDFATYYCQQSHMYPPTFGGGTKVEIK
122
VIQLTQSPSSLSASVGDRVTITCRASQDIGDYLAWFQQRPGKAPKSLIYVASTLQSGVPSRF
BCMA-7 VL Chain (aa)
SGSGSGTHFTLTINSLQPEDFATYYCQQYHSHPWTFGPGTKVDIK
123
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRES
BCMA-8 VL Chain (aa)
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKLEIK
124
SYELTQPPSVSVAPGQTARVTCGANNIGSKSVHWYQQKPGQAPMLVVYDDDDRPSGIPERFS
BCMA-9 VL Chain (aa)
GSNSGNTATLTISGVEAGDEADYFCHVWDRSRDHYVFGTGTKLTVL
125
SYVLTQPPSVSVAPGQTARITCGGNNIERKNVHWYQQKPGQAPVPVVYDDSDRASGIPERFS
BCMA-10 VL Chain (aa)
ASNSGNTATLTISGAQATDEAEYYCQAWDSSSTLYVFGTGTKLTVL
126
QLVLTQPPSASGTPGQRVTISCSGSSSNIGSNAVNWYQQLPGTAPEVLIYNSHQRPSGVPDR
BCMA-11 VL Chain (aa)
FSGSKSGTSASLAINGLQSEDEADYYCAAWDDSLRGYVFGTGTKLTVL
127
QSALTQPPSVSAAPGQKVTISCSGSRSNIGNNYVSWYQQLPGTAPKLLIYDNAKRPSGIPDR
BCMA-12 VL Chain (aa)
FSGSKSGTSATLDIAGLQTGDEADYYCQVWDSSSDHWVFGGGTKLTVL
128
QVQLVQSGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEY
BCMA-1 scFv (aa)
AASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAAWSAPTDYWGQGTLVTVSSGGGGSG
GGGSGGGGSDIVMTQSPDSLSVSPGERATISCKSSQSVLSTSNNKNYLAWYQQKPGQPPRLL
LYWASTREAGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYFSSPYTFGHGTKLEIK
129
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-2 scFv (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGPPSSDIWGQGTMVTVSSGGGGSG
GGGSGGGGSEIVMTQSPATLSVSPGETATLSCRASQSIKTNLAWYQQKPGQAPRLLIYAAST
RATGIPDRFSGSGSGTDFTLTITRLEPEDFAVYYCQQYGSSPTFGRGTKLEIK
130
QVQLVQSGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEY
BCMA-3, 46 scFv (aa)
AASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAAWSAPTDYWGQGTLVTVSSGGGGSG
GGGSGGGGSDIVMTQSPDSLVVSLGERATINCKSSQSVLHSSNNKNYLAWYQQKPGQPPKLL
IYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYTTPLTFGGGTKVEIK
131
QVQLVQSGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEY
BCMA-4 scFv (aa)
AASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAAWSAPTDYWGQGTLVTVSSGGGGSG
GGGSGGGGSAIRMTQSPSSLSASLGDRVTITCRASQDIRNSLAWYQQRPGKAPKLLLSAASR
LESGVPSRFSGTTSGAEYALSISSLQPEDVASYFCQQYYSLPLSFGGGTKVEIK
132
QVQLVQSGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEY
BCMA-5 scFv (aa)
AASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAAWSAPTDYWGQGTLVTVSSGGGGSG
GGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLL
IYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPWTFGQGTKVDIK
133
QVQLVQSGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEY
BCMA-6 scFv (aa)
AASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAAWSAPTDYWGQGTLVTVSSGGGGSG
GGGSGGGGSDIVMTQSPSSLSVSVGERVTITCRASQSISNSLAWYKQRPGEAPKLLIHAASN
VEDGVPSRFSGRGSGTVFTLAISNVQPEDFATYYCQQSHMYPPTFGGGTKVEIK
134
QVQLVQSGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEY
BCMA-7 scFv (aa)
AASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAAWSAPTDYWGQGTLVTVSSGGGGSG
GGGSGGGGSVIQLTQSPSSLSASVGDRVTITCRASQDIGDYLAWFQQRPGKAPKSLIYVAST
LQSGVPSRFSGSGSGTHFTLTINSLQPEDFATYYCQQYHSHPWTFGPGTKVDIK
135
QVQLVQSGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEY
BCMA-8 scFv (aa)
AASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAAWSAPTDYWGQGTLVTVSSGGGGSG
GGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLL
IYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKLEIK
136
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-9 scFv (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVDGDYVDDYWGQGTLVTVSSGGGGSG
GGGSGGGGSSYELTQPPSVSVAPGQTARVTCGANNIGSKSVHWYQQKPGQAPMLVVYDDDDR
PSGIPERFSGSNSGNTATLTISGVEAGDEADYFCHVWDRSRDHYVFGTGTKLTVL
137
EVQLVESGGGLVKPGGSLRLSCAASGFPFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-10 scFv (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGPPSFDIWGQGTMVTVSSGGGGSG
GGGSGGGGSSYVLTQPPSVSVAPGQTARITCGGNNIERKNVHWYQQKPGQAPVPVVYDDSDR
ASGIPERFSASNSGNTATLTISGAQATDEAEYYCQAWDSSSTLYVFGTGTKLTVL
138
QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRRAPGKGLEWVSGISWNSGSIGYAD
BCMA-11 scFv (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGPDYDPDAFDIWGQGTMVTVSSGG
GGSGGGGSGGGGSQLVLTQPPSASGTPGQRVTISCSGSSSNIGSNAVNWYQQLPGTAPEVLI
YNSHQRPSGVPDRFSGSKSGTSASLAINGLQSEDEADYYCAAWDDSLRGYVFGTGTKLTVL
139
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-12 scFv (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGPPSFDIWGQGTMVTVSSGGGGSG
GGGSGGGGSQSALTQPPSVSAAPGQKVTISCSGSRSNIGNNYVSWYQQLPGTAPKLLIYDNA
KRPSGIPDRFSGSKSGTSATLDIAGLQTGDEADYYCQVWDSSSDHWVFGGGTKLTVL
140
SYAMH
BCMA-13 CDR-H1 (aa)
Kabat numbering
141
SYGMH
BCMA-14, -15 CDR-H1
(aa) Kabat numbering
142
SSAMQ
BCMA-16 CDR-H1 (aa)
Kabat numbering
143
SYWMS
BCMA-18 CDR-H1 (aa)
Kabat numbering
144
SYYMH
BCMA-19 CDR-H1 (aa)
Kabat numbering
145
VISYDGSNKYYADSVKG
BCMA-13, -14, -15 CDR-
H2 (aa) Kabat numbering
146
WIVVGSGNTNYAQKFQE
BCMA-16 CDR-H2 (aa)
Kabat numbering
147
HINQDGSEKYYVDSVKG
BCMA-18 CDR-H2 (aa)
Kabat numbering
148
WINPNSGGTNYAQKFQG
BCMA-19 CDR-H2 (aa)
Kabat numbering
149
LPGRDGYPGAFDY
BCMA-13, -14 CDR-H3
(aa)
150
DQYSSSAQRADFDY
BCMA-15 CDR-H3 (aa)
151
APYYDILTGYYL
BCMA-16 CDR-H3 (aa)
152
EADSSADY
BCMA-17, -33 CDR-H3
(aa)
153
WLAVTN
BCMA-18 CDR-H3 (aa)
154
DGGDV
BCMA-19 CDR-H3 (aa)
155
GGLGITPYYFDY
BCMA-20, -27 CDR-H3
(aa)
156
VDGGYTEDY
BCMA-21 CDR-H3 (aa)
157
VDGDYTEDY
BCMA-22, -23, -40 CDR-
H3 (aa)
158
GFTFSSY
BCMA-13, -14, -15, -18
CDR-H1 (aa) Chothia
numbering
159
GFTFTSS
BCMA-16 CDR-H1 (aa)
Chothia numbering
160
GYTFTSY
BCMA-19 CDR-H1 (aa)
Chothia numbering
161
SYDGSN
BCMA-13, -14, -15 CDR-
H2 (aa) Chothia
numbering
162
VVGSGN
BCMA-16 CDR-H2 (aa)
Chothia numbering
163
NQDGSE
BCMA-18 CDR-H2 (aa)
Chothia numbering
164
NPNSGG
BCMA-19 CDR-H2 (aa)
Chothia numbering
165
GFTFSSYAMH
BCMA-13 CDR-H1 (aa)
AbM numbering
166
GFTFSSYGMH
BCMA-14, -15 CDR-H1
(aa) AbM numbering
167
GFTFTSSAMQ
BCMA-16 CDR-H1 (aa)
AbM numbering
168
GFTFSSYWMS
BCMA-18 CDR-H1 (aa)
AbM numbering
169
GYTFTSYYMH
BCMA-19 CDR-H1 (aa)
AbM numbering
170
VISYDGSNKY
BCMA-13, -14, -15 CDR-
H2 (aa) AbM numbering
171
WIVVGSGNTN
BCMA-16 CDR-H2 (aa)
AbM numbering
172
HINQDGSEKY
BCMA-18 CDR-H2 (aa)
AbM numbering
173
WINPNSGGTN
BCMA-19 CDR-H2 (aa)
AbM numbering
174
GSGSNIGSNDVS
BCMA-13, -14, -15, -16,
-18, -21 CDR-L1 (aa)
175
GGNNIGFKGVQ
BCMA-17, -33 CDR-L1
(aa)
176
TGTSSDVGDYNYVA
BCMA-19 CDR-L1 (aa)
177
SGGKTVN
BCMA-20, -27 CDR-L1
(aa)
178
TGSSSDVGKYNLVS
BCMA-22, -23 CDR-L1
(aa)
179
WNDQRPS
BCMA-13, -14, -15, -16,
-18, -21 CDR-L2 (aa)
180
DDSDRPS
BCMA-17, -32, -33 CDR-
L2 (aa)
181
EVINRPS
BCMA-19 CDR-L2 (aa)
182
SNDQRPS
BCMA-20, -27 CDR-L2
(aa)
183
DVNKRPS
BCMA-22, -23, -40 CDR-
L2 (aa)
184
AAWDDSLGGSWV
BCMA-13 CDR-L3 (aa)
185
AAWDDRLNGFWV
BCMA-14 CDR-L3 (aa)
186
AAWDDSLSGWV
BCMA-15 CDR-L3 (aa)
187
ASWDDSLSGWV
BCMA-16 CDR-L3 (aa)
188
QVWDSASDHWV
BCMA-17, -33 CDR-L3
(aa)
189
AAWDDSLNGWV
BCMA-18 CDR-L3 (aa)
190
ISYSRGSTPYV
BCMA-19 CDR-L3 (aa)
191
GSWDDSLNAWV
BCMA-20, -27 CDR-L3
(aa)
192
AAWDDSLNGYV
BCMA-21 CDR-L3 (aa)
193
CSYGGSRSYV
BCMA-22, -40 CDR-L3
(aa)
194
SSYGGSRSYV
BCMA-23 CDR-L3 (aa)
195
QMQLVQYGGGVVQPGRSLRLSCAASGFTFS
BCMA-13 VH FR1 (aa)
196
EVQLLESGGGVVQPGRSLRLSCAASGFTFS
BCMA-14 VH FR1 (aa)
197
QVQLLESGGGLVKPGGSLRLSCAASGFTFS
BCMA-15, -47 VH FR1
(aa)
198
EVQLVQSGPEVKKPGTSVKVSCKASGFTFT
BCMA-16 VH FR1 (aa)
199
QVQLVQSGGGLVKPGGSLRLSCAASGFTFS
BCMA-17, -33, -36, -38,
-41 VH FR1 (aa)
200
EVQLLESGGGLVQPGGSLRLSCAASGFTFS
BCMA-18 VH FR1 (aa)
201
QVQLVQSGAEVKKPGASVKVSCKASGYTFT
BCMA-19 VH FR1 (aa)
202
EVQLLESGGGLVQPGRSLRLSCAASGFTFD
BCMA-20 VH FR1 (aa)
203
EVQLVESGGGLVKPGGSLKLSCAASGFTFS
BCMA-21 VH FR1 (aa)
204
WVRQAPGKGLEWVA
BCMA-13, -14, -15 VH
FR2 (aa)
205
WVRQARGQRLEWIG
BCMA-16 VH FR2 (aa)
206
WIRLAPGKGLEWVS
BCMA-17 VH FR2 (aa)
207
WHRQAPGKGPEWVA
BCMA-18 VH FR2 (aa)
208
WVRQAPGQGLEWMG
BCMA-19 VH FR2 (aa)
209
WVRQAPGKGLEWVS
BCMA-20, -27, -28, -29,
-30, -34, -39 VH FR2 (aa)
210
RFTISRDNSKNTLYLQMNSLKAEDTAVYYCAT
BCMA-13 VH FR3 (aa)
211
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAT
BCMA-14 VH FR3 (aa)
212
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
BCMA-15 VH FR3 (aa)
213
RVTITRDMSTSTAYMELSSLRSEDTAVYYCAA
BCMA-16 VH FR3 (aa)
214
RFTISRDNAESSLYLQMNSLRAEDTAVYYCAR
BCMA-18 VH FR3 (aa)
215
RVTMTRDTSISTAYMELSRLRSDDTAVYYCAR
BCMA-19 VH FR3 (aa)
216
RFTISRDNAKNSLYLQMNSLRAEDTALYYCAK
BCMA-20, -27 VH FR3
(aa)
217
RGQGTLVTVSS
BCMA-13 VH FR4 (aa)
218
RGPGTLVTVSS
BCMA-14 VH FR4 (aa)
219
WGQGTLVNVSS
BCMA-17, -33 VH FR4
(aa)
220
WGQGTTVTVSS
BCMA-19 VH FR4 (aa)
221
QAVLTQPPSASGTPGQRVTISC
BCMA-13, -14 VL FR1
(aa)
222
QSVLTQPPSASGTPGQRVTISC
BCMA-15, -18, -21 VL
FR1 (aa)
223
QSALTQPPSASGTPGQRVTISC
BCMA-16 VL FR1 (aa)
224
QPVLTQPPSVSVAPGKTAMITC
BCMA-17, -33 VL FR1
(aa)
225
QAVLTQPASVSGSPGQSITISC
BCMA-19 VL FR1 (aa)
226
QPVLTQPPSASGTPGQRVTISC
BCMA-20, -27 VL FR1
(aa)
227
QSALTQPASVSGSPGQSITISC
BCMA-22, -23, -40 VL
FR1 (aa)
228
WYQQIPGTAPKLLIY
BCMA-13, -14, -15, -16,
-18, -21 VL FR2 (aa)
229
WYQQKTGQAPVLVVY
BCMA-17, -33 VL FR2
(aa)
230
WYQQHPGKDPKLMIF
BCMA-19 VL FR2 (aa)
231
WFRQVPGTAPQLLIY
BCMA-20, -27 VL FR2
(aa)
232
WYQQPPGKAPKLIIY
BCMA-22, -23, -40 VL
FR2 (aa)
233
GVPDRFSASKSGTSASLAISGLRSEDEADYYC
BCMA-13 VL FR3 (aa)
234
GVPDRFSGSKSGASASLAISGLQSEDEADYYC
BCMA-14 VL FR3 (aa)
235
GVPDRFSGSKSGTSASLVISGLRSEDEADYYC
BCMA-15 VL FR3 (aa)
236
GVPDRFSGSKSGTSASLAISGLQSEDEADYYC
BCMA-16 VL FR3 (aa)
237
GIPERFSGSNSGNTATLTISRVEAGDEADYYC
BCMA-17, -33 VL FR3
(aa)
238
GVPDRFSGSKSGTSASLAISGLRSEDEADYYC
BCMA-18 VL FR3 (aa)
239
GVSDRFSGSKSGNTASLDISGLQPEDEADYYC
BCMA-19 VL FR3 (aa)
240
GVPDRFSGSKSGSSASLDISGLQSEDEAYYYC
BCMA-20, -27 VL FR3
(aa)
241
GVPDRFSGSKSGISASLAISGLRSEDEADYYC
BCMA-21 VL FR3 (aa)
242
GVSNRFSGSKSGNTATLTISGLQGDDEADYYC
BCMA-22, -23, -40 VL
FR3 (aa)
243
FGGGTKVTVL
BCMA-13 VL FR4 (aa)
244
IGTGTKVTVL
BCMA-19 VL FR4 (aa)
245
FGGETKLTVL
BCMA-20, -27 VL FR4
(aa)
246
FGTGTKVTVL
BCMA-21, -22, -23 VL
FR4 (aa)
247
QMQLVQYGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYAD
BCMA-13 VH Chain (aa)
SVKGRFTISRDNSKNTLYLQMNSLKAEDTAVYYCATLPGRDGYPGAFDYRGQGTLVTVSS
248
EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
BCMA-14 VH Chain (aa)
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATLPGRDGYPGAFDYRGPGTLVTVSS
249
QVQLLESGGGLVKPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
BCMA-15 VH Chain (aa)
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDQYSSSAQRADFDYWGQGTLVTVSS
250
EVQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAMQWVRQARGQRLEWIGWIVVGSGNTNYAQ
BCMA-16 VH Chain (aa)
KFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCAAAPYYDILTGYYLWGQGTLVTVSS
251
QVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRLAPGKGLEWVSYISSSGSTIYYAD
BCMA-17 VH Chain (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREADSSADYWGQGTLVNVSS
252
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWHRQAPGKGPEWVAHINQDGSEKYYVD
BCMA-18 VH Chain (aa)
SVKGRFTISRDNAESSLYLQMNSLRAEDTAVYYCARWLAVTNWGQGTLVTVSS
253
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQ
BCMA-19 VH Chain (aa)
KFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDGGDVWGQGTTVTVSS
254
EVQLLESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYAD
BCMA-20 VH Chain (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGGLGITPYYFDYWGQGTLVTVSS
255
EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-21 VH Chain (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGGYTEDYWGQGTLVTVSS
256
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-22,-23,-40 VH
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGDYTEDYWGQGTLVTVSS
Chain (aa)
257
QAVLTQPPSASGTPGQRVTISCSGSGSNIGSNDVSWYQQIPGTAPKLLIYWNDQRPSGVPDR
BCMA-13 VL Chain (aa)
FSASKSGTSASLAISGLRSEDEADYYCAAWDDSLGGSWVFGGGTKVTVL
258
QAVLTQPPSASGTPGQRVTISCSGSGSNIGSNDVSWYQQIPGTAPKLLIYWNDQRPSGVPDR
BCMA-14 VL Chain (aa)
FSGSKSGASASLAISGLQSEDEADYYCAAWDDRLNGFWVFGGGTKLTVL
259
QSVLTQPPSASGTPGQRVTISCSGSGSNIGSNDVSWYQQIPGTAPKLLIYWNDQRPSGVPDR
BCMA-15 VL Chain (aa)
FSGSKSGTSASLVISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVL
260
QSALTQPPSASGTPGQRVTISCSGSGSNIGSNDVSWYQQIPGTAPKLLIYWNDQRPSGVPDR
BCMA-16 VL Chain (aa)
FSGSKSGTSASLAISGLQSEDEADYYCASWDDSLSGWVFGGGTKLTVL
261
QPVLTQPPSVSVAPGKTAMITCGGNNIGFKGVQWYQQKTGQAPVLVVYDDSDRPSGIPERFS
BCMA-17, -33 VL Chain
GSNSGNTATLTISRVEAGDEADYYCQVWDSASDHWVFGGGTKLTVL
(aa)
262
QSVLTQPPSASGTPGQRVTISCSGSGSNIGSNDVSWYQQIPGTAPKLLIYWNDQRPSGVPDR
BCMA-18 VL Chain (aa)
FSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKLTVL
263
QAVLTQPASVSGSPGQSITISCTGTSSDVGDYNYVAWYQQHPGKDPKLMIFEVINRPSGVSD
BCMA-19 VL Chain (aa)
RFSGSKSGNTASLDISGLQPEDEADYYCISYSRGSTPYVIGTGTKVTVL
264
QPVLTQPPSASGTPGQRVTISCSGGKTVNWFRQVPGTAPQLLIYSNDQRPSGVPDRFSGSKS
BCMA-20, -27 VL Chain
GSSASLDISGLQSEDEAYYYCGSWDDSLNAWVFGGETKLTVL
(aa)
265
QSVLTQPPSASGTPGQRVTISCSGSGSNIGSNDVSWYQQIPGTAPKLLIYWNDQRPSGVPDR
BCMA-21 VL Chain (aa)
FSGSKSGISASLAISGLRSEDEADYYCAAWDDSLNGYVFGTGTKVTVL
266
QSALTQPASVSGSPGQSITISCTGSSSDVGKYNLVSWYQQPPGKAPKLIIYDVNKRPSGVSN
BCMA-22 VL Chain (aa)
RFSGSKSGNTATLTISGLQGDDEADYYCCSYGGSRSYVFGTGTKVTVL
267
QSALTQPASVSGSPGQSITISCTGSSSDVGKYNLVSWYQQPPGKAPKLIIYDVNKRPSGVSN
BCMA-23 VL Chain (aa)
RFSGSKSGNTATLTISGLQGDDEADYYCSSYGGSRSYVFGTGTKVTVL
268
QMQLVQYGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYAD
BCMA-13 scFv (aa)
SVKGRFTISRDNSKNTLYLQMNSLKAEDTAVYYCATLPGRDGYPGAFDYRGQGTLVTVSSGG
GGSGGGGSGGGGSQAVLTQPPSASGTPGQRVTISCSGSGSNIGSNDVSWYQQIPGTAPKLLI
YWNDQRPSGVPDRFSASKSGTSASLAISGLRSEDEADYYCAAWDDSLGGSWVFGGGTKVTVL
269
EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
BCMA-14 scFv (aa)
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATLPGRDGYPGAFDYRGPGTLVTVSSGG
GGSGGGGSGGGGSQAVLTQPPSASGTPGQRVTISCSGSGSNIGSNDVSWYQQIPGTAPKLLI
YWNDQRPSGVPDRFSGSKSGASASLAISGLQSEDEADYYCAAWDDRLNGFWVFGGGTKLTVL
270
QVQLLESGGGLVKPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
BCMA-1 5 scFv (aa)
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDQYSSSAQRADFDYWGQGTLVTVSSG
GGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSGSNIGSNDVSWYQQIPGTAPKLL
IYWNDQRPSGVPDRFSGSKSGTSASLVISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVL
271
EVQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAMQWVRQARGQRLEWIGWIVVGSGNTNYAQ
BCMA-16 scFv (aa)
KFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCAAAPYYDILTGYYLWGQGTLVTVSSGGG
GSGGGGSGGGGSQSALTQPPSASGTPGQRVTISCSGSGSNIGSNDVSWYQQIPGTAPKLLIY
WNDQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCASWDDSLSGWVFGGGTKLTVL
272
QVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRLAPGKGLEWVSYISSSGSTIYYAD
BCMA-17 scFv (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREADSSADYWGQGTLVNVSSGGGGSGG
GGSGGGGSQPVLTQPPSVSVAPGKTAMITCGGNNIGFKGVQWYQQKTGQAPVLVVYDDSDRP
SGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSASDHWVFGGGTKLTVL
273
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMSWHRQAPGKGPEWVAHINQDGSEKYYVD
BCMA-18 scFv (aa)
SVKGRFTISRDNAESSLYLQMNSLRAEDTAVYYCARWLAVTNWGQGTLVTVSSGGGGSGGGG
SGGGGSQSVLTQPPSASGTPGQRVTISCSGSGSNIGSNDVSWYQQIPGTAPKLLIYWNDQRP
SGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKLTVL
274
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQ
BCMA-19 scFv (aa)
KFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDGGDVWGQGTTVTVSSGGGGSGGGGS
GGGGSQAVLTQPASVSGSPGQSITISCTGTSSDVGDYNYVAWYQQHPGKDPKLMIFEVINRP
SGVSDRFSGSKSGNTASLDISGLQPEDEADYYCISYSRGSTPYVIGTGTKVTVL
275
EVQLLESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYAD
BCMA-20 scFv (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGGLGITPYYFDYWGQGTLVTVSSGGG
GSGGGGSGGGGSQPVLTQPPSASGTPGQRVTISCSGGKTVNWFRQVPGTAPQLLIYSNDQRP
SGVPDRFSGSKSGSSASLDISGLQSEDEAYYYCGSWDDSLNAWVFGGETKLTVL
276
EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-21 scFv (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGGYTEDYWGQGTLVTVSSGGGGSG
GGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSGSNIGSNDVSWYQQIPGTAPKLLIYWND
QRPSGVPDRFSGSKSGISASLAISGLRSEDEADYYCAAWDDSLNGYVFGTGTKVTVL
277
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-22 scFv (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGDYTEDYWGQGTLVTVSSGGGGSG
GGGSGGGGSQSALTQPASVSGSPGQSITISCTGSSSDVGKYNLVSWYQQPPGKAPKLIIYDV
NKRPSGVSNRFSGSKSGNTATLTISGLQGDDEADYYCCSYGGSRSYVFGTGTKVTVL
278
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-23 scFv (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGDYTEDYWGQGTLVTVSSGGGGSG
GGGSGGGGSQSALTQPASVSGSPGQSITISCTGSSSDVGKYNLVSWYQQPPGKAPKLIIYDV
NKRPSGVSNRFSGSKSGNTATLTISGLQGDDEADYYCSSYGGSRSYVFGTGTKVTVL
279
VDGDDAFDI
VH-3 CDR-H3 (aa)
280
DPLSWDSSGKGPR
VH-4 CDR-H3 (aa)
281
ENYDFWSWRYYYDMDV
VH-5 CDR-H3 (aa)
282
VDGPPSYDI
VH-6 CDR-H3 (aa)
283
GDWDDAFDI
VH-7 CDR-H3 (aa)
284
VDGDYEDY
VH-9 CDR-H3 (aa)
285
DVPSSGDDAFDI
VH-10 CDR-H3 (aa)
286
VDGDDVFDI
VH-11 CDR-H3 (aa)
287
VDGDAFDI
VH-12 CDR-H3 (aa)
288
DYSIN
BCMA-C1 VH CDR-H1
(aa)
289
NFGMN
BCMA-C2 VH CDR-H1
(aa)
290
WINTETREPAYAYDFRG
BCMA-C1 VH CDR-H2
(aa)
291
WINTYTGESYFADDFKG
BCMA-C2 VH CDR-H2
(aa)
292
DYSYAMDY
BCMA-C1 VH CDR-H3
(aa)
293
GEIYYGYDGGFAY
BCMA-C2 VH CDR-H3
(aa)
294
GYTFTDY
BCMA-C1 VH CDR-H1
(aa) Chothia numbering
295
GYTFTNF
BCMA-C2 VH CDR-H1
(aa) Chothia numbering
296
NTETRE
BCMA-C1 VH CDR-H2
(aa) Chothia numbering
297
NTYTGE
BCMA-C2 VH CDR-H2
(aa) Chothia numbering
298
GYTFTDYSIN
BCMA-C1 VH CDR-H1
(aa) AbM numbering
299
GYTFTNFGMN
BCMA-C2 VH CDR-H1
(aa) AbM numbering
300
WINTETREPA
BCMA-C1 VH CDR-H2
(aa) AbM numbering
301
WINTYTGESY
BCMA-C2 VH CDR-H2
(aa) AbM numbering
302
RASESVTILGSHLIH
BCMA-C1 VL CDR-L1
(aa)
303
RASQDVNTAVS
BCMA-C2 VL CDR-L1
(aa)
304
LASNVQT
BCMA-C1 VL CDR-L2
(aa)
305
SASYRYT
BCMA-C2 VL CDR-L2
(aa)
306
LQSRTIPRT
BCMA-C1 VL CDR-L3
(aa)
307
QQHYSTPWT
BCMA-C2 VL CDR-L3
(aa)
308
QIQLVQSGPELKKPGETVKISCKASGYTFT
BCMA-C1 VH FR1 (aa)
309
QIQLVQSGPDLKKPGETVKLSCKASGYTFT
BCMA-C2 VH FR1 (aa)
310
WVKRAPGKGLKWMG
BCMA-C1 VH FR2 (aa)
311
WVKQAPGKGFKWMA
BCMA-C2 VH FR2 (aa)
312
RFAFSLETSASTAYLQINNLKYEDTATYFCAL
BCMA-C1 VH FR3 (aa)
313
RFAFSVETSATTAYLQINNLKTEDTATYFCAR
BCMA-C2 VH FR3 (aa)
314
WGQGTSVTVSS
BCMA-C1 VH FR4 (aa)
315
WGQGTLVTVSA
BCMA-C2 VH FR4 (aa)
316
DIVLTQSPPSLAMSLGKRATISC
BCMA-C1 VL FR1 (aa)
317
DVVMTQSHRFMSTSVGDRVSITC
BCMA-C2 VL FR1 (aa)
318
WYQQKPGQPPTLLIQ
BCMA-C1 VL FR2 (aa)
319
WYQQKPGQSPKLLIF
BCMA-C2 VL FR2 (aa)
320
GVPARFSGSGSRTDFTLTIDPVEEDDVAVYYC
BCMA-C1 VL FR3 (aa)
321
GVPDRFTGSGSGADFTLTISSVQAEDLAVYYC
BCMA-C2 VL FR3 (aa)
322
FGGGTKLEIK
BCMA-C1 VL FR4 (aa)
323
FGGGTKLDIK
BCMA-C2 VL FR4 (aa)
324
QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAY
BCMA-C1 VH Chain (aa)
DFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS
325
QIQLVQSGPDLKKPGETVKLSCKASGYTFTNFGMNWVKQAPGKGFKWMAWINTYTGESYFAD
BCMA-C2 VH Chain (aa)
DFKGRFAFSVETSATTAYLQINNLKTEDTATYFCARGEIYYGYDGGFAYWGQGTLVTVSA
326
DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGV
BCMA-C1 VL Chain (aa)
PARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIK
327
DVVMTQSHRFMSTSVGDRVSITCRASQDVNTAVSWYQQKPGQSPKLLIFSASYRYTGVPDRF
BCMA-C2 VL Chain (aa)
TGSGSGADFTLTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLDIK
328
DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGV
BCMA-C1 VL-VH scFv
PARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGGGGSGGGGSGGG
(aa)
GSQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAY
AYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS
329
QIQLVQSGPDLKKPGETVKLSCKASGYTFTNFGMNWVKQAPGKGFKWMAWINTYTGESYFAD
BCMA-C2 VH-VL scFv
DFKGRFAFSVETSATTAYLQINNLKTEDTATYFCARGEIYYGYDGGFAYWGQGTLVTVSAGG
(aa)
GGSGGGGSGGGGSDVVMTQSHRFMSTSVGDRVSITCRASQDVNTAVSWYQQKPGQSPKLLIF
SASYRYTGVPDRFTGSGSGADFTLTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLDIK
330
CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGTCCCTGAGACTCTC
BCMA-1 scFv (nt)
CTGTACAGCTTCTGGATTCACCTTTGGTGATTATGCTATGAGCTGGTTCCGCCAGGCTCCAG
GGAAGGGGCTGGAGTGGGTAGGTTTCATTAGAAGCAAAGCTTATGGTGGGACAACAGAATAC
GCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCCAAAAGCATCGCCTATCT
GCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTGCGGCCTGGAGTGCCC
CGACTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGC
GGAGGTGGCTCTGGCGGTGGCGGATCGGATATTGTGATGACCCAGTCTCCAGACTCCCTGTC
TGTGTCTCCGGGCGAGAGGGCCACCATCAGCTGCAAGTCCAGCCAGAGTGTTTTATCCACCT
CCAACAATAAGAACTATTTAGCTTGGTATCAGCAGAAACCAGGACAGCCCCCTAGGCTGCTC
CTTTACTGGGCATCTACCCGGGAGGCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGG
GACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCGGTTTATTACTGTC
AACAATATTTCAGTTCTCCGTACACTTTTGGCCACGGGACCAAGCTGGAAATCAAA
331
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTC
BCMA-2 scFv (nt)
CTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAG
GGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGAC
TCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAAT
GAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGTGGATGGCCCTCCTT
CTTCTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGC
GGAGGTGGCTCTGGCGGTGGCGGATCGGAAATAGTGATGACGCAGTCTCCAGCCACCCTGTC
TGTGTCTCCAGGGGAAACAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTATTAAGACCAACT
TGGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGCTGCATCCACC
AGGGCCACTGGCATCCCAGACAGATTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCAC
CATCACCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAATATGGTAGCTCAC
CCACTTTTGGCCGGGGGACCAAGCTGGAAATCAAA
332
CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGTCCCTGAGACTCTC
BCMA-3 scFv (nt)
CTGTACAGCTTCTGGATTCACCTTTGGTGATTATGCTATGAGCTGGTTCCGCCAGGCTCCAG
GGAAGGGGCTGGAGTGGGTAGGTTTCATTAGAAGCAAAGCTTATGGTGGGACAACAGAATAC
GCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCCAAAAGCATCGCCTATCT
GCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTGCGGCCTGGAGTGCCC
CGACTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGC
GGAGGTGGCTCTGGCGGTGGCGGATCGGATATTGTGATGACCCAGTCTCCAGACTCCCTGGT
TGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTACACAGCT
CCAACAATAAGAATTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTC
ATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGGTTCAGTGGCAGCGGGTCTGG
GACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTC
AGCAGTATTATACTACTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAAATCAAA
333
CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGTCCCTGAGACTCTC
BCMA-4 scFv (nt)
CTGTACAGCTTCTGGATTCACCTTTGGTGATTATGCTATGAGCTGGTTCCGCCAGGCTCCAG
GGAAGGGGCTGGAGTGGGTAGGTTTCATTAGAAGCAAAGCTTATGGTGGGACAACAGAATAC
GCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCCAAAAGCATCGCCTATCT
GCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTGCGGCCTGGAGTGCCC
CGACTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGC
GGAGGTGGCTCTGGCGGTGGCGGATCGGCCATCCGGATGACCCAGTCTCCATCCTCCCTGTC
CGCGTCTCTGGGGGACAGAGTCACCATCACTTGCCGGGCGAGTCAGGACATTAGGAATTCTT
TGGCCTGGTATCAGCAGAGGCCAGGGAAAGCCCCTAAACTCCTGCTTTCTGCTGCATCCAGA
TTGGAAAGTGGGGTCCCTTCTAGGTTCAGTGGCACTACTTCTGGGGCGGAGTATGCTCTCAG
CATCAGCAGCCTGCAGCCTGAAGATGTCGCATCTTATTTCTGTCAGCAGTATTATAGTCTCC
CTCTCTCCTTCGGCGGAGGGACCAAGGTGGAAATCAAA
334
CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGTCCCTGAGACTCTC
BCMA-5 scFv (nt)
CTGTACAGCTTCTGGATTCACCTTTGGTGATTATGCTATGAGCTGGTTCCGCCAGGCTCCAG
GGAAGGGGCTGGAGTGGGTAGGTTTCATTAGAAGCAAAGCTTATGGTGGGACAACAGAATAC
GCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCCAAAAGCATCGCCTATCT
GCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTGCGGCCTGGAGTGCCC
CGACTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGC
GGAGGTGGCTCTGGCGGTGGCGGATCGGACATCGTGATGACCCAGTCTCCAGACTCCCTGGC
TGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCT
CCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTC
ATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGG
GACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTC
AGCAATATTATAGTACTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGATATCAAA
335
CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGTCCCTGAGACTCTC
BCMA-6 scFv (nt)
CTGTACAGCTTCTGGATTCACCTTTGGTGATTATGCTATGAGCTGGTTCCGCCAGGCTCCAG
GGAAGGGGCTGGAGTGGGTAGGTTTCATTAGAAGCAAAGCTTATGGTGGGACAACAGAATAC
GCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCCAAAAGCATCGCCTATCT
GCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTGCGGCCTGGAGTGCCC
CGACTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGC
GGAGGTGGCTCTGGCGGTGGCGGATCGGATATTGTGATGACCCAGTCTCCATCGTCCCTGTC
TGTGTCTGTAGGAGAGAGAGTCACCATCACTTGTCGGGCGAGTCAGTCTATAAGTAATTCCT
TAGCCTGGTATAAACAGAGACCGGGAGAAGCCCCTAAACTCCTGATACATGCTGCATCCAAT
GTGGAAGATGGGGTCCCTTCGAGGTTCAGCGGCAGGGGATCTGGGACAGTTTTCACTCTCGC
CATCAGCAATGTACAGCCTGAAGATTTCGCAACTTACTACTGTCAGCAGAGTCACATGTACC
CTCCGACTTTCGGCGGGGGGACCAAGGTGGAAATCAAA
336
CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGTCCCTGAGACTCTC
BCMA-7 scFv (nt)
CTGTACAGCTTCTGGATTCACCTTTGGTGATTATGCTATGAGCTGGTTCCGCCAGGCTCCAG
GGAAGGGGCTGGAGTGGGTAGGTTTCATTAGAAGCAAAGCTTATGGTGGGACAACAGAATAC
GCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCCAAAAGCATCGCCTATCT
GCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTGCGGCCTGGAGTGCCC
CGACTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGC
GGAGGTGGCTCTGGCGGTGGCGGATCGGTCATCCAGTTGACCCAGTCTCCCTCCTCACTGTC
TGCATCTGTAGGGGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGACATTGGCGATTATT
TAGCCTGGTTTCAGCAGAGACCAGGGAAAGCCCCTAAGTCCCTGATCTATGTTGCGTCCACT
TTGCAGAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACACACTTCACTCTCAC
CATCAACAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATCATAGTCACC
CGTGGACGTTCGGCCCAGGGACCAAGGTGGATATCAAA
337
CAGGTCCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGTCCCTGAGACTCTC
BCMA-8 scFv (nt)
CTGTACAGCTTCTGGATTCACCTTTGGTGATTATGCTATGAGCTGGTTCCGCCAGGCTCCAG
GGAAGGGGCTGGAGTGGGTAGGTTTCATTAGAAGCAAAGCTTATGGTGGGACAACAGAATAC
GCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCCAAAAGCATCGCCTATCT
GCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTGCGGCCTGGAGTGCCC
CGACTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGC
GGAGGTGGCTCTGGCGGTGGCGGATCGGATATTGTGATGACCCAGTCTCCAGACTCCCTGGC
TGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCT
CCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTC
ATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGG
GACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTC
AGCAATATTATAGTACTCCGTACACTTTTGGCCAGGGGACCAAGCTGGAAATCAAA
338
GAAGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTC
BCMA-9 scFv (nt)
CTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAG
GGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGAC
TCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAAT
GAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGTGGACGGTGACTACG
TCGATGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGC
GGAGGTGGCTCTGGCGGTGGCGGATCGTCCTATGAGCTGACTCAGCCGCCCTCGGTGTCTGT
GGCCCCAGGACAGACGGCCAGGGTTACCTGTGGGGCAAATAATATTGGAAGCAAAAGTGTCC
ACTGGTACCAGCAGAAGCCAGGCCAGGCCCCCATGCTGGTCGTCTATGATGATGACGACCGG
CCCTCCGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCAT
CAGCGGGGTCGAGGCCGGGGATGAGGCCGACTACTTCTGTCACGTGTGGGATAGAAGTCGTG
ATCATTATGTCTTCGGAACTGGGACCAAGCTGACCGTCCTA
339
GAAGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTC
BCMA-10 scFv (nt)
CTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAG
GGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGAC
TCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAAT
GAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGTGGACGGTGACTACG
TCGATGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGC
GGAGGTGGCTCTGGCGGTGGCGGATCGTCCTATGAGCTGACTCAGCCGCCCTCGGTGTCTGT
GGCCCCAGGACAGACGGCCAGGGTTACCTGTGGGGCAAATAATATTGGAAGCAAAAGTGTCC
ACTGGTACCAGCAGAAGCCAGGCCAGGCCCCCATGCTGGTCGTCTATGATGATGACGACCGG
CCCTCCGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCAT
CAGCGGGGTCGAGGCCGGGGATGAGGCCGACTACTTCTGTCACGTGTGGGATAGAAGTCGTG
ATCATTATGTCTTCGGAACTGGGACCAAGCTGACCGTCCTA
340
CAGGTGCAGCTGGTACAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTC
BCMA-11 scFv (nt)
CTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCGAGCTCCAG
GGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTATGCGGAC
TCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAAT
GAACAGTCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCTGGGGCCCGACT
ACGATCCCGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTTTCCTCAGGTGGA
GGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGCTTGTGCTGACTCAGCCACC
CTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACA
TCGGAAGTAATGCTGTAAACTGGTACCAGCAGCTCCCAGGAACGGCCCCCGAAGTCCTCATC
TATAATAGTCATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCAC
CTCAGCCTCCCTGGCCATCAATGGGCTCCAGTCTGAGGACGAGGCTGATTATTACTGTGCAG
CATGGGATGACAGCCTGAGAGGTTACGTCTTCGGAACTGGGACCAAGCTCACCGTCCTA
341
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTC
BCMA-12 scFv (nt)
CTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAG
GGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGAC
TCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAAT
GAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGTGGATGGCCCTCCTT
CTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGC
GGAGGTGGCTCTGGCGGTGGCGGATCGCAGTCTGCCCTGACGCAGCCGCCCTCAGTGTCTGC
GGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGCCGCTCCAACATTGGGAATAATT
ATGTATCCTGGTACCAACAGCTCCCAGGAACAGCCCCCAAACTCCTCATTTATGACAATGCT
AAGCGACCCTCAGGAATTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCT
GGACATCGCCGGACTCCAGACTGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTA
GTAGTGATCATTGGGTATTCGGCGGAGGGACCAAGCTCACCGTCCTA
342
CAGATGCAGCTGGTGCAGTATGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTC
BCMA-13 scFv (nt)
CTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAG
GCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGAC
TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAAT
GAACAGCCTGAAAGCTGAGGACACGGCTGTGTATTACTGTGCTACCCTACCCGGTAGAGATG
GCTACCCCGGAGCCTTTGACTACAGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGA
GGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGGCTGTGCTGACTCAGCCACC
CTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCGGCTCCAACA
TCGGAAGTAATGATGTCTCCTGGTATCAGCAGATCCCAGGAACGGCCCCCAAACTCCTCATC
TACTGGAATGATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGCCTCCAAGTCTGGCAC
CTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAG
CATGGGATGACAGCCTGGGTGGTTCTTGGGTGTTCGGCGGAGGGACCAAGGTCACCGTCCTA
343
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTC
BCMA-14 scFv (nt)
CTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAG
GCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGAC
TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAAT
GAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCTACCCTACCCGGTAGAGATG
GCTACCCCGGAGCCTTTGACTACAGGGGCCCGGGAACCCTGGTCACCGTCTCCTCAGGTGGA
GGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGGCTGTGCTGACTCAGCCACC
CTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCGGCTCCAACA
TCGGAAGTAATGATGTCTCCTGGTATCAGCAGATCCCAGGAACGGCCCCCAAACTCCTCATC
TACTGGAATGATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCGC
CTCAGCCTCTCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTATTGTGCAG
CATGGGATGACAGGTTGAACGGTTTTTGGGTGTTCGGCGGAGGGACCAAGCTCACCGTCCTA
344
CAGGTGCAGCTGTTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTC
BCMA-15 scFv (nt)
CTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAG
GCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGAC
TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAAT
GAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGATCAGTATAGCAGTA
GCGCACAAAGGGCCGACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGT
GGAGGCGGTTCAGGCGGAGGTGGTTCTGGCGGTGGCGGATCGCAGTCTGTGCTGACGCAGCC
ACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCGGCTCCA
ACATCGGAAGTAATGATGTCTCCTGGTATCAGCAGATCCCAGGAACGGCCCCCAAACTCCTC
ATCTACTGGAATGATCAGCGGCCCTCAGGGGTCCCTGACCGGTTCTCAGGCTCCAAGTCTGG
CACCTCAGCCTCCCTGGTCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTG
CAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA
345
GAGGTCCAGCTGGTACAGTCTGGGCCTGAGGTGAAGAAGCCTGGGACCTCAGTGAAGGTCTC
BCMA-16 scFv (nt)
CTGCAAGGCTTCTGGATTCACCTTTACTAGCTCTGCTATGCAGTGGGTGCGACAGGCTCGTG
GACAACGCCTTGAGTGGATAGGATGGATCGTCGTTGGCAGTGGTAACACAAACTACGCACAG
AAGTTCCAGGAAAGAGTCACCATTACCAGGGACATGTCCACAAGCACAGCCTACATGGAGCT
GAGCAGCCTGAGATCCGAGGACACGGCCGTGTATTACTGTGCGGCAGCTCCGTATTACGATA
TTTTGACTGGTTATTATTTATGGGGCCAGGGAACGCTGGTCACCGTCTCCTCAGGTGGAGGC
GGTTCTGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGTCTGCCCTGACTCAGCCACCCTC
AGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCGGCTCCAACATCG
GAAGTAATGATGTCTCCTGGTATCAGCAGATCCCAGGAACGGCCCCCAAACTCCTCATCTAC
TGGAATGATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTC
AGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCATCAT
GGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA
346
CAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTC
BCMA-17 scFv (nt)
CTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCTGGCTCCAG
GGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGAC
TCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAAT
GAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGAGGCCGATAGTAGCG
CTGACTACTGGGGCCAGGGAACCCTGGTCAACGTCTCCTCAGGTGGAGGCGGTTCAGGCGGA
GGTGGCTCTGGCGGTGGCGGATCGCAGCCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGGC
CCCAGGAAAGACGGCCATGATTACCTGTGGGGGAAACAACATTGGATTTAAAGGTGTGCAGT
GGTACCAGCAGAAGACAGGCCAGGCCCCTGTGCTGGTCGTCTATGATGATAGCGACCGGCCC
TCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAG
CAGGGTCGAAGCCGGGGATGAGGCCGATTATTACTGTCAGGTGTGGGATAGTGCTAGTGATC
ATTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA
347
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTC
BCMA-18 scFv (nt)
CTGTGCAGCCTCTGGATTCACGTTTAGTAGCTATTGGATGAGCTGGCACCGCCAGGCTCCAG
GGAAGGGGCCGGAGTGGGTGGCCCACATAAACCAAGACGGAAGTGAGAAGTACTATGTGGAC
TCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCGAGAGTTCACTGTATCTGCAAAT
GAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGGTGGCTGGCGGTTACTA
ACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGC
TCTGGCGGTGGCGGATCGCAGTCTGTGTTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGG
GCAGAGGGTCACCATCTCTTGTTCTGGAAGCGGCTCCAACATCGGAAGTAATGATGTCTCCT
GGTATCAGCAGATCCCAGGGACGGCCCCCAAACTCCTCATCTACTGGAATGATCAGCGGCCC
TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAG
TGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATG
GTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA
348
CAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTC
BCMA-19 scFv (nt)
CTGCAAGGCTTCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTG
GACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAG
AAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCT
GAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATGGTGGGGACGTCT
GGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCT
GGCGGTGGCGGATCGCAGGCTGTGCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACA
GTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGATTATAACTATGTCGCCT
GGTATCAACAACACCCAGGCAAAGACCCCAAACTCATGATTTTTGAGGTCATTAATCGGCCC
TCAGGGGTTTCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGGACATCTC
TGGGCTCCAGCCTGAGGACGAGGCTGATTATTACTGCATCTCATATTCACGAGGCAGCACTC
CTTATGTCATCGGAACTGGGACCAAGGTGACCGTCCTA
349
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTC
BCMA-20 scFv (nt)
CTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAG
GGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTATGCGGAC
TCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAAT
GAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAGGGGGGCCTAGGAATAA
CCCCATACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGC
GGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGCCTGTGCTGACTCAGCCACCCTC
AGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCGGGAGGCAAGACTGTAAACT
GGTTCCGGCAGGTCCCAGGAACGGCCCCCCAACTCCTCATCTATAGTAATGATCAGCGGCCC
TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCTCCTCAGCCTCCCTGGACATCAG
TGGGCTCCAGTCTGAGGATGAGGCTTATTATTACTGTGGATCATGGGATGACAGCCTCAATG
CTTGGGTGTTCGGCGGAGAGACCAAGCTGACCGTCCTA
350
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAAACTCTC
BCMA-21 scFv (nt)
CTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAG
GGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGAC
TCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAAT
GAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGTAGACGGAGGCTACA
CAGAGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGC
GGAGGTGGCTCTGGCGGTGGCGGATCGCAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGG
GACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCGGCTCCAACATCGGAAGTAATG
ATGTCTCCTGGTATCAGCAGATCCCAGGAACGGCCCCCAAACTCCTCATCTACTGGAATGAT
CAGCGGCCCTCAGGGGTCCCTGACCGGTTCTCAGGCTCCAAGTCTGGCATCTCAGCCTCCCT
GGCCATCAGCGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACA
GCCTGAATGGTTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA
351
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTC
BCMA-22 scFv (nt)
CTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAG
GGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGAC
TCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAAT
GAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGTAGACGGAGACTACA
CAGAGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGC
GGAGGTGGCTCTGGCGGTGGCGGATCGCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGG
GTCTCCTGGACAGTCGATCACTATCTCCTGCACTGGAAGCAGCAGTGATGTTGGCAAATATA
ATCTTGTCTCCTGGTACCAACAGCCCCCAGGCAAAGCCCCCAAGCTCATAATTTATGACGTC
AATAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCAC
CCTGACAATCTCTGGGCTCCAGGGTGACGACGAGGCTGATTATTATTGTTGCTCATATGGAG
GTAGTAGGTCTTATGTCTTCGGAACTGGGACCAAGGTGACCGTCCTA
352
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTC
BCMA-23 scFv (nt)
CTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAG
GGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGAC
TCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAAT
GAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGTAGACGGAGACTACA
CAGAGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGC
GGAGGTGGCTCTGGCGGTGGCGGATCGCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGG
GTCTCCTGGACAGTCGATCACTATCTCCTGCACTGGAAGCAGCAGTGATGTTGGCAAATATA
ATCTTGTCTCCTGGTACCAACAGCCCCCAGGCAAAGCCCCCAAGCTCATAATTTATGACGTC
AATAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCAC
CCTGACAATCTCTGGGCTCCAGGGTGACGACGAGGCTGATTATTATTGTAGCTCATATGGAG
GTAGTAGGTCTTATGTCTTCGGAACTGGGACCAAGGTGACCGTCCTA
353
X1X2X3MX4
Consensus CDR-H1
X1 = D or S;
X2 = Y or S;
X3 = A, G, W, or Y;
X4 = H, Q, or S
354
X1IX2X3X4X5X6X7X8X9X10X11YX12 X13 X14 X15 X16 X17
Consensus CDR-H2
X1 = F, G, H, V, W or Y;
X2 = N, R, S or V;
X3 = P, Q, S, V, W or Y;
X4 = K or null;
X5 = A or null;
X6 = D, G, N, S, or Y;
X7 = G or S;
X8 = G or S;
X9 = E, G, N, T or S;
X10 = I, K, or T;
X11 = E, G, N or Y;
X12 = A or V;
X13 = A, D or Q;
X14 = K or S;
X15 = F or V;
X16 = K or Q;
X17 = E or G
355
X1X2X3X4X5X6X7X8X9X10X11X12X13X14
Consensus CDR-H3
X1 = A, D, E, G, L, V or W;
X2 = A, D, G, L, P, Q or S;
X3 = A, D, G, L or Y;
X4 = D, G, P, R, S, V, Y or null;
X5 = D, I, P, S, T, Y or null;
X6 = A, G, I, S, T, V, Y or null;
X7 = A, D, E, F, L, P, S, Y or null;
X8 = P, Q, T, Y or null;
X9 = D, G, R, Y or null;
X10 = A, F, Y or null;
X11 = D, F or null;
X12 = F or null;
X13 = D, T or Y;
X14 = I, L, N, V or Y
356
X1X2X3X4X5X6X7X8X9X10X11X12X13X14 X15 X16 X17
Consensus CDR-L1
X1 = G, K, R, S or T;
X2 = A, G or S;
X3 = G, N, S or T;
X4 = G, K, N, Q, R or S;
X5 = S or null;
X6 = D, N, V or null;
X7 = L, V or null;
X8 = H, S, Y or null;
X9 = S, T or null;
X10 = S or null;
X11 = D, G, I, N, S or null;
X12 = D, E, G, K, I, N or null;
X13 = F, G, K, N, R, S, Y or null;
X14 = D, K, N, T or null;
X15 = A, D, G, L, N, S, T or Y;
X16 = L or V;
X17 = A, H, N, Q or S
357
X1X2X3X4X5X6X7
Consensus CDR-L2
X1 = A, D, E, N, S, V or W;
X2 = A, D, N, S or V;
X3 = A, D, H, I, N or S;
X4 = D, K, N, Q, R or T;
X5 = L, R or V;
X6 = A, E, P or Q;
X7 = A, D, S or T
358
X1X2X3X4X5X6X7X8X9X10X11X12
Consensus CDR-L3
X1 = A, C, G, H, I, Q or S;
X2 = A, Q, S or V;
X3 = S, W or Y;
X4 = D, F, G, H or Y;
X5 = D, G, M, R, S or T;
X6 = A, G, H, L, R, S, T or Y;
X7 = L, P, R, S or null;
X8 = D, G, N, R, S, T or null;
X9 = A, G, H, L, P or null;
X10 = F, S or null;
X11 = L, P, W or Y;
X12 = S, T or V
359
GGGGS
4GS linker (aa)
360
GGGS
3GS linker (aa)
361
GGGGSGGGGSGGGGS
(4GS)3 linker (aa)
362
GSTSGSGKPGSGEGSTKG
Linker (aa)
363
ESKYGPPCPPCP
Spacer (IgG4hinge) (aa)
364
gaatctaagtacggaccgccctgccccccttgccct
Spacer (IgG4hinge) (nt)
365
ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
Hinge-CH3 spacer (aa)
YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
366
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD
Hinge-CH2-CH3 spacer
GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ
(aa)
PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
367
MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGL
Human BCMA; GenBank
SLIISLAVFVLMFLLRKISSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVE
No. BAB60895.1
ECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR
368
MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCLGL
Human BCMA; NCBI
SLIISLAVFVLMFLLRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVE
No. NP_001183.2
ECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR
369
MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNARSGLLGMANIDLEKSRTGD
Human BCMA Variant;
EIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALS
GenBank No.
ATEIEKSISAR
ABN42510.1
370
MAQQCFHSEYFDSLLHACKPCHLRCSNPPATCQPYCDPSVTSSVKGTYTVLWIFLGLTLVLS
Mouse BCMA; NCBI No.
LALFTISFLLRKMNPEALKDEPQSPGQLDGSAQLDKADTELTRIRAGDDRIFPRSLEYTVEE
NP_035738.1
CTCEDCVKSKPKGDSDHFFPLPAMEEGATILVTTKTGDYGKSSVPTALQSVMGMEKPTHTR
371
MLQMARQCSQNEYFDSLLHDCKPCQLRCSSTPPLTCQRYCNASMTNSVKGMNAILWTCLGLS
Cynomolgus BCMA;
LIISLAVFVLTFLLRKMSSEPLKDEFKNTGSGLLGMANIDLEKGRTGDEIVLPRGLEYTVEE
GenBank No.
CTCEDCIKNKPKVDSDHCFPLPAMEEGATILVTTKTNDYCNSLSAALSVTEIEKSISAR
EHH60172.1
372
GISWNSGSIXYADSVKG
BCMA-28 CDR-H2 (aa)
Kabat numbering
373
YISGSGSTIYYADSVKG
BCMA-33 CDR-H2 (aa)
Kabat numbering
374
YISSSGNTIYYADSVKG
BCMA-41 CDR-H2
Kabat numbering
375
CIPCQLR
human BCMA epitope
(residues 21-27)
376
DLGPPYGDDAFDI
BCMA-24, -28, -29, -39
CDR-H3 (aa)
377
DLDPDDAFDI
BCMA-30 CDR-H3 (aa)
378
VDGDYDDY
BCMA-35 CDR-H3 (aa)
379
SNTPPLTCQR
human BCMA epitope
(residues 30-39)
380
RASQGISNYLA
BCMA-25 CDR-L1 (aa)
381
RSSQSLLHSNGYNYLD
BCMA-28 CDR-L1 (aa)
382
TGTSSDVGSYNLVS
BCMA-29 CDR-L1 (aa)
383
RASQPIRSNLA
BCMA-30 CDR-L1 (aa)
384
KSSQSVLNSSNNKNYVA
BCMA-31 CDR-L1 (aa)
385
GGNNIGSKGVH
BCMA-32 CDR-L1 (aa)
386
RASQSISNYLA
BCMA-34 CDR-L1 (aa)
387
GSSTGPVTSAHSPS
BCMA-36 CDR-L1 (aa)
388
GSSTGAVTNGHSPY
BCMA-37, -38 CDR-L1
(aa)
389
RASQGIRYELX
BCMA-39 CDR-L1 (aa)
390
TGSSSDVSKYNLVS
BCMA-40 CDR-L1 (aa)
391
SGSSSNIGGNSVD
BCMA-41 CDR-L1 (aa)
392
RASQGIGNGLA
BCMA-42 CDR-L1 (aa)
393
SVTNSVK
human BCMA epitope
(residues 44-50)
394
KSSQNLLYSSNNKNYLA
BCMA-44 CDR-L1 (aa)
395
RASQGIGRSLA
BCMA-45 CDR-L1 (aa)
396
GGNNIGSKSVH
BCMA-47, -48 CDR-L1
(aa)
397
GGDQIGRKSVH
BCMA-49 CDR-L1 (aa)
398
RASQNIGDWLA
BCMA-51 CDR-L1 (aa)
399
WGSTRES
BCMA-24 CDR-L2 (aa)
400
SASTLQS
BCMA-25 CDR-L2 (aa)
401
LGSNRAS
BCMA-28 CDR-L2 (aa)
402
EVSKRPS
BCMA-29 CDR-L2 (aa)
403
SASTRAT
BCMA-30 CDR-L2 (aa)
404
DASNRAT
BCMA-34 CDR-L2 (aa)
405
ETTNRHS
BCMA-36 CDR-L2 (aa)
406
DTTNRHS
BCMA-37 CDR-L2 (aa)
407
DTNNRHS
BCMA-38 CDR-L2 (aa)
408
AASTLQS
BCMA-39 CDR-L2 (aa)
409
ANDRRPS
BCMA-41 CDR-L2 (aa)
410
CSQNEYF
human BCMA epitope
(residues 8-15)
411
DASSLRS
BCMA-45 CDR-L2 (aa)
412
YDTDRPS
BCMA-47, -48 CDR-L2
(aa)
413
YDSDRPS
BCMA-49 CDR-L2 (aa)
414
GASILES
BCMA-51 CDR-L2 (aa)
415
QQYISLPWT
BCMA-24 CDR-L3 (aa)
416
QQSYTSRQT
BCMA-25 CDR-L3 (aa)
417
MQALQTPPWT
BCMA-28 CDR-L3 (aa)
418
CSYAGSSTSRDV
BCMA-29 CDR-L3 (aa)
419
RHYAPLT
BCMA-30 CDR-L3 (aa)
420
QQRSNWPPYT
BCMA-34 CDR-L3 (aa)
421
HLWDRSRDHYV
BCMA-26, -35 CDR-L3
(aa)
422
LLSSGDARMV
BCMA-36 CDR-L3 (aa)
423
SLSHAGDRVF
BCMA-37 CDR-L3 (aa)
424
LLSYSDARLA
BCMA-38 CDR-L3 (aa)
425
LQHNSYPLT
BCMA-39 CDR-L3 (aa)
426
ESWDDALNGHV
BCMA-41 CDR-L3 (aa)
427
QQYVEDALT
BCMA-42 CDR-L3 (aa)
428
LLHACIPCQLR
human BCMA epitope
(residues 17-27)
429
QQYYSSPYT
BCMA-44 CDR-L3 (aa)
430
QQLNGYPWT
BCMA-45 CDR-L3 (aa)
431
QLWDSDSDDFA
BCMA-47 CDR-L3 (aa)
432
QVWDSSTGQYVV
BCMA-49 CDR-L3 (aa)
433
QKYDGAPPWT
BCMA-51 CDR-L3 (aa)
434
EVQLLESGGGLVQPGRSLRLSCVASGFTFD
BCMA-27 VH FR1 (aa)
435
QVQLVQSGGGLVQPGRSLRLSCAASGFTFG
BCMA-30 VH FR1 (aa)
436
EVQLVQSGGGLVQPGRSLRLSCTASGFTFG
BCMA-25, -31, -44, -51
VH FR1 (aa)
437
QVQLVESGGGLVKPGGSLRLSCAASGFTFS
BCMA-32, -49 VH FR1
(aa)
438
TGQLVQSGGGLVQPGRSLRLSCAASGFTFD
BCMA-34 VH FR1 (aa)
439
EVQLLESGGGLVQPGRSLRLSCTASGFTFG
BCMA-42 VH FR1 (aa)
440
agctatgagctgacacagcctccaagcgcctctggcacacctggacagcgagtgacaatgag
BCMA-52 scFv (nt)
ctgtagcggcaccagcagcaacatcggcagccacagcgtgaactggtatcagcagctgcctg
(O/SSE)
gcacagcccctaaactgctgatctacaccaacaaccagcggcctagcggcgtgcccgataga
ttttctggcagcaagagcggcacaagcgccagcctggctatttctggactgcagagcgagga
cgaggccgactattattgtgccgcctgggacggctctctgaacggccttgtttttggcggag
gcaccaagctgacagtgctgggatctagaggtggcggaggatctggcggcggaggaagcgga
ggcggcggatctcttgaaatggctgaagtgcagctggtgcagtctggcgccgaagtgaagaa
gcctggcgagagcctgaagatcagctgcaaaggcagcggctacagcttcaccagctactgga
tcggctgggtccgacagatgcctggcaaaggccttgagtggatgggcatcatctaccccggc
gacagcgacaccagatacagccctagctttcagggccacgtgaccatcagcgccgacaagtc
tatcagcaccgcctacctgcagtggtccagcctgaaggcctctgacaccgccatgtactact
gcgccagatactctggcagcttcgacaattggggccagggcacactggtcaccgtgtccagc
441
RFTISRDNAKSSLYLQMNSLRAEDTAVYYCAR
BCMA-37 VH FR3 (aa)
442
SYELTQPPSASGTPGQRVTMSCSGTSSNIGSHSVNWYQQLPGTAPKLLIYTNNQRPSGVPDR
BCMA-52 scFv (aa)
FSGSKSGTSASLAISGLQSEDEADYYCAAWDGSLNGLVFGGGTKLTVLGSRGGGGSGGGGSG
GGGSLEMAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPG
DSDTRYSPSFQGHVTISADKSISTAYLQWSSLKASDTAMYYCARYSGSFDNWGQGTLVTVSS
443
DSPSPGTTPKNSLYLQMNSLRAEDTAVYYCAK
BCMA-47 VH FR3 (aa)
444
GGQGTMVTVSS
BCMA-28 VH FR4 (aa)
445
WRQGTMVTVSS
BCMA-47 VH FR4 (aa)
446
DIQMTQSPAFLSASVGDRVTVTC
BCMA-25 VL FR1 (aa)
447
DIVMTQSPLSLSVTPGEPASISC
BCMA-28 VL FR1 (aa)
448
QPVLTQPASVSGSPGQSITISC
BCMA-29 VL FR1 (aa)
449
EIVLTQSPATLSVSPGERATLSC
BCMA-30 VL FR1 (aa)
450
DVVMTQSPDSLAVSLGERATISC
BCMA-31 VL FR1 (aa)
451
QTVVTQPPSVSVAPGQTARITC
BCMA-32 VL FR1 (aa)
452
EIVMTQSPATLSLSPGDRATLSC
BCMA-34 VL FR1 (aa)
453
NFMLTQPPSVSVAPGQTARITC
BCMA-35 VL FR1 (aa)
454
QSVLTQEPSLTVSPGETVTLTC
BCMA-36 VL FR1 (aa)
455
QLVLTQEPSLTVSPGGTVTLTC
BCMA-37 VL FR1 (aa)
456
QAVLTQEPSLTVSPGGTVTLTC
BCMA-38 VL FR1 (aa)
457
DIQXTQSPSSLSASVGDRVTITC
BCMA-39 VL FR1 (aa)
458
QPVLTQPPSVSGTPGQRVTIPC
BCMA-41 VL FR1 (aa)
459
DIQMTQSPSLVSASVGDRVTITC
BCMA-42 VL FR1 (aa)
460
cagtctgccctgacacagcctgccagcgttagtgctagtcccggacagtctatcgccatcag
BCMA-55 scFv (nt)
ctgtaccggcaccagctctgacgttggctggtatcagcagcaccctggcaaggcccctaagc
(O/SSE)
tgatgatctacgaggacagcaagaggcccagcggcgtgtccaatagattcagcggcagcaag
agcggcaacaccgccagcctgacaattagcggactgcaggccgaggacgaggccgattacta
ctgcagcagcaacacccggtccagcacactggtttttggcggaggcaccaagctgacagtgc
tgggatctagaggtggcggaggatctggcggcggaggaagcggaggcggcggatctcttgaa
atggctgaagtgcagctggtgcagtctggcgccgagatgaagaaacctggcgcctctctgaa
gctgagctgcaaggccagcggctacaccttcatcgactactacgtgtactggatgcggcagg
cccctggacagggactcgaatctatgggctggatcaaccccaatagcggcggcaccaattac
gcccagaaattccagggcagagtgaccatgaccagagacaccagcatcagcaccgcctacat
ggaactgagccggctgagatccgacgacaccgccatgtactactgcgccagatctcagcgcg
acggctacatggattattggggccagggaaccctggtcaccgtgtccagc
461
DVVMTQSPDSLAVSLGERATINC
BCMA-44 VL FR1 (aa)
462
AIRMTQSPSSLSASVGDRVTITC
BCMA-45 VL FR1 (aa)
463
QAVLTQPPSVSVAPGKTATITC
BCMA-47 VL FR1 (aa)
464
QPVLTQPPSVSVAPGKTATITC
BCMA-48 VL FR1 (aa)
465
LPVLTQPPSVSVAPGKTARITC
BCMA-49 VL FR1 (aa)
466
AIQLTQSPSTLSASVGDRVAITC
BCMA-51 VL FR1 (aa)
467
WYQQKPGNAPRLLIY
BCMA-25 VL FR2 (aa)
468
WYLQKPGQSPQLLIY
BCMA-28 VL FR2 (aa)
469
WYQQHPGKAPKLMIY
BCMA-29 VL FR2 (aa)
470
WYQQKPGQAPKLLTY
BCMA-30 VL FR2 (aa)
471
WYKQKPGQPPKLVIS
BCMA-31 VL FR2 (aa)
472
WYRQRPGQAPEVVIY
BCMA-32 VL FR2 (aa)
473
WFQKKPGQAPTTLTY
BCMA-36 VL FR2 (aa)
474
WFQQKPGQAPRTLTY
BCMA-37, -38 VL FR2
(aa)
475
WYQQKPGKAPKLLTY
BCMA-39 VL FR2 (aa)
476
WFQEVPGTAPKLLTY
BCMA-41 VL FR2 (aa)
477
WYQQKPGKAPKLLLF
BCMA-42 VL FR2 (aa)
478
QSALTQPASVSASPGQSTATSCTGTSSDVGWYQQHPGKAPKLMIYEDSKRPSGVSNRFSGSK
BCMA-55 scFv (aa)
SGNTASLTISGLQAEDEADYYCSSNTRSSTLVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLE
MAEVQLVQSGAEMKKPGASLKLSCKASGYTFIDYYVYWMRQAPGQGLESMGWINPNSGGTNY
AQKFQGRVTMTRDTSISTAYMELSRLRSDDTAMYYCARSQRDGYMDYWGQGTLVTVSS
479
WYKQKPGGVPQLLIH
BCMA-45 VL FR2 (aa)
480
WYQRKPGQGPVVVIQ
BCMA-47, -48 VL FR2
(aa)
481
WYQQKPGQAPVLVMS
BCMA-49 VL FR2 (aa)
482
WYQQKPGKAPKLLIF
BCMA-51 VL FR2 (aa)
483
GVPDRFSGSGSGTDFTLTISSLQAEDVAIYHC
BCMA-24 VL FR3 (aa)
484
GVPSRFRGTGYGTEFSLTIDSLQPEDFATYYC
BCMA-25 VL FR3 (aa)
485
GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC
BCMA-28 VL FR3 (aa)
486
GVSNRFSGSKSGNTASPTISGLQAEDEADYYC
BCMA-29 VL FR3 (aa)
487
GIPDRFSGSGSGTDFTLTISRLEHEDFAVYYR
BCMA-30 VL FR3 (aa)
488
GVPDRFSGSNSGNTATLTVRGVEAGDEADYYC
BCMA-32 VL FR3 (aa)
489
GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC
BCMA-34 VL FR3 (aa)
490
WTPARFSGSLLGGKAALTLSGAQPEDEADYYC
BCMA-36 VL FR3 (aa)
491
WTPARFSGSLLGGKAALTLSGAQPEDEAEYYC
BCMA-37 VL FR3 (aa)
492
WTPARFSGSLLGGKAALTLSGAQPEDEADYFC
BCMA-38 VL FR3 (aa)
493
GVPSRFSGSGSGTDFALTIRSLQPEDFATYYC
BCMA-39 VL FR3 (aa)
494
GVPDRFSGTKSGTSASLAIRGLQSDDDAHYYC
BCMA-41 VL FR3 (aa)
495
GVPSRFSGSRSGTDYTLTISSLQPEDVATYYC
BCMA-42 VL FR3 (aa)
496
gaggtgcagctggtgcagtctggagcagaggtgaaaaagcccggggagtctctgaagatctc
BCMA-52 VH chain (nt)
ctgtaagggttctggatacagctttaccagctactggatcggctgggtgcgccagatgcccg
ggaaaggcctggagtggatggggatcatctatcctggtgactctgataccagatacagcccg
tccttccaaggccacgtcaccatctcagctgacaagtccatcagcactgcctacctgcagtg
gagcagcctgaaggcctcggacaccgccatgtattactgtgcgcgctactctggttctttcg
ataactggggtcaaggtactctggtgaccgtctcctcagc
497
GVPSRFSGSGSGTEFTLTISGVQSEDSATYHC
BCMA-45 VL FR3 (aa)
498
GIPERFSGSKSGDTASLTISGVEAGDEADYYC
BCMA-47 VL FR3 (aa)
499
GIPERFSGSNSGNTATLTISRVEAGDEGDYYC
BCMA-48 VL FR3 (aa)
500
GIPERFSGSNSGNTATLTISRVEAGDEAAYYC
BCMA-49 VL FR3 (aa)
501
GVPSRFSGSGSGTDFTLTISSLQPEDVAVYYC
BCMA-51 VL FR3 (aa)
502
FGPGTRLDIK
BCMA-25 VL FR4 (aa)
503
FGXGTKLTVL
BCMA-29 VL FR4 (aa)
504
FGQGTKLDIK
BCMA-31, -34 VL FR4
(aa)
505
FGTGTKLDIK
BCMA-35 VL FR4 (aa)
506
FGGGTKVDIK
BCMA-42 VL FR4 (aa)
507
SYWIG
BCMA-52 CDR-H1 (aa)-
Kabat numbering
508
FGQGTKVEIK
BCMA-24, -28, -51 VL
FR4 (aa)
509
GFTFGDYAMH
BCMA-30 CDR-H1 (aa)
AbM numbering
510
GISWNSGSIX
BCMA-28 CDR-H2 (aa)
AbM numbering
511
YISGSGSTIY
BCMA-33 CDR-H2 (aa)
AbM numbering
512
YISSSGNTIY
BCMA-41 CDR-H2 AbM
numbering
513
IIYPGDSDTRYSPSFQG
BCMA-52 CDR-H2 (aa)-
Kabat numbering
514
SWNSG
BCMA-28 CDR-H2 (aa)
Chothia numbering
515
SGSGST
BCMA-33 CDR-H2 (aa)
Chothia numbering
516
SSSGNT
BCMA-41 CDR-H2
Chothia numbering
517
YSGSFDN
BCMA-52 CDR-H3 (aa)-
Kabat, Chothia, and AbM
numbering
518
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSGISWNSGSIGYAD
BCMA-24 VH chain (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGPPYGDDAFDIWGQGTMVTVSS
519
EVQLVQSGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEY
BCMA-25, -31, -44, -51
AASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAAWSAPTDYWGQGTLVTVSS
VH chain (aa)
520
EVQLLESGGGLVQPGRSLRLSCVASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYAD
BCMA-27 VH chain (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGGLGITPYYFDYWGQGTLVTVSS
521
QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIXYAD
BCMA-28 VH chain (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGPPYGDDAFDIGGQGTMVTVSS
522
QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYAD
BCMA-29, -39 VH chain
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGPPYGDDAFDIWGQGTMVTVSS
(aa)
523
QVQLVQSGGGLVQPGRSLRLSCAASGFTFGDYAMHWVRQAPGKGLEWVSGISWNSGSIGYAD
BCMA-30 VH chain (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLDPDDAFDIWGQGTMVTVSS
524
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-32, -49 VH chain
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGPPSFDIWGQGTMVTVSS
525
QVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISGSGSTIYYAD
BCMA-33 VH chain (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREADSSADYWGQGTLVNVSS
526
TGQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYAD
BCMA-34 VH chain (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGPDYDPDAFDIWGQGTMVTVSS
527
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-35 VH chain (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVDGDYDDYWGQGTLVTVSS
528
QVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-36, 38 VH chain
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVDGDYVDDYWGQGTLVTVSS
(aa)
529
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-37 VH chain (aa)
SVKGRFTISRDNAKSSLYLQMNSLRAEDTAVYYCARVDGDYVDDYWGQGTLVTVSS
530
QVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGNTIYYAD
BCMA-41 VH chain (aa)
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGDYVDDYWGQGTLVTVSS
531
EVQLLESGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEY
BCMA-42 VH chain (aa)
AASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAAWSAPTDYWGQGTLVTVSS
532
GYSFTSY
BCMA-52 CDR-H1 (aa)-
Chothia numbering
533
QVQLLESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-47 VH chain (aa)
SVKGDSPSPGTTPKNSLYLQMNSLRAEDTAVYYCAKVDGPPSFDIWRQGTMVTVSS
534
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWGSTRES
BCMA-24 VL chain (aa)
GVPDRFSGSGSGTDFTLTISSLQAEDVAIYHCQQYISLPWTFGQGTKVEIK
535
DIQMTQSPAFLSASVGDRVTVTCRASQGISNYLAWYQQKPGNAPRLLIYSASTLQSGVPSRF
BCMA-25 VL chain (aa)
RGTGYGTEFSLTIDSLQPEDFATYYCQQSYTSRQTFGPGTRLDIK
536
SYVLTQPPSVSVAPGQTARITCGANNIGSKSVHWYQQKPGQAPMLVVYDDDDRPSGIPERFS
BCMA-26 VL chain (aa)
GSNSGNTATLTISGVEAGDEADYFCHLWDRSRDHYVFGTGTKLTVL
537
DIVMTQSPLSLSVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASG
BCMA-28 VL chain (aa)
VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPPWTFGQGTKVEIK
538
QPVLTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVSKRPSGVSN
BCMA-29 VL chain (aa)
RFSGSKSGNTASPTISGLQAEDEADYYCCSYAGSSTSRDVFGXGTKLTVL
539
EIVLTQSPATLSVSPGERATLSCRASQPIRSNLAWYQQKPGQAPKLLIYSASTRATGIPDRF
BCMA-30 VL chain (aa)
SGSGSGTDFTLTISRLEHEDFAVYYRRHYAPLTFGGGTKVEIK
540
DVVMTQSPDSLAVSLGERATISCKSSQSVLNSSNNKNYVAWYKQKPGQPPKLVISWASTRES
BCMA-31 VL chain (aa)
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKLDIK
541
QTVVTQPPSVSVAPGQTARITCGGNNIGSKGVHWYRQRPGQAPEVVIYDDSDRPSGVPDRFS
BCMA-32 VL chain (aa)
GSNSGNTATLTVRGVEAGDEADYYCQVWDSSSDHWVFGGGTKLTVL
542
EIVMTQSPATLSLSPGDRATLSCRASQSISNYLAWYQQKPGQAPRLLIYDASNRATGIPARF
BCMA-34 VL chain (aa)
SGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPYTFGQGTKLDIK
543
NFMLTQPPSVSVAPGQTARITCGANNIGSKSVHWYQQKPGQAPMLVVYDDDDRPSGIPERFS
BCMA-35 VL chain (aa)
GSNSGNTATLTISGVEAGDEADYFCHLWDRSRDHYVFGTGTKLDIK
544
QSVLTQEPSLTVSPGETVTLTCGSSTGPVTSAHSPSWFQKKPGQAPTTLIYETTNRHSWTPA
BCMA-36 VL chain (aa)
RFSGSLLGGKAALTLSGAQPEDEADYYCLLSSGDARMVFGGGTKLTVL
545
QLVLTQEPSLTVSPGGTVTLTCGSSTGAVTNGHSPYWFQQKPGQAPRTLIYDTTNRHSWTPA
BCMA-37 VL chain (aa)
RFSGSLLGGKAALTLSGAQPEDEAEYYCSLSHAGDRVFFGGGTKLTVL
546
QAVLTQEPSLTVSPGGTVTLTCGSSTGAVTNGHSPYWFQQKPGQAPRTLIYDTNNRHSWTPA
BCMA-38 VL chain (aa)
RFSGSLLGGKAALTLSGAQPEDEADYFCLLSYSDARLAFGGGTKLTVL
547
DIQXTQSPSSLSASVGDRVTITCRASQGIRYELXWYQQKPGKAPKLLIYAASTLQSGVPSRF
BCMA-39 VL chain (aa)
SGSGSGTDFALTIRSLQPEDFATYYCLQHNSYPLTFGRGTKLEIK
548
QSALTQPASVSGSPGQSITISCTGSSSDVSKYNLVSWYQQPPGKAPKLIIYDVNKRPSGVSN
BCMA-40 VL chain (aa)
RFSGSKSGNTATLTISGLQGDDEADYYCCSYGGSRSYVFGTGTKLTVL
549
QPVLTQPPSVSGTPGQRVTIPCSGSSSNIGGNSVDWFQEVPGTAPKLLIYANDRRPSGVPDR
BCMA-41 VL chain (aa)
FSGTKSGTSASLAIRGLQSDDDAHYYCESWDDALNGHVFGGGTKLTVL
550
DIQMTQSPSLVSASVGDRVTITCRASQGIGNGLAWYQQKPGKAPKLLLFAASRLESGVPSRF
BCMA-42 VL chain (aa)
SGSRSGTDYTLTISSLQPEDVATYYCQQYVEDALTFGGGTKVDIK
551
YPGDSD
BCMA-52 CDR-H2 (aa)-
Chothia numbering
552
DVVMTQSPDSLAVSLGERATINCKSSQNLLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRES
BCMA-44 VL chain (aa)
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPYTFGQGTKLEIK
553
AIRMTQSPSSLSASVGDRVTITCRASQGIGRSLAWYKQKPGGVPQLLIHDASSLRSGVPSRF
BCMA-45 VL chain (aa)
SGSGSGTEFTLTISGVQSEDSATYHCQQLNGYPWTFGQGTKVDIK
554
QAVLTQPPSVSVAPGKTATITCGGNNIGSKSVHWYQRKPGQGPVVVIQYDTDRPSGIPERFS
BCMA-47 VL chain (aa)
GSKSGDTASLTISGVEAGDEADYYCQLWDSDSDDFAFGTGTKLTVL
555
QPVLTQPPSVSVAPGKTATITCGGNNIGSKSVHWYQRKPGQGPVVVIQYDTDRPSGIPERFS
BCMA-48 VL chain (aa)
GSNSGNTATLTISRVEAGDEGDYYCQVWDSSSDHWVFGGGTKLTVL
556
LPVLTQPPSVSVAPGKTARITCGGDQIGRKSVHWYQQKPGQAPVLVMSYDSDRPSGIPERFS
BCMA-49 VL chain (aa)
GSNSGNTATLTISRVEAGDEAAYYCQVWDSSTGQYVVFGGGTKLTVL
557
AIQLTQSPSTLSASVGDRVAITCRASQNIGDWLAWYQQKPGKAPKLLIFGASILESGVPSRF
BCMA-51 VL chain (aa)
SGSGSGTDFTLTISSLQPEDVAVYYCQKYDGAPPWTFGQGTKVEIK
558
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSGISWNSGSIGYAD
BCMA-24 scFv sequence
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGPPYGDDAFDIWGQGTMVTVSSGG
(aa)
GGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQP
PKLLIYWGSTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAIYHCQQYISLPWTFGQGTKVE
IK
559
EVQLVQSGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEY
BCMA-25 scFv sequence
AASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAAWSAPTDYWGQGTLVTVSSGGGGSG
(aa)
GGGSGGGGSDIQMTQSPAFLSASVGDRVTVTCRASQGISNYLAWYQQKPGNAPRLLIYSAST
LQSGVPSRFRGTGYGTEFSLTIDSLQPEDFATYYCQQSYTSRQTFGPGTRLDIK
560
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-26 scFv sequence
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGPPSFDIWGQGTMVTVSSGGGGSG
(aa)
GGGSGGGGSSYVLTQPPSVSVAPGQTARITCGANNIGSKSVHWYQQKPGQAPMLVVYDDDDR
PSGIPERFSGSNSGNTATLTISGVEAGDEADYFCHLWDRSRDHYVFGTGTKLTVL
561
EVQLLESGGGLVQPGRSLRLSCVASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYAD
BCMA-27 scFv sequence
SVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGGLGITPYYFDYWGQGTLVTVSSGGG
(aa)
GSGGGGSGGGGSQPVLTQPPSASGTPGQRVTISCSGGKTVNWFRQVPGTAPQLLIYSNDQRP
SGVPDRFSGSKSGSSASLDISGLQSEDEAYYYCGSWDDSLNAWVFGGETKLTVL
562
QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIXYAD
BCMA-28 scFv sequence
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGPPYGDDAFDIGGQGTMVTVSSGG
(aa)
GGSGGGGSGGGGSDIVMTQSPLSLSVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSP
QLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPPWTFGQGTKVE
IK
563
QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYAD
BCMA-29 scFv sequence
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGPPYGDDAFDIWGQGTMVTVSSGG
(aa)
GGSGGGGSGGGGSQPVLTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLM
IYEVSKRPSGVSNRFSGSKSGNTASPTISGLQAEDEADYYCCSYAGSSTSRDVFGXGTKLTV
L
564
QVQLVQSGGGLVQPGRSLRLSCAASGFTFGDYAMHWVRQAPGKGLEWVSGISWNSGSIGYAD
BCMA-30 scFv sequence
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLDPDDAFDIWGQGTMVTVSSGGGGS
(aa)
GGGGSGGGGSEIVLTQSPATLSVSPGERATLSCRASQPIRSNLAWYQQKPGQAPKLLIYSAS
TRATGIPDRFSGSGSGTDFTLTISRLEHEDFAVYYRRHYAPLTFGGGTKVEIK
565
EVQLVQSGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEY
BCMA-31 scFv sequence
AASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAAWSAPTDYWGQGTLVTVSSGGGGSG
(aa)
GGGSGGGGSDVVMTQSPDSLAVSLGERATISCKSSQSVLNSSNNKNYVAWYKQKPGQPPKLV
ISWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYTFGQGTKLDIK
566
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-32 scFv sequence
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGPPSFDIWGQGTMVTVSSGGGGSG
(aa)
GGGSGGGGSQTVVTQPPSVSVAPGQTARITCGGNNIGSKGVHWYRQRPGQAPEVVIYDDSDR
PSGVPDRFSGSNSGNTATLTVRGVEAGDEADYYCQVWDSSSDHWVFGGGTKLTVL
567
QVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISGSGSTIYYAD
BCMA-33 scFv sequence
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREADSSADYWGQGTLVNVSSGGGGSGG
(aa)
GGSGGGGSQPVLTQPPSVSVAPGKTAMITCGGNNIGFKGVQWYQQKTGQAPVLVVYDDSDRP
SGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSASDHWVFGGGTKLTVL
568
TGQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYAD
BCMA-34 scFv sequence
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGPDYDPDAFDIWGQGTMVTVSSGG
(aa)
GGSGGGGSGGGGSEIVMTQSPATLSLSPGDRATLSCRASQSISNYLAWYQQKPGQAPRLLIY
DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPYTFGQGTKLDIK
569
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-35 scFv sequence
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVDGDYDDYWGQGTLVTVSSGGGGSGG
(aa)
GGSGGGGSNFMLTQPPSVSVAPGQTARITCGANNIGSKSVHWYQQKPGQAPMLVVYDDDDRP
SGIPERFSGSNSGNTATLTISGVEAGDEADYFCHLWDRSRDHYVFGTGTKLDIK
570
QVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-36 scFv sequence
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVDGDYVDDYWGQGTLVTVSSGGGGSG
(aa)
GGGSGGGGSQSVLTQEPSLTVSPGETVTLTCGSSTGPVTSAHSPSWFQKKPGQAPTTLIYET
TNRHSWTPARFSGSLLGGKAALTLSGAQPEDEADYYCLLSSGDARMVFGGGTKLTVL
571
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-37 scFv sequence
SVKGRFTISRDNAKSSLYLQMNSLRAEDTAVYYCARVDGDYVDDYWGQGTLVTVSSGGGGSG
(aa)
GGGSGGGGSQLVLTQEPSLTVSPGGTVTLTCGSSTGAVTNGHSPYWFQQKPGQAPRTLIYDT
TNRHSWTPARFSGSLLGGKAALTLSGAQPEDEAEYYCSLSHAGDRVFFGGGTKLTVL
572
QVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-38 scFv sequence
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVDGDYVDDYWGQGTLVTVSSGGGGSG
(aa)
GGGSGGGGSQAVLTQEPSLTVSPGGTVTLTCGSSTGAVTNGHSPYWFQQKPGQAPRTLIYDT
NNRHSWTPARFSGSLLGGKAALTLSGAQPEDEADYFCLLSYSDARLAFGGGTKLTVL
573
QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYAD
BCMA-39 scFv sequence
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDLGPPYGDDAFDIWGQGTMVTVSSGG
(aa)
GGSGGGGSGGGGSDIQXTQSPSSLSASVGDRVTITCRASQGIRYELXWYQQKPGKAPKLLIY
AASTLQSGVPSRFSGSGSGTDFALTIRSLQPEDFATYYCLQHNSYPLTFGRGTKLEIK
574
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-40 scFv sequence
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGDYTEDYWGQGTLVTVSSGGGGSG
(aa)
GGGSGGGGSQSALTQPASVSGSPGQSITISCTGSSSDVSKYNLVSWYQQPPGKAPKLIIYDV
NKRPSGVSNRFSGSKSGNTATLTISGLQGDDEADYYCCSYGGSRSYVFGTGTKLTVL
575
QVQLVQSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGNTIYYAD
BCMA-41 scFv sequence
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGDYVDDYWGQGTLVTVSSGGGGSG
(aa)
GGGSGGGGSQPVLTQPPSVSGTPGQRVTIPCSGSSSNIGGNSVDWFQEVPGTAPKLLIYAND
RRPSGVPDRFSGTKSGTSASLAIRGLQSDDDAHYYCESWDDALNGHVFGGGTKLTVL
576
EVQLLESGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEY
BCMA-42 scFv sequence
AASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAAWSAPTDYWGQGTLVTVSSGGGGSG
(aa)
GGGSGGGGSDIQMTQSPSLVSASVGDRVTITCRASQGIGNGLAWYQQKPGKAPKLLLFAASR
LESGVPSRFSGSRSGTDYTLTISSLQPEDVATYYCQQYVEDALTFGGGTKVDIK
577
GYSFTSYWIG
BCMA-52 CDR-H1 (aa)-
AbM numbering
578
EVQLVQSGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEY
BCMA-44 scFv sequence
AASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAAWSAPTDYWGQGTLVTVSSGGGGSG
(aa)
GGGSGGGGSDVVMTQSPDSLAVSLGERATINCKSSQNLLYSSNNKNYLAWYQQKPGQPPKLL
IYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSSPYTFGQGTKLEIK
579
QVQLVQSGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEY
BCMA-45 scFv sequence
AASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAAWSAPTDYWGQGTLVTVSSGGGGSG
(aa)
GGGSGGGGSAIRMTQSPSSLSASVGDRVTITCRASQGIGRSLAWYKQKPGGVPQLLIHDASS
LRSGVPSRFSGSGSGTEFTLTISGVQSEDSATYHCQQLNGYPWTFGQGTKVDIK
580
QVQLLESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-47 scFv sequence
SVKGDSPSPGTTPKNSLYLQMNSLRAEDTAVYYCAKVDGPPSFDIWRQGTMVTVSSGGGGSG
(aa)
GGGSGGGGSQAVLTQPPSVSVAPGKTATITCGGNNIGSKSVHWYQRKPGQGPVVVIQYDTDR
PSGIPERFSGSKSGDTASLTISGVEAGDEADYYCQLWDSDSDDFAFGTGTKLTVL
581
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-48 scFv sequence
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGPPSFDIWGQGTMVTVSSGGGGSG
(aa)
GGGSGGGGSQPVLTQPPSVSVAPGKTATITCGGNNIGSKSVHWYQRKPGQGPVVVIQYDTDR
PSGIPERFSGSNSGNTATLTISRVEAGDEGDYYCQVWDSSSDHWVFGGGTKLTVL
582
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
BCMA-49 scFv sequence
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGPPSFDIWGQGTMVTVSSGGGGSG
(aa)
GGGSGGGGSLPVLTQPPSVSVAPGKTARITCGGDQIGRKSVHWYQQKPGQAPVLVMSYDSDR
PSGIPERFSGSNSGNTATLTISRVEAGDEAAYYCQVWDSSTGQYVVFGGGTKLTVL
583
EVQLVQSGGGLVQPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEY
BCMA-51 scFv sequence
AASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAAWSAPTDYWGQGTLVTVSSGGGGSG
(aa)
GGGSGGGGSAIQLTQSPSTLSASVGDRVAITCRASQNIGDWLAWYQQKPGKAPKLLIFGASI
LESGVPSRFSGSGSGTDFTLTISSLQPEDVAVYYCQKYDGAPPWTFGQGTKVEIK
584
CAGGTGCAGCTGGTGCAATCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTC
BCMA-41 scFv sequence
CTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAG
(nt)
GGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAATACCATATACTACGCAGAC
TCTGTAAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAAAACTCACTGTATCTGCAAAT
GAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGTGGACGGTGACTACG
TCGATGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGC
GGAGGTGGCTCTGGCGGTGGCGGATCGCAGCCTGTGCTGACTCAGCCACCCTCAGTGTCTGG
GACCCCCGGGCAGAGGGTCACCATCCCTTGTTCTGGAAGCAGCTCCAACATCGGAGGTAACT
CTGTAGACTGGTTCCAGGAGGTCCCAGGGACGGCCCCCAAACTCCTCATCTACGCTAATGAT
CGGCGGCCCTCGGGTGTCCCTGACCGCTTCTCTGGCACCAAGTCGGGCACCTCAGCCTCCCT
GGCCATCAGGGGGCTCCAGTCTGACGATGACGCTCATTATTACTGTGAATCCTGGGACGATG
CCCTGAACGGTCACGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA
585
QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAY
BCMA-C1 VH-VL scFv
DFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSGGGGSGG
(aa)
GGSGGGGSDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQL
ASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIK
586
DVVMTQSHRFMSTSVGDRVSITCRASQDVNTAVSWYQQKPGQSPKLLIFSASYRYTGVPDRF
BCMA-C2 VL-VH scFv
TGSGSGADFTLTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLDIKGGGGSGGGGSGGGGSQI
(aa)
QLVQSGPDLKKPGETVKLSCKASGYTFTNFGMNWVKQAPGKGFKWMAWINTYTGESYFADDF
KGRFAFSVETSATTAYLQINNLKTEDTATYFCARGEIYYGYDGGFAYWGQGTLVTVSA
587
IIYPGDSDTR
BCMA-52 CDR-H2 (aa)-
AbM numbering
588
gaagtgcagctggtgcagtctggcgccgaagtgaagaagcctggcgagagcctgaagatcag
BCMA-52 VH chain (nt)
ctgcaaaggcagcggctacagcttcaccagctactggatcggctgggtccgacagatgcctg
(O/SSE)
gcaaaggccttgagtggatgggcatcatctaccccggcgacagcgacaccagatacagccct
agctttcagggccacgtgaccatcagcgccgacaagtctatcagcaccgcctacctgcagtg
gtccagcctgaaggcctctgacaccgccatgtactactgcgccagatactctggcagcttcg
acaattggggccagggcacactggtcaccgtgtccagc
589
SGTSSNIGSHSVN
BCMA-52 CDR-L1 (aa)-
Kabat, Chothia, and AbM
numbering
590
TNNQRPS
BCMA-52 CDR-L2 (aa)-
Kabat, Chothia, and AbM
numbering
591
AAWDGSLNGLV
BCMA-52 CDR-L3 (aa)-
Kabat, Chothia, and AbM
numbering
592
tcctatgagctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatgtc
BCMA-52 VL chain (nt)
ttgttctggaaccagctccaacatcggaagtcactctgtaaactggtaccagcagctcccag
gaacggcccccaaactcctcatctatactaataatcagcggccctcaggggtccctgaccga
ttctctggctccaagtctggcacctcagcctccctggccatcagtggcctccagtctgagga
tgaggctgattattactgtgcagcatgggatggcagcctgaatggtctggtattcggcggag
ggaccaagctgaccgtcctaggt
593
DYYVY
BCMA-55 CDR-H1 (aa)-
Kabat numbering
594
WINPNSGGTNYAQKFQG
BCMA-55 CDR-H2 (aa)-
Kabat numbering
595
SQRDGYMDY
BCMA-55 CDR-H3 (aa)-
Kabat, Chothia, and AbM
numbering
596
GYTFIDY
BCMA-55 CDR-H1 (aa)-
Chothia numbering
597
NPNSGG
BCMA-55 CDR-H2 (aa)-
Chothia numbering
598
GYTFIDYYVY
BCMA-55 CDR-H1 (aa)-
AbM numbering
599
WINPNSGGTN
BCMA-55 CDR-H2 (aa)-
AbM numbering
600
agctatgagctgacacagcctccaagcgcctctggcacacctggacagcgagtgacaatgag
BCMA-52 VL chain (nt)
ctgtagcggcaccagcagcaacatcggcagccacagcgtgaactggtatcagcagctgcctg
(O/SSE)
gcacagcccctaaactgctgatctacaccaacaaccagcggcctagcggcgtgcccgataga
ttttctggcagcaagagcggcacaagcgccagcctggctatttctggactgcagagcgagga
cgaggccgactattattgtgccgcctgggacggctctctgaacggccttgtttttggcggag
gcaccaagctgacagtgctggga
601
TGTSSDVG
BCMA-55 CDR-L1 (aa)-
Kabat, Chothia, and AbM
numbering
602
EDSKRPS
BCMA-55 CDR-L2 (aa)-
Kabat, Chothia, and AbM
numbering
603
SSNTRSSTLV
BCMA-55 CDR-L3 (aa)-
Kabat, Chothia, and AbM
numbering
604
GYSFTSYW
BCMA-52 CDR-H1 (aa)
605
IYPGDSDT
BCMA-52 CDR-H2 (aa)
606
ARYSGSFDN
BCMA-52 CDR-H3 (aa)
607
SSNIGSHS
BCMA-52 CDR-L1 (aa)
608
TNN
BCMA-52 CDR-L2 (aa)
609
EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSP
BCMA-52 VH chain (aa)
SFQGHVTISADKSISTAYLQWSSLKASDTAMYYCARYSGSFDNWGQGTLVTVSS
610
SYELTQPPSASGTPGQRVTMSCSGTSSNIGSHSVNWYQQLPGTAPKLLIYTNNQRPSGVPDR
BCMA-52 VL chain (aa)
FSGSKSGTSASLAISGLQSEDEADYYCAAWDGSLNGLVFGGGTKLTVLG
611
GYTFIDYY
BCMA-55 CDR-H1 (aa)
612
INPNSGGT
BCMA-55 CDR-H2 (aa)
613
ARSQRDGYMDY
BCMA-55 CDR-H3 (aa)
614
ISCTGTSSD
BCMA-55 CDR-L1 (aa)
615
EDS
BCMA-55 CDR-L2 (aa)
616
tcaattggtacgtgg
predicted splice donor site
617
EVQLVQSGAEMKKPGASLKLSCKASGYTFIDYYVYWMRQAPGQGLESMGWINPNSGGTNYAQ
BCMA-55 VH chain (aa)
KFQGRVTMTRDTSISTAYMELSRLRSDDTAMYYCARSQRDGYMDYWGQGTLVTVSS
618
QSALTQPASVSASPGQSIAISCTGTSSDVGWYQQHPGKAPKLMIYEDSKRPSGVSNRFSGSK
BCMA-55 VL chain (aa)
SGNTASLTISGLQAEDEADYYCSSNTRSSTLVFGGGTKLTVLG
619
atggtgctgcagacccaggtgttcatcagcctgctgctgtggatctccggagcatacgga
human IgG-kappa signal
sequence (nt)
620
MVLQTQVFISLLLWISGAYG
human IgG-kappa signal
peptide (aa)
621
gaatctaagtacggaccgccctgccctccctgccctgctcctcctgtggctggaccaagcgt
IgG4/IgG2 hinge-
gttcctgtttccacctaagcctaaagataccctgatgatttcccgcacacctgaagtgactt
IgG2/IgG4 CH2- IgG4
gcgtggtcgtggacgtgagccaggaggatccagaagtgcagttcaactggtacgtggacggc
CH3 spacer (nt)
gtggaagtccacaatgctaagactaaaccccgagaggaacagtttcagtcaacttaccgggt
cgtgagcgtgctgaccgtcctgcatcaggattggctgaacgggaaggagtataagtgcaaag
tgtctaataagggactgcctagctccatcgagaaaacaattagtaaggcaaaagggcagcct
cgagaaccacaggtgtataccctgccccctagccaggaggaaatgaccaagaaccaggtgtc
cctgacatgtctggtcaaaggcttctatccaagtgacatcgccgtggagtgggaatcaaatg
ggcagcccgagaacaattacaagaccacaccacccgtgctggactctgatggaagtttcttt
ctgtattccaggctgaccgtggataaatctcgctggcaggagggcaacgtgttctcttgcag
tgtcatgcacgaagccctgcacaatcattatacacagaagtcactgagcctgtccctgggca
aa
622
gagtctaaatacggaccgccttgtcctccttgtcccgctcctcctgttgccggaccttccgt
optimized SSE IgG4/IgG2
gttcctgtttcctccaaagcctaaggacaccctgatgatcagcaggacccctgaagtgacct
hinge-IgG2/IgG4 CH2-
gcgtggtggtggatgtgtcccaagaggatcccgaggtgcagttcaactggtatgtggacggc
IgG4 CH3 spacer (nt)
gtggaagtgcacaacgccaagaccaagcctagagaggaacagttccagagcacctacagagt
ggtgtccgtgctgacagtgctgcaccaggattggctgaacggcaaagagtacaagtgcaagg
tgtccaacaagggcctgcctagcagcatcgagaaaaccatctccaaggccaagggccagcca
agagagccccaggtttacacactgcctccaagccaagaggaaatgaccaagaatcaggtgtc
cctgacatgcctggtcaagggcttctacccctccgatatcgccgtggaatgggagagcaatg
gccagcctgagaacaactacaagaccacacctcctgtgctggacagcgacggcagtttcttc
ctgtatagtagactcaccgtggataaatcaagatggcaagagggcaacgtgttcagctgcag
cgtgatgcacgaggccctgcacaaccactacacccagaaaagcctgagcctgtctctgggca
ag
623
atgttttgggtgctggtcgtggtcggaggggtgctggcctgttacagcctgctggtgacagt
CD28 transmembrane
cgctttcatcatcttctgggtg
domain (nt)
624
MFWVLVVVGGVLACYSLLVTVAFIIFWV
CD28 tmnsmembrane
domain (aa)
625
aagcgggggagaaagaaactgctgtatattttcaaacagccctttatgagacctgtgcagac
4-1BB-derived
tacccaggaggaagacggatgcagctgtaggtttcccgaggaagaggaaggaggctgtgagc
intracellular co-signaling
tg
sequence (nt)
626
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
4-1BB-derived
intracellular co-signaling
sequence (aa)
627
agagtcaagttttccaggtccgccgacgctccagcctaccagcaggggcagaaccagctgta
CD3-zeta derived
caacgagctgaacctgggcagaagggaagagtacgacgtcctggataagcggagaggccggg
intracellular signaling
accctgagatgggcggcaagcctcggcggaagaacccccaggaaggcctgtataacgaactg
domain (nt)
cagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcgggg
caagggccacgacggcctgtatcagggcctgtccaccgccaccaaggatacctacgacgccc
tgcacatgcaggccctgcccccaagg
628
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL
CD3-zeta derived
QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
intracellular signaling
domain (aa)
629
attgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatcca
CD28 ectodomain spacer
tgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccc
(nt)
630
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP
CD28 ectodomain spacer
(aa)
631
EGRGSLLTCGDVEENPGP
T2A peptide (aa)
632
atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgat
truncated EGFR (tEGFR)
cccacgcaaagtgtgtaacggaataggtattggtgaatttaaagactcactctccataaatg
sequence (nt)
ctacgaatattaaacacttcaaaaactgcacctccatcagtggcgatctccacatcctgccg
gtggcatttaggggtgactccttcacacatactcctcctctggatccacaggaactggatat
tctgaaaaccgtaaaggaaatcacagggtttttgctgattcaggcttggcctgaaaacagga
cggacctccatgcctttgagaacctagaaatcatacgcggcaggaccaagcaacatggtcag
ttttctcttgcagtcgtcagcctgaacataacatccttgggattacgctccctcaaggagat
aagtgatggagatgtgataatttcaggaaacaaaaatttgtgctatgcaaatacaataaact
ggaaaaaactgtttgggacctccggtcagaaaaccaaaattataagcaacagaggtgaaaac
agctgcaaggccacaggccaggtctgccatgccttgtgctcccccgagggctgctggggccc
ggagcccagggactgcgtctcttgccggaatgtcagccgaggcagggaatgcgtggacaagt
gcaaccttctggagggtgagccaagggagtttgtggagaactctgagtgcatacagtgccac
ccagagtgcctgcctcaggccatgaacatcacctgcacaggacggggaccagacaactgtat
ccagtgtgcccactacattgacggcccccactgcgtcaagacctgcccggcaggagtcatgg
gagaaaacaacaccctggtctggaagtacgcagacgccggccatgtgtgccacctgtgccat
ccaaactgcacctacggatgcactgggccaggtcttgaaggctgtccaacgaatgggcctaa
gatcccgtccatcgccactgggatggtgggggccctcctcttgctgctggtggtggccctgg
ggatcggcctcttcatgtga
633
atgctgctcctcgtgacaagcctgctcctgtgtgaactccctcatccagcttttctgctcat
truncated EGFR (tEGFR)
tcctcggaaagtgtgcaacggcatcggcatcggagagttcaaggacagcctgagcatcaatg
sequence (nt) (O/SSE)
ccaccaacatcaagcacttcaagaattgcaccagcatcagcggcgacctgcacattctgcct
gtggcctttagaggcgacagcttcacccacacacctccactggatccccaagagctggatat
cctgaaaaccgtgaaagagattaccggattcctcctgatccaagcctggccagagaacagaa
ccgatctgcacgccttcgagaacctcgagatcatcagaggccggaccaaacagcacggccag
tttagcctggctgtggtgtctctgaacatcaccagtctgggcctgagaagcctgaaagaaat
ctccgacggcgacgtgatcatctccggaaacaagaacctgtgctacgccaacaccatcaact
ggaagaagctgttcggcacctccggccagaaaacaaagatcatctctaaccggggcgagaac
agctgcaaggccaccggacaagtttgtcacgccctgtgtagccctgaaggctgttggggacc
cgaacctagagactgtgtgtcctgccggaatgtgtcccggggcagagaatgtgtggataagt
gcaacctgctggaaggcgagccccgcgagtttgtggaaaacagcgagtgcatccagtgtcac
cccgagtgtctgccccaggccatgaacattacatgcaccggcagaggccccgacaactgtat
tcagtgcgcccactacatcgacggccctcactgcgtgaaaacatgtccagctggcgtgatgg
gagagaacaacaccctcgtgtggaagtatgccgacgccggacatgtgtgccacctgtgtcac
cctaattgcacctacggctgtaccggacctggcctggaaggatgccctacaaacggccctaa
gatccccagcattgccaccggaatggttggagccctgctgcttctgttggtggtggccctcg
gaatcggcctgttcatgtga
634
MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILP
truncated EGFR (tEGFR)
VAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQ
sequence (aa)
FSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGEN
SCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCH
PECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCH
PNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM
635
ggatctgcgatcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgag
EF1alpha promoter with
aagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactg
HTLV1 enhancer
ggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataa
gtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacagctgaag
cttcgaggggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacg
ccggttgagtcgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtct
aggtaagtttaaagctcaggtcgagaccgggcctttgtccggcgctcccttggagcctacct
agactcagccggctctccacgctttgcctgaccctgcttgctcaactctacgtctttgtttc
gttttctgttctgcgccgttacagatccaagctgtgaccggcgcctac
636
aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctcc
Woodchuck Hepatitis
ttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatgg
Virus (WHP)
ctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggccc
Posttranscriptional
gttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttgggg
Regulatory Element
cattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacgg
(WPRE)
cggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgac
aattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccac
ctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttc
cttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacg
agtcggatctccctttgggccgcctccccgc
637
QNEYF
BCMA-52-scFV-mFc
BCMA binding epitope 1
638
CIPCQL
BCMA-52-scFV-mFc
BCMA binding epitope 2
639
CQRYC
BCMA-52-scFV-mFc
BCMA binding epitope 3
640
MLMAG
BCMA-55-scFV-mFc
BCMA binding epitope 1
641
YFDSLL
BCMA-55-scFV-mFc
BCMA binding epitope 2
642
QLRCSSNTPPL
BCMA-55-scFV-mFc
BCMA binding epitope 3
643
gaagtgcagctggtgcagtctggggctgagatgaagaagcctggggcctcactgaagctctc
BCMA-55 VH chain (nt)
ctgcaaggcttctggatacaccttcatcgactactatgtatactggatgcgacaggcccctg
gacaagggcttgagtccatgggatggatcaaccctaacagtggtggcacaaactatgcacag
aagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacatggagct
gagcaggctgagatctgacgacaccgccatgtattactgtgcgcgctcccagcgtgacggtt
acatggattactggggtcaaggtactctggtgaccgtctcctca
644
gaagtgcagctggtgcagtctggcgccgagatgaagaaacctggcgcctctctgaagctgag
BCMA-55 VH chain (nt)
ctgcaaggccagcggctacaccttcatcgactactacgtgtactggatgcggcaggcccctg
(O/SSE)
gacagggactcgaatctatgggctggatcaaccccaatagcggcggcaccaattacgcccag
aaattccagggcagagtgaccatgaccagagacaccagcatcagcaccgcctacatggaact
gagccggctgagatccgacgacaccgccatgtactactgcgccagatctcagcgcgacggct
acatggattattggggccagggaaccctggtcaccgtgtccagc
645
caatctgccctgactcagcctgcctccgtgtctgcgtctcctggacagtcgatcgccatctc
BCMA-55 VL chain (nt)
ctgcactggaaccagcagtgacgttggttggtatcaacagcacccaggcaaagcccccaaac
tcatgatttatgaggacagtaagcggccctcaggggtttctaatcgcttctctggctccaag
tctggcaacacggcctccctgaccatctctgggctccaggctgaggacgaggctgattatta
ctgcagctcaaatacaagaagcagcactttggtgttcggcggagggaccaagctgaccgtcc
ta
646
cagtctgccctgacacagcctgccagcgttagtgctagtcccggacagtctatcgccatcag
BCMA-55 VL chain (nt)
ctgtaccggcaccagctctgacgttggctggtatcagcagcaccctggcaaggcccctaagc
(O/SSE)
tgatgatctacgaggacagcaagaggcccagcggcgtgtccaatagattcagcggcagcaag
agcggcaacaccgccagcctgacaattagcggactgcaggccgaggacgaggccgattacta
ctgcagcagcaacacccggtccagcacactggtttttggcggaggcaccaagctgacagtgc
tg
647
tcctatgagctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatgtc
BCMA-52 scFv
ttgttctggaaccagctccaacatcggaagtcactctgtaaactggtaccagcagctcccag
gaacggcccccaaactcctcatctatactaataatcagcggccctcaggggtccctgaccga
ttctctggctccaagtctggcacctcagcctccctggccatcagtggcctccagtctgagga
tgaggctgattattactgtgcagcatgggatggcagcctgaatggtctggtattcggcggag
ggaccaagctgaccgtcctaggttctagaggtggtggtggtagcggcggcggcggctctggt
ggtggtggatccctcgagatggccgaggtgcagctggtgcagtctggagcagaggtgaaaaa
gcccggggagtctctgaagatctcctgtaagggttctggatacagctttaccagctactgga
tcggctgggtgcgccagatgcccgggaaaggcctggagtggatggggatcatctatcctggt
gactctgataccagatacagcccgtccttccaaggccacgtcaccatctcagctgacaagtc
catcagcactgcctacctgcagtggagcagcctgaaggcctcggacaccgccatgtattact
gtgcgcgctactctggttctttcgataactggggtcaaggtactctggtgaccgtctcctca
648
caatctgccctgactcagcctgcctccgtgtctgcgtctcctggacagtcgatcgccatctc
BCMA-55 scFv
ctgcactggaaccagcagtgacgttggttggtatcaacagcacccaggcaaagcccccaaac
tcatgatttatgaggacagtaagcggccctcaggggtttctaatcgcttctctggctccaag
totggcaacacggcctccctgaccatotctgggctocaggctgaggacgaggctgattatta
ctgcagctcaaatacaagaagcagcactttggtgttcggcggagggaccaagctgaccgtcc
taggttctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgag
atggccgaagtgcagctggtgcagtctggggctgagatgaagaagcctggggcctcactgaa
gctctcctgcaaggcttctggatacaccttcatcgactactatgtatactggatgcgacagg
cccctggacaagggcttgagtccatgggatggatcaaccctaacagtggtggcacaaactat
gcacagaagtttcagggcagggtcaccatgaccagggacacgtccatcagcacagcctacat
ggagctgagcaggctgagatctgacgacaccgccatgtattactgtgcgcgctcccagcgtg
acggttacatggattactggggtcaaggtactctggtgaccgtctcctca
649
ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG
IgG4/IgG2 hinge-
VEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
IgG2/IgG4 CH2-IgG4
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
CH3 spacer (aa)
LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
650
ggatctgcgatcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgag
modified EF1 alpha
aagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactg
promoter
ggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataa
gtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacagctgaag
cttcgaggggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacg
ccggttgagtcgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtct
aggtaagtttaaagctcaggtcgagaccgggcctttgtccggcgctcccttggagcctacct
agactcagccggctctccacgctttgcctgaccctgcttgctcaactctacgtctttgtttc
gttttctgttctgcgccgttacagatccaagctgtgaccggcgcctacggctagcgcc
651
tttatttagtctccagaaaaaggggggaatgaaagaccccacctgtaggtttggcaagctag
MND promoter
gatcaaggttaggaacagagagacagcagaatatgggccaaacaggatatctgtggtaagca
gttcctgccccggctcagggccaagaacagttggaacagcagaatatgggccaaacaggata
tctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccagatgcggtc
ccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaat
gaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttct
gctccccgagctcaataaaagagccca
652
agagtgaagttcagcagatccgccgacgctccagcctatcagcagggccaaaaccagctgta
CD3-zeta derived
caacgagctgaacctggggagaagagaagagtacgacgtgctggataagcggagaggcagag
intracellular signaling
atcctgaaatgggcggcaagcccagacggaagaatcctcaagagggcctgtataatgagctg
domain (nt)
cagaaagacaagatggccgaggcctacagcgagatcggaatgaagggcgagcgcagaagagg
caagggacacgatggactgtaccagggcctgagcaccgccaccaaggatacctatgacgcac
tgcacatgcaggccctgccacctaga
653
GSGEGRGSLLTCGDVEENPGP
T2A peptide (aa)
654
LEGGGEGRGSLLTCGDVEENPGPR
T2A peptide (aa)
655
ATNFSLLKQAGDVEENPGP
P2A peptide (aa)
656
GSGATNFSLLKQAGDVEENPGP
P2A peptide (aa)
657
QCTNYALLKLAGDVESNPGP
E2A peptide (aa)
658
GSGQCTNYALLKLAGDVESNPGP
E2A peptide (aa)
659
VKQTLNFDLLKLAGDVESNPGP
F2A peptide (aa)
660
GSGVKQTLNFDLLKLAGDVESNPGP
F2A peptide (aa)
661
agtctaaatacggac
Optimized splice donor
site
662
tcaactggtatgtgg
Optimized splice donor
site
663
accatctccaaggcc
Optimized splice donor
site
664
gccccaggtttacac
Optimized splice donor
site
665
tcagcagatccgccg
Optimized splice donor
site
666
ctcctgtgtgaactc
Optimized splice donor
site
667
tcggaaagtgtgcaa
Optimized splice donor
site
668
cagcacggccagttt
Optimized splice donor
site
669
aaccggggcgagaac
Optimized splice donor
site
670
ctggaaggcgagccc
Optimized splice donor
site
671
tgttcatgtgagcgg
Optimized splice donor
site (last 4 nt outside of
coding region)
672
cagtttcttcctgtatagtagactcaccgtggataaatcaa
Optimized splice acceptor
site
673
gggcaacgtgttcagctgcagcgtgatgcacgaggccctgc
Optimized splice acceptor
site
674
cggagtgctggcctgttacagcctgctggttaccgtggcct
Optimized splice acceptor
site
675
gctgagagtgaagttcagcagatccgccgacgctccagcct
Optimized splice acceptor
site
676
acacctccactggatccccaagagctggatatcctgaaaac
Optimized splice acceptor
site
677
accggattcctcctgatccaagcctggccagagaacagaac
Optimized splice acceptor
site
678
acggccagtttagcctggctgtggtgtctctgaacatcacc
Optimized splice acceptor
site
679
aggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgg
CD28 endo (nt)
gcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcc
680
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
CD28 endo (aa)
681
aagcggggcagaaagaagctgctctacatcttcaagcagcccttcatgcggcccgtgcagac
4-1BB-derived
cacacaagaggaagatggctgctcctgcagattccccgaggaagaagaaggcggctgcgagc
intracellular co-signaling
tg
sequence (nt)
682
atggtgctgcagacccaggtgttcatcagcctgctgctgtggatctctggcgcctacggc
human IgG-kappa signal
sequence (nt)
683
atggtgctgcagacccaggtgttcatcagcctgctgctgtggatctctggcgcctatgga
human IgG-kappa signal
sequence (nt)
684
atggtgctgcagacacaggtgttcatctccctgctgctgtggatctctggagcatacgga
human IgG-kappa signal
sequence (nt)
685
atggtgctgcagacacaggtgttcatcagcctgctgctgtggatctccggagcatacgga
human IgG-kappa signal
sequence (nt)
686
ctcgagggcggcggagagggcagaggaagtcttctaacatgcggtgacgtggaggagaatcc
T2A peptide (nt)
cggccctagg
687
cttgaaggtggtggcgaaggcagaggcagcctgcttacatgcggagatgtggaagagaaccc
T2A peptide (nt)
cggacctaga
688
atgttctgggtgctcgtggtcgttggcggagtgctggcctgttacagcctgctggttaccgt
CD28 transmembrane
ggccttcatcatcttttgggtc
domain (nt)
689
cgtctaggtaagttt
Predicted splice donor site
690
gaccaaggtgaccgt
Predicted splice donor site
691
tgcactggtaccagc
Predicted splice donor site
692
taaactggtaccagc
Predicted splice donor site
693
atctcctgtaagggt
Predicted splice donor site
694
ggtcaaggtactctg
Predicted splice donor site
695
gaggacagtaagcgg
Predicted splice donor site
696
ggtcaaggtactctg
Predicted splice donor site
697
tgcctccgtgtctgc
Predicted splice donor site
698
caccaaggtgaccgt
Predicted splice donor site
699
tgaactggtatcagc
Predicted splice donor site
700
atctcttgaaatggt
Predicted splice donor site
701
ggccagggcacactg
Predicted splice donor site
702
gaggacagcaagagg
Predicted splice donor site
703
ggccagggaaccctg
Predicted splice donor site
704
tgccagcgttagtgc
Predicted splice donor site
705
aatctaagtacggac
Predicted splice donor site
706
tcaactggtacgtgg
Predicted splice donor site
707
acaattagtaaggca
Predicted splice donor site
708
accacaggtgtatac
Predicted splice donor site
709
tttccaggtccgccg
Predicted splice donor site
710
ctgctctgtgagtta
Predicted splice donor site
711
acgcaaagtgtgtaa
Predicted splice donor site
712
caacatggtcagttt
Predicted splice donor site
713
aacagaggtgaaaac
Predicted splice donor site
714
ctggagggtgagcca
Predicted splice donor site
715
gaggtgcagctggtggagtccggaggaggcctggtgaagccaggaggctccctgaggctgtc
BCMA-23 scFv (nt)
ttgcgcagccagcggcttcacctttagcgactactatatgtcctggatcagacaggcacctg
gcaagggcctggagtgggtgagctacatcagctcctctggctccacaatctactatgccgac
tctgtgaagggccggtttaccatcagcagagataacgccaagaattccctgtatctgcagat
gaacagcctgagggccgaggacacagccgtgtactattgcgccaaggtggacggcgattaca
ccgaggattattggggccagggcacactggtgaccgtgagctccggcggcggcggctctgga
ggaggaggcagcggcggaggaggctcccagtctgccctgacacagccagccagcgtgtccgg
ctctcccggacagtccatcacaatctcttgtaccggctctagctccgacgtgggcaagtaca
acctggtgtcctggtatcagcagccccctggcaaggcccctaagctgatcatctacgatgtg
aacaagaggccatctggcgtgagcaatcgcttcagcggctccaagtctggcaataccgccac
actgaccatcagcggcctgcagggcgacgatgaggcagattactattgttctagctacggcg
gcagcagatcctacgtgttcggcacaggcaccaaggtgaccgtgctg
716
gaggtgcagctggtgcagagcggaggaggcctggtgcagcctggcaggtccctgcgcctgtc
BCMA-25 scFV(nt)
ttgcaccgccagcggcttcacatttggcgactatgccatgtcctggttcaggcaggcaccag
gcaagggcctggagtgggtgggctttatccgctctaaggcctacggcggcaccacagagtat
gccgccagcgtgaagggccggttcaccatcagccgggacgactctaagagcatcgcctacct
gcagatgaactctctgaagaccgaggacacagccgtgtactattgcgcagcatggagcgccc
caaccgattattggggccagggcaccctggtgacagtgagctccggcggcggcggctctgga
ggaggaggaagcggaggaggaggatccgacatccagatgacacagtcccctgcctttctgtc
cgcctctgtgggcgatagggtgaccgtgacatgtcgcgcctcccagggcatctctaactacc
tggcctggtatcagcagaagcccggcaatgcccctcggctgctgatctacagcgcctccacc
ctgcagagcggagtgccctcccggttcagaggaaccggctatggcacagagttttctctgac
catcgacagcctgcagccagaggatttcgccacatactattgtcagcagtcttacaccagcc
ggcagacatttggccccggcacaagactggatatcaag
717
gaggtgcagctggtgcagagcggaggaggcctggtgcagcctggcaggtccctgcgcctgtc
BCMA-25 scFV(nt)
ttgcaccgccagcggcttcacatttggcgactatgccatgtcctggttcaagcaggcaccag
(O/SSE)
gcaagggcctggagtgggtgggctttatccgctctaaggcctacggcggcaccacagagtat
gccgccagcgtgaagggccggttcaccatcagccgggacgactctaagagcatcgcctacct
gcagatgaactctctgaagaccgaggacacagccgtgtactattgcgcagcatggagcgccc
caaccgattattggggccagggcaccctggtgacagtgagctccggcggcggcggctctgga
ggaggaggaagcggaggaggaggatccgacatccagatgacacagtcccctgcctttctgtc
cgcctctgtgggcgatagggtgaccgtgacatgtcgcgcctcccagggcatctctaactacc
tggcctggtatcagcagaagcccggcaatgcccctcggctgctgatctacagcgcctccacc
ctgcagagcggagtgccctcccggttcagaggaaccggctatggcacagagttttctctgac
catcgacagcctgcagccagaggatttcgccacatactattgtcagcagtcttacaccagcc
ggcagacatttggccccggcacaagactggatatcaag
718
gaggtgcagctggtggagtccggaggaggcctggtgaagccaggaggctctctgaggctgag
BCMA-26 scFV(nt)
ctgcgcagcctccggcttcaccttttctgactactatatgagctggatcaggcaggcaccag
gcaagggcctggagtgggtgtcttacatcagctcctctggcagcacaatctactatgccgac
tccgtgaagggcaggttcaccatctctcgcgataacgccaagaatagcctgtatctgcagat
gaactocctgogggccgaggatacagccgtgtactattgcgccaaggtggacggcocccott
cctttgatatctggggccagggcacaatggtgaccgtgagctccggaggaggaggatccggc
ggaggaggctctggcggcggcggctctagctatgtgctgacccagccaccatccgtgtctgt
ggcacctggacagacagcaaggatcacctgtggagcaaacaatatcggcagcaagtccgtgc
actggtaccagcagaagcctggccaggccccaatgctggtggtgtatgacgatgacgatcgg
cccagcggcatccctgagagattttctggcagcaactccggcaataccgccacactgaccat
ctctggagtggaggcaggcgacgaggcagattacttctgtcacctgtgggaccggagcagag
atcactacgtgttcggcacaggcaccaagctgaccgtgctg
719
gaggtgcagctggtggagtccggaggaggcctggtgaagccaggaggctctctgaggctgag
BCMA-26 scFV(nt)
ctgcgcagcctccggcttcaccttttctgactactatatgagctggatcaggcaggcaccag
(O/SSE)
gcaagggcctggagtgggtgtcttacatcagctcctctggcagcacaatctactatgccgac
tccgtgaagggcaggttcaccatctctcgcgataacgccaagaatagcctgtatctgcagat
gaactccctgcgggccgaggatacagccgtgtactattgcgccaaggtggacggcccccctt
cctttgatatctggggccagggcacaatggtgaccgtgagctccggaggaggaggatccggc
ggaggaggctctggcggcggcggctctagctatgtgctgacccagccaccatccgtgtctgt
ggcacctggacagacagcaaggatcacctgtggagcaaacaatatcggcagcaagtccgtgc
actggtaccagcagaagcctggccaggccccaatgctggtggtgtatgacgatgacgatcgg
cccagcggcatccctgagagattttctggcagcaactccggcaataccgccacactgaccat
ctctggagtggaggcaggcgacgaggcagattacttctgtcacctgtgggaccggagcagag
atcactacgtgttcggcacaggcaccaagctgaccgtgctg
720
tcttcatgtgagcgg
truncated marker
predicted splice donor site
721
tggctccgcctttttcccgagggtgggggagaaccgtatat
promoter predicted splice
acceptor site
722
tgaactgcgtccgccgtctaggtaagtttaaagctcaggtc
promoter predicted splice
acceptor site
723
ttctgttctgcgccgttacagatccaagctgtgaccggcgc
promoter predicted splice
acceptor site
724
ctactacatgagctggatccgccaggctccagggaaggggc
BCMA-23 predicted
splice acceptor site
725
ggctgattattattgtagctcatatggaggtagtaggtctt
BCMA-23 predicted
splice acceptor site
726
ctatgccatgtcctggttcaggcaggcaccaggcaagggcc
BCMA-25 predicted
splice acceptor site
727
gtccgcctctgtgggcgatagggtgaccgtgacatgtcgcg
BCMA-25 predicted
splice acceptor site
728
gtgggctttatccgctctaaggcctacggcggcaccacaga
BCMA-25 predicted
splice acceptor site
729
gtgacatgtcgcgcctcccagggcatctctaactacctggc
BCMA-25 predicted
splice acceptor site
730
tacagcgcctccaccctgcagagcggagtgccctcccggtt
BCMA-25 predicted
splice acceptor site
731
ctggccatcagtggcctccagtctgaggatgaggctgatta
BCMA-52 predicted
splice acceptor site
732
agatacagcccgtccttccaaggccacgtcaccatctcagc
BCMA-52 predicted
splice acceptor site
733
cgaggctgattattactgcagctcaaatacaagaagcagca
BCMA-55 predicted
splice acceptor site
734
gccctcaggggtttctaatcgcttctctggctccaagtctg
BCMA-55 predicted
splice acceptor site
735
ctactatatgtcctggatcagacaggcacctggcaagggcc
BCMA-23 predicted
splice acceptor site
(O/SSE)
736
ggcagattactattgttctagctacggcggcagcagatcct
BCMA-23 predicted
splice acceptor site
(O/SSE)
737
ctatgccatgtcctggttcaagcaggcaccaggcaagggcc
BCMA-25 predicted
splice acceptor site
(O/SSE)
738
ctggctatttctggactgcagagcgaggacgaggccgacta
BCMA-52 predicted
splice acceptor site
(O/SSE)
739
agatacagccctagctttcagggccacgtgaccatcagcgc
BCMA-52 predicted
splice acceptor site
(O/SSE)
740
cgaggccgattactactgcagcagcaacacccggtccagca
BCMA-55 predicted
splice acceptor site
(O/SSE)
741
gcccagcggcgtgtccaatagattcagcggcagcaagagcg
BCMA-55 predicted
splice acceptor site
(O/SSE)
742
aagtttctttctgtattccaggctgaccgtggataaatctc
spacer predicted splice
acceptor site
743
gggcaacgtgttctcttgcagtgtcatgcacgaagccctgc
spacer predicted splice
acceptor site
744
aggggtgctggcctgttacagcctgctggtgacagtcgctt
CD28TM predicted splice
acceptor site
745
gctgagagtcaagttttccaggtccgccgacgctccagcct
4-1BB/CD3 zeta
predicted splice acceptor
site
746
actcctcctctggatccacaggaactggatattctgaaaac
truncated marker
predicted splice acceptor
site
747
acagggtttttgctgattcaggcttggcctgaaaacaggac
truncated marker
predicted splice acceptor
site
748
atggtcagttttctcttgcagtcgtcagcctgaacataaca
truncated marker
predicted splice acceptor
site
749
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
Human IgG2 Fc (Uniprot
YSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPP
P01859)
KPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLT
VVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA
LHNHYTQKSLSLSPGK
750
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
Human IgG4 Fc (Uniprot
YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFP
P01861)
PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE
ALHNHYTQKSLSLSLGK
751
gaggtgcagctggtggagtccggaggaggcctggtgaagccaggaggctccctgaggctgtc
anti-BMCA CAR
ttgcgcagccagcggcttcacctttagcgactactatatgtcctggatcagacaggcacctg
gcaagggcctggagtgggtgagctacatcagctcctctggctccacaatctactatgccgac
tctgtgaagggccggtttaccatcagcagagataacgccaagaattccctgtatctgcagat
gaacagcctgagggccgaggacacagccgtgtactattgcgccaaggtggacggcgattaca
ccgaggattattggggccagggcacactggtgaccgtgagctccggcggcggcggctctgga
ggaggaggcagcggcggaggaggctcccagtctgccctgacacagccagccagcgtgtccgg
ctctcccggacagtccatcacaatctcttgtaccggctctagctccgacgtgggcaagtaca
acctggtgtcctggtatcagcagccccctggcaaggcccctaagctgatcatctacgatgtg
aacaagaggccatctggcgtgagcaatcgcttcagcggctccaagtctggcaataccgccac
actgaccatcagcggcctgcagggcgacgatgaggcagattactattgttctagctacggcg
gcagcagatcctacgtgttcggcacaggcaccaaggtgaccgtgctggaatctaagtacgga
ccgccttgtcctccttgtcccgctcctcctgttgccggaccttccgtgttcctgtttcctcc
aaagcctaaggacaccctgatgatcagcaggacccctgaagtgacctgcgtggtggtggatg
tgtcccaagaggatcccgaggtgcagttcaactggtatgtggacggcgtggaagtgcacaac
gccaagaccaagcctagagaggaacagttccagagcacctacagagtggtgtccgtgctgac
agtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcc
tgcctagcagcatcgagaaaaccatctccaaggccaagggccagccaagagagccccaggtt
tacacactgcctccaagccaagaggaaatgaccaagaatcaggtgtccctgacatgcctggt
caagggcttctacccctccgatatcgccgtggaatgggagagcaatggccagcctgagaaca
actacaagaccacacctcctgtgctggacagcgacggcagtttcttcctgtatagtagactc
accgtggataaatcaagatggcaagagggcaacgtgttcagctgcagcgtgatgcacgaggc
cctgcacaaccactacacccagaaaagcctgagcctgtctctgggcaagatgttctgggtgc
tcgtggtcgttggcggagtgctggcctgttacagcctgctggttaccgtggccttcatcatc
ttttgggtcaagcggggcagaaagaagctgctctacatcttcaagcagcccttcatgcggcc
cgtgcagaccacacaagaggaagatggctgctcctgcagattccccgaggaagaagaaggcg
gctgcgagctgagagtgaagttcagcagatccgccgacgctccagcctatcagcagggccaa
aaccagctgtacaacgagctgaacctggggagaagagaagagtacgacgtgctggataagcg
gagaggcagagatcctgaaatgggcggcaagcccagacggaagaatcctcaagagggcctgt
ataatgagctgcagaaagacaagatggccgaggcctacagcgagatcggaatgaagggcgag
cgcagaagaggcaagggacacgatggactgtaccagggcctgagcaccgccaccaaggatac
ctatgacgcactgcacatgcaggccctgccacctaga
752
gaggtgcagctggtgcagagcggaggaggcctggtgcagcctggcaggtccctgcgcctgtc
anti-BMCA CAR
ttgcaccgccagcggcttcacatttggcgactatgccatgtcctggttcaagcaggcaccag
gcaagggcctggagtgggtgggctttatccgctctaaggcctacggcggcaccacagagtat
gccgccagcgtgaagggccggttcaccatcagccgggacgactctaagagcatcgcctacct
gcagatgaactctctgaagaccgaggacacagccgtgtactattgcgcagcatggagcgccc
caaccgattattggggccagggcaccctggtgacagtgagctccggcggcggcggctctgga
ggaggaggaagcggaggaggaggatccgacatccagatgacacagtcccctgcctttctgtc
cgcctctgtgggcgatagggtgaccgtgacatgtcgcgcctcccagggcatctctaactacc
tggcctggtatcagcagaagcccggcaatgcccctcggctgctgatctacagcgcctccacc
ctgcagagcggagtgccctcccggttcagaggaaccggctatggcacagagttttctctgac
catcgacagcctgcagccagaggatttcgccacatactattgtcagcagtcttacaccagcc
ggcagacatttggccccggcacaagactggatatcaaggagtctaaatacggaccgccttgt
cctccttgtcccgctcctcctgttgccggaccttccgtgttcctgtttcctccaaagcctaa
ggacaccctgatgatcagcaggacccctgaagtgacctgcgtggtggtggatgtgtcccaag
aggatcccgaggtgcagttcaactggtatgtggacggcgtggaagtgcacaacgccaagacc
aagcctagagaggaacagttccagagcacctacagagtggtgtccgtgctgacagtgctgca
ccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgcctagca
gcatcgagaaaaccatctccaaggccaagggccagccaagagagccccaggtttacacactg
cctccaagccaagaggaaatgaccaagaatcaggtgtccctgacatgcctggtcaagggctt
ctacccctccgatatcgccgtggaatgggagagcaatggccagcctgagaacaactacaaga
ccacacctcctgtgctggacagcgacggcagtttcttcctgtatagtagactcaccgtggat
aaatcaagatggcaagagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaa
ccactacacccagaaaagcctgagcctgtctctgggcaagatgttctgggtgctcgtggtcg
ttggcggagtgctggcctgttacagcctgctggttaccgtggccttcatcatcttttgggtc
aagcggggcagaaagaagctgctctacatcttcaagcagcccttcatgcggcccgtgcagac
cacacaagaggaagatggctgctcctgcagattccccgaggaagaagaaggcggctgcgagc
tgagagtgaagttcagcagatccgccgacgctccagcctatcagcagggccaaaaccagctg
tacaacgagctgaacctggggagaagagaagagtacgacgtgctggataagcggagaggcag
agatcctgaaatgggcggcaagcccagacggaagaatcctcaagagggcctgtataatgagc
tgcagaaagacaagatggccgaggcctacagcgagatcggaatgaagggcgagcgcagaaga
ggcaagggacacgatggactgtaccagggcctgagcaccgccaccaaggatacctatgacgc
actgcacatgcaggccctgccacctaga
753
gaggtgcagctggtggagtccggaggaggcctggtgaagccaggaggctctctgaggctgag
anti-BMCA CAR
ctgcgcagcctccggcttcaccttttctgactactatatgagctggatcaggcaggcaccag
gcaagggcctggagtgggtgtcttacatcagctcctctggcagcacaatctactatgccgac
tccgtgaagggcaggttcaccatctctcgcgataacgccaagaatagcctgtatctgcagat
gaactccctgcgggccgaggatacagccgtgtactattgcgccaaggtggacggcccccctt
cctttgatatctggggccagggcacaatggtgaccgtgagctccggaggaggaggatccggc
ggaggaggctctggcggcggcggctctagctatgtgctgacccagccaccatccgtgtctgt
ggcacctggacagacagcaaggatcacctgtggagcaaacaatatcggcagcaagtccgtgc
actggtaccagcagaagcctggccaggccccaatgctggtggtgtatgacgatgacgatcgg
cccagcggcatccctgagagattttctggcagcaactccggcaataccgccacactgaccat
ctctggagtggaggcaggcgacgaggcagattacttctgtcacctgtgggaccggagcagag
atcactacgtgttcggcacaggcaccaagctgaccgtgctggaatctaagtacggaccgcct
tgtcctccttgtcccgctcctcctgttgccggaccttccgtgttcctgtttcctccaaagcc
taaggacaccctgatgatcagcaggacccctgaagtgacctgcgtggtggtggatgtgtccc
aagaggatcccgaggtgcagttcaactggtatgtggacggcgtggaagtgcacaacgccaag
accaagcctagagaggaacagttccagagcacctacagagtggtgtccgtgctgacagtgct
gcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaagggcctgccta
gcagcatcgagaaaaccatctccaaggccaagggccagccaagagagccccaggtttacaca
ctgcctccaagccaagaggaaatgaccaagaatcaggtgtccctgacatgcctggtcaaggg
cttctacccctccgatatcgccgtggaatgggagagcaatggccagcctgagaacaactaca
agaccacacctcctgtgctggacagcgacggcagtttcttcctgtatagtagactcaccgtg
gataaatcaagatggcaagagggcaacgtgttcagctgcagcgtgatgcacgaggccctgca
caaccactacacccagaaaagcctgagcctgtctctgggcaagatgttctgggtgctcgtgg
tcgttggcggagtgctggcctgttacagcctgctggttaccgtggccttcatcatcttttgg
gtcaagcggggcagaaagaagctgctctacatcttcaagcagcccttcatgcggcccgtgca
gaccacacaagaggaagatggctgctcctgcagattccccgaggaagaagaaggcggctgcg
agctgagagtgaagttcagcagatccgccgacgctccagcctatcagcagggccaaaaccag
ctgtacaacgagctgaacctggggagaagagaagagtacgacgtgctggataagcggagagg
cagagatcctgaaatgggcggcaagcccagacggaagaatcctcaagagggcctgtataatg
agctgcagaaagacaagatggccgaggcctacagcgagatcggaatgaagggcgagcgcaga
agaggcaagggacacgatggactgtaccagggcctgagcaccgccaccaaggatacctatga
cgcactgcacatgcaggccctgccacctaga
754
agctatgagctgacacagcctccaagcgcctctggcacacctggacagcgagtgacaatgag
anti-BMCA CAR
ctgtagcggcaccagcagcaacatcggcagccacagcgtgaactggtatcagcagctgcctg
gcacagcccctaaactgctgatctacaccaacaaccagcggcctagcggcgtgcccgataga
ttttctggcagcaagagcggcacaagcgccagcctggctatttctggactgcagagcgagga
cgaggccgactattattgtgccgcctgggacggctctctgaacggccttgtttttggcggag
gcaccaagctgacagtgctgggatctagaggtggcggaggatctggcggcggaggaagcgga
ggcggcggatctcttgaaatggctgaagtgcagctggtgcagtctggcgccgaagtgaagaa
gcctggcgagagcctgaagatcagctgcaaaggcagcggctacagcttcaccagctactgga
tcggctgggtccgacagatgcctggcaaaggccttgagtggatgggcatcatctaccccggc
gacagcgacaccagatacagccctagctttcagggccacgtgaccatcagcgccgacaagtc
tatcagcaccgcctacctgcagtggtccagcctgaaggcctctgacaccgccatgtactact
gcgccagatactctggcagcttcgacaattggggccagggcacactggtcaccgtgtccagc
gagtctaaatacggaccgccttgtcctccttgtcccgctcctcctgttgccggaccttccgt
gttcctgtttcctccaaagcctaaggacaccctgatgatcagcaggacccctgaagtgacct
gcgtggtggtggatgtgtcccaagaggatcccgaggtgcagttcaactggtatgtggacggc
gtggaagtgcacaacgccaagaccaagcctagagaggaacagttccagagcacctacagagt
ggtgtccgtgctgacagtgctgcaccaggattggctgaacggcaaagagtacaagtgcaagg
tgtccaacaagggcctgcctagcagcatcgagaaaaccatctccaaggccaagggccagcca
agagagccccaggtttacacactgcctccaagccaagaggaaatgaccaagaatcaggtgtc
cctgacatgcctggtcaagggcttctacccctccgatatcgccgtggaatgggagagcaatg
gccagcctgagaacaactacaagaccacacctcctgtgctggacagcgacggcagtttcttc
ctgtatagtagactcaccgtggataaatcaagatggcaagagggcaacgtgttcagctgcag
cgtgatgcacgaggccctgcacaaccactacacccagaaaagcctgagcctgtctctgggca
agatgttctgggtgctcgtggtcgttggcggagtgctggcctgttacagcctgctggttacc
gtggccttcatcatcttttgggtcaagcggggcagaaagaagctgctctacatcttcaagca
gcccttcatgcggcccgtgcagaccacacaagaggaagatggctgctcctgcagattccccg
aggaagaagaaggcggctgcgagctgagagtgaagttcagcagatccgccgacgctccagcc
tatcagcagggccaaaaccagctgtacaacgagctgaacctggggagaagagaagagtacga
cgtgctggataagcggagaggcagagatcctgaaatgggcggcaagcccagacggaagaatc
ctcaagagggcctgtataatgagctgcagaaagacaagatggccgaggcctacagcgagatc
ggaatgaagggcgagcgcagaagaggcaagggacacgatggactgtaccagggcctgagcac
cgccaccaaggatacctatgacgcactgcacatgcaggccctgccacctaga
755
cagtctgccctgacacagcctgccagcgttagtgctagtcccggacagtctatcgccatcag
anti-BMCA CAR
ctgtaccggcaccagctctgacgttggctggtatcagcagcaccctggcaaggcccctaagc
tgatgatctacgaggacagcaagaggcccagcggcgtgtccaatagattcagcggcagcaag
agcggcaacaccgccagcctgacaattagcggactgcaggccgaggacgaggccgattacta
ctgcagcagcaacacccggtccagcacactggtttttggcggaggcaccaagctgacagtgc
tgggatctagaggtggcggaggatctggcggcggaggaagcggaggcggcggatctcttgaa
atggctgaagtgcagctggtgcagtctggcgccgagatgaagaaacctggcgcctctctgaa
gctgagctgcaaggccagcggctacaccttcatcgactactacgtgtactggatgcggcagg
cccctggacagggactcgaatctatgggctggatcaaccccaatagcggcggcaccaattac
gcccagaaattccagggcagagtgaccatgaccagagacaccagcatcagcaccgcctacat
ggaactgagccggctgagatccgacgacaccgccatgtactactgcgccagatctcagcgcg
acggctacatggattattggggccagggaaccctggtcaccgtgtccagcgagtctaaatac
ggaccgccttgtcctccttgtcccgctcctcctgttgccggaccttccgtgttcctgtttcc
tccaaagcctaaggacaccctgatgatcagcaggacccctgaagtgacctgcgtggtggtgg
atgtgtcccaagaggatcccgaggtgcagttcaactggtatgtggacggcgtggaagtgcac
aacgccaagaccaagcctagagaggaacagttccagagcacctacagagtggtgtccgtgct
gacagtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaagg
gcctgcctagcagcatcgagaaaaccatctccaaggccaagggccagccaagagagccccag
gtttacacactgcctccaagccaagaggaaatgaccaagaatcaggtgtccctgacatgcct
ggtcaagggcttctacccctccgatatcgccgtggaatgggagagcaatggccagcctgaga
acaactacaagaccacacctcctgtgctggacagcgacggcagtttcttcctgtatagtaga
ctcaccgtggataaatcaagatggcaagagggcaacgtgttcagctgcagcgtgatgcacga
ggccctgcacaaccactacacccagaaaagcctgagcctgtctctgggcaagatgttctggg
tgctcgtggtcgttggcggagtgctggcctgttacagcctgctggttaccgtggccttcatc
atcttttgggtcaagcggggcagaaagaagctgctctacatcttcaagcagcccttcatgcg
gcccgtgcagaccacacaagaggaagatggctgctcctgcagattccccgaggaagaagaag
gcggctgcgagctgagagtgaagttcagcagatccgccgacgctccagcctatcagcagggc
caaaaccagctgtacaacgagctgaacctggggagaagagaagagtacgacgtgctggataa
gcggagaggcagagatcctgaaatgggcggcaagcccagacggaagaatcctcaagagggcc
tgtataatgagctgcagaaagacaagatggccgaggcctacagcgagatcggaatgaagggc
gagcgcagaagaggcaagggacacgatggactgtaccagggcctgagcaccgccaccaagga
tacctatgacgcactgcacatgcaggccctgccacctaga
756
cagtctgccctgacacagcctgccagcgttagtgctagtcccggacagtctatcgccatcag
anti-BMCA CAR
ctgtaccggcaccagctctgacgttggctggtatcagcagcaccctggcaaggcccctaagc
tgatgatctacgaggacagcaagaggcccagcggcgtgtccaatagattcagcggcagcaag
agcggcaacaccgccagcctgacaattagcggactgcaggccgaggacgaggccgattacta
ctgcagcagcaacacccggtccagcacactggtttttggcggaggcaccaagctgacagtgc
tgggatctagaggtggcggaggatctggcggcggaggaagcggaggcggcggatctcttgaa
atggctgaagtgcagctggtgcagtctggcgccgagatgaagaaacctggcgcctctctgaa
gctgagctgcaaggccagcggctacaccttcatcgactactacgtgtactggatgcggcagg
cccctggacagggactcgaatctatgggctggatcaaccccaatagcggcggcaccaattac
gcccagaaattccagggcagagtgaccatgaccagagacaccagcatcagcaccgcctacat
ggaactgagccggctgagatccgacgacaccgccatgtactactgcgccagatctcagcgcg
acggctacatggattattggggccagggaaccctggtcaccgtgtccagcgagtctaaatac
ggaccgccttgtcctccttgtcccgctcctcctgttgccggaccttccgtgttcctgtttcc
tccaaagcctaaggacaccctgatgatcagcaggacccctgaagtgacctgcgtggtggtgg
atgtgtcccaagaggatcccgaggtgcagttcaactggtatgtggacggcgtggaagtgcac
aacgccaagaccaagcctagagaggaacagttccagagcacctacagagtggtgtccgtgct
gacagtgctgcaccaggattggctgaacggcaaagagtacaagtgcaaggtgtccaacaagg
gcctgcctagcagcatcgagaaaaccatctccaaggccaagggccagccaagagagccccag
gtttacacactgcctccaagccaagaggaaatgaccaagaatcaggtgtccctgacatgcct
ggtcaagggcttctacccctccgatatcgccgtggaatgggagagcaatggccagcctgaga
acaactacaagaccacacctcctgtgctggacagcgacggcagtttcttcctgtatagtaga
ctcaccgtggataaatcaagatggcaagagggcaacgtgttcagctgcagcgtgatgcacga
ggccctgcacaaccactacacccagaaaagcctgagcctgtctctgggcaagatgttctggg
tgctcgtggtcgttggcggagtgctggcctgttacagcctgctggttaccgtggccttcatc
atcttttgggtcaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactcc
ccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcag
cctatcgctccagagtgaagttcagcagatccgccgacgctccagcctatcagcagggccaa
aaccagctgtacaacgagctgaacctggggagaagagaagagtacgacgtgctggataagcg
gagaggcagagatcctgaaatgggcggcaagcccagacggaagaatcctcaagagggcctgt
ataatgagctgcagaaagacaagatggccgaggcctacagcgagatcggaatgaagggcgag
cgcagaagaggcaagggacacgatggactgtaccagggcctgagcaccgccaccaaggatac
ctatgacgcactgcacatgcaggccctgccacctaga
757
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
anti-BMCA CAR
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGDYTEDYWGQGTLVTVSSGGGGSG
GGGSGGGGSQSALTQPASVSGSPGQSITISCTGSSSDVGKYNLVSWYQQPPGKAPKLIIYDV
NKRPSGVSNRFSGSKSGNTATLTISGLQGDDEADYYCSSYGGSRSYVFGTGTKVTVLESKYG
PPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN
AKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV
YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL
TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFII
FWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQ
NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE
RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
758
EVQLVQSGGGLVQPGRSLRLSCTASGFTFGDYAMSWFKQAPGKGLEWVGFIRSKAYGGTTEY
anti-BMCA CAR
AASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCAAWSAPTDYWGQGTLVTVSSGGGGSG
GGGSGGGGSDIQMTQSPAFLSASVGDRVTVTCRASQGISNYLAWYQQKPGNAPRLLIYSAST
LQSGVPSRFRGTGYGTEFSLTIDSLQPEDFATYYCQQSYTSRQTFGPGTRLDIKESKYGPPC
PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT
KPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL
PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD
KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFIIFWV
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQL
YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR
GKGHDGLYQGLSTATKDTYDALHMQALPPR
759
EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYAD
anti-BMCA CAR
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVDGPPSFDIWGQGTMVTVSSGGGGSG
GGGSGGGGSSYVLTQPPSVSVAPGQTARITCGANNIGSKSVHWYQQKPGQAPMLVVYDDDDR
PSGIPERFSGSNSGNTATLTISGVEAGDEADYFCHLWDRSRDHYVFGTGTKLTVLESKYGPP
CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK
TKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT
LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV
DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFIIFW
VKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQ
LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR
RGKGHDGLYQGLSTATKDTYDALHMQALPPR
760
SYELTQPPSASGTPGQRVTMSCSGTSSNIGSHSVNWYQQLPGTAPKLLIYTNNQRPSGVPDR
anti-BMCA CAR
FSGSKSGTSASLAISGLQSEDEADYYCAAWDGSLNGLVFGGGTKLTVLGSRGGGGSGGGGSG
GGGSLEMAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPG
DSDTRYSPSFQGHVTISADKSISSTAYLQWSSLKASDTAMYYCARYSGSFDNWGQGTLVTVS
ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG
VEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVT
VAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA
YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI
GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
761
QSALTQPASVSASPGQSIAISCTGTSSDVGWYQQHPGKAPKLMIYEDSKRPSGVSNRFSGSK
anti-BMCA CAR
SGNTASLTISGLQAEDEADYYCSSNTRSSTLVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLE
MAEVQLVQSGAEMKKPGASLKLSCKASGYTFIDYYVYWMRQAPGQGLESMGWINPNSGGTNY
AQKFQGRVTMTRDTSISTAYMELSRLRSDDTAMYYCARSQRDGYMDYWGQGTLVTVSSESKY
GPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH
NAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ
VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR
LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFI
IFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQG
QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG
ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
762
QSALTQPASVSASPGQSIAISCTGTSSDVGWYQQHPGKAPKLMIYEDSKRPSGVSNRFSGSK
anti-BMCA CAR
SGNTASLTISGLQAEDEADYYCSSNTRSSTLVFGGGTKLTVLGSRGGGGSGGGGSGGGGSLE
MAEVQLVQSGAEMKKPGASLKLSCKASGYTFIDYYVYWMRQAPGQGLESMGWINPNSGGTNY
AQKFQGRVTMTRDTSISTAYMELSRLRSDDTAMYYCARSQRDGYMDYWGQGTLVTVSSESKY
GPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH
NAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ
VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR
LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFI
IFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQ
NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE
RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
763
ctttttcgcaacgggtttgc
EF1a/HTLV promoter
forward primer
764
gatatcgaattcctgcagcc
Reverse primer just 5′ of
WPRE
765
cgccttgtcctccttgtccagctcctcctgttgccggacct
predicted splice acceptor
site
766
cgccttgtcctccttgtcccgctcctcctgttgccggacct
optimized splice acceptor
site
767
cagtttcttcctgtatagtagactcaccgtggataaatcaa
predicted splice acceptor
site
768
accggattcctcctgattcaggcctggccagagaacagaac
predicted splice acceptor
site
769
LPVLTQPPSTSGTPGQRVTVSCSGSSSNIGSNVVFWYQQLPGTAPKLVIYRNNQRPSGVPDR
anti-BMCA scFv
FSVSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGYVFGTGTKVTVLGSRGGGGSGGGGSG
GGGSLEMAEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPI
LGIANYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARSGYSKSIVSYMDYWGQGT
LVTVSS
770
QAVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVFWYQQLPGTAPKLLIYSNNQRPSGVPDR
anti-BCMA scFV
FSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSASYVFGTGTKVTVLGSRGGGGSGGGGS
GGGGSLEMAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIP
ILGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSGYGSYRWEDSWGQGTL
VTVSS
771
QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGFDVHWYQQLPGTAPKLLIYGNSNRPSGVPD
anti-BCMA scFV
RFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGYVFGTGTKVTVLGSRGGGGSGGGGS
GGGGSLEMAQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQRLEWMGWINP
NSGGTNYAQKFQDRITVTRDTSSNTGYMELTRLRSDDTAVYYCARSPYSGVLDKWGQGTLVT
VSS
772
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQ
anti-BMCA VH
KFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCARSGYSKSIVSYMDYWGQGTLVTVSS
773
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGTANYAQ
anti-BMCA VH
KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSGYGSYRWEDSWGQGTLVTVSS
774
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQRLEWMGWINPNSGGTNYAQ
anti-BMCA VH
KFQDRITVTRDTSSNTGYMELTRLRSDDTAVYYCARSPYSGVLDKWGQGTLVTVSS
775
LPVLTQPPSTSGTPGQRVTVSCSGSSSNIGSNVVFWYQQLPGTAPKLVIYRNNQRPSGVPDR
anti-BCMA VL
FSVSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGYVFGTGTKVTVLG
776
QAVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVFWYQQLPGTAPKLLIYSNNQRPSGVPDR
anti-BMCA VL
FSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSASYVFGTGTKVTVLG
777
QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGFDVHWYQQLPGTAPKLLIYGNSNRPSGVPD
anti-BMCA VL
RFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGYVFGTGTKVTVLG
778
SRGGGGSGGGGSGGGGSLEMA
linker
779
YFDSL
BCMA epitope
780
QNEYFDSLL
BMCA epitope
781
LPVLTQPPSASGTPGQRVTISCSGRSSNIGSNSVNWYRQLPGAAPKLLIYSNNQRPPGVPVR
anti-BCMA scFv
FSGSKSGTSASLAISGLQSEDEATYYCATWDDNLNVHYVFGTGTKVTVLGSRGGGGSGGGGS
GGGGSLEMAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIP
ILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYYSHDMWSEDWGQGT
LVTVSS
782
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYAD
anti-BCMA scFv
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAEMGAVFDIWGQGTMVTVSSGSTSGS
GKPGSGEGSTKGEIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYD
ASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRISWPFTFGGGTKVEIK
783
EIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPARF
anti-BCMA scFv
SGSGSGTDFTLTISSLEPEDFAVYYCQQRISWPFTFGGGTKVEIKRGSTSGSGKPGSGEGST
KGEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYY
ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAEMGAVFDIWGQGTMVTVSS
784
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
anti-BCMA scFv
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGTYLGGLWYFDLWGRGTLVTVSSGS
TSGSGKPGSGEGSTKGDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPG
QSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLGLPLTFGGGTK
VEIK
785
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASG
anti-BCMA scFv
VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLGLPLTFGGGTKVEIKRGSTSGSGKPGS
GEGSTKGQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDG
SNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGTYLGGLWYFDLWGRGTL
VTVSS
786
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPGGGSTSYAQ
anti-BCMA scFv
KFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARESWPMDVWGQGTTVTVSSGSTSGSGK
PGSGEGSTKGEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGAS
TRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYAAYPTFGGGTKVEIK
787
EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARF
anti-BCMA scFv
SGSGSGTEFTLTISSLQSEDFAVYYCQQYAAYPTFGGGTKVEIKRGSTSGSGKPGSGEGSTK
GQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPGGGSTSYA
QKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARESWPMDVWGQGTTVTVSS
788
QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSISYSGSTYYN
anti-BCMA scFv
PSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRGYATSLAFDIWGQGTMVTVSSGS
TSGSGKPGSGEGSTKGEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL
LIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRHVWPPTFGGGTKVEIK
789
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARF
anti-BCMA scFv
SGSGSGTDFTLTISSLEPEDFAVYYCQQRHVWPPTFGGGTKVEIKRGSTSGSGKPGSGEGST
KGQLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSISYSGSTY
YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRGYATSLAFDIWGQGTMVTVSS
790
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSTISSSSSTIYYAD
anti-BCMA scFv
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGSQEHLIFDYWGQGTLVTVSSGSTSG
SGKPGSGEGSTKGEIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIY
DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRFYYPWTFGGGTKVEIK
791
EIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPARF
anti-BCMA scFv
SGSGSGTDFTLTISSLEPEDFAVYYCQQRFYYPWTFGGGTKVEIKRGSTSGSGKPGSGEGST
KGEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSTISSSSSTIYY
ADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGSQEHLIFDYWGQGTLVTVSS
792
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
anti-BCMA scFv
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTDFWSGSPPGLDYWGQGTLVTVSSGS
TSGSGKPGSGEGSTKGDIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKL
LIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQIYTFPFTFGGGTKVEIK
793
DIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSLQSGVPSRF
anti-BCMA scFv
SGSGSGTDFTLTISSLQPEDFATYYCQQIYTFPFTFGGGTKVEIKRGSTSGSGKPGSGEGST
KGQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTDFWSGSPPGLDYWGQGTLVTVSS
794
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQ
anti-BCMA scFv
KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARTPEYSSSIWHYYYGMDVWGQGTTVTV
SSGSTSGSGKPGSGEGSTKGDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWY
QQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFAHTPFTF
GGGTKVEIK
795
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRES
anti-BCMA scFv
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFAHTPFTFGGGTKVEIKRGSTSGSGKPG
SGEGSTKGQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPI
FGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARTPEYSSSIWHYYYGMDVW
GQGTTVTVSS
796
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
anti-BCMA scFv
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGPLQEPPYDYGMDVWGQGTTVTVSSG
STSGSGKPGSGEGSTKGEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPR
LLIYSASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQHHVWPLTFGGGTKVEIK
797
EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYSASTRATGIPARF
anti-BCMA scFv
SGSGSGTEFTLTISSLQSEDFAVYYCQQHHVWPLTFGGGTKVEIKRGSTSGSGKPGSGEGST
KGQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYY
ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGPLQEPPYDYGMDVWGQGTTVTVS
S
798
DIVLTQSPASLAVSLGERATINCRASESVSVIGAHLIHWYQQKPGQPPKLLIYLASNLETGV
anti-BCMA scFv
PARFSGSGSGTDFTLTISSLQAEDAAIYYCLQSRIFPRTFGQGTKLEIKGSTSGSGKPGSGE
GSTKGQVQLVQSGSELKKPGASVKVSCKASGYTFTDYSINWVRQAPGQGLEWMGWINTETRE
PAYAYDFRGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDYSYAMDYWGQGTLVTVSS
799
DIVLTQSPASLAVSLGERATINCRASESVSVIGAHLIHWYQQKPGQPPKLLIYLASNLETGV
anti-BCMA scFv
PARFSGSGSGTDFTLTISSLQAEDAAIYYCLQSRIFPRTFGQGTKLEIKGSTSGSGKPGSGE
GSTKGQVQLVQSGSELKKPGASVKVSCKASGYTFTDYSINWVRQAPGQGLEWMGWINTETRE
PAYAYDFRGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDYSYAMDYWGQGTLVTVSS
800
DIVLTQSPASLAVSLGERATINCRASESVSVIGAHLIHWYQQKPGQPPKLLIYLASNLETGV
anti-BCMA scFv
PARFSGSGSGTDFTLTISSLQAEDAAIYYCLQSRIFPRTFGQGTKLEIKGSTSGSGKPGSGE
GSTKGQVQLVQSGSELKKPGASVKVSCKASGYTFTDYSINWVRQAPGQGLEWMGWINTETRE
PAYAYDFRGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDYSYAMDYWGQGTLVTVSS
801
EVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWVSGIVYSGSTYYAAS
anti-BCMA scFv
VKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCSAHGGESDVWGQGTTVTVSSASGGGGSGG
RASGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSL
QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIK
802
QVQLVESGGGLVQPGRSLRLSCAASGFTFSNYAMSWVRQAPGKGLGWVSGISRSGENTYYAD
anti-BCMA scFv
SVKGRFTISRDNSKNTLYLQMNSLRDEDTAVYYCARSPAHYYGGMDVWGQGTTVTVSSASGG
GGSGGRASGGGGSDIVLTQSPGTLSLSPGERATLSCRASQSISSSFLAWYQQKPGQAPRLLI
YGASRRATGIPDRFSGSGSGTDFTLTISRLEPEDSAVYYCQQYHSSPSWTFGQGTKLEIK
803
QVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWVSGIVYSGSTYYAAS
anti-BCMA scFv
VKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCSAHGGESDVWGQGTTVTVSSASGGGGSGG
RASGGGGSDIRLTQSPSPLSASVGDRVTITCQASEDINKFLNWYHQTPGKAPKLLIYDASTL
QTGVPSRFSGSGSGTDFTLTINSLQPEDIGTYYCQQYESLPLTFGGGTKVEIK
804
EVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWVSGIVYSGSTYYAAS
anti-BCMA scFv
VKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCSAHGGESDVWGQGTTVTVSSASGGGGSGG
RASGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSIGSSSLAWYQQKPGQAPRLLMYGASS
RASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYAGSPPFTFGQGTKVEIK
805
QIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAY
anti-BCMA scFv
DFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSSGGGGSGG
GGSGGGGSDIVLTQSPASLAMSLGKRATISCRASESVSVIGAHLIHWYQQKPGQPPKLLIYL
ASNLETGVPARFSGSGSGTDFTLTIDPVEEDDVAIYSCLQSRIFPRTFGGGTKLEIK
806
QIQLVQSGPELKKPGETVKISCKASGYTFRHYSMNWVKQAPGKGLKWMGRINTESGVPIYAD
anti-BCMA scFv
DFKGRFAFSVETSASTAYLVINNLKDEDTASYFCSNDYLYSLDFWGQGTALTVSSGGGGSGG
GGSGGGGSDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIYWYQQKPGQPPTLLIQL
ASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIK
807
QIQLVQSGPELKKPGETVKISCKASGYTFTHYSMNWVKQAPGKGLKWMGRINTETGEPLYAD
anti-BCMA scFv
DFKGRFAFSLETSASTAYLVINNLKNEDTATFFCSNDYLYSCDYWGQGTTLTVSSGGGGSGG
GGSGGGGSDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIYWYQQKPGQPPTLLIQL
ASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIK
808
QVQLVQSGAEVKKPGASVKVSCKASGYSFPDYYINWVRQAPGQGLEWMGWIYFASGNSEYNQ
anti-BCMA scFv
KFTGRVTMTRDTSINTAYMELSSLTSEDTAVYFCASLYDYDWYFDVWGQGTMVTVSSGGGGS
GGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCKSSQSLVHSNGNTYLHWYLQKPGQSPQLL
IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCSQSSIYPWTFGQGTKLEIK
809
QVQLVQSGAEVKKPGASVKVSCKASGYSFPDYYINWVRQAPGQGLEWMGWIYFASGNSEYNQ
anti-BCMA scFv
KFTGRVTMTRDTSINTAYMELSSLTSEDTAVYFCASLYDYDWYFDVWGQGTMVTVSSGGGGS
GGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCKSSQSLVHSNGNTYLHWYLQKPGQSPQLL
IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCSQSSIYPWTFGQGTKLEIK
810
QVQLVQSGAEVKKPGASVKVSCKASGYSFPDYYINWVRQAPGQGLEWMGWIYFASGNSEYNQ
anti-BCMA scFv
KFTGRVTMTRDTSINTAYMELSSLTSEDTAVYFCASLYDYDWYFDVWGQGTMVTVSSGGGGS
GGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCKSSQSLVHSNGNTYLHWYLQKPGQSPQLL
IYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCSQSSIYPWTFGQGTKLEIK
811
QVQLVQSGAEVKKPGASVKVSCKASGYSFPDYYINWVRQAPGQGLEWMGWIYFASGNSEYNQ
anti-BCMA scFv
KFTGRVTMTRDTSSSTAYMELSSLRSEDTAVYFCASLYDYDWYFDVWGQGTMVTVSSGGGGS
GGGGSGGGGSDIVMTQTPLSLSVTPGEPASISCKSSQSLVHSNGNTYLHWYLQKPGQSPQLL
IYKVSNRFSGVPDRFSGSGSGADFTLKISRVEAEDVGVYYCAETSHVPWTFGQGTKLEIK
812
QVQLVQSGAEVKKPGASVKVSCKASGYSFPDYYINWVRQAPGQGLEWMGWIYFASGNSEYNQ
anti-BCMA scFv
KFTGRVTMTRDTSSSTAYMELSSLRSEDTAVYFCASLYDYDWYFDVWGQGTMVTVSSGGGGS
GGGGSGGGGSDIVMTQTPLSLSVTPGEPASISCKSSQSLVHSNGNTYLHWYLQKPGQSPQLL
IYKVSNRFSGVPDRFSGSGSGADFTLKISRVEAEDVGVYYCAETSHVPWTFGQGTKLEIK
813
QVQLVQSGAEVKKPGASVKVSCKASGYSFPDYYINWVRQAPGQGLEWMGWIYFASGNSEYNQ
anti-BCMA scFv
KFTGRVTMTRDTSSSTAYMELSSLRSEDTAVYFCASLYDYDWYFDVWGQGTMVTVSSGGGGS
GGGGSGGGGSDIVMTQTPLSLSVTPGEPASISCKSSQSLVHSNGNTYLHWYLQKPGQSPQLL
IYKVSNRFSGVPDRFSGSGSGADFTLKISRVEAEDVGVYYCAETSHVPWTFGQGTKLEIK
814
QVQLVESGGGLVQPGGSLRLSCEASGFTLDYYAIGWFRQAPGKEREGVICISRSDGSTYYAD
anti-BCMA VH
SVKGRFTISRDNAKKTVYLQMISLKPEDTAAYYCAAGADCSGYLRDYEFRGQGTQVTVSS
815
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQ
anti-BMCA VH
KFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGGYYSHDMWSEDWGQGTLVTVSS
816
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYAD
anti-BMCA VH
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAEMGAVFDIWGQGTMVTVSS
817
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
anti-BMCA VH
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGTYLGGLWYFDLWGRGTLVTVSS
818
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPGGGSTSYAQ
anti-BMCA VH
KFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARESWPMDVWGQGTTVTVSS
819
QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSISYSGSTYYN
anti-BMCA VH
PSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRGYATSLAFDIWGQGTMVTVSS
820
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSTISSSSSTIYYAD
anti-BMCA VH
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGSQEHLIFDYWGQGTLVTVSS
821
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
anti-BMCA VH
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTDFWSGSPPGLDYWGQGTLVTVSS
822
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQ
anti-BMCA VH
KFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARTPEYSSSIWHYYYGMDVWGQGTTVTV
SS
823
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYAD
anti-BMCA VH
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGPLQEPPYDYGMDVWGQGTTVTVSS
824
QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSINWVRQAPGQGLEWMGWINTETREPAYAY
anti-BMCA VH
DFRGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDYSYAMDYWGQGTLVTVSS
825
EVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWVSGIVYSGSTYYAAS
anti-BMCA VH
VKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCSAHGGESDVWGQGTTVTVSS
826
QVQLVESGGGLVQPGRSLRLSCAASGFTFSNYAMSWVRQAPGKGLGWVSGISRSGENTYYAD
anti-BMCA VH
SVKGRFTISRDNSKNTLYLQMNSLRDEDTAVYYCARSPAHYYGGMDVWGQGTTVTVSS
827
QVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWVSGIVYSGSTYYAAS
anti-BMCA VH
VKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCSAHGGESDVWGQGTTVTVSS
828
EVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWVSGIVYSGSTYYAAS
anti-BMCA VH
VKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCSAHGGESDVWGQGTTVTVSS
829
QIQLVQSGPELKKPGETVKISCKASGYTFRHYSMNWVKQAPGKGLKWMGRINTESGVPIYAD
anti-BMCA VH
DFKGRFAFSVETSASTAYLVINNLKDEDTASYFCSNDYLYSLDFWGQGTALTVSS
830
QIQLVQSGPELKKPGETVKISCKASGYTFTHYSMNWVKQAPGKGLKWMGRINTETGEPLYAD
anti-BMCA VH
DFKGRFAFSLETSASTAYLVINNLKNEDTATFFCSNDYLYSCDYWGQGTTLTVSS
831
QVQLVQSGAEVKKPGASVKVSCKASGYSFPDYYINWVRQAPGQGLEWMGWIYFASGNSEYNQ
anti-BMCA VH
KFTGRVTMTRDTSINTAYMELSSLTSEDTAVYFCASLYDYDWYFDVWGQGTMVTVSS
832
QVQLVQSGAEVKKPGASVKVSCKASGYSFPDYYINWVRQAPGQGLEWMGWIYFASGNSEYNQ
anti-BMCA VH
KFTGRVTMTRDTSSSTAYMELSSLRSEDTAVYFCASLYDYDWYFDVWGQGTMVTVSS
833
LPVLTQPPSASGTPGQRVTISCSGRSSNIGSNSVNWYRQLPGAAPKLLIYSNNQRPPGVPVR
anti-BCMA VL
FSGSKSGTSASLAISGLQSEDEATYYCATWDDNLNVHYVFGTGTKVTVLG
834
EIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPARF
anti-BCMA VL
SGSGSGTDFTLTISSLEPEDFAVYYCQQRISWPFTFGGGTKVEIK
835
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASG
anti-BCMA VL
VPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLGLPLTFGGGTKVEIK
836
EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARF
anti-BCMA VL
SGSGSGTEFTLTISSLQSEDFAVYYCQQYAAYPTFGGGTKVEIK
837
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARF
anti-BCMA VL
SGSGSGTDFTLTISSLEPEDFAVYYCQQRHVWPPTFGGGTKVEIK
838
EIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPARF
anti-BCMA VL
SGSGSGTDFTLTISSLEPEDFAVYYCQQRFYYPWTFGGGTKVEIK
839
DIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSLQSGVPSRF
anti-BCMA VL
SGSGSGTDFTLTISSLQPEDFATYYCQQIYTFPFTFGGGTKVEIK
840
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRES
anti-BCMA VL
GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFAHTPFTFGGGTKVEIK
841
EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYSASTRATGIPARF
anti-BCMA VL
SGSGSGTEFTLTISSLQSEDFAVYYCQQHHVWPLTFGGGTKVEIK
842
DIVLTQSPASLAVSLGERATINCRASESVSVIGAHLIHWYQQKPGQPPKLLIYLASNLETGV
anti-BCMA VL
PARFSGSGSGTDFTLTISSLQAEDAAIYYCLQSRIFPRTFGQGTKLEIK
843
DIQLTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF
anti-BCMA VL
SGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKVEIK
844
DIVLTQSPGTLSLSPGERATLSCRASQSISSSFLAWYQQKPGQAPRLLIYGASRRATGIPDR
anti-BCMA VL
FSGSGSGTDFTLTISRLEPEDSAVYYCQQYHSSPSWTFGQGTKLEIK
845
DIRLTQSPSPLSASVGDRVTITCQASEDINKFLNWYHQTPGKAPKLLIYDASTLQTGVPSRF
anti-BCMA VL
SGSGSGTDFTLTINSLQPEDIGTYYCQQYESLPLTFGGGTKVEIK
846
EIVLTQSPGTLSLSPGERATLSCRASQSIGSSSLAWYQQKPGQAPRLLMYGASSRASGIPDR
anti-BCMA VL
FSGSGSGTDFTLTISRLEPEDFAVYYCQQYAGSPPFTFGQGTKVEIK
847
DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIYWYQQKPGQPPTLLIQLASNVQTGV
anti-BCMA VL
PARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIK
848
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSG
anti-BCMA VL
VPDRFSGSGSGTDFTLKISRVEAEDVGIYYCSQSSIYPWTFGQGTKLEIK
849
DIVMTQTPLSLSVTPGEPASISCKSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSG
anti-BCMA VL
VPDRFSGSGSGADFTLKISRVEAEDVGVYYCAETSHVPWTFGQGTKLEIK
850
atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgat
GMCSFR alpha chain signal
ccca
sequence
851
MLLLVTSLLLCELPHPAFLLIP
GMCSFR alpha chain signal
peptide
852
MALPVTALLLPLALLLHA
CD8 alpha signal peptide
853
MPLLLLLPLLWAGALA
CD33 signal peptide
854
aagtttctttctgtattccagactgaccgtggataaatctc
Optimized splice acceptor
site
855
GAGTCTAAATACGGACCGCCTTGTCCTCCTTGTCCAGCTCCTCCTGTTGCCGGACCTTCCGT
Spacer - codon optimized
GTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGGACCCCTGAAGTGACCT
(nt)
GCGTGGTGGTGGATGTGTCCCAAGAGGATCCCGAGGTGCAGTTCAATTGGTACGTGGACGGC
GTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTTCCAGAGCACCTACAGAGT
GGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGG
TGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCA
AGAGAGCCCCAGGTTTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAATCAGGTGTC
CCTGACATGCCTGGTCAAGGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGAGCAATG
GCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCAGTTTCTTC
CTGTATAGTAGACTCACCGTGGATAAATCAAGATGGCAAGAGGGCAACGTGTTCAGCTGCAG
CGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAAAGCCTGAGCCTGTCTCTGGGCA
AA
856
gaatctaagtacggaccgccttgtcctccttgtcccgctcctcctgttgccggaccttccgt
Alternative CO/SSE spacer
gttcctgtttcctccaaagcctaaggacaccctgatgatcagcaggacccctgaagtgacct
(nt)
gcgtggtggtggatgtgtcccaagaggatcccgaggtgcagttcaactggtatgtggacggc
gtggaagtgcacaacgccaagaccaagcctagagaggaacagttccagagcacctacagagt
ggtgtccgtgctgacagtgctgcaccaggattggctgaacggcaaagagtacaagtgcaagg
tgtccaacaagggcctgcctagcagcatcgagaaaaccatctccaaggccaagggccagcca
agagagccccaggtttacacactgcctccaagccaagaggaaatgaccaagaatcaggtgtc
cctgacatgcctggtcaagggcttctacccctccgatatcgccgtggaatgggagagcaatg
gccagcctgagaacaactacaagaccacacctcctgtgctggacagcgacggcagtttcttc
ctgtatagtagactcaccgtggataaatcaagatggcaagagggcaacgtgttcagctgcag
cgtgatgcacgaggccctgcacaaccactacacccagaaaagcctgagcctgtctctgggca
ag
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16178571
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juno therapeutics, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Mar 30th, 2022 06:04PM
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Mar 30th, 2022 06:04PM
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Bristol-Myers Squibb
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Health Care
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Pharmaceuticals & Biotechnology
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nyse:bmy
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Juno Therapeutics
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Mar 8th, 2022 12:00AM
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Dec 3rd, 2015 12:00AM
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https://www.uspto.gov?id=US11266739-20220308
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Methods and compositions for adoptive cell therapy
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Provided are methods for multiple administrations of cells for adoptive cell therapy, and for administering cells to subjects having received prior administrations, and compositions and articles of manufacture for use in the methods. The cells generally express recombinant molecules such as recombinant receptors, e.g., chimeric antigen receptors (CARs) and/or other transgenic receptors. The methods can involve administering cells expressing a first or prior receptor(s) and cells expressing a second or subsequent receptor(s), the second or subsequent receptor(s) being distinct from the first, and which generally do not express the first receptor, and/or administering the cells expressing the second receptor to a subject having received the first administration. The methods can provide various advantages, such as improved efficacy in the context an immune response in the subject against the first or prior receptor and/or in the context of antigen loss, downregulation, or modification, following a first or prior administration.
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11266739
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1. A method of treatment, comprising:
(a) administering to a subject T cells expressing a first chimeric antigen receptor (CAR) that specifically binds to a first antigen associated with a disease or condition in the subject, wherein the disease or condition comprises a B cell acute lymphoblastic leukemia (B-ALL) and the first antigen is CD19; and
(b) subsequently administering, to a subject in which the disease or condition has relapsed following the administration of the first CAR in (a), T cells expressing a second CAR that specifically binds to a second antigen that is CD22, wherein:
at the time of, or immediately prior to, the administration of T cells expressing the second CAR, the subject is in the relapse following the administration of T cells expressing the first CAR; and
the T cells expressing the second CAR do not express the first CAR, and the second CAR comprises at least one region identical in amino acid sequence to a corresponding region of the first CAR.
2. A method of treatment, comprising administering T cells expressing a second chimeric antigen receptor (CAR) to a subject that has previously received an administration of T cells expressing a first chimeric antigen receptor (CAR) and has relapsed following the previous administration of the first CAR, wherein:
said T cells expressing the second CAR do not express the first CAR;
said first CAR specifically binds to a first antigen associated with a disease or condition in the subject, wherein the disease or condition comprises a B cell acute lymphoblastic leukemia (B-ALL) and the first antigen is CD19;
said second CAR specifically binds to a second antigen that is CD22, and said second CAR comprises at least one region identical in amino acid sequence to a corresponding region of said first CAR; and
at the time of, or immediately prior to, the administration of T cells expressing the second CAR, the subject is in the relapse following the administration of T cells expressing the first CAR.
3. The method of claim 1, wherein:
the time between the initiation of administration of T cells expressing the first CAR and the initiation of administration of T cells expressing the second CAR is at least about 28 days, at least about 35 days, at least about 42 days, at least about 49 days, or at least about 60 days.
4. The method of claim 2, wherein:
the time between initiation of the administration of T cells expressing the first CAR and initiation of the administration of T cells expressing the second CAR is at least about 28 days, at least about 35 days, at least about 42 days, at least about 49 days, or at least about 60 days.
5. The method of claim 2, wherein said subject has not received a dose of T cells expressing the second CAR prior to the administration of cells expressing the second CAR.
6. The method of claim 2, wherein:
the administration of the T cells expressing the second CAR comprises administration of the cells in an amount sufficient for reduction in burden of the disease or condition in the subject; or
the administration of the T cells expressing the second CAR effects a reduction in burden of the disease or condition in the subject, thereby treating the disease or condition.
7. The method of claim 2, wherein:
the administration of the T cells expressing the first CAR comprises administration of the cells in an amount sufficient for reduction in burden of the disease or condition in the subject; or
the administration of the T cells expressing the first CAR and/or the administration of the cells expressing the second CAR effects a reduction in burden of the disease or condition in the subject, thereby treating the disease or condition.
8. The method of claim 2, wherein the T cells expressing the second CAR are autologous to the subject.
9. The method of claim 2, wherein the administration of the T cells expressing the first CAR and/or the administration of the T cells expressing the second CAR independently comprises administration of from or from about 1×106 to about 1×108 of the CAR-expressing T cells.
10. The method of claim 2, wherein administration of the T cells expressing the second CAR comprise administration of from or from about 1×106 to about 1×108 of the CAR-expressing T cells.
11. The method of claim 2, wherein the T cells expressing the first CAR are autologous to the subject.
12. The method of claim 1, wherein the T cells expressing the first CAR and the T cells expressing the second CAR are autologous to the subject.
13. The method of 1, wherein the administration of the T cells expressing the first CAR and/or the administration of the T cells expressing the second CAR independently comprises administration of from or from about 1×106 to about 1×108 of the CAR-expressing T cells.
14. The method of claim 1, wherein administration of the T cells expressing the second CAR comprise administration of from or from about 1×106 to about 1×108 of the CAR-expressing T cells.
15. The method of claim 2, wherein said subject has not received a dose of T cells expressing a CAR that binds to the second antigen prior to the administration of T cells expressing the second CAR.
16. A method of treatment, comprising:
(a) administering, to a subject having a disease or condition that is a B cell malignancy, T cells expressing a first chimeric antigen receptor (CAR) that specifically binds to a first antigen that is CD19; and
(b) subsequently administering, to a subject in which the disease or condition has relapsed following the administration of the first CAR in (a), T cells expressing a second CAR that specifically binds to a second antigen that is CD22, wherein the T cells expressing the second CAR do not express the first CAR, and the second CAR comprises at least one region identical in amino acid sequence to a corresponding region of the first CAR, wherein:
at the time of, or immediately prior to, initiation of the administration of T cells expressing the second CAR, the subject is in the relapse following the administration of T cells expressing the first CAR.
17. The method of claim 16, wherein the disease or condition is a leukemia or a lymphoma.
18. The method of claim 16, wherein the T cells expressing the first CAR and the T cells expressing the second CAR are autologous to the subject.
19. The method of claim 16, wherein the administration of the T cells expressing the first CAR and/or the administration of the cells expressing the second CAR independently comprises administration of from or from about 1×106 to about 1×108 of the CAR-expressing T cells.
20. The method of claim 16, wherein the time between the initiation of administration of the T cells expressing the first CAR and the initiation of administration of the T cells expressing the second CAR is at least about 28 days, at least about 35 days, at least about 42 days, at least about 49 days, or at least about 60 days.
21. A method of treatment, comprising administering, to a subject having a disease or condition that is a B cell malignancy, T cells expressing a second chimeric antigen receptor (CAR), wherein:
the subject has previously received an administration of T cells expressing a first chimeric antigen receptor (CAR) that specifically binds to a first antigen that is CD19 and has relapsed following the previous administration of the first CAR;
the second CAR specifically binds to a second antigen that is CD22, wherein the T cells expressing the second CAR do not express the first CAR, and the second CAR comprises at least one region identical in amino acid sequence to a corresponding region of the first CAR; and
at the time of, or immediately prior to, initiation of the administration of T cells expressing the second CAR, the subject is in the relapse following the administration of T cells expressing the first CAR.
22. The method of claim 21, wherein the T cells expressing the second CAR are autologous to the subject.
23. The method of claim 21, wherein the administration of the T cells expressing the first CAR and/or the administration of the T cells expressing the second CAR independently comprise from or from about 1×106 to about 1×108 of the CAR-expressing T cells.
24. The method of claim 21, wherein the time between the initiation of administration of T cells expressing the first CAR and the initiation of administration of T cells expressing the second CAR is at least about 28 days, at least about 35 days, at least about 42 days, at least about 49 days, or at least about 60 days.
25. The method of claim 21, wherein said subject has not received a dose of T cells expressing the second CAR prior to the administration of T cells expressing the second CAR.
26. The method of claim 21, wherein said subject has not received a dose of cells expressing a CAR that binds to the second antigen prior to the administration of cells expressing the second CAR.
27. The method of claim 16, wherein administration of the T cells expressing the second CAR comprise administration of from or from about 1×106 to about 1×108 of the CAR-expressing T cells.
28. The method of claim 21, wherein administration of the T cells expressing the second CAR comprise administration of from or from about 1×106 to about 1×108 of the CAR-expressing T cells.
29. The method of claim 1, wherein the at least one region identical in sequence is selected from the group consisting of an intracellular costimulatory signaling domain, an ITAM-containing domain, a transmembrane domain, and a combination thereof.
30. The method of claim 29, wherein the at least one region identical in amino acid sequence is an ITAM-containing domain that is a human CD3zeta signaling domain.
31. The method of claim 29, wherein the at least one region identical in amino acid sequence is a costimulatory signaling domain that is a CD28 signaling domain or a 4-1BB signaling domain.
32. The method of claim 30, wherein the at least one region identical in amino acid sequence further comprises a costimulatory signaling domain that is a 4-1BB signaling domain.
33. The method of claim 2, wherein the at least one region identical in amino acid sequence is selected from the group consisting of an intracellular costimulatory signaling domain, an ITAM-containing domain, a transmembrane domain, and a combination thereof.
34. The method of claim 33, wherein the at least one region identical in amino acid sequence is an ITAM-containing domain that is a human CD3zeta signaling domain.
35. The method of claim 33, wherein the at least one region identical in amino acid sequence is a costimulatory signaling domain that is a CD28 signaling domain or a 4-1BB signaling domain.
36. The method of claim 34, wherein the at least one region identical in amino acid sequence further comprises a costimulatory signaling domain that is a 4-1BB signaling domain.
37. The method of claim 16, wherein the at least one region identical in amino acid sequence is selected from the group consisting of an intracellular costimulatory signaling domain, an ITAM-containing domain, a transmembrane domain, and a combination thereof.
38. The method of claim 37, wherein the at least one region identical in amino acid sequence is an ITAM-containing domain that is a human CD3zeta signaling domain.
39. The method of claim 37, wherein the at least one region identical in amino acid sequence is a costimulatory signaling domain that is a CD28 signaling domain or a 4-1BB signaling domain.
40. The method of claim 38, wherein the at least one region identical in amino acid sequence further comprises a costimulatory signaling domain that is a 4-1BB signaling domain.
41. The method of claim 21, wherein the at least one region identical in amino acid sequence is selected from the group consisting of an intracellular costimulatory signaling domain, an ITAM-containing domain, a transmembrane domain, and a combination thereof.
42. The method of claim 41, wherein the at least one region identical in amino acid sequence is an ITAM-containing domain that is a human CD3zeta signaling domain.
43. The method of claim 41, wherein the at least one region identical in amino acid sequence is a costimulatory signaling domain that is a CD28 signaling domain or a 4-1BB signaling domain.
44. The method of claim 42, wherein the at least one region identical in amino acid sequence further comprises costimulatory signaling domain that is a 4-1BB signaling domain.
45. The method of claim 21, wherein the B cell malignancy is a leukemia or a lymphoma.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S. provisional application No. 62/087,224 filed Dec. 3, 2014, entitled “Methods and Compositions for Adoptive Cell Therapy,” the contents of which are incorporated by reference in their entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042001200SeqList.txt, created Dec. 3, 2015, which is 65,266 bytes in size. The information in electronic format of the Sequence Listing is incorporated by reference in its entirety.
FIELD
The present disclosure relates to adoptive cell therapy involving the administration of multiple doses of cells expressing genetically engineered (recombinant) receptors, e.g., via multiple administration steps and/or by administration to subjects having received a prior administration. In general, cells administered in connection with certain different administration steps express distinct receptors. The recombinant receptors may include chimeric receptors, e.g., chimeric antigen receptors (CARs), and/or other transgenic receptors such as transgenic T cell receptors (TCRs). Features of the methods provide various advantages, such as improved efficacy, for example, due to increased exposure of the treated subject to administered cells expressing receptors that target disease-associated antigens. A subsequent administration of cells expressing a receptor distinct from that expressed by cells in a first or prior administration can improve efficacy. For example, it can minimize the risk of reduced exposure to the cells, which can result from specific anti-receptor immune response in the subject, and/or allow for effective targeting in cases of antigen loss and/or downregulation or modification of the antigen or epitope targeted by the first or prior receptor.
BACKGROUND
Various methods are available for adoptive cell therapy using engineered cells expressing recombinant receptors, such as chimeric antigen receptor (CARs). Improved methods are needed, for example, to improve efficacy of such therapies, for example, by increasing exposure of the subject to the administered cells. Such methods are needed, for example, that improve expansion and/or persistence of the administered cells, provide the ability to treat refractory or relapsed subjects, and/or that reduce the risk of toxicity or other unwanted outcomes. Provided are methods, compositions, and articles of manufacture that meet such needs.
SUMMARY
Provided are methods for administering cells to subjects, such as for adoptive cell therapy, for example, in treating cancer and other diseases, conditions, or disorders, as well as cells, compositions, and articles of manufacture for use in such methods. The cells generally express one or more recombinant receptors, such as chimeric antigen receptor (CARs), other antigen receptors, and/or other chimeric receptors. In some embodiments, the methods increase exposure of the subject to the administered cells, such as by improving expansion and/or persistence of the administered cells, provide the ability to treat refractory or relapsed subjects and/or subjects displaying loss, downregulation, or modification of a targeted antigen or epitope thereof. In some embodiments, the methods reduce the risk of toxicity or other unwanted outcomes compared with other methods of cell therapy.
In some embodiments, provided are methods of treatment, carried out by administering cells to a subject, where the cells express a second (or subsequent) receptor, such as a second (or subsequent) chimeric antigen receptor (CAR) or transgenic TCR, where the subject has previously received an administration or dose of cells expressing a first (or prior) receptor, such as a first (or prior) CAR or TCR. The second or subsequent receptor is generally distinct from the first or prior receptor. In some embodiments, the methods further include administering the cells expressing the first or prior receptor prior to the administration of the cells expressing the second or subsequent receptor. For example, in some embodiments, the methods are carried out by (a) administering to the subject the cells expressing the first or prior receptor (e.g., first or prior CAR), and (b) administering to the subject cells expressing a first or prior receptor, e.g., CAR; and (b) administering to the subject cells expressing a second or subsequent receptor, e.g., CAR.
In some embodiments, the cells expressing the second or subsequent receptor, e.g., CAR, do not express the first or prior receptor, e.g., CAR. In some embodiments, the first (or prior) and/or second (or subsequent) receptor is an antigen receptor, such as a CAR or a transgenic TCR. In some such embodiments, the first or prior receptor, e.g., CAR, specifically binds to an antigen associated with a disease or condition or disorder in the subject. In some embodiments, the second or subsequent receptor, e.g., CAR, specifically binds to the antigen specifically bound by the first or prior receptor. In some embodiments, the first or prior receptor, e.g., CAR, and the second or subsequent receptor, e.g., CAR, specifically bind to the same epitope of the antigen. In some embodiments, the first or prior receptor competes for binding to the antigen with the second or subsequent receptor, or vice versa. In some embodiments, the first or prior receptor and the second or subsequent receptor specifically bind to distinct epitopes or portions of the antigen.
In some embodiments, the second or subsequent receptor specifically binds to a different antigen associated with the disease or condition or disorder in the subject. For example, in some embodiments, the antigen recognized or bound by the first receptor is CD19 and the antigen specifically bound or recognized by the second or subsequent receptor is a B-cell specific or B-cell associated antigen (or antigen associated with or specific for B cell disease(s), e.g., B cell malignancy), that is distinct from CD19, such as CD22 or CD20.
In some embodiments, the second or subsequent receptor, e.g., CAR, does not specifically bind to the antigen specifically bound by the first or prior receptor, e.g., CAR. In some embodiments, the cells expressing the second or subsequent receptor do not include a receptor that specifically binds to the antigen specifically bound by the first or prior receptor.
In some embodiments, at the time of, prior to, and/or immediately prior to, the administration of cells expressing the second or subsequent receptor, the subject exhibits a detectable humoral and/or cell-mediated immune response specific for the first or prior receptor. In some embodiments, the subject does not exhibit a detectable humoral or cell-mediated immune response against the second or subsequent receptor, e.g., CAR within about 30 days, within about 60 days, or within about 90 days, of the administration of the cells expressing the second or subsequent receptor, such as the administration in (b).
In some embodiments, at the time of, prior to, and/or immediately prior to, the administration of cells expressing the second or subsequent receptor, the disease or condition persists in the subject; and/or the disease or condition has relapsed in the subject.
In some embodiments, at the time of, prior to, and/or immediately prior to, the administration of cells expressing the second or subsequent receptor, the subject exhibits downregulation, loss, or modification of the antigen specifically bound by the first or prior receptor.
In some embodiments, the time between the administration of the cells expressing the first or prior receptor and the administration of the cells expressing the second or subsequent receptor is at least about 28 days; at least about 35 days; at least about 42 days; at least about 49 days; or at least about 60 days. In some embodiments, the time is at least about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 days, such as at least 14 days or at least 21 days.
In some embodiments, the first or prior receptor, e.g., CAR, includes at least one immunoreactive epitope that is not present in the second or subsequent receptor, e.g., CAR. In some embodiments, the at least one immunoreactive epitope includes at least one B cell epitope or epitope recognized by the humoral immune system; and/or includes at least one T cell epitope or epitope recognized by a cell-mediated response such as one recognized by a cytotoxic and/or helper T cell.
In some embodiments, the subject has not received a dose of cells expressing the first or prior receptor prior to the administration in (a); and/or has not received a dose of cells expressing the second or subsequent receptor prior to the administration in (b) or prior to the initiation of the method.
In some embodiments, the disease or condition is a tumor. In some embodiments, it is or is associated with an infectious disease. In some embodiments, it is or is associated with an autoimmune disease or disorder.
The second or subsequent receptor, e.g., second or subsequent CAR, generally includes one or more differences in amino acid sequence compared to the first or prior receptor, e.g., the first or prior CAR. In some embodiments, the one or more differences includes at least one amino acid sequence difference compared to a region of the first or prior receptor, e.g., CAR, to which a detectable immune response is generated in the subject following the administration in (a) or the prior administration of cells expressing the first or prior receptor, e.g., CAR. In some embodiments, such one or more differences include at least one amino acid sequence difference compared to each region of the first or prior receptor to which a detectable immune response is generated in the subject following the administration in (a) or the prior administration. In some embodiments, such an immune response is detected in connection with the methods.
In some embodiments, the methods further include, prior to the administration of the cells expressing the second or subsequent receptor or prior to the administration in (b), detecting the presence of a receptor-specific, e.g., CAR-specific, immune response in the subject. In some embodiments, the detection comprises identifying at least a region of the first or prior receptor, e.g., CAR, to which the subject exhibits a specific immune response, such as a specific antibody- or cell-mediated immune response.
In some embodiments, the second or subsequent receptor, e.g., CAR contains one or more amino acid sequence differences compared to the region of the first or prior receptor, e.g., CAR, for which an immune response in the subject, such as a detectable immune response in the subject, is specific. In some such embodiments, such region of the first or prior receptor is or includes a junction between two endogenous sequences or domains. In some embodiments, it is or includes a region within one or more CAR portions selected from the group consisting of an scFv portion, a linker portion, an amino acid sequence not endogenous to the subject, a sequence derived from a difference species than that of the subject, and/or junction between two CAR domains. In some embodiments, it is or includes a framework region (FR) within the scFv portion, a heavy chain FR sequence, a heavy chain CDR sequence, a light chain FR sequence, and/or a light chain CDR sequence.
In some embodiments, the subsequent or second receptor, e.g., CAR, includes at least one region that is identical in amino acid sequence to a corresponding region of the first or prior receptor, e.g., CAR. In some embodiments, such corresponding region of the first or prior receptor, e.g., CAR, is a region to which the subject does not exhibit a detectable humoral or cell-mediated immune response, e.g., prior to or at the time of the administration of cells expressing the second receptor. In some embodiments, it is or includes an endogenous sequence. In some embodiments, it is or includes a region within a CAR portion selected from the group consisting of a costimulatory domain, an ITAM-containing domain, a transmembrane domain, a transduction or expression marker, a sequence endogenous to the host, and/or an antibody domain derived from the same species as the host.
In some embodiments, the methods result in an increase or enhancement of exposure of the subject to cells compared with other methods. In some embodiments, the maximum number of CAR-expressing cells, the area under the curve (AUC) for CAR-expressing cells over time, and/or the duration of detectable CAR-expressing cells in the subject following the administration of the cells expressing the second or subsequent receptor is greater as compared to that achieved via a method using an alternative dosing regimen involving administration of the cells expressing the first or prior receptor, e.g., the administration in (a), and a second or subsequent administration of cells expressing the first or prior receptor, which is carried out at the same point in time and/or otherwise under the same conditions as the administration in the provided method of the cells expressing the subsequent or second receptor, e.g., as the administration in (b).
In some embodiments, the method results in a maximum concentration or number of receptor-expressing, e.g., CAR-expressing, cells in the blood of the subject of at least at or about 10 receptor-expressing (e.g., CAR-expressing) cells per microliter, at least 50% of the total number of peripheral blood mononuclear cells (PBMCs), at least at least about 1×105 CAR-expressing cells, or at least 1,000, 2,000, 3,000, 4,000, or 5,000 copies of CAR-encoding DNA per micrograms DNA. In some embodiments, at day 30, at day 60, or at day 90 following the initiation of the administration of cells expressing the second or subsequent receptor, e.g., the administration in (b), receptor-expressing, e.g., CAR-expressing, cells are detectable in the blood or serum of the subject; and/or the blood of the subject contains at least 20% receptor-expressing (e.g., CAR-expressing) cells, at least 10 receptor-expressing (e.g., CAR-expressing) cells per microliter or at least 1×104 receptor-expressing (e.g., CAR-expressing) cells.
In some embodiments, any of the above embodiments may involve multiple subsequent administrations. For example, in some embodiments, the methods are carried out in an iterative fashion, in which multiple administrations of cells, each expressing a further subsequent receptor (e.g., administrations of cells expressing third, fourth, fifth, sixth, and so-forth receptors, each distinct in some say from the first or prior receptor(s)).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows results from an exemplary chromium release assay detecting the presence of a cytolytic immune response specific for CAR-expressing cells following administration of anti-CD19 CAR-expressing cells in a human subject. Results are shown for mixed-lymphocyte cultures containing peripheral blood mononuclear cells (PBMCs) derived from the subject pre-infusion (left panel) and post-infusion (right panel) with the CAR-expressing cells, in the presence of either CAR-expressing (“CD19-CAR”) and non-CAR-expressing (“Mock”). “E/T”=effector to target cell ratio.
FIG. 2 shows results from an exemplary ELISpot analysis confirming immune responses to certain overlapping peptides representing particular regions of a CAR in an exemplary human subject. Numbers labeled with “pep” represent various overlapping peptides along the length of the CAR sequence, with corresponding regions indicated above the chart.
FIG. 3 shows an epitope affinity map for predicted binding affinities of peptides of an exemplary region of a chimeric receptor for binding to HLA-A2:01, including a series of overlapping 8mer to 14mer peptides of an exemplary junction region having an amino acid sequence CYSLLVTVAFIIFWVKRGRKKLLYIFKQPF (SEQ ID NO: 6) where residues 1-15 correspond to an exemplary CD28 transmembrane domain and residues 16-30 correspond to an exemplary 4-1BB costimulatory domain. The figure also depicts predicted binding affinities of a series of overlapping 8mer to 14mer peptides of a variant junction region having an amino acid sequence CYSLLVTVAFIIFWNNVKRGRKKLLYIFKQPF (SEQ ID NO: 13), containing inserted asparagine residues between the CD28 transmembrane domain and 4-1BB costimulatory domain.
FIG. 4A and FIG. 4B depict algorithm-based T cell epitope predictions for HLA class I and HLA class II alleles, respectively, showing the total number of sequences in the dataset including each position along the length of the sequence with a predicted IC50 of less than 50 nm weighted according to the frequency of the individual HLA alleles in the population.
FIG. 5 depicts algorithm-based T cell epitope predictions for HLA class I and HLA class II alleles of a series of variant peptides. Scores were determined and weighted as described in Example 2. Triangles and a dotted line indicated class I weighted scores. Circles and a solid line indicate class II weighted scores.
FIG. 6 depicts the amino acid sequence of an exemplary CD28-4-1BB sequence of SEQ ID NO: 5. Amino acids corresponding to the CD28 transmembrane domain are indicated by a solid line with arrows indicating the beginning and end positions; amino acids of the exemplary 4-1BB costimulatory domain are indicated by a dashed line with arrows indicating the beginning and end positions; amino acids of the exemplary junction region are indicated by a dashed and dotted line with arrows indicating the beginning and end positions and by italics. The two amino acids immediately flanking the junction site are indicated by a box. Exemplary amino acids that in some embodiments are targeted for modification, including K28, R31, and L34, are bolded and underlined. Regions of acidic residues that may be involved in 4-1BB-mediated TRAF-binding and signaling are indicated by a double underline.
DETAILED DESCRIPTION
I. Methods of Treatment with Cells Expressing Recombinant Receptors
Provided are methods, compositions, and articles of manufacture for use in cell therapy, for example, for the treatment of various diseases and conditions such as tumors. The methods involve administering to a subject engineered cells expressing recombinant molecules, typically recombinant receptors designed to recognize and/or specifically bind to molecules associated with the disease or condition and/or to promote a particular therapeutic effect. Such binding can result in a response, such as an immune response targeting such molecules. The recombinant receptors may include chimeric receptors, e.g., chimeric antigen receptors (CARs), and/or other transgenic receptors, such as transgenic antigen receptors including transgenic T cell receptors (TCRs).
In particular, the methods involve multiple administrations of such cells or the administration of cells to a subject having received a prior administration or dose. Typically, the cells administered in a first or prior administration (e.g., in a first or prior dose) are distinct from those administered in the second or subsequent administration(s) or dose(s). Typically, the cells are distinct at least in part by way of their expression of distinct recombinant molecules, e.g., distinct recombinant receptors. In some embodiments, the cells of the second or subsequent administration or dose do not express the receptor expressed by those of the first dose or administration. In some embodiments, such cells express a receptor that is distinct from that of the first administration or dose. Thus, in some embodiments, the methods involve administering second (and/or third, fourth, fifth, and so forth) dose of cells to subjects having received a first dose, and/or administering to the subject the first and second (and/or third, fourth, fifth, and so forth) dose, in which the cells administered in the second dose express a receptor that is distinct from the receptor expressed by the cells administered in the first dose. The methods may be carried out in an iterative fashion.
In some aspects, the provided embodiments are based on observations herein that increased exposure of the subject to administered cells expressing the recombinant receptors (e.g., increased number of cells or duration over time) can improve efficacy and therapeutic outcomes in adoptive cell therapy. Preliminary analysis conducted following the administration of different CD19-targeting CAR-expressing T cells to subjects with various CD19-expressing cancers in multiple clinical trials revealed a correlation between greater and/or longer degree of exposure to the CAR-expressing cells and treatment outcomes.
Such outcomes included patient survival and remission, even in individuals with severe or significant tumor burden. Nonetheless, exposure may be limited by host immune responses against the recombinant receptors expressed by the administered cells, which may prematurely eliminate the cells. Once such a host immune response develops, either acquired or innate, it may not be feasible or effective to attempt to increase exposure or provide retreatment of subjects by administering a subsequent dose of cells expressing the same recombinant receptor. Once such an immune response has developed against the receptor, administration of such a second or subsequent dose of cells expressing the same receptor or one with similar immunogenic epitopes may result in rapid elimination of the cells before they have had a chance to expand and/or persist to an effective or substantial degree. Provided are embodiments that address these challenges.
In some embodiments, by providing a second (and/or other subsequent) dose of cells that expresses a second (and/or other subsequent) receptor (e.g., CAR) distinct from the first or prior receptor expressed by a first or prior dose, the provided methods address the problem of reduced exposure due to a host immune response against the first or prior receptor. In particular, because the cells of the second or subsequent dose do not express the same receptor expressed by the cells of the first or prior dose, the risk of the subject having mounted an immune response specific for a molecule present on the cells of the second or subsequent dose is reduced.
In some aspects, the provided embodiments are based on observations of antigen loss, mutation, modification, and/or downregulation in the context of immunotherapy, e.g., adoptive cellular immunotherapy. For example, CD19-negative disease has been observed in certain subjects having been administered CD19-targeted therapy, including anti-CD19 CAR-expressing T cells. In some embodiments, the provided methods offer advantages in such contexts of antigen downregulation or loss or modification. For example, administration of cells expressing a second or subsequent receptor which specifically binds to a different antigen as compared to the antigen or epitope targeted by a first or prior receptor—where the subject experiences or displays loss or modification of such epitope or antigen—can allow for continued treatment of the disease or condition and/or for improved efficacy. The different antigen in some embodiments is another antigen specific to or associated with the same disease or condition to or with which the first antigen is specific or associated. In some embodiments, it is a variant of the first antigen, such as a splice variant or mutated version expressed in the subject, e.g., during or subsequent to therapeutic intervention. Thus, also among the advantages of certain methods and compositions provided herein include the ability to provide continued, effective treatment in a subject experiencing antigen loss, downregulation, or modification which renders a first treatment approach less effective.
In some embodiments, the subject exhibits an immune response against the first or prior receptor following the first or prior administration, e.g., at the time of or immediately prior to the second or subsequent administration, such that further administration of cells expressing the first receptor or a method with similar immunogenicity, may not be efficacious. In some embodiments, the subject does not exhibit an immune response or a particular type or degree of immune response, against the second receptor following the administration of the cells expressing the second receptor, or does not exhibit such a response within a certain time period, such as within about 60 days of the administration of those cells. The type of immune response may be a detectable immune response, a humoral immune response, and/or a cell-mediated immune response. In some embodiments, the presence or absence of such an immune response after the first or prior administration is detected, and can inform which differences are designed to be present in the second or subsequent receptor as compared to the first or prior receptor. Such detection may include identifying at least a region of the first or otherwise prior receptor (e.g., CAR) to which the subject exhibits a specific immune response. Such an identified region may be varied in the second or otherwise subsequent receptor, e.g., a receptor selected in the second administration that differs in that one or more region.
In particular, the second molecule, e.g., second receptor (e.g., the second CAR), generally differs to some degree, e.g., in amino acid sequence and/or immunological epitope(s), from the first receptor (e.g. first CAR). Thus, the first or prior receptor generally includes at least one immunoreactive epitope that is not present in the second or subsequent receptor, such as at least one B cell epitope and/or at least one T cell epitope, which may be recognized by the immune system of the subject to which the cells are administered. In particular embodiments, the second (or subsequent) receptor, e.g., CAR, includes one or more differences in amino acid sequence compared to the first or prior receptor.
Such differences may include at least one difference compared to a region of the first or prior receptor to which a detectable immune response is exhibited in the subject following the first or prior administration, e.g., a difference in a region in the second or subsequent receptor that corresponds to such a region in the first or prior receptor. Regions including the difference(s) may include an antigen-binding portion, such as an scFv portion, including framework region(s) (FRs) within an scFv or variable region portion, such as FR1, FR2, FR3, e.g., of the VH, a heavy and/or light chain variable region portion, a linker portion, a hinge portion, a junction between two CAR domains, a transduction or expression marker, and/or a sequence of amino acids within the CAR that is non-endogenous, e.g., is not identical to a sequence present in an endogenous molecule of the host, such as a junctional region between two domains not naturally associated with one another in a single amino acid sequence in the natural setting, e.g., a junction between two endogenous sequences within a chimeric receptor or antibody/antibody fragment.
Although one or more differences is generally present in the second or other subsequent receptor as compared to the first or otherwise prior receptor, the receptors may also include regions of similarity, e.g., regions of amino acid sequence identity. In some embodiments, the region(s) of identity are ones to which the subject does not or is unlikely to exhibit an immune response following the first or prior administration. Such regions may include regions within a costimulatory domain, an ITAM-containing domain, a transmembrane domain, a CDR, and/or a transduction or expression marker. Where an scFv and/or variable region differs between the different receptors, it may be that the respective scFv or variable regions are derived from the same species (e.g., mouse or human), derived from different species, and/or combinations thereof.
In some embodiments, the noted differences are the only differences or substantially or essentially the only differences, between the recombinant molecule, e.g., receptor, in the cells of the first dose or administration as compared to the second dose or administration. In some embodiments, aside from differences in the receptor and/or other noted differences, the cells and/or cell populations administered in a prior and subsequent administration are identical or essentially or substantially identical. In some embodiments, the ratio of cells expressing detectable surface levels of one or more markers is the same or similar in one administration as compared to the subsequent administration. In some embodiments, the percentages of populations and/or sub-populations of cells in the different doses or administrations are the same or substantially or essentially the same. The different doses may contain the same percentage of T cells, CD8+ and/or CD4+ T cells, T cells of a particular lineage or activation state or experience, such as relative percentages of effector, naïve, and/or memory T cells, and/or sub-populations thereof such as TCM, TEM, TSCM cells and/or the cells may be derived from the same subject, sample, tissue, and/or fluid or compartment. In some embodiments, another portion of the same composition of cells used to engineer the cells of the first dose, e.g., by transduction with a vector encoding the recombinant receptor, is used to engineer the cells of the second administration. In some embodiments, the composition is preserved, e.g., by cryopreservation, prior to the second administration.
In some embodiments, the doses are administered in particular amounts and/or according to particular timing parameters. In some embodiments, the second or otherwise subsequent dose of cells expressing the second (or third, fourth, fifth, etc.) receptor is given at a time after an immune response has developed, had a chance to develop, and/or has been detected or otherwise confirmed to be present, against the receptor in the first or other prior dose, such as at or about or at least at or about 28 days or 35 days following the first or other prior dose.
The subsequent dose may be used for retreatment upon relapse, and/or to prevent recurrence of the targeted disease or disorder, and/or to address or prevent a reduction in exposure to cells expressing a recombinant receptor targeting the disease or condition or antigen of interest following a first or prior dose. For example, the subsequent dose in some embodiments is administered after or upon detection of a decline in persistence or expansion of such cells or in total or relative numbers of such cells in the subject or organ or fluid thereof. Thus, in some embodiments one or more of these parameters is measured, detected, or assessed in the time between the first or other prior dose and the second or other subsequent dose, and the timing or decision to administer the subsequent dose is made based on the outcome of such assessment. For example, the second dose may be administered at a time at which it is determined that the number or concentration of the receptor-expressing cells is below a desired level or has declined below a certain percentage of maximum or other measured concentration or number.
The recombinant receptors, e.g., CARs or transgenic TCRs, generally specifically bind to one or more antigen expressed by, associated with, and/or specific for a disease or condition in the subject and/or cell(s) or tissue(s) thereof. Such diseases may include tumors, cancers, other proliferative diseases, autoimmune diseases or disorders, and/or infectious agents or disease. In some embodiments, the first (or other prior) and the second (or other subsequent) receptors, although distinct, specifically bind to the same such antigen. The binding may be to a similar or the same epitope. In some embodiments, the binding of one receptor to antigen competes for binding to the antigen with the other. The binding in some embodiments is to an entirely different epitope, not competing with that of the other receptor, and/or to a completely different antigen. In this respect, the methods in some embodiments may be useful in treating subjects whose disease or condition has become resistant to treatments targeting a particular epitope or antigen, such as resulting from target downregulation or mutation by the disease or condition or cells thereof, and/or experiencing antigen loss. For example, in some embodiments, the antigen recognized or bound by the first receptor is CD19 and the antigen specifically bound or recognized by the second or subsequent receptor is a B-cell specific or B-cell associated antigen (or antigen associated with or specific for B cell disease(s), e.g., B cell malignancy), that is distinct from CD19, such as CD22 or CD20.
Thus, by offering the ability to target a similar but distinct disease-associated epitope or antigen, the methods in some embodiments improve efficacy not only by increasing overall persistence of engineered cells in the subject, but also by allowing the cells and/or form of therapy to function even in the context of downregulation or mutation of the original target. In some embodiments, the second receptor binds to the same antigen and a different antigen in the same disease, and/or the cell contains multiple receptors, each binding to a different antigen or epitope, one or more of which may be distinct from or the same as that recognized by the first receptor. In some embodiments, the second or subsequent receptor binds to a variant, e.g., a different splice variant or a modified version, of the antigen recognized by the first receptor.
In some embodiments, the different receptors have domain(s) (such as antigen-binding domains, e.g., sFvs, and/or or other domains of chimeric receptors, e.g., other CAR domains) having sequences with origins in the same species and/or those having sequences with origins in different species. For example, in some embodiments, the different receptors contain two distinct binding domains derived from the same species, such as two mouse-derived scFv domains or two scFv domains with framework region (FR) sequences derived from mouse, such as those derived from FMC63 and SJ25C1, respectively. In other embodiments, the different receptors contain two distinct binding domains for the same or different antigens derived from different species, such as a first receptor having an scFv or other binding domain derived from a murine sequence, such as FMC63 or SJ25C1 and another receptor with a domain derived in whole or in part from another species, such as a human or humanized sequence, such as one that binds to the same antigen, e.g., to a same or similar or distinct epitope, or to a distinct antigen.
In some embodiments, the receptor is a receptor other than an antigen receptor, such as one of a pair of binding partners and/or variant thereof, the other partner of which is specifically expressed in the context of a disease or condition or cells or tissues thereof, and/or expression of which is associated with the disease or condition. In some embodiments, such receptors are chimeric receptors. In some embodiments, such chimeric receptors contain extracellular binding portions that specifically interact with such a binding partner, and contain, for example, transmembrane and/or intracellular signaling domain(s) capable of potentiating an immunostimulatory signal or signals, such as an activating and/or costimulatory domains such as those present in certain chimeric antigen receptors.
In some embodiments, the provided methods are for long-term or continuous treatment or management of the disease or disorder in the subject, involving first, second, third, and/or multiple additional subsequent administrations of engineered cells, in which one or more of the doses includes cells expressing recombinant receptors distinct from those in other dose(s), but targeting the same disease or condition in the subject, such as distinct receptors targeting the same or different disease-specific or disease-associate antigen, at the same or different epitope(s). The long-term or chronic treatment or management in some embodiments is an iterative process, in which the subject is monitored for immunogenicity and/or drop in exposure, presence, persistence, numbers, and/or percentages of the cells, and a next subsequent administration (e.g., next subsequent receptor) is introduced if and when a particular indicator of loss of efficacy or risk thereof with respect to the first or prior receptor or cells is detected. In some embodiments, each subsequent administration is initiated upon detection of one or more indicators of a risk of loss of efficacy, such as reduced persistence of, expansion of, or exposure to the cells in the prior dose, an immune response specific thereto in the subject, relapse, resistance, and/or downregulation or change in the target antigen.
Exemplary Methods of Dosing with a Second Chimeric Receptor
In some embodiments, the methods include administration of a second chimeric receptor to a subject that has developed an immune response and/or is likely to be immunogenic to the first chimeric receptor. In some embodiments, the first chimeric receptor contains a junction region of a first and second domain that is immunogenic. In some embodiments, the immunogenic region includes one or more peptide epitopes (also called a T cell epitope). In some cases, a junction region that contains potential peptide epitopes spanning the junction of the two domains can be immunogenic and result in the generation of an immune response upon administration to a subject of a chimeric receptor containing the junction region. In some embodiments, the junction region can include a plurality of individual overlapping peptide fragments of contiguous sequence of about 8 to 24 amino (e.g. 8 to 15 amino acids or 8 to 13 amino acids, such as about or 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids) directly C-terminal of the junction that joins a first domain and a second domain of the chimeric receptor and/or of about 8 to 24 amino acids (e.g. 8 to 15 amino acids or 8 to 13 amino acids, such as about or 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids) directly N-terminal of the junction, which peptide fragments each can include or span the junction of the two domains. Thus, in some cases, the junction region can contain a plurality of potential peptide epitopes that may exhibit a binding affinity for an HLA molecule and/or be capable of inducing an immune response.
In some embodiments, an immunogenic region, such as a junction region, of a chimeric receptor can be identified. In some embodiments, the immunogenic region can be identified by its ability to bind to an MHC molecule or by its ability to elicit an immune response under certain conditions. In some embodiments, overlapping peptides of a chimeric receptor, such as overlapping 8mer to 20 mer peptides, such as 9mers, 10mers, 11mers, 12mers, 13mers, 14mers or 15mers can be assessed for MHC binding using algorithmic or other computational methods, such as described below. In some embodiments, a chimeric receptor can be assessed to determine if it is immunogenic by assessing an immune response in a subject to which it has been administered, such as a subject administered cells genetically engineered with the chimeric receptor (e.g. CAR). Exemplary methods of assessing immune responses are described below.
In some embodiments, the at least one peptide epitope is capable of binding to a major histocompatibility complex (MHC) molecule, such as a class I or class II protein, which are molecules that contain a polymorphic peptide binding site or binding groove that can, in some cases, complex with peptide fragments of polypeptides, including peptides processed by the cell machinery. In some embodiments, the peptide epitope is capable of binding to an MHC molecule that is a human MHC molecule. In some embodiments, the MHC molecule is a human leukocyte antigen (HLA) molecule. In some embodiments, the at least one peptide epitope exhibits a binding affinity (e.g. IC50) for an HLA molecule, such as an HLA class I molecule or an HLA class II molecule. In some embodiments, the junction region of the reference chimeric receptor contains a peptide epitope that exhibits a binding affinity of less than 1000 nM, less than 500 nM or less than 50 nM.
In some embodiments, at least one or more peptide epitopes of a junction region of a reference chimeric receptor is an MHC class II epitope. In some embodiments, peptides that bind to MHC class II molecules can be between 8 and 20 amino acids in length, including between 10 and 17 amino acids in length. In some embodiments, the peptides that bind to MHC class II molecules can be longer than 20 amino acids. In some embodiments, the peptide lies in an extended conformation along the MHC II peptide-binding groove. In some embodiments, the MHC II peptide-binding groove is open at both ends. In some embodiments, the peptide is held in place at least in part by main-chain atom contacts with conserved residues that line the peptide-binding groove. In some embodiments, the MHC class II allele can be any known to be present in a subject, such as a human subject. In some embodiments, the MHC allele can be, but is not limited to, DR1, DR3, DR4, DR7, DR52, DQ1, DQ2, DQ4, DQ8 and DP1. In some embodiments, the MHC class II allele can be any set forth in Tables 1B. In some embodiments, the MHC class II allele is an HLA-DRB1*0101, an HLA-DRB*0301, HLA-DRB*0701, HLA-DRB*0401 an HLA-DQB1*0201.
In some embodiments, the at least one peptide epitope of a junction region of a reference chimeric receptor is an MHC class I epitope. In some embodiments, peptides that bind to MHC class I molecules can be between 7 to 15 amino acids in length. In some embodiments, peptides that bind to MHC class I molecule can be between 8 to 13 amino acids in length. In some embodiments, the binding of the peptide is stabilized at its two ends by contacts between atoms in the main chain of the peptide and invariant sites in the peptide-binding groove of all MHC class I molecules. In some embodiments, there are invariant sites at both ends of the groove which bind the amino and carboxy termini of the peptide. In some embodiments, variations in peptide length can be accommodated by a kink in the peptide backbone. In some embodiments, the kink includes proline or glycine residues, which may allow flexibility. In some embodiments, the MHC class I allele can be any known to be present in a subject, such as a human subject. In some embodiments, the MHC class I allele is an HLA-A2, HLA-A1, HLA-A3, HLA-A24, HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45 or HLA-Cw8 allele. In some embodiments, the MHC class I allele can be any set forth in Tables 1A, which are among the most frequent MHC class I alleles (Solberg et al., (2008) Hum Immunol. 2008 July; 69(7):443-6). In some embodiments, the HLA class I allele is HLA-A*02:01, HLA-A*03:01, HLA-A*11:01 or HLA-B*08:01.
In some embodiments, the MHC class I allele is an HLA-A2 allele, which in some populations is expressed by approximately 50% of the population. In some embodiments, the HLA-A2 allele can be an HLA-A*0201, *0202, *0203, *0206, or *0207 gene product. In some cases, there can be differences in the frequency of subtypes between different populations. For example, in some embodiments, more than 95% of the HLA-A2 positive Caucasian population is HLA-A*0201, whereas in the Chinese population the frequency has been reported to be approximately 23% HLA-A*0201, 45% HLA-A*0207, 8% HLA-A*0206 and 23% HLA-A*0203. In some embodiments, the MHC molecule is HLA-A*0201.
In some embodiments, the second chimeric receptor is a variant chimeric receptor containing a modified junction region compared to a junction region of reference chimeric receptor, which can be the first chimeric receptor, in which one or more amino acid residues at a position 8 to 24 amino acids (e.g. 8 to 15 amino acids or 8 to 13 amino acids, such as about or 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids) directly C-terminal of the junction that joins a first domain and a second domain of the reference chimeric receptor and/or at a position 8 to 24 amino acids (e.g. 8 to 15 amino acids or 8 to 13 amino acids, such as about or 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids) directly N-terminal of the junction are modified, such as by insertion, deletion or amino acid replacement. In some embodiments, the variant chimeric receptor contains up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid differences or modifications in the modified junction region compared to the junction region in the reference chimeric receptor.
In some embodiments, the variant chimeric receptor contains a domain of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the first domain of the reference chimeric receptor and/or contains a domain of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the second domain of the reference chimeric receptor. In some embodiments, the variant chimeric receptor contains a domain that is identical in sequence to the first domain of the reference chimeric receptor and contains a domain of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the second domain of the reference chimeric receptor. In some embodiments, the variant chimeric receptor contains a domain of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the first domain of the reference chimeric receptor and contains a domain that is identical in sequence to the second domain of the reference chimeric receptor. In some embodiments, at least one or both of the domains present in the variant chimeric receptor is modified compared to the first domain and/or the second domain of the reference chimeric receptor in the portion containing the modified junction region.
In some embodiments, the variant chimeric receptor has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference chimeric receptor. In some embodiments, the variant chimeric receptor contains up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid differences or modifications (e.g. amino acid insertions, deletions or replacements) compared to the reference chimeric receptor.
In some embodiments, the first and/or second domain of the reference chimeric receptor (e.g. reference CAR) is a domain of a natural endogenous human protein or a domain having 100% identity with a domain or function portion thereof of a natural or endogenous protein. In some embodiments, the first domain and second domain are not present in the same molecule in vivo in a human subject. In some embodiments, the first domain and second domain are not present in a single natural or endogenous human protein or polypeptide.
In some embodiments, the first and/or second domain is or comprises an extracellular binding domain, a hinge domain, a transmembrane domain, or an intracellular signaling domain or functional portions thereof. In some embodiments, the intracellular signaling domain is or comprises a costimulatory signaling domain, such as a CD28, 4-1BB, or ICOS co-stimulatory signaling domain. In some embodiments, the intracellular signaling domain is or comprises an activating cytoplasmic signaling domain, such as a domain that is or includes a T cell receptor (TCR) component and/or that contains an immunoreceptor tyrosine-based activation motif (ITAM). In some cases, the activating cytoplasmic domain is or comprises a cytoplasmic signaling domain of a zeta chain of a CD3-zeta (CD3) chain or a functional variant or signaling portion thereof.
In some embodiments, the reference chimeric receptor is a CAR. In some embodiments, the chimeric receptors, such as a CAR, contains from its N-terminus to C-terminus in order: an extracellular ligand-binding domain, a transmembrane domain and an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or includes an activating signaling domain (e.g. a components of TCR and/or containing an ITAM, for example a CD3-zeta signaling domain). In some embodiments, the intracellular signaling domain is or includes a costimulatory signaling domain (e.g. a CD28, 4-1BB or ICOS signaling domain). In some embodiments, the intracellular signaling domain contains only one of the costimulatory signaling domain or activating signaling domain or contains both domains in either order. In some embodiments, the intracellular signaling domain contains both the costimulatory signaling domain and activating signaling domain.
In some embodiments, the variant chimeric receptor can contain from its N-terminus to C-terminus in order: an extracellular ligand-binding domain, a transmembrane domain and an intracellular signaling domain, which optionally can include a costimulatory signaling domain (e.g. CD28, 4-1BB or ICOS) and/or an activating signaling domain (e.g. a components of TCR and/or containing an ITAM, for example a CD3-zeta signaling domain) each alone as part of the intracellular signaling domain or in either order, in which the variant chimeric receptor contains a modification at one or more amino acid residues within a contiguous portion of 8 to 24 amino acids (e.g. 8 to 15 amino acids or 8 to 13 amino acids, such as about or 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids) on either side (N-terminal and/or C-terminal) of the junction.
In some embodiments, the features of a reference chimeric receptor can be any described in subsection III below. In some embodiments, the features of a variant chimeric receptor also can be any as described in subsection III below, except that the variant chimeric receptor contains one or more modifications (e.g. insertions, deletions or replacements) in a modified junction region as described.
In some embodiments, the variant chimeric receptor contains a modified junction region containing one or more modifications (e.g. insertions, deletions or replacements) in a junction region of a reference chimeric receptor as described, wherein the reference chimeric receptor contains a first domain that is or comprises an extracellular ligand binding domain or a portion thereof and a second domain that is or comprises a hinge domain or a portion thereof, joined in contiguous sequence at a junction.
In some embodiments, the variant chimeric receptor contains a modified junction region containing one or more modifications (e.g. insertions, deletions or replacements) in a junction region of a reference chimeric receptor as described, wherein the reference chimeric receptor contains a first domain that is or comprises a hinge domain or a portion thereof and a second domain that is or comprises a transmembrane domain or a portion thereof, joined in contiguous sequence at a junction.
In some embodiments, the variant chimeric receptor contains a modified junction region containing one or more modifications (e.g. insertions, deletions or replacements) in a junction region of a reference chimeric receptor as described, wherein the reference chimeric receptor contains a first domain that is or comprises a transmembrane domain or a portion thereof and a second domain that is or comprises a costimulatory signaling domain or a portion thereof, joined in contiguous sequence at a junction.
In some embodiments, the variant chimeric receptor contains a modified junction region containing one or more modifications (e.g. insertions, deletions or replacements) in a junction region of a reference chimeric receptor as described, wherein the reference chimeric receptor contains a first domain that is or comprises a costimulatory signaling domain or a portion thereof and a second domain that is or comprises an activating cytoplasmic signaling domain or a portion thereof, joined in contiguous sequence at a junction.
In some embodiments, the first domain of the reference chimeric receptor is or comprises a transmembrane domain or a portion thereof. In some embodiments, the transmembrane domain include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154 and/or transmembrane regions containing functional variants thereof such as those retaining a substantial portion of the structural, e.g., transmembrane, properties thereof. In some embodiments, the transmembrane domain is a transmembrane domain derived from CD4, CD28, or CD8, e.g., CD8alpha, or functional variant thereof. In some embodiments, the second domain of the reference chimeric receptor is or comprises a costimulatory signaling domain, which is directly linked or joined to the transmembrane domain. In some embodiments, the costimulatory signaling domain is or comprises a signaling domain of CD28, 4-1BB, OX40, DAP10, and ICOS.
In some embodiments, the variant chimeric receptor contains a modified junction region containing one or more modifications (e.g. insertions, deletions or replacements) in a junction region of a reference chimeric receptor as described, wherein the reference chimeric receptor contains a first domain that is or comprises a CD28 transmembrane domain or a portion thereof and a second domain that is or comprises a 4-1BB costimulatory signaling domain or a portion thereof, joined in contiguous sequence at a junction. In some embodiments, the CD28 transmembrane domain is or comprises the sequence of amino acids set forth in SEQ ID NO:2, 103 or 104 or is a functional portion or variant thereof comprising a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2, 103 or 104. In some embodiments the 4-1BB signaling domain is or comprises the sequence of amino acids set forth in SEQ ID NO:3 or a functional portion or variant thereof comprising a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:3. In some embodiments, the first domain and second domain together comprise or have the sequence of amino acids set forth in SEQ ID NO:5 or a functional portion or variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:5. In some embodiments, the first domain and second domain of the reference chimeric receptor together have or comprise the sequence of amino acids set forth in SEQ ID NO:5.
In some embodiments, the variant chimeric receptor comprises a modified junction region that is less than 100% sequence identity to SEQ ID NO:137 but greater than 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96% to SEQ ID NO:137 and includes the modifications. In some embodiments, the variant chimeric receptor has or comprises a sequence of amino acids that is less than 100% sequence identity to SEQ ID NO:5 but greater than 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96% to SEQ ID NO:5 and includes the modifications. In some embodiments, the variant chimeric receptor has or comprises a modified junction region comprising the sequence of amino acids set forth in any of SEQ ID NOS: 138-157, a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96% sequence identity to the sequence of amino acids set forth in any of SEQ ID NOS: 138-157, or a functional portion thereof, each that include the modification(s).
In some embodiments, the variant chimeric receptor does not contain a modification at or of a hydrophobic amino acid residue or within a hydrophobic portion in the transmembrane domain, such as the CD28 transmembrane domain. In some embodiments, the variant chimeric receptor contains one or more modifications at or of a hydrophobic amino acid residues or within a hydrophobic portion in the transmembrane domain, such as the CD28 transmembrane domain. In some embodiments, the one or more modifications is or comprises a substitution of the hydrophobic amino acid with another different hydrophobic amino acid residue. In some embodiments, the one or more modifications is not or does not comprise a modification at or of a hydrophobic amino acid residue or within a hydrophobic portion in the transmembrane domain other than a substitution with another hydrophobic amino acid residue.
In some cases, transmembrane domains contain one or more tryptophan residues that interact with the lipid bilayers of a membrane. In some cases, the one or more tryptophan residues can be located near the lipid-water interface. In some cases, the one or more tryptophan residues anchor or assist in anchoring the transmembrane domain within the membrane. See e.g. de Jesus and Allen, Biochim Biophys Acta. 2013 February; 1828(2):864-76. In some embodiments, the variant chimeric receptor does not contain a modification at one or both of a tryptophan residue in the transmembrane domain, such as the CD28 transmembrane domain. In some embodiments, the chimeric receptor does not contain a modification at an amino acid position corresponding to position 2 and/or position 26 with reference to numbering of SEQ ID NO: 5, each of which corresponds to a tryptophan in the reference chimeric receptor.
In some embodiments, the domain in the variant chimeric receptor that corresponds to the transmembrane domain of the reference chimeric receptor has a substantially hydrophobic hydropathy profile and/or has a grand average of hydopathy (GRAVY) value of greater than 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2 or greater.
In some embodiments, the variant chimeric receptor does not contain a modification at or of an amino acid residue involved in or necessary for the signaling of the costimulatory signaling domain, such as at or of an amino acid residue involved in or necessary for 4-1BB signaling. In general, costimulatory signaling involves interactions with TRAF molecules. In some embodiments, the variant chimeric receptor does not contain a modification at or of an amino acid residue that interacts with or is part of a binding motif for binding to a TRAF molecule. In some embodiments, the variant chimeric receptor does not contain a modification at or of an amino acid residue in the costimulatory signaling domain of the reference chimeric receptor that comprises the motif (P/S/A/T)X(Q/E)E. In some embodiments, the TRAF molecule is TRAF 1, TRAF2 and/or TRAF3. In some embodiments, the domain in the variant chimeric receptor that corresponds to the costimulatory signaling domain of the reference chimeric receptor is capable of inducing the activation or cellular localization of a TRAF and/or is capable of inducing TRAF-mediated signaling. In some embodiments, the variant chimeric receptor contains amino acids TTQE at positions corresponding to 49-52 and/or amino acids PEEE at positions corresponding to residues 60-63, each with reference to numbering set forth in SEQ ID NO:5.
In some embodiments, the variant chimeric receptor contains one or more amino acid modification within a portion between residue 13 and 42 or between amino acid residue 15 and 40, with reference to numbering set forth in SEQ ID NO:5.
In some embodiments, the modification is or includes insertion of one or more amino acid residues. In some embodiments, the one or more insertion is between amino acid residues adjacent to the junction between the domains. In some embodiments, the one or more amino acid insertions is between amino acid residues 27 and 28 with reference to numbering set forth in SEQ ID NO:5. In some embodiments, the one or more insertions can include insertion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues. In some embodiments, the insertion is of 1, 2, 3, 4, or 5 amino acid residues. In some embodiments, the insertion is to any amino acid residues. In some embodiments, the insertion of is insertion of an asparagine (N).
In some embodiments, the modification is or includes one or more amino acid replacements at a residue corresponding to residue 28, 31 or 34 with reference to numbering set forth in SEQ ID NO:5. In some embodiments, the amino acid replacement can be to any other amino acid. In some embodiments, the amino acid replacement is to an amino acid residue that is leucine (L), asparagine (N), glutamine (Q), alanine (A), serine (S) or histidine (H). In some embodiments, the amino acid replacement is or corresponds to one or more of K28A, K28H, K28L, K28Q, K28S, R31A, R31H, R31L, R31N, L34A and L34S, with reference to numbering set forth in SEQ ID NO:5.
In some embodiments, the variant chimeric receptor contains a modified junction region with two or more, such as up to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid modifications compared to a junction region of a reference chimeric receptor. In some embodiments, the amino acid replacements are or correspond to amino acid replacements selected from among K28Q/R31A, K28Q/R31N, K28Q/R31S, K28Q/L34A, K28Q/L34S, R31N/L34A, R31N/L34S, K28Q/R31N/L34A, K28Q/R31N/L34S.
In some embodiments, the variant chimeric receptor has or comprises a modified junction region that has the sequence of amino acids set forth in any of SEQ ID NOS: 138-157, a a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96% sequence identity to the sequence of amino acids set forth in any of SEQ ID NOS: 138-157, or a functional portion thereof, each that includes the modification(s).
In some embodiments, the variant chimeric receptor has or comprises the sequence of amino acids set forth in any of SEQ ID NOS: 114-134, a functional variant thereof comprising a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96% sequence identity to the sequence of amino acids set forth in any of SEQ ID NOS: 114-134, or a functional portion thereof, each that includes the modification(s).
In some embodiments, the variant chimeric receptor contains a modified junction region such that peptide fragments of such region exhibit a lower binding affinity for a human leukocyte antigen (HLA) and/or the region exhibits reduced immunogenicity, including following administration to a subject.
In some embodiments, a peptide fragment having the sequence of an 8-15 amino acid portion of the modified junction region has a binding affinity for a human leukocyte antigen (HLA) molecule that is lower than the binding affinity, for the same HLA molecule, of a peptide fragment having the sequence of the corresponding portion of the junction region of the reference chimeric receptor. In some embodiments, the peptide fragment of the corresponding portion of the junction region of the reference chimeric receptor has a binding affinity of less than 1000 nM, less than 500 nM or less than 50 nM.
In some embodiments, the average of the binding affinities of all 8-15 amino acid fragments, or of all 8, 9, 10, 11, 12, 13, 14, or 15 amino acid fragments, within the modified junction region for a human HLA molecule is lower than the average of the binding affinities of all 8-15 amino acid fragments, or of all 8, 9, 10, 11, 12, 13, 14, or 15 amino acid fragments, within the junction region of the reference chimeric receptor. In some embodiments, the binding affinity or average of binding affinities is more than 2-fold, more than 5-fold, more than 10-fold, more than 25-fold, more than 50-fold or more than 100-fold lower.
In some embodiments, the number of peptide fragments having the sequence of an 8-15 amino acid portion of the modified junction region that has a binding affinity for a human leukocyte antigen (HLA) of less than 1000 nM is reduced compared to the number of peptide fragments having the sequence of an 8-15 amino acid portion of the junction region of the reference chimeric receptor that has the same affinity for binding the same HLA. In some embodiments, the number of peptide fragments having the sequence of an 8-15 amino acid portion of the modified junction region that has a binding affinity for a human leukocyte antigen (HLA) of less than 500 nM is reduced compared to the number of peptide fragments having the sequence of an 8-15 amino acid portion of the junction region of the reference chimeric receptor that has the same affinity for binding the same HLA. In some embodiments, the number of peptide fragments having the sequence of an 8-15 amino acid portion of the modified junction region that has a binding affinity for a human leukocyte antigen (HLA) of less than 50 nM is reduced compared to the number of peptide fragments having the sequence of an 8-15 amino acid portion of the junction region of the reference chimeric receptor that has the same affinity for binding the same HLA.
In some embodiments, the binding affinity can be determined experimentally or algorithmically. In some embodiments, a peptide binding affinity for an MHC can be determined computationally, such as by using algorithms based on quantitative binding affinity models (Lafuente and Reche (2009) Current Pharmaceutical Design, 15:3209-3220). In some embodiments, the binding affinity can be determined in an in vitro assay.
In some embodiments, determining a peptide's binding affinity to an MHC molecule involves radioactivity or fluorescence competition binding assays. See, e.g. Ettinger et al., J. Immunol. 160:2365 (1998). In some embodiments, the competition assay yield a comparison of binding affinities of different peptides. Some MHC binding studies utilize detergent solubilized class I molecules from EBV transformed cell lines (see, e.g. Sette, A., et al., Mol Immunol, 31(11):813-22 (1994). In some embodiments, the competitive assay involves naturally loaded MHC, and the MHC molecule of interest can be purified away from other MHC molecules in the detergent lysate or be used in a mixture with other MHC molecules. In some embodiments, radiolabeled peptides can be identified that have a high affinity for the MHC molecule in question. In some embodiments, the affinity of additional “test” peptides for the MHC molecule in question is then determined by their ability to compete with the high affinity radiolabeled peptide.
In some embodiments, determining peptide affinity can involve a reconstitution assay, e.g. using “T2” cells, in which cells expressing an appropriate MHC allele are “stripped” of a native binding peptide by incubating at pH 2-3 for a short period of time. In some embodiments, to determine the binding affinity of a putative MHC-binding peptide for the same MHC allele, the stripped MHC monomer can be combined in solution with the putative MHC-binding peptide, beta2-microglobulin and a conformation-dependent monoclonal antibody. In some embodiments, the difference in fluorescence intensity determined between cells incubated with and without the test binding peptide after labeling, for example, either directly with the labeled monoclonal antibody or a fluorescence-labeled secondary antibody, can be used to determine binding of the test peptide.
In some embodiments, the binding affinity for an MHC (e.g. HLA) molecule is represented by an IC50, which is the concentration of peptide in a binding assay at which 50% inhibition of binding of a reference peptide is observed. In some cases, such assays can be run under conditions in which IC50 values approximate KD values (i. e., limiting HLA proteins and labeled peptide concentrations). In some embodiments, binding can be expressed relative to a reference peptide.
In some embodiments, the binding affinity can be predicted using in silico methods. Exemplary in silico methods for predicting binding affinity for MHC binding using algorithmic or other computational methods are known in the art, See, for example, Marsh, et al., The HLA Factsbook (Academic Press, 2000). In some embodiments, an algorithm can be used to predict if a peptide of interest should bind to a given MHC molecule. See, e.g., Southwood, et al., J. Immunol. 160:3363 (1998); Honeyman, et al., Nat. Biotechnol. 16:966-969 (1998); Breisie, et al., Bioinformatics 14:121-131 (1998), as well as the “SYFPEITHI” algorithm (Hans-Georg Rammensee, et al., Immunogenetics (1999) 50: 213-219), Zhang et al., PLoS ONE 7(2): e30483. doi: 10.1371/journal.pone.0030483, the Immune Epitope and Analysis Resource (IEDB) (Peters B, et al. PLoS Biology 3: 379 (2005)), and the “BIMAS” algorithm (Parker, K. C., M. A. Bednarek, and J. E. Coligan. J. Immunol. 152:163 (1994).
In some embodiments, algorithm prediction tools, including those available from IEDB, use one or more predictions using ANN (Nielsen et al. (2003) Protein Sci., 12:1007-1017 and Lundegaard et al. (2008) NAR, 36:W509-512), SMM (Peters and Sette (2005) BMC Bioinformatics, 6:132) and comblib (Sidney et al. (2008) Immunome Res. 4:2), or the Consensus tool (see Kim, et al. (2012) Immune epitope database analysis resource, NAR, combining predictions from any of the foregoing).
In some embodiments, prediction of antigen processing can be accomplished using an algorithm for proteosomal cleavage (PaProC). See Kuttler et al., J. Mol. Biol. 298 (2000), 417-429 and Nussbaum et al., Immunogenetics 53 (2001), 87-94.
In some embodiments, the variant chimeric receptor, which can be the second chimeric receptor, exhibits a reduction in a detectable immune response compared to the reference chimeric receptor, which can be the first chimeric receptor. In some embodiments, the immune response is a humoral immune response. In some embodiments, the immune response is a cell-mediated immune response. In some embodiments, the immune response is reduced greater than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold or more. In some embodiments, the immune response is assessed in vitro. In some embodiments, the immune response is assessed in vivo upon administration of a chimeric receptor to a subject, such as administration of cells expressing a chimeric receptor. In some embodiments, a host immune response to the chimeric receptor is assessed as described below.
II. Administration of Cells in Adoptive Cell Therapy
The provided methods generally involve administering multiple doses of cells expressing recombinant molecules such as recombinant receptors, such as CARs, other chimeric receptors, or other antigen receptors, such as transgenic TCRs, to subjects having a disease or condition, such as a disease or condition a component of which is specifically recognized by and/or treated by the recombinant molecules, e.g., receptors. The administrations generally effect an improvement in one or more symptoms of the disease or condition and/or treat or prevent the disease or condition or symptom thereof.
As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human. In some embodiments, the subject has been treated with a therapeutic agent targeting the disease or condition prior to one or more of the administrations or doses. In some aspects, the subject is or becomes refractory or non-responsive to the other therapeutic agent. In some embodiments, the subject has not become refractory or non-responsive but the administration of the cells expressing the second or subsequent receptor is carried out prophylactically, for example, to prevent the subject from becoming refractory or resistant to treatment.
In some embodiments, the subject at the time or immediately prior to one or more of the administrations has persistent or relapsed disease. For example, disease may have relapsed following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT or become refractory to such treatment. The disease or condition may have relapsed or become refractory to the cells of the first administration or dose prior to or at the time of the second administration. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy, such as a therapy other than adoptive cell therapy, or an adoptive cell therapy, such as the first administration of cells expressing a distinct receptor.
In some embodiments, the subject is responsive to the other therapeutic agent or cell administration and treatment with the therapeutic agent or administration reduces disease burden. In some aspects, the subject is initially responsive to the therapeutic agent or administration, but exhibits a relapse of the disease or condition over time, e.g., at which point administration of the cell therapy or second dose or administration is carried out. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk for relapse, such as at a high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.
In some embodiments, the subject has not received prior treatment with another therapeutic agent. In some embodiments, the subject has not received a dose of cells expressing a receptor, e.g. CAR, prior to the administration of the first dose and/or has not received a dose of cells expressing the CAR or other receptor expressed by such cells or expressing any recombinant receptor targeting the same molecule or antigen. In some embodiments, the subject has not received a dose of cells expressing the receptor of the first dose prior to the administration of the first dose. In other embodiments, multiple doses of the cells of the first and/or second administration are given.
Among the diseases, conditions, and disorders are tumors, including solid tumors, hematologic malignancies, and melanomas, and including localized and metastatic tumors, infectious diseases, such as infection with a virus or other pathogen, e.g., HIV, HCV, HBV, CMV, HPV, and parasitic disease, and autoimmune and inflammatory diseases. In some embodiments, the disease or condition is a tumor, cancer, malignancy, neoplasm, or other proliferative disease or disorder. Such diseases include but are not limited to leukemia, lymphoma, e.g., chronic lymphocytic leukemia (CLL), ALL, non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, refractory follicular lymphoma, mantle cell lymphoma, indolent B cell lymphoma, B cell malignancies, cancers of the colon, lung, liver, breast, prostate, ovarian, skin, melanoma, bone, and brain cancer, ovarian cancer, epithelial cancers, renal cell carcinoma, pancreatic adenocarcinoma, Hodgkin lymphoma, cervical carcinoma, colorectal cancer, glioblastoma, neuroblastoma, Ewing sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and/or mesothelioma.
In some embodiments, the disease or condition is a tumor and the subject has a large tumor burden prior to the administration of the first dose, such as a large solid tumor or a large number or bulk of disease-associated, e.g., tumor, cells. In some aspects, the subject has a high number of metastases and/or widespread localization of metastases. In some aspects, the tumor burden in the subject is low and the subject has few metastases. In some embodiments, the size or timing of the doses is determined by the initial disease burden in the subject. For example, whereas in some aspects the subject may be administered a relatively low number of cells in the first dose, in context of lower disease burden the dose may be higher.
In some embodiments, the disease or condition is an infectious disease or condition, such as, but not limited to, viral, retroviral, bacterial, and protozoal infections, immunodeficiency, Cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus. In some embodiments, the disease or condition is an autoimmune or inflammatory disease or condition, such as arthritis, e.g., rheumatoid arthritis (RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Grave's disease, Crohn's disease, multiple sclerosis, asthma, and/or a disease or condition associated with transplant.
In some embodiments, the antigen associated with the disease or disorder is selected from the group consisting of orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, OEPHa2, ErbB2, 3, or 4, FBP, fetal acethycholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1), a cyclin, such as cyclin A1 (CCNA1), and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens.
As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply necessarily complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.
As used herein, “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.
“Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided cells and compositions are used to delay development of a disease or to slow the progression of a disease.
As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, cells that suppress tumor growth reduce the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the cells.
An “effective amount” of an agent, e.g., a pharmaceutical formulation, cells, or composition, in the context of administration, refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.
A “therapeutically effective amount” of an agent, e.g., a pharmaceutical formulation or cells, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered. In some embodiments, the provided methods involve administering the cells and/or compositions at effective amounts, e.g., therapeutically effective amounts.
A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. In the context of lower tumor burden, the prophylactically effective amount in some aspects will be higher than the therapeutically effective amount.
Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.
In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.
In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical or similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.
The cells can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intrathoracic, intracranial, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration of the cells. In some embodiments, it is administered by multiple bolus administrations of the cells, for example, over a period of no more than 3 days, or by continuous infusion administration of the cells.
For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.
In some embodiments, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or other agent, such as a cytotoxic or therapeutic agent. Thus, the cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents includes a cytokine, such as IL-2 or other cytokine, for example, to enhance persistence.
In some embodiments, the methods comprise administration of a chemotherapeutic agent, e.g., a conditioning chemotherapeutic agent, for example, to reduce tumor burden prior to the dose administrations.
Once the cells are administered to the subject (e.g., human), the biological activity of the engineered cell populations in some aspects is measured by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells also can be measured by assaying expression and/or secretion of certain cytokines, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load. In some aspects, toxic outcomes, persistence and/or expansion of the cells, and/or presence or absence of a host immune response, are assessed.
In certain embodiments, engineered cells are modified in any number of ways, such that their therapeutic or prophylactic efficacy is increased. For example, the engineered CAR or TCR expressed by the population can be conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating compounds, e.g., the CAR or TCR, to targeting moieties is known in the art. See, for instance, Wadwa et al., J. Drug Targeting 3: 1 1 1 (1995), and U.S. Pat. No. 5,087,616.
III. Recombinant Receptors Expressed by the Cells
The cells generally express recombinant receptors. The receptors expressed by the cells of the different doses typically are distinct from one another, at least in part. The receptors may include antigen receptors, such as functional non-TCR antigen receptors, including chimeric antigen receptors (CARs), and other antigen-binding receptors such as transgenic T cell receptors (TCRs). The receptors may also include other chimeric receptors, such as receptors binding to particular ligands and having transmembrane and/or intracellular signaling domains similar to those present in a CAR.
Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in international patent application publication numbers WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the antigen receptors include a CAR as described in U.S. Pat. No. 7,446,190, and those described in International Patent Application Publication No.: WO/2014055668 A1. Examples of the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190, 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also International Patent Publication No.: WO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, 7,446,190, and 8,389,282, and U.S. patent application Publication No. US 2013/0149337. Among the chimeric receptors are chimeric antigen receptors (CARs). The chimeric receptors, such as CARs, generally include an extracellular antigen binding domain, such as a portion of an antibody molecule, generally a variable heavy (VH) chain region and/or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment.
In some embodiments, the binding domain(s), e.g., the antibody, e.g., antibody fragment, portion of the recombinant receptor further includes at least a portion of an immunoglobulin constant region, such as a hinge region, e.g., an IgG4 hinge region, and/or a CH1/CL and/or Fc region. In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgG1. In some aspects, the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. Exemplary spacers, e.g., hinge regions, include those described in international patent application publication number WO2014031687. In some examples, the spacer is or is about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, international patent application publication number WO2014031687, U.S. Pat. No. 8,822,647 or published app. No. US2014/0271635.
In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgG1. In some embodiments, the spacer has the sequence ESKYGPPCPPCP (set forth in SEQ ID NO: 1), and is encoded by the sequence set forth in SEQ ID NO: 158. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 107. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 108. In some embodiments, the constant region or portion is of IgD. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 109. In some embodiments, the spacer has a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 1, 107, 108 or 109.
This antigen recognition domain generally is linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, and/or signal via another cell surface receptor. The signal may be immunostimulatory and/or costimulatory in some embodiments. In some embodiments, it may be suppressive, e.g., immunosuppressive. Thus, in some embodiments, the antigen-binding component (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154 and/or transmembrane regions containing functional variants thereof such as those retaining a substantial portion of the structural, e.g., transmembrane, properties thereof. In some embodiments, the transmembrane domain is a transmembrane domain derived from CD4, CD28, or CD8, e.g., CD8alpha, or functional variant thereof. The transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and/or transmembrane domain(s).
Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
The receptor, e.g., the CAR, generally includes at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the antigen-binding portion is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR or other chimeric receptor includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25 or CD16.
In some embodiments, upon ligation of the CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptors to initiate signal transduction following antigen receptor engagement.
In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.
T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.
In some aspects, the CAR includes a primary cytoplasmic signaling sequence derived from a signaling molecule or domain that promotes primary activation of a TCR complex in a natural setting. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from the CD3 zeta chain, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.
In some embodiments, the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some aspects, the same CAR includes both the activating and costimulatory components.
In some embodiments, the activating domain is included within one CAR, whereas the costimulatory component is provided by another CAR recognizing another antigen, present on the same cell. In some embodiments, the CARs include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see WO2014/055668). In some aspects, the cells include one or more stimulatory or activating CAR and/or a costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than the one associated with and/or specific for the disease or condition whereby an activating signal delivered through the disease-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.
In some embodiments, the intracellular signaling component of the recombinant receptor, such as CAR, comprises a CD3 zeta intracellular domain and a costimulatory signaling region. In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD 137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.
In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.
In some embodiments, the CAR or other antigen receptor further includes a marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor, such as a truncated version of a cell surface receptor, such as truncated EGFR (tEGFR). In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. For example, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence. An exemplary polypeptide for a truncated EGFR (e.g. tEGFR) comprises the sequence of amino acids set forth in SEQ ID NO: 111 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 111. An exemplary T2A linker sequence comprises the sequence of amino acids set forth in SEQ ID NO: 110 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 110.
In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.
In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self” by the immune system of the host into which the cells will be adoptively transferred.
In some embodiments, the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive transfer and encounter with ligand.
In some cases, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR is one that includes multiple costimulatory domains of different costimulatory receptors.
In some embodiments, the chimeric antigen receptor includes an extracellular portion containing an antigen-binding domain, such as an antibody or antigen-binding antibody fragment, such as an scFv or Fv. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment includes an scFv and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain includes a signaling domain of a zeta chain of a CD3-zeta (CD3) chain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some aspects, the transmembrane domain contains a transmembrane portion of CD28. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. The extracellular domain and transmembrane domain can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein. In some embodiments, the receptor contains extracellular portion of the molecule from which the transmembrane domain is derived, such as a CD28 extracellular portion. In some embodiments, the chimeric antigen receptor contains an intracellular domain derived from a T cell costimulatory molecule or a functional variant thereof, such as between the transmembrane domain and intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.
For example, in some embodiments, the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some embodiments, the CAR contains an antibody, e.g., antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4-1BB or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some such embodiments, the receptor further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4 hinge, such as a hinge-only spacer.
In some embodiments, the transmembrane domain of the recombinant receptor, e.g., the CAR, is or includes a transmembrane domain of human CD28 (e.g. Accession No. P01747.1) or variant thereof, such as a transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 2 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2; in some embodiments, the transmembrane-domain containing portion of the recombinant receptor comprises the sequence of amino acids set forth in SEQ ID NO: 104 or a sequence of amino acids having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.
In some embodiments, the intracellular signaling component(s) of the recombinant receptor, e.g. the CAR, contains an intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as a domain with an LL to GG substitution at positions 186-187 of a native CD28 protein. For example, the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 112 or 113 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 112 or 113. In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of 4-1BB (e.g. (Accession No. Q07011.1) or functional variant or portion thereof, such as the sequence of amino acids set forth in SEQ ID NO: 3 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 3.
In some embodiments, the intracellular signaling domain of the recombinant receptor, e.g. the CAR, comprises a human CD3 zeta stimulatory signaling domain or functional variant thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human CD3 (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. Nos. 7,446,190 or 8,911,993. For example, in some embodiments, the intracellular signaling domain comprises the sequence of amino acids as set forth in SEQ ID NO: 4, 105 or 159 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 4, 105 or 159.
In some aspects, the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgG1, such as the hinge only spacer set forth in SEQ ID NO: 1. In other embodiments, the spacer is or contains an Ig hinge, e.g., an IgG4-derived hinge, optionally linked to a CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains, such as set forth in SEQ ID NO: 108. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a CH3 domain only, such as set forth in SEQ ID NO: 107. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers.
For example, in some embodiments, the CAR includes an antibody such as an antibody fragment, including scFvs, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR includes an antibody or fragment, such as scFv, a spacer such as any of the Ig-hinge containing spacers, a CD28-derived transmembrane domain, a 4-1BB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.
In some embodiments, nucleic acid molecules encoding such CAR constructs further includes a sequence encoding a T2A ribosomal skip element and/or a tEGFR sequence, e.g., downstream of the sequence encoding the CAR. In some embodiments, the sequence encodes a T2A ribosomal skip element set forth in SEQ ID NO: 110, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 110. In some embodiments, T cells expressing an antigen receptor (e.g. CAR) can also be generated to express a truncated EGFR (EGFRt) as a non-immunogenic selection epitope (e.g. by introduction of a construct encoding the CAR and EGFRt separated by a T2A ribosome switch to express two proteins from the same construct), which then can be used as a marker to detect such cells (see e.g. U.S. Pat. No. 8,802,374). In some embodiments, the sequence encodes an tEGFR sequence set forth in SEQ ID NO:111, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 111.
The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided receptors and other polypeptides, e.g., linkers or peptides, may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
The recombinant receptors, such as CARs, expressed by the cells administered to the subject in the various doses generally recognize or specifically bind to a molecule that is expressed in, associated with, and/or specific for the disease or condition or cells thereof being treated. Upon specific binding to the molecule, e.g., antigen, the receptor generally delivers an immunostimulatory signal, such as an ITAM-transduced signal, into the cell, thereby promoting an immune response targeted to the disease or condition. For example, in some embodiments, the cells in the first dose express a receptor, e.g., CAR, that specifically binds to an antigen expressed by a cell or tissue of the disease or condition or associated with the disease or condition.
In some embodiments, the cells in the subsequent dose express a receptor, e.g., CAR, that specifically binds to an antigen expressed by a cell or tissue of the disease or condition or associated with the disease or condition. In some aspects, the receptor expressed by the cells of the second dose specifically binds the same antigen as, or competes for binding with, the receptor of the first dose. In other embodiments, the receptor expressed by the cells of the second dose specifically binds to a different antigen than that bound by the receptor of the first dose.
Thus, in some embodiments, the second receptor (e.g., the second CAR) differs to some degree, e.g., in amino acid sequence and/or immunological epitope(s), from the first receptor (e.g. first CAR). For example, in some aspects, the receptor, e.g., the CAR, expressed by the cells administered in the first dose contains at least one immunoreactive epitope that is not expressed by the cells of the subsequent dose. In some embodiments, the receptor, e.g., CAR, expressed by the cells of the second dose, does not contain an immunoreactive epitope expressed by the cells of the first dose. Exemplary immunoreactive epitopes include B cell epitopes and T cell epitopes, which may be recognized by the immune system of the subject to which the cells are administered.
Thus, in some embodiments, one or more component of the CAR of the subsequent dose is distinct from the CAR of the first dose. In some embodiments, the second (or subsequent) receptor, e.g., CAR, includes one or more differences in amino acid sequence compared to the first or prior receptor. For example, in some aspects, the CAR expressed by the cells of the subsequent dose contains a distinct scFv, distinct signaling domains, and/or distinct junctions as compared to the CAR expressed by the cells of the first dose. In some embodiments, sequences in the first and/or second receptor or other molecule that are non-endogenous to the host, e.g., not present as such in a molecule present naturally in the host. Exemplary of such non-endogenous sequences are sequences spanning the junctions of non-naturally associated or fused domains within a chimeric molecule, such as a CAR. In some embodiments, the CAR expressed by the cells of the subsequent dose contains distinct costimulatory, stimulatory, transmembrane, and/or other domains from that of the first dose.
Such differences may include at least one difference compared to a region of the first or prior receptor to which a detectable immune response is exhibited in the subject following the first or prior administration, e.g., a difference in a region in the second or subsequent receptor that corresponds to such a region in the first or prior receptor. Regions including the difference(s) may include an antigen-binding portion, such as an scFv portion, including framework region(s) within an scFv or variable region portion, such as a heavy and/or light chain variable region portion, a linker portion, a hinge portion, a junction between two CAR domains, and/or a transduction or expression marker.
In some embodiments, the first or other prior and second or other subsequent receptors may include regions of similarity, e.g., regions of amino acid sequence identity. For example, in some embodiments, the CAR expressed by the cells of the subsequent dose contains the same scFv, the same signaling domains, and/or the same junctions as the CAR expressed by the cells of the first dose. In some embodiments, it further contains the same costimulatory, stimulatory, transmembrane, and/or other domains as that of the first dose.
In some aspects, the region(s) of identity are ones to which the subject does not or is unlikely to exhibit an immune response following the first or prior administration. Such regions may include regions within a costimulatory domain, an ITAM-containing domain, a transmembrane domain, a CDR, and/or a transduction or expression marker.
In some aspects, the antigen-binding domains, such as the antibody or antibody fragments and/or domains thereof, e.g., light and/or heavy chain variable regions, e.g., scFvs, of the second receptor, e.g., second CAR, is or are derived from a different species than that or those of the first receptor, e.g., first CAR. Exemplary species from which such domains or fragments may be derived include human and non-human species, such as mouse. For example, in some embodiments, an scFv of a first or prior CAR is derived from mouse antibody or antibody with a murine-derived portion, such as an FMC63 or SJ25C1 scFv, and the scFv of the second or subsequent CAR is derived from a human antibody or antibody fragment, or vice versa.
In some embodiments, the antigen-binding domain, e.g., antibody portion or fragment, e.g., scFv, of the first CAR and that of the second CAR are derived from the same species. In some such embodiments, the domain of the first and second receptors are derived from the same species but contain one or more differences in sequence. In some aspects, the scFv of the first CAR is derived from a mouse sequence, e.g., FMC63, and the scFv of the second CAR is derived from a distinct mouse sequence, e.g., SJ25C1, or vice versa.
In some aspects, the receptor, e.g., CAR, of the second dose contains the same antigen binding domain, e.g., antibody fragment or portion, e.g., scFv, as the receptor, e.g., CAR, of the first dose. In some embodiments, such a subsequent or second receptor contains one or more distinct junctional region as compared to the CAR of the first or prior dose.
In some such embodiments, administration of cells expressing the second or subsequent CAR results in reduced elimination of the cells of the second or subsequent dose as compared to a method in which the second CAR contains the same junction or junctions as the cells of the first dose and/or contains non-endogenous sequence(s) present in the receptor of the first dose.
In some aspects, the first and second receptor target the same antigen. In some embodiments, the first and second receptor target the same epitope of the antigen. For example, in some embodiments, the first and second receptor target the same or an overlapping epitope and/or compete for binding to the antigen with one another, but are derived from different species, such as mouse and human, respectively. In other embodiments, the first and second receptor target different epitopes on the same antigen.
In some embodiments, the second molecule, e.g., receptor, e.g., CAR, does not include one or more immunogenic portion(s) contained in the first; in some embodiments, the second receptor does not contain any immunogenic portions in the first receptor (or portions deemed to be immunogenic by a specified assay). In some such embodiments, the second receptor, e.g., CAR, is specifically chosen and/or designed so that it does not include an immunogenic portion(s) contained in the first receptor and/or does not contain a portion deemed to be immunogenic, e.g., in a particular subject and/or to which a specific immune response has been detected, e.g., in the subject being treated. In some aspects, the immunogenic portion(s) of the first receptor, e.g., CAR, has/have been replaced with a distinct sequence or distinct sequences.
In some embodiments, such as where the subject has become resistant to treatment targeting the antigen or other binding partner targeted by the first receptor or molecule, and/or where the antigen or binding partner is or has been down-regulated or mutated in the subject or disease tissue (e.g., tumor), the receptor (e.g., CAR) of the second dose is designed or chosen to target a distinct antigen as compared to that targeted by the receptor of the first dose. In some such aspects, administration of the second dose (i.e. the cells expressing the second molecule, e.g., second receptor) effects a larger reduction in disease burden in the subject, e.g., tumor burden, than administration of a subsequent dose of the same cells, cells expressing the same receptor, and/or containing cells expressing a receptor that targets the same antigen.
In this respect, the methods in some embodiments may be useful in treating subjects whose disease or condition has become resistant to treatments targeting a particular epitope or antigen or other disease target, such as resulting from down-regulation or mutation by the disease or condition or cells thereof. Thus, by offering the ability to target a similar but distinct disease-associated epitope or disease, the methods in some embodiments improve efficacy not only by increasing overall exposure of the subject to cells expressing the receptors or other recombinant molecules, e.g., via increased expansion or persistence of such engineered cells in the subject, but also by allowing the cells to function even in the context of downregulation or mutation of the original target.
IV. Host Immune Responses to Administered Cells
In some embodiments, the efficacy of adoptive cell therapy may be limited by the development of an immune response in the subject to the cells and/or construct administered. It is observed herein that even in certain subjects having B cell malignancies, who often are immunocompromised, immune responses can be detected that are specific for regions of receptors expressed by cells administered in adoptive cell therapy. Additionally, in some contexts, loss, downregulation, and/or modification, of a disease-specific or disease-associated antigen being targeted by cell therapy can occur in a subject, which can impair efficacy of therapy targeting that antigen or epitope. For example, CD19-negative disease have been observed in certain subjects having been treated with anti-CD19 immunotherapy. In some embodiments, the provided methods provide offer improved efficacy in one or more of such contexts.
In some embodiments of the provided methods, one or more of the doses or administrations, e.g., the subsequent dose(s) or administration of cells expressing the second receptor, is administered at a time at which an immune response, e.g., an adaptive or specific immune response to the first recombinant receptor and/or cells, in the subject is present, detectable, or detectable above a certain level. The presence or degree of a specific immune response to the recombinant molecule can be related to the immunogenic properties of the receptor, e.g., the CAR or transgenic TCR, expressed by the cells, and/or the time during which the subject has been exposed thereto. For example, in some embodiments, an immune response, e.g., a specific humoral and/or cell-mediated immune response against the receptor, is detected at or about 28 days, at or about 35 days, or at or about 42 days following the first exposure of the subject to the cells expressing the first receptor. Thus, in some embodiments, the subsequent dose of receptor-expressing cells that do not express the receptor expressed by the cells of the first dose, is administered after an immune response, an adaptive or specific immune response, a detectable immune response, and/or a memory response against the first recombinant receptor or cells of the first or prior dose has developed in the subject. In this regard, the ability of cells of the subsequent dose to expand and/or persist in the subject is improved in comparison to other methods in which the cells of the subsequent dose express the same receptor as the first dose. In some embodiments, the second or subsequent dose is administered at a point in time that is at least or is greater than at or about 28 days, 35 days, or 42 days. In some embodiments, it is administered at or about or at least at or about 14 or 21 days.
The methods may involve the detection of the presence or absence or level of such an immune response or indicator thereof, for example, following the administration of a first or second dose and before the administration of the subsequent or next subsequent dose.
In some embodiments, the decision of when and/or whether to administer the subsequent dose depends on whether the subject exhibits such an immune response or detectable readout thereof, e.g., a detectable specific or adaptive host immune response specific for the cells or recombinant receptor, e.g., CAR, expressed by the cells of the first dose, and/or whether such a response is detected above a certain level. In some embodiments, where such a response is detected, the subject is administered the subsequent dose.
In general, the subsequent dose is administered at a time at which the subject exhibits a specific or adaptive, e.g., humoral or cell-mediated, immune response against the receptor, e.g., CAR, expressed by the cells of the first dose, or exhibits such a response or indicator thereof at a detectable level or above an acceptable level. In some aspects, at the time of administration of the subsequent dose, the subject exhibits a humoral or cell-mediated immune response against the receptor, e.g., CAR, expressed by the cells of the first dose.
In some embodiments, the host immune response is or comprises a humoral immune response. The humoral immune response may be indicated by the presence of antibodies specific for the cells or receptors expressed thereby in the serum, other bodily fluid, and/or organ or tissue of the subject. In some embodiments, such antibodies of a particular isotype are present, such as IgM or IgG, e.g., IgG1, IgG2, IgG3, and/or IgG4; in some embodiments they include IgE.
In some embodiments, the immune response is or comprises a cell-mediated component. A cell-mediated response may be indicated by the presence of cells, e.g., T cells, e.g., helper or cytotoxic T cells, that specifically recognize one or more epitopes of the recombinant receptor or cells via a T cell receptor.
In some embodiments the immune response is a primary immune response; in some aspects, the immune response is a memory response.
In some of any of the above embodiments, a detectable immune response refers to an amount detectable by any of a number of known methods for assessing specific immune responses to particular antigens and cells. For example, in some embodiments, the immune response of the specified type is detectable by performing ELISpot, ELISAs, or cell-based antibody detection methods, for example, by flow cytometry, on serum from the subject to detect the presence of antibodies that specifically bind to and/or neutralize antigens present on the cells, e.g., binding to epitopes of the recombinant receptor, e.g., CAR. In some such assays, isotype of the detected antibody is determined and may indicate the type of response and/or whether the response is a memory response.
In some embodiments, the specified immune response is detectable by cytotoxic T-lymphocyte (CTL) assays for detection of CD8+ T cells that specifically bind to and induce cytotoxicity in response to epitopes in the recombinant receptor, and/or a mixed lymphocyte reaction, using cells, e.g., irradiated cells, expressing the recombinant receptor, as stimulator cells.
In some aspects, the detectable immune response is one that is detected by such a method above or significantly above the level of a control sample, such as a non-coated well or well coated with a control peptide or cells not expressing the recombinant receptor and/or levels detected based on pre-treatment serum or blood sample from the subject prior to treatment with the cells expressing the recombinant receptors.
In some aspects, the presence or absence of such a host immune response and/or quantity, degree, or extent thereof, is detected or measured, for example, following the administration of the first dose or subsequent dose(s).
Humoral immune responses may be detected by any of a number of well-known assays for detection of antibodies specific for particular antigens or cells, including binding assays, immunoassays, and including cell-based assays. The assays may include those designed to assess the presence or absence of particular functions of the antibodies, such as their ability to carry out a particular effector function upon binding to the antigen, such as neutralizing antibody assays. In some embodiments, outcomes of humoral immune responses, such as antigen-specific antibodies, e.g., neutralizing antibodies, are detected using cell-based assays, e.g., by incubating pre- and post-treatment cells from the subject with cells expressing the recombinant receptor (and control cells) and detecting antigen-specific binding and/or other outcomes, such as neutralizing outcomes, e.g., by flow cytometry or enzymatic assays. In some embodiments, ELISA, and/or ELISpot assays are used to detect and quantify antibodies specific for the recombinant receptors, such as CARs, and epitopes mapped using known techniques, such as those using individual peptides representing portions of the receptor. See, e.g., Berger et al. Blood. 2006 March; 107(6): 2294-2302, Berger et al. J Virol. 2001 January 75(2): 799-808, Riddell et al. Nature Medicine. 1996 February 2(2): 216-223, Berger et al. Blood. 2005 February 105(4): 1640-1647, Jensen et al. Biol Blood Marrow Transplant. 2010 September; 16(9): 1245-1256. In some embodiments isotype of the detected antibodies are assessed, for example by using detection antibodies specific for particular isotypes, e.g., human isotypes.
Cellular or cell-based immune response to the cells and/or receptors may be detected and/or measured using any of a number of well-known techniques. Such techniques may include cytotoxic T-lymphocyte (CTL) assays for detection of CD8+ T cells that specifically bind to and induce cytotoxicity in response to epitopes in the recombinant receptor, e.g., CAR, and/or cells administered. In some embodiments, the assay is a mixed lymphocyte reaction, such as those using PBMCs or other host-derived cells from blood or other organ or tissue as responder cells, and cells induced to express the recombinant receptor, e.g., irradiated T cells expressing the CAR, as stimulator cells. The stimulator cells generally are autologous and may be the same cells administered to the subject, and may be irradiated. Non-transduced cells or cells not expressing the transgene of interest may be used as negative controls in place of the stimulator cells in control samples. Likewise, responder cell samples from pre-treated time points or other subjects may be used in control samples. In some aspects, such assays assess the ability of host cells to carry out one or more effector functions, e.g., antigen-specific cell lysis, e.g., using a chromium release assay to detect cytotoxic T cells present in the subject which specifically recognize and antigens present on or in the administered cells and induce a cytotoxic response. In some embodiments, peripheral blood cells, e.g., PBMCs, are obtained from a subject before and after administration of the cells, and each used in an assay, such as a cell lysis assay, using autologous T cells modified to express the recombinant receptor, which generally are irradiated. Specific lysis indicates the presence of receptor-specific cell-mediated immune response. Epitope mapping may be carried out using panels of peptides representing portions of the recombinant receptor. See, e.g., Berger et al. Blood. 2006 March; 107(6): 2294-2302, Berger et al. J Virol. 2001 January 75(2): 799-808, Riddell et al. Nature Medicine. 1996 February 2(2): 216-223, Berger et al. Blood. 2005 February 105(4): 1640-1647, Lamers, Blood 2011 117: 72-82. HLA tetramer binding assays may be used for the enumeration of antigen-specific T cells. In some aspects, lymphoproliferative assays (LPAs) and/or assays to assess for secreted cytokines, such as ELISAs and/or intracellular staining and assessment by flow cytometry, are used for detection of transgene-specific CD4+ T cells.
In some embodiments, the presence or absence of a specific immune response against the cells or receptor in the subject is assessed, for example, by any of the assays described herein, e.g., epitope mapping. In some aspects, immunogenic epitopes and/or regions within the first receptor, e.g., to which an immune response has developed or is likely to have developed in the subject following the first administration, are determined and a second receptor chosen that does not contain such immunogenic epitopes or regions and/or that contains amino acid differences in such regions.
In some embodiments, the presence or absence of such an immune response after the first or prior administration is detected, and informs which differences are designed to be present in the second or subsequent receptor as compared to the first or prior receptor. Such detection may include identifying at least a region of the first or otherwise prior receptor (e.g., CAR) to which the subject exhibits a specific immune response.
Thus, in some embodiments, the cells of the second dose are selected based on the receptor that they express. In some embodiments, the second dose contains cells expressing a receptor (e.g., CAR) that does not contain a particular immunoreactive epitope for which the subject has developed an immune response following administration of the first or prior dose and/or does not contain any such immunoreactive epitope or any such epitope determined to be immunogenic. In some aspects, a subject may be administered a second or subsequent dose of cells expressing a receptor that is distinct from the receptor expressed by the cells of the first dose in one or more regions determined to be immunogenic. Thus, in some embodiments, the selection of the receptor-expressing cells to be administered in the second (or other subsequent) dose is patient-specific.
In some embodiments, administration of the second dose containing cells that express a receptor, e.g. CAR, that is distinct from the receptor expressed by the cells of the first dose, does not elicit a detectable humoral or cell-mediated immune response specific for the receptor of the first dose. In some aspects, the second dose does not elicit a detectable humoral or cell-mediated immune response against the receptor expressed by the cells of the second dose.
Thus, in some embodiments, the subject does not exhibit an immune response, such as a detectable immune response, e.g., a humoral or cell-mediated immune response, against the second receptor following the administration of the cells expressing the second receptor, or does not exhibit such a response within a certain time period, such as within about 60 days of the administration of those cells.
In some embodiments, the method prevents the induction of or reduces the level of antibodies against the receptor expressed by the cells of the second dose. For example, antibody titers of anti-receptor, e.g. anti-CAR, antibodies, for example, as measured in the serum of the subject by ELISA, are decreased following administration of the subsequent dose, as compared to methods in which a subsequent dose of cells expressing the same receptor as the cells of the first dose is administered. Thus, in some embodiments, the methods improve efficacy by increasing exposure of the subject to the administered cells by preventing or reducing host immune responses that would otherwise clear or prevent expansion of the administered cells.
V. Dosing
The methods generally are designed to improve efficacy of adoptive cell therapy, such as by providing increased exposure of the subject to the cells, e.g., over time. The methods involve administering a first dose, generally followed by a second and/or one or more additional subsequent doses, with particular time frames between certain different doses.
In the context of adoptive cell therapy, administration of a given “dose” encompasses administration of the given amount or number of cells as a single composition and/or single uninterrupted administration, e.g., as a single injection or continuous infusion, and also encompasses administration of the given amount or number of cells as a split dose, provided in multiple individual compositions or infusions, over a specified period of time, which is no more than 3 days. Thus, in some contexts, the dose is a single or continuous administration of the specified number of cells, given or initiated at a single point in time. In some contexts, however, the dose is administered in multiple injections or infusions over a period of no more than three days, such as once a day for three days or for two days or by multiple infusions over a single day period.
Thus, in some aspects, the cells are administered in a single pharmaceutical composition.
In some embodiments, the cells are administered in a plurality of compositions, collectively containing the cells of a single dose.
The term “split dose” refers to a dose that is split so that it is administered over more than one infusion, e.g., over more than one day. This type of dosing is encompassed by the present methods and is considered to be a single dose. The split dose is infused over a period of no more than three days.
Thus, one or more of the doses in some aspects may be administered as a split dose. For example, in some embodiments, the dose may be administered to the subject over 2 days or over 3 days. Exemplary methods for split dosing include administering 25% of the dose on the first day and administering the remaining 75% of the dose on the second day. In other embodiments 33% of the dose may be administered on the first day and the remaining 67% administered on the second day. In some aspects, 10% of the dose is administered on the first day, 30% of the dose is administered on the second day, and 60% of the dose is administered on the third day. In some embodiments, the split dose is not spread over more than 3 days.
In some embodiments, multiple doses are given, in some aspects using the same timing guidelines as those with respect to the timing between the first and second doses, e.g., by administering a first and multiple subsequent doses, with each subsequent dose given at a point in time that is greater than about 28 days after the administration of the first or prior dose.
As used herein, “first dose” is used to describe the timing of a given dose being prior to the administration of a subsequent or second dose. The term does not necessarily imply that the subject has never before received a dose of cell therapy or even that the subject has not before received a dose of the same cells or cells expressing the same or different recombinant receptor or targeting the same or different antigen. For example, in some embodiments, the first dose as used herein represents the second or greater infusion of the cells to the subject.
As used herein, “second dose” is used to describe the timing of a given dose being subsequent to the administration of a prior, e.g., first, dose. The term does not necessarily imply that the subject has only before received one dose of cell therapy or that the subject has only before received doses of cells expressing the same recombinant receptor or targeting the same antigen. In some embodiments, multiple doses are administered between the first and second doses, and/or prior to the first or subsequent to the second dose. For example, multiple doses of cells of the first dose (or cells expressing the receptor of the first does) may be administered, followed by multiple doses of the cells or receptor of the second dose. The terms first and second are merely used to describe different doses relative in time to one another.
With reference to a prior dose, such as a first dose, the term “subsequent dose” refers to a dose that is administered to the same subject after the prior, e.g., first, dose. In some aspects, the subsequent dose is the second, third, fourth, and so forth, dose. Neither the term “subsequent” nor a particular numerical value (e.g., “second”), when describing a dose, implies the absence of intervening doses.
In some embodiments, one or more consecutive doses of cells expressing the same receptor, e.g., CAR, as the first dose, may be administered to the subject.
With reference to a prior dose, such as a first dose, the term “consecutive dose” refers to a dose that is administered to the same subject after the prior, e.g., first, dose without any intervening doses having been administered to the subject in the interim. Nonetheless, the term does not encompass the second, third, and/or so forth, injection or infusion in a series of infusions or injections comprised within a single split dose. Thus, unless otherwise specified, a second infusion within a one, two or three-day period is not considered to be a “consecutive” dose as used herein. Likewise, a second, third, and so-forth in the series of multiple doses within a split dose also is not considered to be an “intervening” dose in the context of the meaning of “consecutive” dose. Thus, unless otherwise specified, a dose administered a certain period of time, greater than three days, after the initiation of a first or prior dose, is considered to be a “consecutive” dose even if the subject received a second or subsequent injection or infusion of the cells following the initiation of the first dose, so long as the second or subsequent injection or infusion occurred within the three-day period following the initiation of the first or prior dose.
Thus, unless otherwise specified, multiple administrations of the same cells over a period of up to 3 days is considered to be a single dose, and administration of cells within 3 days of an initial administration is not considered a consecutive dose and is not considered to be an intervening dose for purposes of determining whether a second dose is “consecutive” to the first.
In some embodiments, multiple consecutive doses are given. The multiple consecutive doses may be administered, for example, using the same timing guidelines as those specified for the timing between a first and first consecutive dose, such as by administering a first and multiple consecutive doses, with each consecutive dose given within a period of time that is greater than about 14 and less than about 28 days, e.g., about 21 days, after the administration of the first or immediately prior dose. In some embodiments, with respect to the first dose, the consecutive doses each include the same cells as those administered in the first dose, and/or the same recombinant receptor expressed by those in the first dose. In some such embodiments, administration of such consecutive dose(s) is followed by administration of a second dose, and in some cases additional consecutive dose(s).
Thus, in some aspects, the subject may be administered multiple doses of cells expressing the same receptor. In some embodiments, multiple consecutive doses containing cells expressing a first receptor may be administered to the subject prior to the subsequent administration of cells expressing a distinct receptor, such as prior to the administration of the second dose, which may in some embodiments also be followed by a consecutive administration of cells of the second dose. In some aspects, the multiple consecutive doses of cells expressing the second (or third, fourth, fifth, and so forth) receptor may be administered to the subject.
In some embodiments, for example, in the context of multiple clinical trials, a subject may be administered a first dose of cells expressing a receptor being investigated in a first clinical trial and a second (or other subsequent) dose of cells expressing a receptor being investigated in a second clinical trial.
In some embodiments, the provided methods are for long-term or continuous treatment or management of the disease or disorder in the subject, involving first, second, third, and/or multiple additional subsequent administrations of engineered cells, each expressing distinct recombinant receptors targeting the same disease or condition in the subject. Such long-term treatment or management may involve an iterative process, in which the subject is monitored and a next subsequent administration (e.g., next subsequent receptor) is introduced if and when a particular indicator of loss of efficacy or risk thereof is detected. In some embodiments, each subsequent administration is initiated upon detection of one or more indicators of a risk of loss of efficacy, such as reduced persistence of, expansion of, or exposure to the cells in the prior dose, an immune response specific thereto in the subject, relapse, resistance, and/or downregulation or change in the target antigen.
In some embodiments, the subsequent dose is administered for retreatment upon relapse, and/or to prevent recurrence of the targeted disease or disorder, and/or to address or prevent a reduction in exposure to cells expressing the recombinant receptors following a first dose, for example, upon detection of a decline in persistence or expansion of such cells or in numbers of such cells. Thus, in some embodiments one or more of these parameters is measured, detected, or assessed in the time between the first or other prior dose and the second or other subsequent dose, and the timing or decision to administer the subsequent dose is made based on the outcome of such assessment. For example, the second dose may be administered at a time at which it is determined that the number or concentration of the receptor-expressing cells is below a desired level or has declined below a certain percentage of maximum or other measured concentration or number.
Dosage Amount or Size
In some embodiments, the first or subsequent dose contains a number of cells, number of recombinant receptor (e.g., CAR)-expressing cells, number of T cells, or number of peripheral blood mononuclear cells (PBMCs) in the range from about 105 to about 106 of such cells per kilogram body weight of the subject, and/or a number of such cells that is no more than about 105 or about 106 such cells per kilogram body weight of the subject. For example, in some embodiments, the first or subsequent dose includes less than or no more than at or about 1×105, at or about 2×105, at or about 5×105, or at or about 1×106 of such cells per kilogram body weight of the subject. In some embodiments, the first dose includes at or about 1×105, at or about 2×105, at or about 5×105, or at or about 1×106 of such cells per kilogram body weight of the subject, or a value within the range between any two of the foregoing values. In particular embodiments, the numbers and/or concentrations of cells refer to the number of recombinant receptor (e.g., CAR)-expressing cells. In other embodiments, the numbers and/or concentrations of cells refer to the number or concentration of all cells, T cells, or peripheral blood mononuclear cells (PBMCs) administered.
In some embodiments, for example, where the subject is a human, the first or subsequent dose includes fewer than about 1×108 total recombinant receptor (e.g., CAR)-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), e.g., in the range of about 1×106 to 1×108 such cells, such as 2×106, 5×106, 1×107, 5×107, or 1×108 or total such cells, or the range between any two of the foregoing values.
In some embodiments, the first or subsequent dose contains fewer than about 1×108 total recombinant receptor (e.g., CAR)-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs) cells per m2 of the subject, e.g., in the range of about 1×106 to 1×108 such cells per m2 of the subject, such as 2×106, 5×106, 1×107, 5×107, or 1×108 such cells per m2 of the subject, or the range between any two of the foregoing values.
In certain embodiments, the number of cells, recombinant receptor (e.g., CAR)-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs) in the first or subsequent dose is greater than about 1×106 such cells per kilogram body weight of the subject, e.g., 2×106, 3×106, 5×106, 1×107, 5×107, 1×108, 1×109, or 1×1010 such cells per kilogram of body weight and/or, 1×108, or 1×109, 1×1010 such cells per m2 of the subject or total, or the range between any two of the foregoing values.
In some embodiments, the number of cells administered in the subsequent dose is the same as or similar to the number of cells administered in the first dose in any of the embodiments herein, such as less than or no more than at or about 1×105, at or about 2×105, at or about 5×105, or at or about 1×106 of such cells per kilogram body weight of the subject. In some embodiments, the subsequent dose(s) contains at or about 1×105, at or about 2×105, at or about 5×105, or at or about 1×106 of such cells per kilogram body weight of the subject, or a value within the range between any two of the foregoing values. In some embodiments, such values refer to numbers of recombinant receptor-expressing cells; in other embodiments, they refer to number of T cells or PBMCs or total cells administered. In some aspects, the subsequent dose is larger than the first dose. For example, in some embodiments, the subsequent dose contains more than about 1×106 cells, recombinant receptor (e.g. CAR)-expressing cells, T cells, and/or PBMCs per kilogram body weight of the subject, such as about 3×106, 5×106, 1×107, 1×108, or 1×109 such cells per kilogram body weight of the subject. In some embodiments, the amount or size of the subsequent dose is sufficient to reduce disease burden or an indicator thereof, and/or one or more symptoms of the disease or condition. In some embodiments, the second (or other subsequent) dose is of a size effective to improve survival of the subject, for example, to induce survival, relapse-free survival, or event-free survival of the subject for at least 6 months, or at least 1, 2, 3, 4, or 5 years. In some embodiments, the number of cells, recombinant receptor (e.g. CAR)-expressing cells, T cells, and/or PBMCs administered and/or number of such cells administered per body weight of the subject in the subsequent dose is at least 2-fold, 5-fold, 10-fold, 50-fold, or 100-fold or more greater than the number administered in the first dose. In some embodiments, disease burden, tumor size, tumor volume, tumor mass, and/or tumor load or bulk is reduced following the subsequent dose by at least at or about 50, 60, 70, 80, 90% or more compared to that immediately prior to the administration of the first dose or of the second (or other subsequent) dose.
In other embodiments, the number of cells administered in the subsequent dose is lower than the number of cells administered in the first dose.
In some embodiments, multiple subsequent doses are administered following the first dose, such that an additional dose or doses are administered following administration of the second (or other subsequent) dose. In some aspects, the number of cells administered to the subject in the additional subsequent dose or doses (i.e., the third, fourth, fifth, and so forth) is the same as or similar to the first dose, the second dose, and/or other subsequent dose. In some embodiments, the additional dose or doses are larger than prior doses.
In some aspects, the size of the first and/or subsequent dose is determined based on one or more criteria such as response of the subject to prior treatment, e.g. chemotherapy, disease burden in the subject, such as tumor load, bulk, size, or degree, extent, or type of metastasis, stage, and/or likelihood or incidence of the subject developing toxic outcomes, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or a host immune response against the cells and/or recombinant receptors being administered.
In some aspects, the size of the first and/or subsequent dose is determined by the burden of the disease or condition in the subject. For example, in some aspects, the number of cells administered in the first dose is determined based on the tumor burden that is present in the subject immediately prior to administration of the first dose. In some embodiments, the size of the first and/or subsequent dose is inversely correlated with disease burden. In some aspects, as in the context of a large disease burden, the subject is administered a low number of cells, for example less than about 1×106 cells per kilogram of body weight of the subject. In other embodiments, as in the context of a lower disease burden, the subject is administered a larger number of cells, such as more than about 1×106 cells per kilogram body weight of the subject.
In some aspects, the number of cells administered in the subsequent dose is determined based on the tumor burden that is present in the subject following administration of the first dose. In some embodiments, e.g. where the first dose has decreased disease burden or has done so below a particular threshold amount or level, e.g., one above which there is an increased risk of toxic outcome, the subsequent dose is large, e.g. more than 1×106 cells (e.g., total cells, receptor-expressing cells, T cells, or PBMCs) per kilogram body weight, and/or is larger than the first dose. In other aspects, the number of cells administered in the subsequent dose is low, e.g. less than about 1×106, e.g. the same as or lower than the first dose, where the first dose has reduced tumor burden to a small extent or where the first dose has not led to a detectable reduction in tumor burden.
In some embodiments, the number of cells administered in the first dose is lower than the number of cells administered in other methods, such as those in which a large single dose of cells is administered, such as to administer the cells in before an immune response develops. Thus, in some embodiments, the methods reduce toxicity or toxic outcomes as compared to other methods that involve administration of a larger dose.
In some embodiments, the first dose includes the cells in an amount that does not cause or reduces the likelihood of toxicity or toxic outcomes, such as cytokine release syndrome (CRS), severe CRS (sCRS), macrophage activation syndrome, tumor lysis syndrome, fever of at least at or about 38 degrees Celsius for three or more days and a plasma level of CRP of at least at or about 20 mg/dL, and/or neurotoxicity. In some aspects, the number of cells administered in the first dose is determined based on the likelihood that the subject will exhibit toxicity or toxic outcomes, such as CRS, sCRS, and/or CRS-related outcomes following administration of the cells. For example, in some embodiments, the likelihood for the development of toxic outcomes in a subject is predicted based on tumor burden. In some embodiments, the methods include detecting or assessing the toxic outcome and/or disease burden prior to the administration of the dose.
In some embodiments, the second (or other subsequent) dose is administered at a time point at which a clinical risk for developing cytokine-release syndrome (CRS), macrophage activation syndrome, or tumor lysis syndrome, or neurotoxicity is not present or has passed or has subsided following the first administration, such as after a critical window after which such events generally have subsided and/or are less likely to occur, e.g., in 60, 70, 80, 90, or 95% of subjects with a particular disease or condition.
Timing of Doses
In some aspects, the timing of the second or subsequent dose is measured from the initiation of the first dose to the initiation of the subsequent dose. In other embodiments, the timing of the subsequent dose is measured from the completion of the first dose, or from the median day of administration of the first dose, e.g. in the context of split dosing, described herein, where a dose is administered over more than one day, e.g. over 2 days or over 3 days.
In some embodiments, whether a subsequent dose of cells expressing a receptor, e.g. CAR, that is distinct from that expressed by the cells of the first dose is administered, is determined based on the presence or degree of an immune response or detectable immune response in the subject to the cells of the first dose or recombinant receptor expressed thereby. In some aspects, a subsequent dose containing cells expressing a different receptor than the cells of the first dose will be administered to a subject with a detectable host adaptive immune response, or an immune response that has become established or reached a certain level, stage, or degree.
In some embodiments, the second (or other subsequent) dose is administered at a point in time at which a second administration of cells expressing the first receptor (e.g., CAR) is likely to be or is predicted to be eliminated by the host immune system. The likeliness of developing an immune response may be determined by measuring receptor-specific immune responses in the subject following administration of the first dose, as described herein.
For example, in some embodiments, subjects may be tested following the first (or other prior) dose and prior to the second (or other subsequent) dose to determine whether an immune response is detectable in the subject after the first dose. In some such embodiments, the detection of an immune response to the first dose may trigger the need to administer the second dose.
In some aspects, samples from the subjects may be tested as described herein to determine if there is a decline in or lower than desired exposure, for example, less than a certain number or concentration of cells, as described herein, in the subject after the first or prior dose. In some such aspects, the detection of a decline in the exposure of the subject to the cells may trigger the need to administer the second dose.
In some embodiments, the subsequent dose is administered at a point in time at which the disease or condition in the subject has not relapsed following the reduction in disease burden in response to the first or prior dose. In some embodiments, the disease burden reduction is indicated by a reduction in one or more factors, such as load or number of disease cells in the subject or fluid or organ or tissue thereof, the mass or volume of a tumor, or the degree or extent of metastases. Such a factor is deemed to have relapsed if after reduction in the factor in response to an initial treatment or administration, the factor subsequently increases.
In some embodiments, the second dose is administered at a point in time at which the disease has relapsed. In some embodiments, the relapse is in one or one or more factors, or in the disease burden generally. In some aspects, the subsequent dose is administered at a point in time at which the subject, disease burden, or factor thereof has relapsed as compared to the lowest point measured or reached following the first or prior administration, but still is lower compared to the time immediately prior to the first dose. In some embodiments, the subject is administered the subsequent dose at a point in time at which disease burden or factor indicative thereof has not changed, e.g. at a time when an increase in disease burden has been prevented.
In some embodiments, the subsequent dose is administered at a time when a host adaptive immune response is detected, has become established, or has reached a certain level, degree, or stage. In some aspects, the subsequent dose is administered following the development of a memory immune response in the subject.
In some aspects, the time between the administration of the first dose and the administration of the subsequent dose is about 28 to about 35 days, about 29 to about 35 days, or more than about 35 days. In some embodiments, the administration of the second dose is at a time point more than about 28 days after the administration of the first dose. In some aspects, the time between the first and subsequent dose is about 28 days.
In some embodiments, an additional dose or doses, e.g. subsequent doses, are administered following administration of the second dose. In some aspects, the additional dose or doses are administered at least about 28 days following administration of a prior dose. In some embodiments, no dose is administered less than about 28 days following the prior dose.
In some aspects, the present methods are advantageous in that they allow administration of receptor-expressing cells at time points that extend beyond the time range that other methods may administer a second dose containing cells that express the receptor of the first dose, e.g., after an immune response to the cells of the first dose is detected.
In some embodiments, the methods reduce toxicity or toxic outcomes as compared to other methods, for example, by allowing the second administration to occur after toxic outcomes following the first dose have cleared, e.g., which may be at a point in time at which a second administration of cells expressing the first receptor would be cleared by an immune response to the first receptor.
In some aspects, the methods allow for administration of a second dose at a point in time at which a relapse has occurred, for example, when the subject has initially responded to treatment but develops relapse, for example, at a point in time at which a second administration of cells expressing the first receptor would be cleared by an immune response to the first receptor.
In some embodiments, e.g. where one or more consecutive doses expressing the receptor expressed by the cells of the first dose are administered to the subject, the consecutive doses may be separated by about 14, about 15, about 21, about 27, or about 28 days. In some aspects, the consecutive dose is administered 21 days following a prior dose. In some embodiments, the consecutive dose is administered between 14 and 28 days following administration of a prior dose.
In any of the embodiments, the methods in some cases include the administration of the first or prior dose and the subsequent dose(s), and in other cases include the administration of the subsequent dose(s) to a subject who has previously received the first or prior dose but do not include the administration of the first or prior dose itself. Thus, the methods in some cases involve the administration of consolidating treatment, such as by administering a consolidating subsequent dose to a subject that has previously received a dose of recombinant receptor-expressing, e.g., CAR-expressing, cells.
In some embodiments, disease burden, tumor size, tumor volume, tumor mass, and/or tumor load or bulk is reduced following the subsequent dose by at least at or about 50, 60, 70, 80, 90% or more compared to that immediately prior to the administration of the first or prior dose or of the second or subsequent dose.
VI. Cell Exposure and Persistence
In some embodiments, the provided methods increase exposure of the subject to the administered cells (e.g., increased number of cells or duration over time) and/or improve efficacy and therapeutic outcomes in adoptive cell therapy. In some aspects, the methods are advantageous in that a greater and/or longer degree of exposure to the cells expressing the recombinant receptors, e.g., CAR-expressing cells, improves treatment outcomes as compared with other methods. Such outcomes may include patient survival and remission, even in individuals with severe tumor burden.
In some embodiments, the presence and/or amount of cells expressing the recombinant receptor (e.g., CAR-expressing cells) in the subject following the first dose and/or following the subsequent dose is detected. In some aspects, quantitative PCR (qPCR) is used to assess the quantity of cells expressing the recombinant receptor (e.g., CAR-expressing cells) in the blood or serum or organ or tissue (e.g., disease site) of the subject. In some aspects, persistence is quantified as copies of DNA or plasmid encoding the receptor, e.g., CAR, per microgram of DNA, or as the number of receptor-expressing, e.g., CAR-expressing, cells per microliter of the sample, e.g., of blood or serum, or per total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or T cells per microliter of the sample.
In some embodiments, the cells are detected in the subject at or at least at 4, 14, 15, 27, or 28 days following the administration of the second (or other subsequent) dose. In some aspects, the cells are detected at or at least at 2, 4, or 6 weeks following, or 3, 6, or 12, 18, or 24, or 30 or 36 months, or 1, 2, 3, 4, 5, or more years, following administration of the second (or other subsequent) dose.
In some embodiments, the persistence of receptor, e.g., CAR, -expressing cells in the subject by the methods, following administration of the subsequent dose, is greater as compared to that which would be achieved by alternative methods such as those involving the administration of a single dose or administration of a subsequent dose of cells expressing the same receptor, e.g. CAR, as the cells of the first (or other prior) dose.
In some embodiments, the persistence and/or expansion and/or presence of recombinant receptor-expressing, e.g., CAR-expressing, cells in the subject following administration of the second dose is greater as compared to that achieved via a method using an alternative dosing regimen, such as one involving the administration of a single dose of receptor-expressing cells or one involving a second dose or multiple doses of cells expressing the same receptor as expressed by the cells of the first dose.
The exposure, e.g., number of cells, indicative of expansion and/or persistence, may be stated in terms of maximum numbers of the cells to which the subject is exposed, duration of detectable cells or cells above a certain number or percentage, area under the curve for number of cells over time, and/or combinations thereof and indicators thereof. Such outcomes may be assessed using known methods, such as qPCR to detect copy number of nucleic acid encoding the recombinant receptor compared to total amount of nucleic acid or DNA in the particular sample, e.g., blood or serum, and/or flow cytometric assays detecting cells expressing the receptor generally using antibodies specific for the receptors. Cell-based assays may also be used to detect the number or percentage of functional cells, such as cells capable of binding to and/or neutralizing and/or inducing responses, e.g., cytotoxic responses, against cells of the disease or condition or expressing the antigen recognized by the receptor.
In some aspects, increased exposure of the subject to the cells includes increased expansion of the cells. In some embodiments, the receptor-(e.g., CAR-)expressing cells expand in the subject following administration of the first dose and/or following administration of the subsequent dose. In some aspects, the methods result in greater expansion of the cells compared with other methods, such as those involving the administration a single dose of cells or a subsequent dose or doses of cells expressing the same receptor as the first dose.
In some aspects, the method results in high in vivo proliferation of the administered cells, for example, as measured by flow cytometry. In some aspects, high peak proportions of the cells are detected. For example, in some embodiments, at a peak or maximum level following the first or subsequent administration, in the blood or disease-site of the subject or white blood cell fraction thereof, e.g., PBMC fraction or T cell fraction, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells express the recombinant receptor, e.g., the CAR.
In some embodiments, the method results in a maximum concentration, in the blood or serum or other bodily fluid or organ or tissue of the subject, of at least 100, 500, 1000, 1500, 2000, 5000, 10,000 or 15,000 copies of or nucleic acid encoding the receptor, e.g., the CAR per microgram of DNA, or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 receptor-expressing, e.g., CAR-expressing cells per total number of peripheral blood mononuclear cells (PBMCs), total number of mononuclear cells, total number of T cells, or total number of microliters. In some embodiments, the cells expressing the receptor are detected as at least 10, 20, 30, 40, 50, or 60% of total PBMCs in the blood of the subject, and/or at such a level for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, or 52 weeks following the first or subsequent administration or for 1, 2, 3, 4, or 5, or more years following such administration.
In some aspects, the method results in at least a 2-fold, at least a 4-fold, at least a 10-fold, or at least a 20-fold increase in copies of nucleic acid encoding the recombinant receptor, e.g., CAR, per microgram of DNA, e.g., in the serum of the subject.
In some embodiments, cells expressing the receptor are detectable in the blood or serum of the subject, e.g., by a specified method, such as qPCR or flow cytometry-based detection method, at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 or more days following administration of the first dose or after administration of the second (or other subsequent) dose, or for at least at or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 or more weeks following the administration of the first dose or subsequent dose(s).
In some aspects, at least about 1×102, at least about 1×103, at least about 1×104, at least about 1×105, or at least about 1×106 or at least about 5×106 or at least about 1×107 or at least about 5×107 or at least about 1×108 recombinant receptor-expressing, e.g., CAR-expressing cells, and/or at least 10, 25, 50, 100, 200, 300, 400, or 500, or 1000 receptor-expressing cells per microliter, e.g., at least 10 per microliter, are detectable or are present in the subject or fluid, tissue, or compartment thereof, such as in the blood, e.g., peripheral blood, or disease site thereof. In some embodiments, such a number or concentration of cells is detectable in the subject for at least about 20 days, at least about 40 days, or at least about 60 days, or at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 2 or 3 years, following administration of the first dose or following the administration of the subsequent dose(s). Such cell numbers may be as detected by flow cytometry-based or quantitative PCR-based methods and extrapolation to total cell numbers using known methods. See, e.g., Brentjens et al., Sci Transl Med. 2013 5(177), Park et al, Molecular Therapy 15(4):825-833 (2007), Savoldo et al., JCI 121(5):1822-1826 (2011), Davila et al. (2013) PLoS ONE 8(4):e61338, Davila et al., Oncoimmunology 1(9):1577-1583 (2012), Lamers, Blood 2011 117:72-82, Jensen et al. Biol Blood Marrow Transplant 2010 September; 16(9): 1245-1256, Brentjens et al., Blood 2011 118(18):4817-4828.
In some aspects, the copy number of nucleic acid encoding the recombinant receptor, e.g., vector copy number, per 100 cells, for example in the peripheral blood or bone marrow or other compartment, as measured by immunohistochemistry, PCR, and/or flow cytometry, is at least 0.01, at least 0.1, at least 1, or at least 10, at about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or at least about 6 weeks, or at least about 2, 3, 4, 5, 6, 7, 8. 9, 10, 11, or 12 months or at least 2 or 3 years following administration of the cells, e.g., the first or subsequent dose(s). In some embodiments, the copy number of the vector expressing the receptor, e.g. CAR, per microgram of genomic DNA is at least 100, at least 1000, at least 5000, or at least 10,000, or at least 15,000 or at least 20,000 at a time about 1 week, about 2 weeks, about 3 weeks, or at least about 4 weeks following administration of the first dose or subsequent dose(s) of receptor-expressing, e.g. CAR-expressing, cells, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or at least 2 or 3 years following such administration.
In some aspects, the receptor, e.g. CAR, expressed by the cells, is detectable by quantitative PCR (qPCR) or by flow cytometry in the subject, blood thereof, and/or disease site thereof, at a time that is at least about 3 months, at least about 6 months, at least about 12 months, at least about 1 year, at least about 2 years, at least about 3 years, or more than 3 years, following the administration of the cells, e.g., following the initiation of the administration of the first dose or the second or subsequent dose.
In some embodiments, the area under the curve (AUC) for concentration of receptor-(e.g., CAR-)expressing cells in a fluid, tissue, or organ, e.g., blood, of the subject over time following the administration of the first dose is greater as compared to that achieved via an alternative dosing regimen where the subject is administered a single dose of cells or multiple doses of cells expressing the same receptor.
In some aspects, the area under the curve (AUC) for concentration of receptor-(e.g., CAR-)expressing cells in a fluid, tissue, or organ, e.g., blood, of the subject over time over time following the administration of the subsequent dose is greater as compared to that achieved via an alternative dosing regimen where the subject is administered a single dose of cells or multiple doses of cells expressing the same receptor.
VII. Disease Burden
The administration of the doses generally reduces or prevents the expansion or burden of the disease or condition in the subject. For example, where the disease or condition is a tumor, the methods generally reduce tumor size, bulk, or metastasis, and/or improve prognosis or survival or other symptom associated with tumor burden. In some embodiments, administration of the second or subsequent dose is timed with respect to the development of a transgene-specific immune response and/or relapse following the first or prior dose.
Disease burden can encompass a total number of cells of the disease in the subject or in an organ, tissue, or bodily fluid of the subject, such as the organ or tissue of the tumor or another location, e.g., which would indicate metastasis. For example, tumor cells may be detected and/or quantified in the blood or bone marrow in the context of certain hematological malignancies. Disease burden can include, in some embodiments, the mass of a tumor and/or the number or extent of metastases.
In some embodiments, the administration of the two or more doses effects a reduction in disease burden. In some aspects, administration of the doses may prevent an increase in disease burden, and this may be evidenced by no change in disease burden.
In some aspects, the disease or condition persists following administration of the first dose and/or administration of the first dose is not sufficient to eradicate the disease or condition in the subject.
In some aspects, administration of the second dose reduces disease burden as compared to disease burden at a time immediately prior to the first dose, or at a time immediately prior to the second dose. In some aspects, for example in the context of relapse, administration of the second dose effects a reduction in disease burden as compared to the peak level of disease burden following administration of the first dose.
In some embodiments, the method reduces the burden of the disease or condition, e.g., number of tumor cells, size of tumor, duration of patient survival or event-free survival, to a greater degree and/or for a greater period of time as compared to the reduction that would be observed with a method using an alternative dosing regimen, such as one in which the subject receives a single dose of cells or multiple doses of cells expressing the same receptor, e.g., CAR. In some embodiments, disease burden is reduced to a greater extent or for a greater duration following the second dose compared to the reduction that would be effected by administering a second dose of cells expressing the same receptor, e.g., CAR.
In some embodiments, the burden of disease or condition in the subject is detected, assessed, or measured. Disease burden may be detected in some aspects by detecting the total number of disease or disease-associated cells, e.g., tumor cells, in the subject, or in an organ, tissue, or bodily fluid of the subject, such as blood or serum. In some embodiments, disease burden, e.g. tumor burden, is assessed by measuring the mass of a solid tumor and/or the number or extent of metastases. In some aspects, survival of the subject, survival within a certain time period, extent of survival, presence or duration of event-free or symptom-free survival, or relapse-free survival, is assessed. In some embodiments, any symptom of the disease or condition is assessed. In some embodiments, the measure of disease or condition burden is specified.
In some aspects, disease burden is measured or detected prior to administration of the first dose, following the administration of the first dose but prior to administration of the second dose, and/or following administration of the second or subsequent dose. In the context of multiple subsequent doses, disease burden in some embodiments may be measured prior to or following any of the subsequent doses, or at a time between administration of subsequent doses.
In some aspects, administration of the doses effects a reduction in disease burden, e.g. tumor burden, such as a at or about 10, 20, 30, 40, 50, 60, 70, 90, or 100 percent decrease in burden compared to immediately prior to the administration of the second dose or overall compared to immediately prior to the first dose. In some embodiments, disease burden, tumor size, tumor volume, tumor mass, and/or tumor load or bulk is reduced following the second dose by at least at or about 50, 60, 70, 80, 90% or more compared to that immediately prior to the administration of the first dose or of the subsequent dose.
In some embodiments, reduction of disease burden by the method comprises an induction in morphologic complete remission, for example, as assessed at 1 month, 2 months, 3 months, or more than 3 months, after administration of, e.g., initiation of, the first or any subsequent dose. In some aspects, an assay for minimal residual disease, for example, as measured by multiparametric flow cytometry, is negative, or the level of minimal residual disease is less than about 0.3%, less than about 0.2%, less than about 0.1%, or less than about 0.05%.
In some embodiments, the event-free survival rate or overall survival rate of the subject is improved by the methods, as compared with other methods. For example, in some embodiments, event-free survival rate or probability for subjects treated by the methods at 6 months following the first dose is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some aspects, overall survival rate is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some embodiments, the subject treated with the methods exhibits event-free survival, relapse-free survival, or survival to at least 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some embodiments, the time to progression is improved, such as a time to progression of greater than at or about 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
In some embodiments, following treatment by the method, the probability of relapse is reduced as compared to other methods. For example, in some embodiments, the probability of relapse at 6 months following the first dose is less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10%.
VIII. Toxicity and Toxic Outcomes
In some embodiments, the methods reduce or prevent toxicity or an outcome or symptom thereof, for example, compared to administration of cells as a single dose, administration of a subsequent dose of cells that express the same receptor as the first dose, and/or administration of the subsequent dose at a time which is earlier than the time between the first and subsequent doses specified by the method.
Administration of adoptive T cell therapy, such as treatment with T cells expressing chimeric antigen receptors, can induce toxic effects or outcomes such as cytokine release syndrome and neurotoxicity. In some examples, such effects or outcomes parallel high levels of circulating cytokines, which may underlie the observed toxicity.
In some aspects, the present methods may reduce toxicity or toxic outcomes as compared to other methods by allowing administration of a smaller first dose than other methods, for example, where a single large dose is administered, for example, where multiple smaller doses of cells expressing the same receptor may be eliminated by a host immune response.
In some embodiments, the present methods may reduce toxicity or toxic outcomes as compared to other methods by allowing administration of a subsequent dose more than 28 days after the administration of the first dose, for example, at a time point at which an immune response to the first receptor has developed such that cells expressing the first receptor would be eliminated if administered again. Thus, in some aspects, the methods reduce toxicity as compared to methods that administer multiple doses at a point in time at which the subject is at risk for developing CRS.
In some aspects, the toxic outcome is or is associated with or indicative of cytokine release syndrome (CRS) or severe CRS (sCRS). CRS, e.g., sCRS, can occur in some cases following adoptive T cell therapy and administration to subjects of other biological products. See Davila et al., Sci Transl Med 6, 224ra25 (2014); Brentjens et al., Sci. Transl. Med. 5, 177ra38 (2013); Grupp et al., N. Engl. J. Med. 368, 1509-1518 (2013); and Kochenderfer et al., Blood 119, 2709-2720 (2012); Xu et al., Cancer Letters 343 (2014) 172-78.
CRS may be treated using anti-inflammatory therapy such as an anti-IL-6 therapy, e.g., anti-IL-6 antibody, e.g., tocilizumab, or antibiotics. In some embodiments, the subject is treated with such a therapy following the first administration and the subsequent dose is administered only if and when the CRS-associated symptom(s) are reduced or declining or declined below an acceptable level following such treatment.
Outcomes, signs and symptoms of CRS are known and include those described herein. In some embodiments, where a particular dosage regimen or administration effects or does not effect a given CRS-associated outcome, sign, or symptom, particular outcomes, signs, and symptoms and/or quantities or degrees thereof may be specified.
The method of measuring or detecting the various outcomes may be specified.
In some aspects, prior to the administration of the first dose, subsequent to the administration of the first dose and before administration of the subsequent dose, or following the administration of the subsequent dose, a CRS-associated outcome is assessed in the subject. In some embodiments, the level of the toxic outcome, e.g. the CRS-related outcome, e.g. the serum level of an indicator of CRS, is measured by ELISA.
In some aspects, the toxic outcome is or is associated with neurotoxicity. In some embodiments, the methods reduce symptoms associated with neurotoxicity compared to other methods. For example, subjects treated according to the present methods may have reduced symptoms of neurotoxicity, such as limb weakness or numbness, loss of memory, vision, and/or intellect, uncontrollable obsessive and/or compulsive behaviors, delusions, headache, cognitive and behavioral problems including loss of motor control, cognitive deterioration, and autonomic nervous system dysfunction, and sexual dysfunction, compared to subjects treated by other methods.
In some embodiments, the methods reduce outcomes associated with neurotoxicity including damages to the nervous system and/or brain, such as the death of neurons. In some aspects, the methods reduce the level of factors associated with neurotoxicity such as beta amyloid (Aβ), glutamate, and oxygen radicals.
In some embodiments, subjects administered doses according to the methods have reduced symptoms, outcomes, or factors associated with neurotoxicity compared to administration of a single dose, administration of a subsequent dose of cells that expresses the same receptor, e.g., CAR, as the first dose, and/or administration of the subsequent dose at a time which is earlier than the time between the first and subsequent doses specified by the method.
IX. Cells
The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells, e.g., those derived from human subjects and engineered, for example, to express the recombinant receptors. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, and re-introducing them into the same subject, before or after cryopreservation.
Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naïve T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
In some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.
Vectors and Methods for Genetic Engineering
Also provided are methods, compositions, and kits, for producing the genetically engineered cells expressing recombinant receptors. The genetic engineering generally involves introduction of a nucleic acid encoding the recombinant or engineered component into the cell, such as by retroviral transduction, transfection, or transformation.
In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.
In some contexts, overexpression of a stimulatory factor (for example, a lymphokine or a cytokine) may be toxic to a subject. Thus, in some contexts, the engineered cells include gene segments that cause the cells to be susceptible to negative selection in vivo, such as upon administration in adoptive immunotherapy. For example in some aspects, the cells are engineered so that they can be eliminated as a result of a change in the in vivo condition of the subject to which they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell II: 223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).
In some aspects, the cells further are engineered to promote expression of cytokines or other factors. Various methods for the introduction of genetically engineered components, e.g., antigen receptors, e.g., CARs, are well known and may be used with the provided methods and compositions. Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.
In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557.
In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
Methods of lentiviral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.
In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).
Other approaches and vectors for transfer of the nucleic acids encoding the recombinant products are those described, e.g., in international patent application, Publication No.: WO2014055668, and U.S. Pat. No. 7,446,190.
Among additional nucleic acids, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.
Preparation of Cells for Engineering
In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for introduction of the nucleic acid encoding the transgenic receptor such as the CAR, may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
In some aspects, the cells of the second dose are derived from the same apheresis product as the cells of the first dose. In some embodiments, the cells of multiple doses, e.g., first, second, third, and so forth, are derived from the same apheresis product.
In other embodiments, the cells of the second (or other subsequent) dose are derived from an apheresis product that is distinct from that from which the cells of the first (or other prior) dose are derived.
In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig.
In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.
In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++/Mg++ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.
In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques.
For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (markerhigh) on the positively or negatively selected cells, respectively.
In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.
In embodiments, memory T cells are present in both CD62L+ and CD62L− subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L−CD8+ and/or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.
In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.
CD4+ T helper cells are sorted into naïve, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L− and CD45RO−.
In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher© Humana Press Inc., Totowa, N.J.).
In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.
In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.
The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.
In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.
In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.
In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.
In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Number WO2009/072003, or US 20110003380 A1.
In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.
In some aspects, the separation and/or other steps is carried out using CliniMACS system (Miltenyi Biotic), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.
The CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.
In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.
In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.
In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are generally then frozen to −80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.
In some embodiments, the provided methods include cultivation, incubation, culture, and/or genetic engineering steps. For example, in some embodiments, provided are methods for incubating and/or engineering the depleted cell populations and culture-initiating compositions.
Thus, in some embodiments, the cell populations are incubated in a culture-initiating composition. The incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.
In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agents include IL-2 and/or IL-15, for example, an IL-2 concentration of at least about 10 units/mL.
In some aspects, incubation is carried out in accordance with techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, the T cells are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.
In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.
In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigen-specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.
X. Compositions and Formulations
Also provided are compositions including the cells, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.
The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
In some aspects, the choice of carrier is determined in part by the particular cell and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).
Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.
The cells and compositions may be administered using standard administration techniques, formulations, and/or devices. Administration of the cells can be autologous or heterologous. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyoi (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.
Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
XI. Articles of Manufacture
Also provided are articles of manufacture, such as kits and devices, for the administration of the cells to subjects in according to the provided methods for adoptive cell therapy, and for storage and administration of the cells and compositions.
The articles of manufacture include one or more containers, typically a plurality of containers, packaging material, and a label or package insert on or associated with the container or containers and/or packaging, generally including instructions for administration of the cells to a subject.
The containers generally contain the cells to be administered, e.g., one or more unit doses thereof. The article of manufacture typically includes a plurality of containers, each containing a single unit dose of the cells. The unit dose may be an amount or number of the cells to be administered to the subject in the first dose or twice the number (or more) the cells to be administered in the first or subsequent dose(s). It may be the lowest dose or lowest possible dose of the cells that would be administered to the subject in connection with the administration method. In some embodiments, the unit dose is the minimum number of cells or number of cells that would be administered in a single dose to any subject having a particular disease or condition or any subject, according to the methods herein. For example, the unit dose in some aspects may include a minimum number of cells that would be administered to a patient of a relatively lower body weight and/or with relatively high disease burden, such that in some cases more than one unit dose is administered to a given subject as a first dose and one or more than one unit dose is administered to a given subject in one or more subsequent dose, e.g., according to the provided methods. In some embodiments, the number of cells in the unit dose is the number of cells or number of recombinant receptor-expressing or CAR-expressing cells that it is desired to administer to a particular subject in a first dose, such as a subject from which the cells have been derived. In some embodiments, the cells have been derived from the subject to be treated by methods as provided herein or in need thereof.
In some embodiments, one or more of the unit doses contains cells that express the same receptor, e.g., CAR. In some aspects, one or more of the unit doses contains cells that express a different receptor, e.g., CAR, than one or more of the other unit doses.
In some embodiments, each of the containers individually comprises a unit dose of the cells that express the first, or second, or third, and so forth, receptor, that contains the same or substantially the same number of cells. Thus in some embodiments, each of the containers comprises the same or approximately or substantially the same number of cells or number of recombinant receptor-expressing cells. In some embodiments, the unit dose includes less than about 1×108, less than about 5×107, less than about 1×106 or less than about 5×105 of the engineered cells, of total cells, of T cells, or PBMCs, per kg of the subject to be treated and/or from which the cells have been derived. In some embodiments, each unit dose contains at or about 2×106, 5×106, 1×107, 5×107, or 1×108 engineered cells, total cells, T cells, or PBMCs.
Suitable containers include, for example, bottles, vials, syringes, and flexible bags, such as infusion bags. In particular embodiments, the containers are bags, e.g., flexible bags, such as those suitable for infusion of cells to subjects, e.g., flexible plastic or PVC bags, and/or IV solution bags. The bags in some embodiments are sealable and/or able to be sterilized, so as to provide sterile solution and delivery of the cells and compositions. In some embodiments, the containers, e.g., bags, have a capacity of at or about or at least at or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000 ml capacity, such as between at or about 10 and at or about 100 or between at or about 10 and at or about 500 mL capacity. In some embodiments, the containers, e.g., bags, are and/or are made from material which is stable and/or provide stable storage and/or maintenance of cells at one or more of various temperatures, such as in cold temperatures, e.g. below at or about or at or about −20° C., −80° C., −120° C., 135° C. and/or temperatures suitable for cryopreservation, and/or other temperatures, such as temperatures suitable for thawing the cells and body temperature such as at or about 37° C., for example, to permit thawing, e.g., at the subject's location or location of treatment, e.g., at bedside, immediately prior to treatment.
The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container has one or more port, e.g., sterile access ports, for example, for connection of tubing or cannulation to one or more tubes, e.g., for intravenous or other infusion and/or for connection for purposes of transfer to and from other containers, such as cell culture and/or storage bags or other containers. Exemplary containers include infusion bags, intravenous solution bags, vials, including those with stoppers pierceable by a needle for injection.
The article of manufacture may further include a package insert or label with one or more pieces of identifying information and/or instructions for use. In some embodiments, the information or instructions indicates that the contents can or should be used to treat a particular condition or disease, and/or providing instructions therefor. The label or package insert may indicate that the contents of the article of manufacture are to be used for treating the disease or condition. In some embodiments, the label or package insert provides instructions to treat a subject, e.g., the subject from which the cells have been derived, via a method involving the administration of a first and one or more subsequent doses of the cells, e.g., according to any of the embodiments of the provided methods. In some embodiments, the instructions specify administration, in a first dose, of one unit dose, e.g., the contents of a single individual container in the article of manufacture, followed by one or more subsequent doses at a specified time point or within a specified time window and/or after the detection of the presence or absence or amount or degree of one or more factors or outcomes in the subject.
In some embodiments, the instructions specify administering a plurality of the unit doses to the subject by carrying out a first administration and a subsequent administration. In some embodiments, the first administration comprises delivering one of said unit doses containing cells expressing a first receptor, e.g., CAR, to the subject and the subsequent administration comprises administering one or a plurality of said unit doses containing cells expressing the same receptor, e.g. CAR, as the first administration to the subject.
In some aspects, the first administration comprises delivering one of said unit doses containing cells expressing a first receptor, e.g., CAR, to the subject and the subsequent administration comprises administering one or a plurality of said unit doses containing cells expressing a distinct receptor, e.g. CAR, to the subject. In some embodiments, whether the patient receives a second administration that contains cells expressing the same receptor as the first administration or receives a second administration that contains cells expressing a receptor, e.g., CAR, that is distinct from that expressed by the cells of the first administration, may be determined based on any of the parameters of the methods described herein. For example, in some aspects, e.g., where the patient has developed an immune response to the first receptor, a unit dose containing cells that express a receptor that is distinct from the first receptor may be administered.
In some embodiments, the instructions specify that the second (or other subsequent) administration is to be carried out at a time at least about 28 or at least about 35 days following the first administration, e.g., following the initiation of the first administration or the prior administration. In some embodiments, the instructions specify that the subsequent dose is to be administered at a time after which it has been determined that the subject exhibits a detectable adaptive host immune response specific for the receptor, e.g., CAR, expressed by the cells of the first (or other prior) dose.
In some embodiments, the label or package insert or packaging comprises an identifier to indicate the specific identity of the subject from which the cells are derived and/or are to be administered. In the case of autologous transfer, the identity of the subject from which the cells are derived is the same as the identity of the subject to which the cells are to be administered. Thus, the identifying information may specify that the cells are to be administered to a particular patient, such as the one from which the cells were originally derived. Such information may be present in the packaging material and/or label in the form of a bar code or other coded identifier, or may indication the name and/or other identifying characteristics of the subject.
The article of manufacture in some embodiments includes one or more, typically a plurality, of containers containing compositions comprising the cells, e.g., individual unit dose forms thereof, and further include one or more additional containers with a composition contained therein which includes a further agent, such as a cytotoxic or otherwise therapeutic agent, for example, which is to be administered in combination, e.g., simultaneously or sequentially in any order, with the cells. Alternatively, or additionally, the article of manufacture may further include another or the same container comprising a pharmaceutically-acceptable buffer. It may further include other materials such as other buffers, diluents, filters, tubing, needles, and/or syringes.
The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include “consisting” and/or “consisting essentially of” aspects and variations.
Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.
The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.
As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
As used herein, a statement that a cell or population of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.
As used herein, a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.
The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
XII. Exemplary Embodiments
Among the provided embodiments are:
1. A method of treatment, comprising:
(a) administering to a subject cells expressing a first chimeric antigen receptor (CAR) that specifically binds to an antigen associated with a disease or condition in the subject; and
(b) administering to the subject cells expressing a second CAR, which is distinct from said first CAR, and not expressing the first CAR.
2. The method of embodiment 1, wherein, at the time of or immediately prior to the administration of cells expressing the second CAR:
the subject exhibits a detectable humoral and/or cell-mediated immune response specific for the first CAR;
the disease or condition persists in the subject; and/or
the disease or condition has relapsed in the subject.
3. The method of embodiment 1 or embodiment 2, wherein:
the time between the administration of cells expressing the first CAR and the administration of cells expressing the second CAR is at least about 28 days, at least about 35 days, at least about 42 days, at least about 49 days, and/or at least about 60 days.
4. The method of any of embodiments 1-3, wherein said first CAR comprises at least one immunoreactive epitope that is not present in said second CAR.
5. The method of embodiment 4, wherein:
said at least one immunoreactive epitope comprises at least one B cell epitope; and/or
said at least one immunoreactive epitope comprises at least one T cell epitope.
6. The method of any of embodiments 1-5, wherein:
said subject has not received a dose of cells expressing the first CAR prior to said administration in (a); and/or
said subject has not received a dose of cells expressing the second CAR prior to the administration in (b).
7. The method of any of embodiments 1-6, wherein the second CAR specifically binds to the same antigen as the first CAR.
8. The method of any of embodiments 1-7, wherein the disease or condition is a tumor.
9. The method of any of embodiments 1-8, wherein the disease or condition is a B cell malignancy.
10. The method of embodiment 9, wherein the first CAR and the second CAR specifically bind to the same epitope of said antigen.
11. The method of any of embodiments 7-10, wherein the first CAR competes for binding to said antigen with the second CAR.
12. The method of any of embodiments 7-9 and 11, wherein the first CAR and the second CAR specifically bind to distinct epitopes of said antigen.
13. The method of any of embodiments 1-12, wherein:
the second CAR specifically binds to another antigen associated with said disease or condition compared to the antigen bound by the first CAR; or
the second CAR does not specifically bind to the antigen specifically bound by the first CAR.
14. The method of any of embodiments 9-13, wherein the first CAR specifically binds to an antigen associated with a B cell malignancy that is selected from CD19, CD22 or CD20 and the second CAR binds to another antigen from among CD19, CD22 or CD20 that is distinct from the antigen bound by the first CAR.
15. The method of embodiment 14, wherein the first CAR specifically binds to CD19 and the second CAR specifically binds to CD22.
16. The method of any of embodiments 1-6, wherein the cells expressing the second CAR do not comprise a receptor that specifically binds to said antigen specifically bound by the first CAR.
17. The method of any of embodiments 1-16, wherein the subject does not exhibit a detectable humoral or cell-mediated immune response against the second CAR within about 30 days, within about 60 days, or within about 90 days, of the administration of cells expressing the second CAR.
18. The method of any of embodiments 1-17, wherein the second CAR comprises one or more differences in amino acid sequence compared to the first CAR.
19. The method of embodiment 18, wherein
the one or more differences comprise at least one amino acid sequence difference compared to a region of the first CAR to which a detectable immune response is generated in the subject following the administration of cells expressing the first CAR; and/or
the one or more differences comprise at least one amino acid sequence difference compared to each region of the first CAR to which a detectable immune response is generated in the subject following the administration of cells expressing the first CAR.
20. The method of any of embodiments 1-19, further comprising, prior to the administration of cells expressing the second CAR, detecting the presence of a CAR-specific immune response in the subject.
21. The method of embodiment 20, wherein the detection comprises identifying at least a region of the first CAR to which the subject exhibits a specific immune response.
22. The method of embodiment 21, wherein the second CAR contains one or more amino acid sequence differences compared to said region of the first CAR for which the immune response is specific.
23. The method of any of embodiments 19-22, wherein the region of the first CAR comprises a region within one or more CAR portions selected from the group consisting of an scFv portion, a linker portion, an amino acid sequence not endogenous to the subject, a sequence derived from a different species than that of the subject, and/or a junction between two CAR domains; and/or where the region of the first CAR is a junction region comprising amino acids on each side of a junction between two domains.
24. The method of embodiment 23, wherein:
the region comprises a framework region (FR) within the scFv portion,
the region comprises a heavy chain FR sequence
the region comprises a heavy chain CDR sequence,
the region comprises a light chain FR sequence, and/or
the region comprises a light chain CDR sequence.
25. The method of embodiment 24, wherein the region comprises a junction region, wherein the junction region comprises up to 15 contiguous amino acids directly C-terminal of a junction that joins a first domain and a second domain of the first CAR and/or up to 15 contiguous amino acids directly N-terminal of the junction, and optionally further comprises the junction.
26. The method of embodiment 25, wherein:
the first domain and/or second domain comprise a domain of a natural or endogenous human protein or a domain having 100% identity with a domain or functional portion thereof of a natural or endogenous human protein, wherein the natural or endogenous human protein optionally is expressed by the subject to be treated; and/or
the first domain and/or second domain comprises an extracellular binding domain, a hinge domain, a transmembrane domain, or an intracellular signaling domain, which intracellular signaling domain is, optionally, a costimulatory signaling domain or an activating cytoplasmic signaling domain.
27. The method of embodiment 26, wherein the first domain and second domain are not present in the same molecule in vivo in a human subject, or are not present in a single natural or endogenous human protein or polypeptide.
28. The method of embodiment 26 or embodiment 27, wherein the first domain and second domain are or comprise, respectively, an extracellular ligand binding domain and a hinge domain, a hinge domain and a transmembrane domain, a transmembrane domain and an intracellular costimulatory signaling domain, and an intracellular costimulatory signaling domain and an activating cytoplasmic signaling domain, which can include functional portions of such domains.
29. The method of any of embodiments 26-28, wherein the first domain is or comprises a transmembrane domain or a functional portion thereof and the second domain is or comprises a costimulatory signaling domain or a functional portion thereof.
30. The method of embodiment 29, wherein the transmembrane domain is a CD28 transmembrane domain or a functional portion thereof and the costimulatory signaling domain is a 4-1BB signaling domain or a functional portion thereof.
31. The method of any of embodiments 26-30, wherein the second CAR comprises:
a domain of at least 95% sequence identity to the first domain and/or a domain of at least 95% sequence identity to the second domain;
a domain identical in sequence to the first domain and a domain of at least 95% sequence identity to the second domain; or
a domain of at least 95% sequence identity to the first domain and a domain identical in sequence to the second domain,
wherein at least one or both of the domains present in the second CAR comprises at least one or more amino acid sequence differences compared to one or both of the first domain and second domain of the first CAR in the portion comprising the modified junction region.
32. The method of any of embodiments 29-31, wherein:
the first CAR comprises a CD28 transmembrane domain and a 4-1BB co-stimulatory domain that together comprise the sequence of amino acids set forth in SEQ ID NO:5 or a variant or functional portion thereof comprising a sequence of amino acids that is at least 95% identical to SEQ ID NO:5 and comprises the junction region; and
the second CAR comprises a sequence that is modified compared to the first CAR, the modification comprising at least one amino acid sequence difference in a portion comprising a sequence of between residue 13 and 42 or between 15 and 40, with reference to numbering set forth in SEQ ID NO:5.
33. The method of any of embodiments 18-32, wherein the second CAR comprises no more than 20 amino acid sequence differences compared to the first CAR or the second CAR comprises at least 95% amino acid sequence identity to the first CAR.
34. The method of any of embodiments 18-33, wherein a region of the second CAR containing at least one of the one or more sequence differences:
contains fewer 8-15 amino acid portions, as compared to the corresponding region of the first CAR, that has a binding affinity for a human leukocyte antigen (HLA) molecule of an IC50 of less than 1000 nM; or
has a binding affinity for a human leukocyte antigen (HLA) molecule that is lower than the binding affinity for the same HLA molecule of a peptide fragment having the sequence of the corresponding portion of the junction region of the reference chimeric receptor; or
has a binding affinity of at least one peptide fragment within the region, or a reduced average binding affinity of all peptide fragments having the sequence of an 8-15 amino acid portion within the region, for a human leukocyte antigen (HLA) molecule, as compared to the corresponding region of the first CAR.
35. The method of any of embodiments 18-34, wherein a reduced detectable immune response is generated in the subject following the administration of cells expressing the second CAR to the corresponding region of the second CAR that comprises at least one amino acid sequence difference compared to the immune response generated in the subject to the region in the first CAR following its administration to the subject.
36. The method of any of any of embodiments 1-17, wherein:
the first CAR comprises a CD28 transmembrane domain or a functional portion thereof and a 4-1BB costimulatory signaling domain or a function portion thereof; and
the second CAR comprises a transmembrane domain and a costimulatory signaling domain that is distinct from one or both of such domains in the first CAR.
37. The method of any of embodiments 1-36, wherein the second CAR comprises at least one region identical in amino acid sequence to a corresponding region of the first CAR.
38. The method of embodiment 37, wherein the corresponding region of the first CAR is a region to which the subject does not exhibit a detectable humoral or cell-mediated immune response at the time of the administration of the cells expressing the second CAR.
39. The method of embodiment 37 or embodiment 38, wherein the corresponding region of the first CAR comprises a region within a CAR portion selected from the group consisting of a costimulatory domain, an ITAM-containing domain, a transmembrane domain, a transduction or expression marker, a sequence endogenous to the host, and/or an antibody domain derived from the same species as the host.
40. The method of any of embodiments 1-39, wherein the maximum number of CAR-expressing cells, the area under the curve (AUC) for CAR-expressing cells over time, and/or the duration of detectable CAR-expressing cells in the subject following the administration of cells expressing the second CAR is greater as compared to that achieved via a method comprising an alternative dosing regimen comprising performing the administration of cells expressing the first CAR followed by performing a second administration of cells expressing the first CAR, said second administration being carried out at the same point in time as the administration of cells expressing the second CAR.
41. The method of any of embodiments 1-40, wherein:
the method results in a maximum concentration or number of CAR-expressing cells in the blood of the subject of at least at or about 10 CAR-expressing cells per microliter, at least 50% of the total number of peripheral blood mononuclear cells (PBMCs), at least at least about 1×105 CAR-expressing cells, or at least 1,000, or at least 2,000, or at least 3,000, or at least 4,000, or at least 5,000 copies of CAR-encoding DNA per micrograms DNA; and/or
at day 30, at day 60, or at day 90 following the initiation of the administration of cells expressing the second CAR, CAR-expressing cells are detectable in the blood or serum of the subject; and/or
at day 30, at day 60, or at day 90 following the initiation of the administration of cells expressing the second CAR, the blood of the subject contains at least 20% CAR-expressing cells, at least 10 CAR-expressing cells per microliter or at least 1×104 CAR-expressing cells.
42. The method of any of embodiments 1-41, wherein the dose of cells expressing the first CAR and/or the dose of cells expressing the second CAR independently comprise cells in an amount sufficient for reduction in burden of a disease or condition in the subject.
43. The method of any of embodiments 1-42, wherein the administration of cells expressing the first CAR and/or the administration of cells expressing the second CAR effects a reduction in burden of the disease or condition in the subject, thereby treating the disease or condition.
44. The method of any of embodiments 1-43, wherein the cells are T cells.
45. The method of any of embodiments 1-44, wherein the T cells are autologous to the subject.
46. A method of treatment, comprising administering cells to a subject, wherein said cells do not express a first chimeric antigen receptor (CAR) and express a second CAR, wherein:
said subject has previously received an administration of cells expressing the first CAR;
said first CAR specifically binds to an antigen associated with a disease or condition in the subject; and
said second CAR specifically binds to the antigen specifically bound by the first CAR or a different antigen associated with the disease or condition in the subject.
47. The method of embodiment 46, wherein prior to administering cells expressing the second CAR, administering to the subject cells expressing the first CAR.
48. The method of any of embodiments 46-47, wherein, at the time of or immediately prior to the administration of cells expressing the second CAR:
the subject exhibits a detectable humoral and/or cell-mediated immune response specific for the first CAR;
the disease or condition persists in the subject; and/or
the disease or condition has relapsed in the subject.
49. The method of any of embodiments 46-48, wherein:
the time between the administration of cells expressing the first CAR and the administration of cells expressing the second CAR is at least about 28 days, at least about 35 days, at least about 42 days, at least about 49 days, and/or at least about 60 days.
50. The method of any of embodiments 46-49, wherein said first CAR comprises at least one immunoreactive epitope that is not present in said second CAR.
51. The method of embodiment 50, wherein:
said at least one immunoreactive epitope comprises at least one B cell epitope; and/or
said at least one immunoreactive epitope comprises at least one T cell epitope.
52. The method of any of embodiments 46-51, wherein said subject has not received a dose of cells expressing the second CAR prior to the administration.
53. The method of any of embodiments 46-52, wherein the second CAR specifically binds to the same antigen as the first CAR.
54. The method of any of embodiments 46-53, wherein the disease or condition is a tumor.
55. The method of any of embodiments 46-54, wherein the disease or condition is a B cell malignancy.
56. The method of embodiment 53, wherein the first CAR and the second CAR specifically bind to the same epitope of said antigen.
57. The method of any of embodiments 53-56, wherein the first CAR competes for binding to said antigen with the second CAR.
58. The method of any of embodiments 53-55 and 57, wherein the first CAR and the second CAR specifically bind to distinct epitopes of said antigen.
59. The method of any of embodiments 46-58, wherein:
the second CAR specifically binds to another antigen associated with said disease or condition compared to the antigen bound by the first CAR; or the second CAR does not specifically bind to the antigen specifically bound by the first CAR.
60. The method of any of embodiments 55-59, wherein the first CAR specifically binds to an antigen associated with a B cell malignancy that is selected from CD19, CD22 or CD20 and the second CAR binds to another antigen from among CD19, CD22 or CD20 that is distinct from the antigen bound by the first CAR.
61. The method of embodiment 60, wherein the first CAR specifically binds to CD19 and the second CAR specifically binds to CD22.
62. The method of any of embodiments 46-52, wherein the cells expressing the second CAR do not comprise a receptor that specifically binds to said antigen specifically bound by the first CAR.
63. The method of any of embodiments 46-62, wherein the subject does not exhibit a detectable humoral or cell-mediated immune response against the second CAR within about 30 days, within about 60 days, or within about 90 days, of the administration of cells expressing the second CAR.
64. The method of any of embodiments 46-63, wherein the second CAR comprises one or more differences in amino acid sequence compared to the first CAR.
65. The method of embodiment 64, wherein
the one or more differences comprise at least one amino acid sequence difference compared to a region of the first CAR to which a detectable immune response is generated in the subject following the administration of cells expressing the first CAR; and/or
the one or more differences comprise at least one amino acid sequence difference compared to each region of the first CAR to which a detectable immune response is generated in the subject following the administration of cells expressing the first CAR.
66. The method of any of embodiments 46-65, further comprising, prior to the administration of cells expressing the second CAR, detecting the presence of a CAR-specific immune response in the subject.
67. The method of embodiment 66, wherein the detection comprises identifying at least a region of the first CAR to which the subject exhibits a specific immune response.
68. The method of embodiment 67, wherein the second CAR contains one or more amino acid sequence differences compared to said region of the first CAR for which the immune response is specific.
69. The method of any of embodiments 65-68, wherein the region of the first CAR comprises a region within one or more CAR portions selected from the group consisting of an scFv portion, a linker portion, an amino acid sequence not endogenous to the subject, a sequence derived from a different species than that of the subject, and/or a junction between two CAR domains; and/or where the region of the first CAR is a junction region comprising amino acids on each side of a junction between two domains.
70. The method of embodiment 69, wherein:
the region comprises a framework region (FR) within the scFv portion,
the region comprises a heavy chain FR sequence
the region comprises a heavy chain CDR sequence,
the region comprises a light chain FR sequence, and/or
the region comprises a light chain CDR sequence.
71. The method of embodiment 69, wherein the region comprises a junction region, wherein the junction region comprises up to 15 contiguous amino acids directly C-terminal of a junction that joins a first domain and a second domain of the first CAR and/or up to 15 contiguous amino acids directly N-terminal of the junction, and optionally further comprises the junction.
72. The method of embodiment 71, wherein:
the first domain and/or second domain comprise a domain of a natural or endogenous human protein or a domain having 100% identity with a domain or functional portion thereof of a natural or endogenous human protein, wherein the natural or endogenous human protein optionally is expressed by the subject to be treated; and/or
the first domain and/or second domain comprises an extracellular binding domain, a hinge domain, a transmembrane domain, or an intracellular signaling domain, which intracellular signaling domain is, optionally, a costimulatory signaling domain or an activating cytoplasmic signaling domain.
73. The method of embodiment 72, wherein the first domain and second domain are not present in the same molecule in vivo in a human subject, or are not present in a single natural or endogenous human protein or polypeptide.
74. The method of embodiment 72 or embodiment 73, wherein the first domain and second domain are or comprise, respectively, an extracellular ligand binding domain and a hinge domain, a hinge domain and a transmembrane domain, a transmembrane domain and an intracellular costimulatory signaling domain, and an intracellular costimulatory signaling domain and an activating cytoplasmic signaling domain, which can include functional portions of such domains.
75. The method of any of embodiments 72-74, wherein the first domain is or comprises a transmembrane domain or a functional portion thereof and the second domain is or comprises a costimulatory signaling domain or a functional portion thereof.
76. The method of embodiment 75, wherein the transmembrane domain is a CD28 transmembrane domain or a functional portion thereof and the costimulatory signaling domain is a 4-1BB signaling domain or a functional portion thereof.
77. The method of any of embodiments 72-76, wherein the second CAR comprises:
a domain of at least 95% sequence identity to the first domain and/or a domain of at least 95% sequence identity to the second domain;
a domain identical in sequence to the first domain and a domain of at least 95% sequence identity to the second domain; or
a domain of at least 95% sequence identity to the first domain and a domain identical in sequence to the second domain,
wherein at least one or both of the domains present in the second CAR comprises at least one or more amino acid sequence differences compared to one or both of the first domain and second domain of the first CAR in the portion comprising the modified junction region.
78. The method of any of embodiments 75-77, wherein:
the first CAR comprises a CD28 transmembrane domain and a 4-1BB co-stimulatory domain that together comprise the sequence of amino acids set forth in SEQ ID NO:5 or a variant or functional portion thereof comprising a sequence of amino acids that is at least 95% identical to SEQ ID NO:5 and comprises the junction region; and
the second CAR comprises a sequence that is modified compared to the first CAR, the modification comprising at least one amino acid sequence difference in a portion comprising a sequence of between residue 13 and 42 or between 15 and 40, with reference to numbering set forth in SEQ ID NO:5.
79. The method of any of embodiments 64-78, wherein the second CAR comprises no more than 20 amino acid sequence differences compared to the first CAR or the second CAR comprises at least 95% amino acid sequence identity to the first CAR.
80. The method of any of embodiments 64-79, wherein a region of the second CAR containing at least one of the one or more sequence differences:
contains fewer 8-15 amino acid portions, as compared to the corresponding region of the first CAR, that has a binding affinity for a human leukocyte antigen (HLA) molecule of an 1050 of less than 1000 nM; or
has a binding affinity for a human leukocyte antigen (HLA) molecule that is lower than the binding affinity for the same HLA molecule of a peptide fragment having the sequence of the corresponding portion of the junction region of the reference chimeric receptor; or
has a binding affinity of at least one peptide fragment within the region, or a reduced average binding affinity of all peptide fragments having the sequence of an 8-15 amino acid portion within the region, for a human leukocyte antigen (HLA) molecule, as compared to the corresponding region of the first CAR.
81. The method of any of embodiments 64-80, wherein a reduced detectable immune response is generated in the subject following the administration of cells expressing the second CAR to the corresponding region of the second CAR that comprises at least one amino acid sequence difference compared to the immune response generated in the subject to the region in the first CAR following its administration to the subject.
82. The method of any of any of embodiments 46-63, wherein:
the first CAR comprises a CD28 transmembrane domain or a functional portion thereof and a 4-1BB costimulatory signaling domain or a function portion thereof; and
the second CAR comprises a transmembrane domain and a costimulatory signaling domain that is distinct from one or both of such domains in the first CAR.
83. The method of any of embodiments 46-82, wherein the second CAR comprises at least one region identical in amino acid sequence to a corresponding region of the first CAR.
84. The method of embodiment 83, wherein the corresponding region of the first CAR is a region to which the subject does not exhibit a detectable humoral or cell-mediated immune response at the time of the administration of the cells expressing the second CAR.
85. The method of embodiment 83 or embodiment 84, wherein the corresponding region of the first CAR comprises a region within a CAR portion selected from the group consisting of a costimulatory domain, an ITAM-containing domain, a transmembrane domain, a transduction or expression marker, a sequence endogenous to the host, and/or an antibody domain derived from the same species as the host.
86. The method of any of embodiments 46-85, wherein the maximum number of CAR-expressing cells, the area under the curve (AUC) for CAR-expressing cells over time, and/or the duration of detectable CAR-expressing cells in the subject following the administration of cells expressing the second CAR is greater as compared to that achieved via a method comprising an alternative dosing regimen comprising performing the administration of cells expressing the first CAR followed by performing a second administration of cells expressing the first CAR, said second administration being carried out at the same point in time as the administration of cells expressing the second CAR.
87. The method of any of embodiments 46-86, wherein:
the method results in a maximum concentration or number of CAR-expressing cells in the blood of the subject of at least at or about 10 CAR-expressing cells per microliter, at least 50% of the total number of peripheral blood mononuclear cells (PBMCs), at least at least about 1×105 CAR-expressing cells, or at least 1,000, or at least 2,000, or at least 3,000, or at least 4,000, or at least 5,000 copies of CAR-encoding DNA per micrograms DNA; and/or
at day 30, at day 60, or at day 90 following the initiation of the administration of cells expressing the second CAR, CAR-expressing cells are detectable in the blood or serum of the subject; and/or
at day 30, at day 60, or at day 90 following the initiation of the administration of cells expressing the second CAR, the blood of the subject contains at least 20% CAR-expressing cells, at least 10 CAR-expressing cells per microliter or at least 1×104 CAR-expressing cells.
88. The method of any of embodiments 46-87, wherein the dose of cells expressing the first CAR and/or the dose of cells expressing the second CAR independently comprise cells in an amount sufficient for reduction in burden of a disease or condition in the subject.
89. The method of any of embodiments 46-88, wherein the administration of cells expressing the first CAR and/or the administration of cells expressing the second CAR effects a reduction in burden of the disease or condition in the subject, thereby treating the disease or condition.
90. The method of any of embodiments 46-89, wherein the cells are T cells.
91. The method of any of embodiments 46-90, wherein the T cells are autologous to the subject.
XIII. EXAMPLES
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1
Analysis of Transgene Product-Specific Host Immune Responses
Pre- and post-treatment peripheral blood mononuclear cells (PBMC) samples were obtained from four (4) subjects with B cell malignancies treated with autologous T cells expressing a CD19-specific CAR. The CAR included an anti-CD19 scFv derived from murine antibody, a hinge domain, a CD28 transmembrane domain, a 4-1BB intracellular signaling domain, a CD3-zeta intracellular signaling domain, a T2A domain, and a truncated EGFR (EGFRt) portion.
Pre- and post (day 42)-infusion PBMCs obtained from the subjects were assessed to detect the presence or absence of specific anti-CAR immune responses essentially as described by Berger et al. Blood. 2006 March; 107(6): 2294-2302, Berger et al. J Virol. 2001 January 75(2): 799-808, Riddell et al. Nature Medicine. 1996 February 2(2): 216-223, Berger et al. Blood. 2005 February 105(4): 1640-1647. Briefly, PBMCs (responders) were stimulated in vitro with autologous gamma-irradiated cells transduced with the CAR expressed by the administered cells (stimulators at a 1:1 or 2:1 responder-to-stimulator ratio). The cultures then were assessed in a chromium release assay for cytotoxicity against autologous 51Cr-labeled CAR-transduced (“CD19 CAR”) and non-transduced (“Mock”) T cells (targets) at various effector-to-target (E/T) ratios. Following co-incubation, release of chromium was quantified and the percentage of maximum achievable lysis in each sample determined.
The results for samples derived from one exemplary patient are shown in FIG. 1, which depicts the cytolytic activity of PBMCs pre-infusion and post-infusion at day 42. Whereas no cytolytic activity specific for CAR-transduced target cells was detected in any pre-infusion PBMC-derived cultures, in two of the four subjects assessed, CAR-specific lytic activity was detected in cultures derived from post-infusion PBMC samples. These results indicate that CAR-specific immune responses can develop following a single infusion of CAR-expressing T cells.
Epitope mapping was carried out to assess region(s) of the CAR recognized by the specific immune responses. Pre- and post-infusion PBMC samples were stimulated in the presence of individual pools of multiple 15-mer peptides, with sequences representing overlapping portions (11 amino acid overlap) of the entire length of an approximately 500 amino acid sequence of the CAR expressed by the administered cells. Cells were stained with antibodies to detect CD8 and CD4 surface expression and intracellular expression of cytokine. Twenty-three (23) pools were assessed, each containing ten (10) peptides each and collectively including 125 individual overlapping peptides, with each peptide represented in at least two of the pools.
This design permitted the generation of an analytic grid to assess responses specific for individual peptides, whereby a peptide present in more than one pool detected as hits in this assay was deemed a potentially immunogenic peptide hit. For the two patients in whom a CAR-specific immune response had been detected, six and three peptide hits, respectively, were identified.
Individual ELISpot assays were performed using an anti-cytokine capture antibody to assess the presence or absence of a specific immune response for each of these individual hits (see Berger et al. (2006); Berger et al. (2001); Riddell et al. (1996); and Berger et al. (2005), supra). The results of an exemplary assay for one patient are shown in FIG. 2. Specific immune responses against peptides with sequences within the VH portion of the scFv of the CAR were detected in both patients assessed (including regions within the FR1, CDR1, and FR2 regions for one patient and within the FR3 for the other). For the first patient, specific immune responses also were detected against two overlapping 15-mer peptides, each containing the junction between the transmembrane domain and costimulatory domain of the CAR (labeled “fusion site” in FIG. 2). These two overlapping 15-mer peptides had the amino acid sequences AFIIFWVKRGRKKLL (SEQ ID NO: 8) and FWVKRGRKKLLYIFK (SEQ ID NO: 9), respectively. In another study following administration with a different CAR having a murine scFv, CD28 transmembrane and costimulatory domains and a CD3 zeta domain, using similar methods, an immune response also was detected for one subject against a pool containing VH portions of an anti-CD19 scFv and for another subject in a pool containing junction portions.
No specific immune responses were detected in the patients by this assay against peptides within other regions. For example, in this assay, no specific responses were detected against peptides having sequences within other CDRs or framework regions of the scFv, peptides within regions of costimulatory or transmembrane domain but not spanning the junction between the two, or peptides within the EGFRt or CD3-t region of the CAR. Specific immune responses were not detected against endogenous sequences.
Example 2
In Silico Analysis of Peptides Derived from Junction Regions of a CAR for Binding to HLA Class I and HLA Class II
T cell epitope prediction tools, available from the Immune Epitope Database and analysis resource (IEDB), were used for in silico analysis to predict MHC-binding affinities and other properties related to potential immunogenicity for each of a series of overlapping peptide sequences within a portion of an exemplary CAR sequence. The portion included a spacer having an immunoglobulin-derived hinge domain, a human CD28 transmembrane domain, a human 4-1BB costimulatory domain, and a human CD3zeta signaling domain. In the portion assessed, the hinge domain was a human IgG4 hinge domain, the CD28 transmembrane domain comprised a sequence set forth in SEQ ID NO:2 and the 4-1BB costimulatory domain contained the sequence set forth in SEQ ID NO:3. This portion thus contained three junctions between different domains derived from human sequences (which junctions may have represented sites of potential immunogenicity against a CAR upon administration to a human subject): the junction between the spacer region and transmembrane domain, the junction between the transmembrane domain and costimulatory domain, and the junction between the costimulatory domain and intracellular signaling domain (see FIGS. 3A and 3B).
To identify portions of the sequence that may have particular properties making them more likely to be presented to T cells, affinities for binding to 27 individual HLA class I alleles and 56 individual HLA class II alleles were predicted for overlapping peptides along the length of the portion, of 8-14 amino acids in length and of 15 amino acids in length (containing 9-mer binding core), respectively. These alleles, collectively representing HLA alleles present in greater than 99% of the worldwide population, and their approximate frequency in the United States population are listed in Tables 1A and 1B.
TABLE 1A
HLA class I
Frequency
Class I
allele
in population
1
HLA-A*01:01
12.94
2
HLA-A*02:01
42.88
3
HLA-A*02:03
0.19
4
HLA-A*02:06
1.55
5
HLA-A*03:01
13.50
6
HLA-A*11:01
11.60
7
HLA-A*23:01
8.30
8
HLA-A*24:02
22.56
9
HLA-A*26:01
5.36
10
HLA-A*30:01
6.29
11
HLA-A*30:02
5.21
12
HLA-A*31:01
6.87
13
HLA-A*32:01
3.71
14
HLA-A*33:01
2.62
15
HLA-A*68:01
6.36
16
HLA-A*68:02
4.79
17
HLA-B*07:02
12.96
18
HLA-B*08:01
9.23
19
HLA-B*15:01
6.54
20
HLA-B*35:01
13.03
21
HLA-B*40:01
9.79
22
HLA-B*44:02
7.22
23
HLA-B*44:03
8.96
24
HLA-B*51:01
8.51
25
HLA-B*53:01
7.26
26
HLA-B*57:01
3.49
27
HLA-B*58:01
4.82
TABLE 1B
HLA class II
Class
Frequency
II
allele
in population
1
HLA-DRB1*01:01
13.62
2
HLA-DRB1*15:01
22.86
3
HLA-DRB1*03:01
21.82
4
HLA-DRB1*04:01
15.54
5
HLA-DRB1*11:01
10.92
6
HLA-DRB1*13:01
9.86
7
HLA-DRB1*07:01
19.84
8
HLA-DRB1*01:01
4.06
9
HLA-DRB1*01:02
1.85
10
HLA-DRB1*04:02
6.28
11
HLA-DRB1*04:05
1.22
12
HLA-DRB1*04:07
2.78
13
HLA-DRB1*04:08
1.26
14
HLA-DRB1*08:04
0.86
15
HLA-DRB1*09:01
5.33
16
HLA-DRB1*10:01
2.78
17
HLA-DRB1*11:02
0.94
18
HLA-DRB1*11:03
0.74
19
HLA-DRB1*11:04
4.76
20
HLA-DRB1*15:02
0.78
21
HLA-DRB1*15:03
1.22
22
HLA-DRB1*16:01
4.06
23
HLA-DRB1*16:02
0.84
24
HLA-DRB3*02:02
0.00
25
HLA-DRB3*03:01
0.00
26
HLA-DRB5*01:01
0.00
27
HLA-
30.57
DQA1*01:01/DQB1
*05:01
28
HLA-
76.17
DQA1*05:01/DQB1
*02:01
29
HLA-DQA1*01:02/DQB1*05:02
21.13
30
HLA-DQA1*01:02/DQB1*06:02
30.74
31
HLA-DQA1*03:01/DQB1*03:02
31.56
32
HLA-DQA1*01:02/DQB1*06:04
19.00
33
HLA-DQA1*05:01/DQB1*03:01
80.58
34
HLA-DQA1*02:01/DQB1*02:02
27.99
35
HLA-DQA1*03:01/DQB1*03:01
49.92
36
HLA-DQA1*02:01/DQB1*03:03
23.32
37
HLA-DQA1*03:03/DQB1*03:03
20.22
38
HLA-DPA1*01:03/DPB1*01:01
99.83
39
HLA-DPA1*01:03/DPB1*02:01
99.83
40
HLA-DPA1*01:03/DPB1*03:01
99.82
41
HLA-DPA1*01:03/DPB1*04:01
99.88
42
HLA-DPA1*01:03/DPB1*04:02
99.86
43
HLA-DPA1*01:03/DPB1*05:01
99.81
44
HLA-DPA1*02:01/DPB1*01:01
23.54
45
HLA-DPA1*02:01/DPB1*02:01
24.11
46
HLA-DPA1*02:01/DPB1*03:01
17.63
47
HLA-DPA1*02:01/DPB1*04:01
46.73
48
HLA-DPA1*02:01/DPB1*04:02
38.04
49
HLA-DPA1*02:01/DPB1*05:01
13.24
50
HLA-DPA1*02:01/DPB1*06:01
8.59
51
HLA-DPA1*02:01/DPB1*09:01
7.26
52
HLA-DPA1*02:01/DPB1*11:01
9.98
53
HLA-DPA1*02:01/DPB1*13:01
11.55
54
HLA-DPA1*02:01/DPB1*14:01
7.98
55
HLA-DPA1*02:01/DPB1*15:01
7.73
56
HLA-DPA1*02:01/DPB1*17:01
10.40
Algorithm-based T cell epitope prediction tools available from the IEDB were used to predict IC50 values for binding to HLA class I molecules for each 8-14 amino acid peptide in the dataset using ANN (Nielsen et al. (2003) Protein Sci., 12:1007-1017 and Lundegaard et al. (2008) NAR, 36:W509-512) and, in some cases, one or more additional prediction using SMM (Peters and Sette (2005) BMC Bioinformatics, 6:132) and comblib (Sidney et al. (2008) Immunome Res. 4:2, or the Consensus tool (see Kim, et al. (2012) Immune epitope database analysis resource, NAR (combining predictions from any of the foregoing). Predictions for IC50 values for binding to HLA class II for each 15 amino acid peptide in the dataset was made using the NetMHCIIpan method (Karosiene et al. (2013) Immunogenetics 65(10):711; Nielsen et al. (2008) PLoS Comput Biol. 4(7)e1000107). For each individual position within the portion of the CAR amino acid sequence, the total number of sequences in the dataset that included the position and was predicted to bind to any of the class I or class II alleles with a predicted IC50 of less than 50 nm was determined. FIGS. 4A (HLA class I) and 4B (HLA class II), depict the results for class I and class II alleles, respectively, showing positional coverage along the length of the sequence, based on the determined total number, weighted according to the frequency of the individual HLA alleles in the population. The area under the curve (AUC) across the entire assessed region was approximately 1321 for HLA class I binding and 2943 for HLA class II binding. The AUC for the transmembrane-costimulatory domain region was approximately 931 for HLA class I binding and 2212 for HLA class II binding.
As shown in FIGS. 4A and 4B, certain portions of the sequence were predicted by this method to contain fragments more likely to bind well in MHC complexes and thus be presented as epitopes for potential recognition by T cells. Binding affinity for HLA alleles alone does not necessarily predict immunogenicity. Given that the individual domains (e.g., transmembrane, costimulatory) in this exemplary CAR were human-derived, upon administration to a human subject, immunogenic responses were less likely to develop against an epitope within any one of these individual regions alone (as opposed to an epitope spanning multiple regions not ordinarily associated with one another, and/or including a junction between such regions). For example, even for a peptide predicted to bind well to and be presented in the context of an MHC molecule, if the peptide was derived entirely from an endogenous protein, it may be recognized as “self” and thus may fail to induce a productive immune response. For example, whereas certain regions entirely within a single transmembrane or cytoplasmic domain scored highly on the HLA-binding affinity prediction, in the results described in Example 1, no immune responses were detected against peptide sequences solely within either one of these domains of a similar CAR sequence. Accordingly, while various “hot spots” were observed with respect to predicted HLA-binding affinity, subsequent assessment and alteration focused on those areas that not only had higher predicted IC50 values, but also included potential epitopes that spanned the junction between different domains derived from two different proteins.
In particular, a junction region that includes one or more potential peptide epitopes spanning the junction of the CD28 transmembrane domain and 4-1BB signaling domain of the exemplary CAR was further assessed. With respect to the sequence set forth in SEQ ID NO:5, which includes the exemplary human CD28 transmembrane domain (SEQ ID NO:2) and exemplary human 4-1BB costimulatory domain (SEQ ID NO:3), the assessed junction region contained 13 amino acids on either side of the junction spanning the CD28 transmembrane and 4-1BB costimulatory domains as follows: FWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCEL (SEQ ID NO:5), in which a 26 amino acid junction region is indicated by bold, and the two amino acids just C′ and N′ of the junction between the domains is indicated by underline. The assessed 26 amino acid junction region is set forth in SEQ ID NO:137 and corresponds to amino acid residues 15 to 40 of the sequence of amino acids set forth in SEQ ID NO:5.
In silico modeling was carried out to identify one or more amino acid modifications (mutations) within the 26-amino acid junction region set forth in SEQ ID NO: 137 resulting in peptide fragments that were predicted to bind with high IC50 values to class I and class II alleles, and thus that were likely to reduce the potential for inducing immunogenicity against a CAR containing this region. Specifically, predictions were made for variant peptide fragments of the junction region containing one or more mutations at amino acid residue positions corresponding to positions 14, 17 and 20 with numbering with reference to SEQ ID NO:137 (which correspond to one or more mutations at amino acid positions corresponding to positions 28, 31 and 34 with numbering with reference to SEQ ID NO:5). In this exemplary study, these residues were chosen for further analysis following in silico mutagenesis and binding predictions of all high affinity epitopes in which all possible single amino acid replacements across that epitope were surveyed for their impact on the predicted IC50 values. Residues that resulted in greater IC50 predictions (decrease in the binding) were identified, which identified the above residues as being sensitive to replacements.
A series of different variant junction regions were assessed, each containing one or more amino acid replacement at the assessed position(s), as compared to the non-mutated junction region within the exemplary CAR sequence. An exemplary subset of amino acid replacements at the identified positions were chosen that may be less disruptive to the structure or function of either the transmembrane region of the costimulatory signaling domain. Also, replacements were chosen that may be able to impact more than one epitope at a time, since the epitopes overlap. Specifically, individual variant junction regions contained the following modifications (amino acid replacements): K28A, K28H, K28L, K28Q, K28S, R32A, R31H, R31L, R31N, L34A, L34S, K28Q/R31A, K28Q/R31N, K28Q/R31S, K28Q/L34A, K28Q/L34S, R31N/L34A, R31N/L34S, K28Q/R31N/L34A, K28Q/R31N/L34S, with numbering with reference to SEQ ID NO:5.
For the non-variant and variant junction regions, weighted immunogenicity scores were obtained for class I and class II alleles, using the T cell epitope prediction tools available from IEDB. Scores were derived using predicted IC50 values for each of a series of 8-mer to 14-mer overlapping peptides (for each of the 27 HLA class I alleles, individually) and a series of 15-mer overlapping peptides (for each of the 56 HLA class II alleles, individually) within the respective (variant or non-variant) 26-amino acid junction region, and were weighted based on relative frequency in the population of the individual HLA class I and class II alleles. A higher relative score is indicative of a higher degree of predicted binding.
The results are set forth in FIG. 5. The results demonstrate the ability to decrease in the overall predicted HLA class I immunogenicity score within a CAR junction region by modifying amino acids within the region. The results also confirm the ability to reduce predicted HLA class I binding affinity (and hence reduced predicted immunogenicity score) without resulting in a substantial increase in the predicted immunogenicity score for HLA class II binding. Thus, in general, the results showed that amino acid modification(s) within a region spanning a junction between a CD28 transmembrane domain and a 4-1BB costimulatory domain of a CAR could be made and effect an overall reduction in the predicted affinity for human HLA binding, which would be consistent with a reduction in potential for immunogenicity, upon administration to a human subject, of a chimeric receptor identical to a receptor having this region, but containing the modification or combination of modifications in this region.
Example 3
Comparison of In Silico Analysis and In Vitro Binding of Peptides Derived from Junction Regions of a CAR for Binding to HLA Class I
Actual binding affinities for certain HLA class I alleles (A*02:01, A*03:01, A*11:01, and B*08:01) were assessed in vitro for exemplary overlapping 9 amino acid peptide sequences within a portion of the 26 amino acid junction region spanning the CD28 and 4-1BB junction. Specifically, assessment was of a series of overlapping 9-mer peptides derived from the sequence VAFIIFWVKRGRKKLL (set forth in SEQ ID NO: 7), which contains a portion of the CD28 transmembrane domain and 4-1BB costimulatory domain spanning the junction between the domains (bond joining the two amino acids noted in underline). In addition, a series of overlapping 9-mer peptides of each of a number of different variants of this portion also were assessed, each variant containing a mutation or mutations in this region as described in Example 2.
The various 9-mer overlapping peptides were synthesized and their purity tested by MALDI-TOF Mass Spectrometry. The synthetic peptides were then incubated with recombinant MHC molecules to assess binding properties using the REVEAL Epitope Discovery System, which is a high-throughput binding assay that measures the degree to which each peptide is able to stabilize a ternary MHC-peptide complex (ProImmune, Oxford, United Kingdom). Each peptide was separately tested for this ability with respect to each of the HLA class I alleles, normalized to the degree observed for a positive control (known T cell epitope for the relevant allele). The results are reported as a score, in which the binding was normalized to the positive control peptide set at 100%. In this analysis, a score of greater than 50 generally was considered to represent good or high affinity binding.
The results were compared to predicted binding (IC50) values obtained for binding of the same peptide:MHC complex, using the in silico prediction methods as described in Example 2. Since the maximum IC50 value predicted was about 50,000, the IC50 values were log transformed, subtracted from LOG(50000) and divided by LOG(50000) to obtain a normalized in silico score ((Log(50000)−log IC50)/Log(50000)). In this analysis, an in silico binding prediction score of greater than 2.0 generally was considered to represent predicted good or high affinity binding.
The results are set forth in Table 2A (HLA-A*02:01 and HLA-A*03:01) and Table 2B (HLA-A*11:01 and HLA-B*08:01). In general, the in silico binding predictions were predictive of the actual in vitro binding results. In some cases, a relatively higher binding was predicted in silico, but not observed in the in vitro assay.
The results also were consistent with predicted binding affinity being generally predictive of affinity as measured in the in vitro assay. Additionally, the results demonstrated successful reduction of binding affinity to an HLA by modifications within a junction region, and that it was possible to modify the sequence in a way that resulted in a lower predicted or actual binding affinity or score of one of the overlapping potential epitopes, without increasing (or while also reducing) binding affinity or score for another of the overlapping epitopes containing the same residue. In some embodiments, such mutations or modifications may be particularly advantageous. As a non-limiting example of the results, modifications K29L, R31H, L34S and/or L34A, with reference to numbering set forth in SEQ ID NO:5, generally resulted in a reduced predicted or actual binding affinity or score for at least one HLA allele and/or for at least one peptide within the region assessed, without resulting in a higher binding affinity to another HLA allele and/or without resulting in a higher binding affinity for another peptide.
TABLE 2A
In Silico and In Vitro MHC binding of Variant Peptides
Over-
A*03:01
lapping
A*02:01
SEQ
Reference
Peptide
IEDB
In Silico
In Vitro
IEDB
In
In
ID
Peptide
Sequence
Mutation
IC50
Score
Score
IC50
Silico
Vitro
NO.
1
VAFIIFWVK
none
19617
0.41
0.70
262
2.28
1.50
16
2
AFIIFWVKR
none
25314
0.30
0.50
17846
0.45
5.70
17
3
FIIFWVKRG
none
7967
0.80
10.10
23693
0.32
1.40
18
4
IIFWVKRGR
none
24769
0.31
1.70
406
2.09
24.30
19
5
IFWVKRGRK
none
30463
0.22
4.50
3482
1.16
20.50
20
6
FWVKRGRKK
none
28878
0.24
3.50
17327
0.46
2.20
21
7
WVKRGRKKL
none
27956
0.25
1.40
22961
0.34
13.10
22
1
VAFIIFWVS
K29S
12273
0.61
70.60
22660
0.34
0.70
23
2
AFIIFWVSR
K29S
23924
0.32
4.50
18157
0.44
0.30
24
3
FIIFWVSRG
K29S
3382
1.17
0.20
21751
0.36
0.10
25
4
IIFWVSRGR
K29S
21442
0.37
2.60
155
2.51
25.30
26
5
IFWVSRGRK
K29S
30615
0.21
1.30
1880
1.42
39.20
27
6
FWVSRGRKK
K29S
28679
0.24
1.50
17832
0.45
2.90
28
7
WVSRGRKKL
K29S
23551
0.33
2.30
22394
0.35
2.10
29
1
VAFIIFWVL
K29L
1336
1.57
0.20
20145
0.39
0.00
30
2
AFIIFWVLR
K29L
22444
0.35
5.70
15583
0.51
0.30
31
3
FIIFWVLRG
K29L
2037
1.39
7.20
20853
0.38
0.10
32
4
IIFWVLRGR
K29L
17613
0.45
8.40
238
2.32
2.80
33
5
IFWVLRGRK
K29L
30293
0.22
2.90
3675
1.13
19.50
34
6
FWVLRGRKK
K29L
28857
0.24
3.30
16996
0.47
0.40
35
7
WVLRGRKKL
K29L
19522
0.41
2.40
23063
0.34
0.70
36
1
VAFIIFWVH
K29H
23252
0.33
3.10
10359
0.68
0.30
37
2
AFIIFWVHR
K29H
22819
0.34
0.30
18506
0.43
0.30
38
3
FIIFWVHRG
K29H
1691
1.47
37.30
22930
0.34
0.00
39
4
IIFWVHRGR
K29H
23573
0.33
4.70
326
2.19
30.10
40
5
IFWVHRGRK
K29H
29930
0.22
7.10
1062
1.67
37.80
41
6
FWVHRGRKK
K29H
28189
0.25
1.60
17052
0.47
3.60
42
7
WVHRGRKKL
K29H
25035
0.30
1.60
22278
0.35
3.40
43
1
VAFIIFWVA
K29A
2733
1.26
28.50
21010
0.38
0.00
44
2
AFIIFWVAR
K29A
23072
0.34
2.20
17755
0.45
0.50
45
3
FIIFWVARG
K29A
1902
1.42
22486
0.35
102
4
IIFWVARGR
K29A
22077
0.36
9.60
206
2.39
18.60
46
5
IFWVARGRK
K29A
30251
0.22
1.00
3893
1.11
53.70
47
6
FWVARGRKK
K29A
28353
0.25
13.50
16210
0.49
5.00
48
7
WVARGRKKL
K29A
22630
0.34
8.80
22548
0.35
1.30
49
1
VAFIIFWVQ
K29Q
17921
0.45
5.30
22927
0.34
0.20
50
2
AFIIFWVQR
K29Q
24165
0.32
0.40
19279
0.41
0.20
51
3
FIIFWVQRG
K29Q
4152
1.08
16.80
23136
0.33
0.40
52
4
IIFWVQRGR
K29Q
21783
0.36
5.80
231
2.34
18.00
53
5
IFWVQRGRK
K29Q
30704
0.21
6.10
3177
1.20
36.70
54
6
FWVQRGRKK
K29Q
29501
0.23
5.20
18619
0.43
6.40
55
7
WVQRGRKKL
K29Q
24480
0.31
8.10
23630
0.33
4.90
56
4
IIFWVKRGS
R32S
18902
0.42
13.60
19450
0.41
16.20
57
5
IFWVKRGSK
R32S
29391
0.23
2.40
3348
1.17
10.40
58
6
FWVKRGSKK
R32S
28317
0.25
1.90
14227
0.55
5.90
59
7
WVKRGSKKL
R32S
23485
0.33
3.00
22637
0.34
1.90
60
4
IIFWVKRGL
R32L
2692
1.27
82.20
16661
0.48
15.90
61
5
IFWVKRGLK
R32L
29250
0.23
2.40
1973
1.40
26.90
62
6
FWVKRGLKK
R32L
27554
0.26
9.40
14434
0.54
2.70
63
7
WVKRGLKKL
R32L
20709
0.38
24.50
22985
0.34
10.00
64
4
IIFWVKRGH
R32H
27453
0.26
5.30
1665
1.48
26.60
65
5
IFWVKRGHK
R32H
29806
0.22
2.20
3806
1.12
9.00
66
6
FWVKRGHKK
R32H
27689
0.26
2.60
16743
0.48
4.10
67
7
WVKRGHKKL
R32H
25923
0.29
3.90
22313
0.35
1.40
68
4
IIFWVKRGA
R32A
6107
0.91
85.90
16069
0.49
42.90
69
5
IFWVKRGAK
R32A
29354
0.23
13.80
3470
1.16
22.00
70
6
FWVKRGAKK
R32A
28151
0.25
4.30
17066
0.47
4.70
71
7
WVKRGAKKL
R32A
24746
0.31
4.70
22982
0.34
4.10
72
4
IIFWVKRGN
R32N
25979
0.28
9.30
18552
0.43
4.30
73
5
IFWVKRGNK
R32N
29978
0.22
3.50
2669
1.27
8.00
74
6
FWVKRGNKK
R32N
28430
0.25
8.70
17713
0.45
3.60
75
7
WVKRGNKKL
R32N
24790
0.30
2.40
22813
0.34
0.90
76
4
IIFWVQRGS
K29Q/R32S
14326
0.54
22.40
17741
0.45
4.50
77
5
IFWVQRGSK
K29Q/R32S
29540
0.23
33.50
3052
1.212
0.30
78
6
FWVQRGSKK
K29Q/R32S
28907
0.24
0.80
15992
0.50
1.90
79
7
WVQRGSKKL
K29Q/R32S
18121
0.44
4.40
23279
0.33
1.20
80
4
IIFWVQRGA
K29Q/R32A
2658
1.27
95.30
13467
0.57
2.70
81
5
IFWVQRGAK
K29Q/R32A
29482
0.23
11.70
3205
1.191
7.00
82
6
FWVQRGAKK
K29Q/R32A
28792
0.24
2.20
18649
0.43
2.70
83
7
WVQRGAKKL
K29Q/R32A
20217
0.39
3.00
23651
0.33
2.00
84
4
IIFWVQRGN
K29Q/R32N
23103
0.34
13.00
16590
0.48
3.60
85
5
IFWVQRGNK
K29Q/R32N
30164
0.22
16.10
2438
1.31
23.20
86
6
FWVQRGNKK
K29Q/R32N
29113
0.23
3.10
19165
0.42
1.30
87
7
WVQRGNKKL
K29Q/R32N
19790
0.40
4.30
23457
0.33
1.50
88
7
WVKRGRKKS
L35S
30812
0.21
3.90
25365
0.29
0.90
89
7
WVKRGRKKA
L35A
28556
0.24
4.50
24086
0.32
0.90
90
7
WVQRGNKKS
K29Q/L35S
26883
0.27
1.20
25680
0.29
0.70
91
7
WVQRGNKKA
K29Q/L35A
21998
0.36
1.90
24564
0.31
1.20
92
1
VAFIIFWVR
K29R
20045
0.40
1.20
5746
0.94
0.70
100
2
AFIIFWVRR
K29R
25059
0.30
1.40
17924
1.00
101
TABLE 2B
In Silico and In Vitro MHC binding of Variant Peptides
A*11:01
B*08:01
Overlapping
In
In
In
In
SEQ
Reference
Peptide
IEDB
Silico
Vitro
IEDB
Silico
Vitro
ID
Peptide
Sequence
Mutation
IC50
Score
Score
IC50
Score
Score
NO.
1
VAFIIFWVK
wt
19
3.42
109.20
15806
0.50
0.30
16
2
AFIIFWVKR
wt
3271
1.18
25.90
23244
0.33
1.90
17
3
FIIFWVKRG
wt
22946
0.34
0.40
18081
0.44
0.10
18
4
IIFWVKRGR
wt
706
1.85
54.50
23025
0.34
0.00
19
5
IFWVKRGRK
wt
9701
0.71
29.30
23513
0.33
0.40
20
6
FWVKRGRKK
wt
22175
0.35
6.50
21764
0.36
0.00
21
7
WVKRGRKKL
wt
24020
0.32
4.80
225
2.35
28.50
22
1
VAFIIFWVS
K29S
12403
0.61
25.10
13796
0.56
0.10
23
2
AFIIFWVSR
K29S
2175
1.36
54.00
21862
0.36
0.30
24
3
FIIFWVSRG
K29S
20651
0.38
5.10
17854
0.45
0.40
25
4
IIFWVSRGR
K29S
162
2.49
68.90
23445
0.33
0.00
26
5
IFWVSRGRK
K29S
5778
0.94
39.20
23448
0.33
0.00
27
6
FWVSRGRKK
K29S
21126
0.37
6.30
23067
0.34
0.00
28
7
WVSRGRKKL
K29S
23684
0.32
4.40
3208
1.19
6.20
29
1
VAFIIFWVL
K29L
14191
0.55
0.20
1196
1.62
0.00
30
2
AFIIFWVLR
K29L
850
1.77
17.20
23012
0.34
0.20
31
3
FIIFWVLRG
K29L
19162
0.42
2.40
17427
0.46
0.20
32
4
IIFWVLRGR
K29L
185
2.43
35.20
23298
0.33
0.40
33
5
IFWVLRGRK
K29L
6386
0.89
29.30
23324
0.33
0.00
34
6
FWVLRGRKK
K29L
21862
0.36
5.00
23437
0.33
0.00
35
7
WVLRGRKKL
K29L
23725
0.32
3.20
1342
1.57
4.00
36
1
VAFIIFWVH
K29H
1209
1.62
12.30
16202
0.49
0.00
37
2
AFIIFWVHR
K29H
4939
1.01
34.90
22682
0.34
0.30
38
3
FIIFWVHRG
K29H
19850
0.40
0.90
17464
0.46
0.20
39
4
IIFWVHRGR
K29H
196
2.41
77.50
22662
0.34
0.20
40
5
IFWVHRGRK
K29H
7133
0.85
36.60
22030
0.36
0.10
41
6
FWVHRGRKK
K29H
22214
0.35
5.60
23778
0.32
0.00
42
7
WVHRGRKKL
K29H
23844
0.32
5.60
814
1.79
19.20
43
1
VAFIIFWVA
K29A
12499
0.60
2.80
5131
0.99
0.00
44
2
AFIIFWVAR
K29A
2784
1.25
51.80
21850
0.36
0.20
45
3
FIIFWVARG
K29A
20922
0.38
18463
0.43
102
4
IIFWVARGR
K29A
239
2.32
70.60
23580
0.33
0.30
46
5
IFWVARGRK
K29A
8772
0.76
34.90
23612
0.33
0.00
47
6
FWVARGRKK
K29A
20762
0.38
8.90
23035
0.34
0.00
48
7
WVARGRKKL
K29A
23920
0.32
13.80
2821
1.25
32.40
49
1
VAFIIFWVQ
K29Q
15477
0.51
3.90
14875
0.53
0.20
50
2
AFIIFWVQR
K29Q
2174
1.36
15.00
23016
0.34
0.00
51
3
FIIFWVQRG
K29Q
22161
0.35
1.50
17653
0.45
0.60
52
4
IIFWVQRGR
K29Q
361
2.14
72.80
23548
0.33
0.10
53
5
IFWVQRGRK
K29Q
9561
0.72
128.50
23670
0.32
0.00
54
6
FWVQRGRKK
K29Q
22394
0.35
5.70
22604
0.34
1.90
55
7
WVQRGRKKL
K29Q
23688
0.32
12.80
2512
1.30
45.10
56
4
IIFWVKRGS
R32S
18923
0.42
100.00
22325
0.35
0.40
57
5
IFWVKRGSK
R32S
7476
0.83
47.90
21691
0.36
1.20
58
6
FWVKRGSKK
R32S
19910
0.40
5.60
20823
0.38
0.10
59
7
WVKRGSKKL
R32S
24090
0.32
11.80
585
1.93
56.10
60
4
IIFWVKRGL
R32L
20182
0.39
32.40
17368
0.46
1.70
61
5
IFWVKRGLK
R32L
4201
1.08
61.10
23537
0.33
58.30
62
6
FWVKRGLKK
R32L
17309
0.46
25.30
20839
0.38
6.30
63
7
WVKRGLKKL
R32L
24095
0.32
22.10
765
1.82
65.70
64
4
IIFWVKRGH
R32H
7117
0.85
19.50
23227
0.33
0.10
65
5
IFWVKRGHK
R32H
10783
0.67
30.90
23461
0.33
3.20
66
6
FWVKRGHKK
R32H
17635
0.45
27.60
20754
0.38
0.40
67
7
WVKRGHKKL
R32H
23924
0.32
2.80
269
2.27
47.60
68
4
IIFWVKRGA
R32A
19134
0.42
22.60
19585
0.41
0.90
69
5
IFWVKRGAK
R32A
8311
0.78
69.70
21592
0.36
2.20
70
6
FWVKRGAKK
R32A
20234
0.39
13.20
21762
0.36
0.30
71
7
WVKRGAKKL
R32A
23857
0.32
4.60
1366
1.56
34.10
72
4
IIFWVKRGN
R32N
19351
0.41
42.10
22864
0.34
0.90
73
5
IFWVKRGNK
R32N
6780
0.87
45.90
24238
0.31
0.10
74
6
FWVKRGNKK
R32N
20732
0.38
7.30
21565
0.37
0.10
75
7
WVKRGNKKL
R32N
24036
0.32
4.10
1181
1.63
65.90
76
4
IIFWVQRGS
K29Q/R32S
18031
0.44
16.90
22961
0.34
0.10
77
5
IFWVQRGSK
K29Q/R32S
7300
0.84
103.30
22846
0.34
0.00
78
6
FWVQRGSKK
K29Q/R32S
20419
0.39
6.80
21853
0.36
0.00
79
7
WVQRGSKKL
K29Q/R32S
23740
0.32
5.90
5230
0.98
6.80
80
4
IIFWVQRGA
K29Q/R32A
18055
0.44
56.50
20759
0.38
0.20
81
5
IFWVQRGAK
K29Q/R32A
8237
0.78
61.30
22801
0.34
0.20
82
6
FWVQRGAKK
K29Q/R32A
20696
0.38
18.30
22612
0.34
0.00
83
7
WVQRGAKKL
K29Q/R32A
23552
0.33
3.80
8314
0.78
11.40
84
4
IIFWVQRGN
K29Q/R32N
18437
0.43
29.00
23425
0.33
0.00
85
5
IFWVQRGNK
K29Q/R32N
6566
0.88
67.50
24059
0.32
0.10
86
6
FWVQRGNKK
K29Q/R32N
21228
0.37
5.90
22436
0.35
0.00
87
7
WVQRGNKKL
K29Q/R32N
23751
0.32
10.70
7869
0.80
32.00
88
7
WVKRGRKKS
L35S
23906
0.32
6.70
10345
0.68
4.30
89
7
WVKRGRKKA
L35A
23864
0.32
3.10
1225
1.61
3.10
90
7
WVQRGNKKS
K29Q/L35S
23612
0.33
5.00
21235
0.37
0.00
91
7
WVQRGNKKA
K29Q/L35A
23576
0.33
0.80
14247
0.55
0.70
92
1
VAFIIFWVR
K29R
108
2.67
34.60
16290
0.49
0.10
100
2
AFIIFWVRR
K29R
3387
1.17
1.50
22717
0.34
0.30
101
Example 4
Analysis of Peptides Derived from Junction Region of a CAR for Binding to HLA-A2:01
In order to identify CAR-derived peptides potentially capable of inducing immunogenic responses, a series of overlapping peptides within the non-variant (reference) sequence containing the junction between the CD28 transmembrane domain and 4-1BB costimulatory domain of a CAR were assessed in silico. Algorithms were used to predict binding affinities for the peptide groove of a common human MHC class I molecule (HLA-A2:01) using in silico analysis to predict affinity for binding. As set forth in FIG. 3, the assessed portion of the CAR had the sequence CYSLLVTVAFIIFWVKRGRKKLLYIFKQPF (set forth in SEQ ID NO: 6), which contains a portion of the of the CD28 transmembrane domain (set forth in SEQ ID NO:2) and a portion of the 4-1BB costimulatory domain (set forth in SEQ ID NO:3), with the residues spanning the junction of the domains shown by underline. Predicted HLA-A2:01 binding affinity was assessed in silico for a series of 140 overlapping peptides of 8-14 amino acids of the sequence set forth in SEQ ID NO:6. Thirty-five (35) of the peptides contained only sequence from the transmembrane domain portion; 35 of the peptides contained only from the costimulatory domain portion, and 70 of the peptides had a junction or fusion region sequence, containing amino acid residues bridging the junction between the domains. For this assessment, peptide fragments predicted to bind to HLA-A2:01 with a dissociation constant of 0 nM to 50 nM were considered predicted to bind with high affinity. Peptide fragments predicted to bind with a dissociation constant of 51 nM to 1000 nM were considered predicted to bind with low affinity. Peptide fragments predicted to bind with a predicted affinity of 1000 nM to 5000 nM were considered predicted to bind with rare affinity. The results are presented in FIG. 3.
As shown in FIG. 3, two of the peptides derived from the reference sequence in this region, each containing a sequence with an overlapping region spanning the junction between the domains were predicted to exhibit low binding affinity for HLA-A2:01. Specifically, a 14-mer peptide having the sequence FIIFWVKRGRKKLL (SEQ ID NO: 10), was predicted to bind with a dissociation constant of 294 nM, and a 13-mer peptide having the sequence of FIIFWVKRGRKKL (SEQ ID NO: 11) was predicted to bind with a dissociation constant of 618 nM. These peptides each included a portion of the 15-mer peptide set forth in SEQ ID NO:1 and identified in Example 1. Shorter 8-mer to 12-mer peptides within this sequence were not predicted to exhibit binding to HLA-A2:01. Another 13-mer peptide containing the amino acid sequence IIFWVKRGRKKLL (SEQ ID NO: 12) was predicted to have a rare binding affinity with a predicted dissociation constant of approximately 3000 nM. None of the remaining fragments that bridged the junction between the two domains were predicted by this assay to exhibit binding affinity for HLA-A2:01 (all had a predicted dissociation constant of far greater than 5000 nM, and in most cases higher than 14,000 nm or 20,000 nM or greater). In each of the peptides predicted to bind to HLA-A2:01, neither of the two junction-spanning residues (VK) themselves was predicted to be an anchor residue; rather, such peptides contained these residues in non-flanking positions.
Approximately 15 of the peptides containing sequence derived only from the transmembrane domain were predicted to have a dissociation constant for HLA-A2:01 of less than 5000 nM. Two peptides containing sequence only from the co-stimulatory domain were predicted to have a dissociation constant for HLA-A2:01 binding of less than 5000 nM. The costimulatory domain and transmembrane domain in the assessed sequence are derived from endogenous human sequences, which generally are less likely to be immunogenic to a human subject. For example, in the study described in Example 1, no immune responses were detected that were specific for peptide sequences solely within either one of these domains of the CAR. Accordingly, variants of peptides containing sequence spanning the junction region were assessed.
To generate variant peptides predicted to have reduced binding affinities to HLA-A2:01 and/or reduced immunogenicity in a human subject having this HLA allele, a variant sequence was generated in silico, containing mutations in the junction region as compared to the sequence set forth in SEQ ID NO:6. Given that peptides containing the junction-spanning “VK” residues (at non-anchor positions) were predicted to exhibit high binding affinities for HLA-A2:01, two asparagine residues were inserted in the junction between the CD28 transmembrane and 4-1BB co-stimulatory domains. The variant contained the sequence CYSLLVTVAFIIFWVNNKRGRKKLLYIFKQPF (set forth in SEQ ID NO: 13, the sequence flanking the junction that was generated by insertion of the asparagine residues is shown in underline). The exemplary variant sequence of SEQ ID NO: 13 was assessed by the same predictive methods. To assess predicted binding affinities for this variant sequence, a series of 154 overlapping fragments of 8-14 amino acids of the sequence set forth in SEQ ID NO: 13 were assessed by in silico analysis as described above, whereby 35 peptides had a sequence only in the transmembrane portion, 35 peptides had a sequence only in the costimulatory domain portion and 84 peptides contained a junction region sequence containing amino acids bridging the domains, including one or both of the inserted asparagine residues.
The results are depicted in FIG. 3. As shown, overall, the HLA-A2:01 binding affinities of overlapping peptides within the variant region containing the junction, collectively, were substantially reduced as compared to the non-variant sequence. In particular, the predicted dissociation constant for binding to HLA-A2:01 of peptides in the portion of the junction region previously predicted to be immunogenic was substantially reduced. For example, peptide variants IIFWVNNKRGRKKL (SEQ ID NO: 14) and IIFWVNNKRGRKK (SEQ ID NO: 15), which included altered flanking residues compared to peptides identified as set forth in SEQ ID NOS:10 and 11, respectively, were predicted to exhibit no detectable binding affinity to HLA-A2:01. Two 14-mer peptides, FIIFWVNNKRGRKK (SEQ ID NO:11) and IFWVNNKRGRKKLL (SEQ ID NO:12), were predicted to exhibit a dissociation constant for binding to this HLA indicating a rare binding affinity, within the range of 1000 nM to 5000 nM. All other peptides containing the modified junction region sequence were predicted to exhibit a dissociation constant of greater than 5000 nM, and in most cases higher than 14,000 nM or 20,000 nM or greater, and thus were not predicted to exhibit binding affinity for HLA-A2:01 by this assessment. Additionally, the modification of the junction region sequence did not create any new peptides predicted to have higher binding affinities for HLA-A2:01 within the costimulatory or transmembrane domain regions.
Example 5
Administration of Anti-CD22 CAR-Expressing Cells to Subjects Previously Treated with Anti-CD19 CAR
Six subjects with relapsed/refractory CD22+ B cell acute lymphoblastic leukemia (ALL) were administered autologous T cells expressing an anti-CD22 chimeric antigen receptor (CAR). The CAR included a human anti-CD22 scFv antibody, a CD8alpha transmembrane domain, a 4-1BB intracellular signaling domain, and a CD3zeta intracellular signaling domain.
All subjects had previously undergone at least one prior allogeneic hematopoietic stem cell transplant and had received treatment with one of various CD19-directed CAR-T cell therapies. Five of the subjects had relapsed with ALL on which CD19 was not detected (“CD19 neg”) and one subject was otherwise a non-responder to the prior CD19 CAR therapy.
Table 3 summarizes the characteristics of the treated patients.
TABLE 3
Patient Characteristics
Pre-HCT
disease
CD22
burden (%
Prior
Prior anti-
CD19 neg
site
leukemia
ID
Age/Sex
HCT
CD19 CAR
relapse
density
in aspirate)
1
22/M
Y
Y
Y
2084
>95%
2
20/F
Y (2)
Y
Y
13452
5%
3
22/M
Y
Y
Y
846
>90%
4
22/M
Y
Y
N
2589
95%
5
7/F
Y
Y
Y
2839
32%
6
17/F
Y
Y
Y
2185
1%
HCT: hematopoietic cell transplantation.
Prior to administration of the cells, patients underwent autologous leukapheresis to harvest peripheral blood mononuclear cells (PBMCs). T cells were isolated from the harvested PBMCs by immunoaffinity-based enrichment for CD3 expression and cultured in the presence of anti-CD3/-CD28 beads, followed by transduction with a lentiviral vector encoding the anti-CD22 CAR. The cells were cultured for 7-10 days. Subjects received induction chemotherapy with 25 mg/m2 fludarabine on Days −4, −3 and −2 and 900 mg/m2 cyclophosphamide on day −2 (cell infusion on Day 0). Each patient received an initial CAR T cell dose of 3×105 transduced T-cells/recipient weight (kg) by intravenous infusion. The second subject enrolled developed grade 3 diarrhea, meeting the criteria for dose-limiting toxicity (DLT), which led to dose expansion at the first dose-level to treat a total of 6 subjects. No subsequent DLTs were seen at this dosage. Two subjects developed grade 1 cytokine release syndrome (CRS), one subject developed grade 2 CRS, and in two subjects CRS was not present.
The number of CAR-T cells in peripheral blood, bone marrow or cerebrospinal fluid was determined at certain timepoints post-treatment by incubating cells with CD22-Fc. For patients in which expansion was observed, evidence for CAR-T cell expansion was seen in peripheral blood, bone marrow and cerebrospinal fluid, beginning at about day 7. The maximum or peak CAR-T cell expansion was generally observed between about day 12 and about day 15 post-infusion. Table 7 sets forth the maximum or peak percentage of anti-CD22 CAR-T cells observed in this assessment period as a percentage of total T cells in each sample for the treated subjects. Clinical responses were evaluated at day 28 (+/−4 days) post-infusion.
As shown in Table 4, the results were consistent with responses being generally correlated to degree of CAR-T cell expansion. For three subjects that exhibited no or low CAR-T cell expansion also showed evidence of disease progression. Two other subjects had stable disease, and one was observed with complete remission with no MRD. Flow cytometric CAR persistence was detected out to 47 days post-infusion in this subject, with remission maintained for 3 months post-infusion. The results demonstrate safe, feasible, and clinically active anti-CD22 CAR T-cell therapy in subjects having undergone (and having become non-responsive to, e.g., due to epitope/antigen loss) previous anti-CD19 CAR therapy.
TABLE 4
Treatment response
Maximum CAR expansion (flow)
ID
PB
Marrow
CSF
CRS
Best Response
1
0
0
n/a
None
PD
2
52.3%
19.5%
0%
Gr 1
MRD neg CR
3
73%
36%
32%
Gr 1
SD
4
6%
1%
0%
Gr 2
SD
5
0%
1.3%
0%
None
PD
6
1.8%
2%
0%
None
PD
PB: peripheral blood;
CSF: cerebrospinal fluid;
CRS: cytokine release syndrome;
PD: progressive disease:
MRD: minimal residual disease;
CR: complete remission;
SD: stable disease.
The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.
TABLE 5
Sequences
SEQ
ID NO
Sequence
Note
1
ESKYGPPCPPCP
IgG4 hinge
2
FWVLVVVGGVLACYSLLVTVAFIIFWV
CD28 transmembrane
domain
3
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
4-1BB costimulatory
domain
(amino acids 214-255 of
Q07011.1)
Homo sapien
4
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP
CD3-zeta intracellular
RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK
signaling domain
DTYDALHMQALPPR
5
FWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTT
CD28-4-1BB
QEEDGCSCRFPEEEEGGCEL
6
CYSLLVTVAFIIFWVKRGRKKLLYIFKQPF
Peptide
7
VAFIIFWVKRGRKKLL
Peptide
8
AFIIFWVKRGRKKLL
Peptide
9
FWVKRGRKKLLYIFK
Peptide
10
FIIFWVKRGRKKLL
Peptide
11
FIIFWVKRGRKKL
Peptide
12
IIFWVKRGRKKLL
Peptide
13
CYSLLVTVAFIIFWVNNKRGRKKLLYIFKQPF
Variant junction region
14
IIFWVNNKRGRKKL
Variant peptide
15
IIFWVNNKRGRKK
Variant peptide
16
VAFIIFWVK
Synthetic peptide
17
AFIIFWVKR
Synthetic peptide
18
FIIFWVKRG
Synthetic peptide
19
IIFWVKRGR
Synthetic peptide
20
IFWVKRGRK
Synthetic peptide
21
FWVKRGRKK
Synthetic peptide
22
WVKRGRKKL
Synthetic peptide
23
VAFIIFWVS
Synthetic peptide
K28S
24
AFIIFWVSR
Synthetic peptide
K28S
25
FIIFWVSRG
Synthetic peptide
K28S
26
IIFWVSRGR
Synthetic peptide
K28S
27
IFWVSRGRK
Synthetic peptide
K28S
28
FWVSRGRKK
Synthetic peptide
K28S
29
WVSRGRKKL
Synthetic peptide
K28S
30
VAFIIFWVL
Synthetic peptide
K28L
31
AFIIFWVLR
Synthetic peptide
K28L
32
FIIFWVLRG
Synthetic peptide
K28L
33
IIFWVLRGR
Synthetic peptide
K28L
34
IFWVLRGRK
Synthetic peptide
K28L
35
FWVLRGRKK
Synthetic peptide
K28L
36
WVLRGRKKL
Synthetic peptide
K28L
37
VAFIIFWVH
Synthetic peptide
K28H
38
AFIIFWVHR
Synthetic peptide
K28H
39
FIIFWVHRG
Synthetic peptide
K28H
40
IIFWVHRGR
Synthetic peptide
K28H
41
IFWVHRGRK
Synthetic peptide
K28H
42
FWVHRGRKK
Synthetic peptide
K28H
43
WVHRGRKKL
Synthetic peptide
K28H
44
VAFIIFWVA
Synthetic peptide
K28A
45
AFIIFWVAR
Synthetic peptide
K28A
46
IIFWVARGR
Synthetic peptide
K28A
47
IFWVARGRK
Synthetic peptide
K28A
48
FWVARGRKK
Synthetic peptide
K28A
49
WVARGRKKL
Synthetic peptide
K28A
50
VAFIIFWVQ
Synthetic peptide
K28Q
51
AFIIFWVQR
Synthetic peptide
K28Q
52
FIIFWVQRG
Synthetic peptide
K28Q
53
IIFWVQRGR
Synthetic peptide
K28Q
54
IFWVQRGRK
Synthetic peptide
K28Q
55
FWVQRGRKK
Synthetic peptide
K28Q
56
WVQRGRKKL
Synthetic peptide
K28Q
57
IIFWVKRGS
Synthetic peptide
R31S
58
IFWVKRGSK
Synthetic peptide
R31S
59
FWVKRGSKK
Synthetic peptide
R31S
60
WVKRGSKKL
Synthetic peptide
R31S
61
IIFWVKRGL
Synthetic peptide
R31L
62
IFWVKRGLK
Synthetic peptide
R31L
63
FWVKRGLKK
Synthetic peptide
R31L
64
WVKRGLKKL
Synthetic peptide
R31L
65
IIFWVKRGH
Synthetic peptide
R31H
66
IFWVKRGHK
Synthetic peptide
R31H
67
FWVKRGHKK
Synthetic peptide
R31H
68
WVKRGHKKL
Synthetic peptide
R31H
69
IIFWVKRGA
Synthetic peptide
R31A
70
IFWVKRGAK
Synthetic peptide
R31A
71
FWVKRGAKK
Synthetic peptide
R31A
72
WVKRGAKKL
Synthetic peptide
R31A
73
IIFWVKRGN
Synthetic peptide
R31N
74
IFWVKRGNK
Synthetic peptide
R31N
75
FWVKRGNKK
Synthetic peptide
R31N
76
WVKRGNKKL
Synthetic peptide
R31N
77
IIFWVQRGS
Synthetic peptide
K28Q/R31S
78
IFWVQRGSK
Synthetic peptide
K28Q/R31S
79
FWVQRGSKK
Synthetic peptide
K28Q/R31S
80
WVQRGSKKL
Synthetic peptide
K28Q/R31S
81
IIFWVQRGA
Synthetic peptide
K28Q/R31A
82
IFWVQRGAK
Synthetic peptide
K28Q/R31A
83
FWVQRGAKK
Synthetic peptide
K28Q/R31A
84
WVQRGAKKL
Synthetic peptide
K28Q/R31A
85
IIFWVQRGN
Synthetic peptide
K28Q/R31N
86
IFWVQRGNK
Synthetic peptide
K28Q/R31N
87
FWVQRGNKK
Synthetic peptide
K28Q/R31N
88
WVQRGNKKL
Synthetic peptide
K28Q/R31N
89
WVKRGRKKS
Synthetic peptide
L34S
90
WVKRGRKKA
Synthetic peptide
L34A
91
WVQRGNKKS
Synthetic peptide
K28Q/L34S
92
WVQRGNKKA
Synthetic peptide
K28Q/L34A
93
MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCP
4-1BB costimulatory
PNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAG
domain
CSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVL
(Accession No.
VNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTS
Q07011.1)
TALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF
Homo sapien
PEEEEGGCEL
94
MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCP
CD28 transmembrane
PNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAG
domain
CSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVL
(Accession No. P10747)
VNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTS
Homo sapien
TALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF
PEEEEGGCEL
95
MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTAL
CD3 zeta chain
FLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG
(Accession No. P20963)
KPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST
Homo sapien
ATKDTYDALHMQALPPR
96
FIIFWVNNKRGRKK
Synthetic peptide
97
IFWVNNKRGRKKLL
Synthetic peptide
98
FIIFWVNNKRGRKK
Synthetic peptide
99
IFWVNNKRGRKKLL
Synthetic peptide
100
VAFIIFWVR
Synthetic peptide
K28R
101
AFIIFWVRR
Synthetic peptide
K28R
102
FIIFWVARG
Synthetic peptide
K28A
103
MFWVLVVVGGVLACYSLLVTVAFIIFWV
CD28 transmembrane
domain
(amino acids 153-179 of
Accession No. P10747)
Homo sapien
104
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGV
CD28, including
LACYSLLVTVAFIIFWV
transmembrane
(amino acids 114-179 of
Accession No. P10747)
Homo sapien
105
RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP
CD3 zeta
RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY
Homo sapien
QGLSTATKDTYDALHMQALPPR
106
GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT
spacer (IgG4hinge) (nt)
homo sapien
107
ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI
Hinge-CH3 spacer
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS
Homo sapien
VMHEALHNHYTQKSLSLSLGK
108
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
Hinge-CH2-CH3 spacer
QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG
Homo sapien
KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL
TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK
SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
109
RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKE
IgD-hinge-Fc
KEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSD
Homo sapien
LKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGT
SVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLC
EVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVP
APPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH
110
LEGGGEGRGSLLTCGDVEENPGPR
T2A
artificial
111
MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFK
tEGFR
NCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQA
artificial
WPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISD
GDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCH
ALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECI
QCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNT
LVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGAL
LLLLVVALGIGLFM
112
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
CD28 cytoplasmic
domain
(amino acids 180-220
of P10747)
Homo sapien
113
RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
CD28 cytoplasmic
domain variant
(LL to GG)
Homo sapien
114
FWVLVVVGGVLACYSLLVTVAFIIFWVARGRKKLLYIFKQPFMRPVQTT
CD28-4-1BB K28A
QEEDGCSCRFPEEEEGGCEL
variant
115
FWVLVVVGGVLACYSLLVTVAFIIFWVHRGRKKLLYIFKQPFMRPVQTT
CD28-4-1BB K28H
QEEDGCSCRFPEEEEGGCEL
variant
116
FWVLVVVGGVLACYSLLVTVAFIIFWVLRGRKKLLYIFKQPFMRPVQTT
CD28-4-1BB K28L
QEEDGCSCRFPEEEEGGCEL
variant
117
FWVLVVVGGVLACYSLLVTVAFIIFWVQRGRKKLLYIFKQPFMRPVQTT
CD28-4-1BB K28Q
QEEDGCSCRFPEEEEGGCEL
variant
118
FWVLVVVGGVLACYSLLVTVAFIIFWVSRGRKKLLYIFKQPFMRPVQTT
CD28-4-1BB K28S
QEEDGCSCRFPEEEEGGCEL
variant
119
FWVLVVVGGVLACYSLLVTVAFIIFWVKRGAKKLLYIFKQPFMRPVQTT
CD28-4-1BB R31A
QEEDGCSCRFPEEEEGGCEL
variant
120
FWVLVVVGGVLACYSLLVTVAFIIFWVKRGHKKLLYIFKQPFMRPVQTT
CD28-4-1BB R31H
QEEDGCSCRFPEEEEGGCEL
variant
121
FWVLVVVGGVLACYSLLVTVAFIIFWVKRGLKKLLYIFKQPFMRPVQTT
CD28-4-1BB R31L
QEEDGCSCRFPEEEEGGCEL
variant
122
FWVLVVVGGVLACYSLLVTVAFIIFWVKRGNKKLLYIFKQPFMRPVQTT
CD28-4-1BB R31N
QEEDGCSCRFPEEEEGGCEL
variant
123
FWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKALYIFKQPFMRPVQTT
CD28-4-1BB L34A
QEEDGCSCRFPEEEEGGCEL
variant
124
FWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKSLYIFKQPFMRPVQTT
CD28-4-1BB L34S
QEEDGCSCRFPEEEEGGCEL
variant
125
FWVLVVVGGVLACYSLLVTVAFIIFWVQRGAKKLLYIFKQPFMRPVQTT
CD28-4-1BB
QEEDGCSCRFPEEEEGGCEL
K28Q/R31A variant
126
FWVLVVVGGVLACYSLLVTVAFIIFWVQRGNKKLLYIFKQPFMRPVQTT
CD28-4-1BB
QEEDGCSCRFPEEEEGGCEL
K28Q/R31N variant
127
FWVLVVVGGVLACYSLLVTVAFIIFWVQRGSKKLLYIFKQPFMRPVQTT
CD28-4-1BB
QEEDGCSCRFPEEEEGGCEL
K28Q/R31S variant
128
FWVLVVVGGVLACYSLLVTVAFIIFWVQRGRKKALYIFKQPFMRPVQTT
CD28-4-1BB
QEEDGCSCRFPEEEEGGCEL
K28Q/L34A variant
129
FWVLVVVGGVLACYSLLVTVAFIIFWVQRGRKKSLYIFKQPFMRPVQTT
CD28-4-1BB
QEEDGCSCRFPEEEEGGCEL
K28Q/L34S variant
130
FWVLVVVGGVLACYSLLVTVAFIIFWVKRGNKKALYIFKQPFMRPVQTT
CD28-4-1BB
QEEDGCSCRFPEEEEGGCEL
R31N/L34A variant
131
FWVLVVVGGVLACYSLLVTVAFIIFWVKRGNKKSLYIFKQPFMRPVQTT
CD28-4-1BB
QEEDGCSCRFPEEEEGGCEL
R31N/L34S variant
132
FWVLVVVGGVLACYSLLVTVAFIIFWVQRGNKKALYIFKQPFMRPVQTT
CD28-4-1BB
QEEDGCSCRFPEEEEGGCEL
K28Q/R31N/L34A
variant
133
FWVLVVVGGVLACYSLLVTVAFIIFWVQRGNKKSLYIFKQPFMRPVQTT
CD28-4-1BB
QEEDGCSCRFPEEEEGGCEL
K28Q/R31N/L34S
variant
134
FWVLVVVGGVLACYSLLVTVAFIIFWVNNKRGRKKLLYIFKQPFMRPVQ
CD28-4-1BB with
TTQEEDGCSCRFPEEEEGGCEL
variant junction region
with NN insertion
135
MFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQT
CD28-4-1BB
TQEEDGCSCRFPEEEEGGCEL
136
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGV
CD28-4-1BB
LACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF
PEEEEGGCEL
137
SLLVTVAFIIFWVKRGRKKLLYIFKQ
CD28-4-1BB junction
region
138
SLLVTVAFIIFWVARGRKKLLYIFKQ
CD28-4-1BB junction
region K14A variant
139
SLLVTVAFIIFWVHRGRKKLLYIFKQ
CD28-4-1BB junction
region K14H variant
140
SLLVTVAFIIFWVLRGRKKLLYIFKQ
CD28-4-1BB junction
region K14L variant
141
SLLVTVAFIIFWVQRGRKKLLYIFKQ
CD28-4-1BB junction
region K14Q variant
142
SLLVTVAFIIFWVSRGRKKLLYIFKQ
CD28-4-1BB junction
region K14S variant
143
SLLVTVAFIIFWVKRGAKKLLYIFKQ
CD28-4-1BB junction
region R17A variant
144
SLLVTVAFIIFWVKRGHKKLLYIFKQ
CD28-4-1BB junction
region R17H variant
145
SLLVTVAFIIFWVKRGLKKLLYIFKQ
CD28-4-1BB junction
region R17L variant
146
SLLVTVAFIIFWVKRGNKKLLYIFKQ
CD28-4-1BB junction
region R17N variant
147
SLLVTVAFIIFWVKRGRKKALYIFKQ
CD28-4-1BB junction
region L20A variant
148
SLLVTVAFIIFWVKRGRKKSLYIFKQ
CD28-4-1BB junction
region L20S variant
149
SLLVTVAFIIFWVQRGAKKLLYIFKQ
CD28-4-1BB junction
region K14Q/R17A
variant
150
SLLVTVAFIIFWVQRGNKKLLYIFKQ
CD28-4-1BB junction
region K14Q/R17N
variant
151
SLLVTVAFIIFWVQRGSKKLLYIFKQ
CD28-4-1BB junction
region K14Q/R17S
variant
152
SLLVTVAFIIFWVQRGRKKALYIFKQ
CD28-4-1BB junction
region K14Q/L20A
variant
153
SLLVTVAFIIFWVQRGRKKSLYIFKQ
CD28-4-1BB junction
region K14Q/L20S
variant
154
SLLVTVAFIIFWVKRGNKKALYIFKQ
CD28-4-1BB junction
region R17N/L20A
variant
155
SLLVTVAFIIFWVKRGNKKSLYIFKQ
CD28-4-1BB junction
region R17N/L20S
variant
156
SLLVTVAFIIFWVQRGNKKALYIFKQ
CD28-4-1BB junction
region
K14Q/R17N/L20A
variant
157
SLLVTVAFIIFWVQRGNKKSLYIFKQ
CD28-4-1BB junction
region
K14Q/R17N/L20S
variant
158
G A A T C T A A G T A C G G A C C G C C C T G C C
spacer (IgG4hinge)
C C C C T T G C C C T
(nucleotide)
homo sapien
159
RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP
CD3 Zeta
RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK
DTYDALHMQALPPR
|
14958919
|
juno therapeutics, inc.
|
USA
|
B2
|
Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
|
Open
|
|
|
Mar 30th, 2022 06:04PM
|
Mar 30th, 2022 06:04PM
|
Bristol-Myers Squibb
|
Health Care
|
Pharmaceuticals & Biotechnology
|
|
nyse:bmy
|
Juno Therapeutics
|
Jan 14th, 2020 12:00AM
|
Aug 28th, 2015 12:00AM
|
https://www.uspto.gov?id=US10533055-20200114
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Antibodies and chimeric antigen receptors specific for CD19
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Provided are CD19 binding molecules, including anti-CD19 antibodies, including antibody fragments such as single-chain fragments, and chimeric receptors including the antibodies, such as chimeric antigen receptors (CARs). Among the antibodies are human antibodies, including those that compete for binding to CD19 with reference antibodies, such as murine antibodies. In some embodiments, the antibodies display similar functional properties to the reference antibodies, such as comparable binding affinities and/or competitive inhibition properties. Also provided are genetically engineered cells expressing the chimeric receptors, and uses of the binding molecules and cells adoptive cell therapy.
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10533055
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1. A chimeric antigen receptor (CAR) comprising:
a) an extracellular portion comprising an anti-CD19 antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment comprises:
a heavy chain variable (VH) region, wherein the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO: 18; a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 81; and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 20; and
a light chain variable (VL) region, wherein the VL region comprises:
a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO: 21, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or
a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 24; or
a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 27; or
a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 30; or
a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 33;
and
b) an intracellular signaling domain, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).
2. The chimeric antigen receptor of claim 1, wherein the antibody or antigen binding fragment thereof comprises:
a VH region comprising the amino acid sequence set forth in SEQ ID NO: 11, or a sequence that has at least 90% amino acid sequence identity to SEQ ID NO: 11; and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 13;
a VH region comprising the amino acid sequence set forth in SEQ ID NO: 11, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:11, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 14, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:14;
a VH region comprising the amino acid sequence set forth in SEQ ID NO: 11, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:11, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 16, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:16;
a VH region comprising the amino acid sequence set forth in SEQ ID NO: 12, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:12, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 17, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:17; or
a VH region comprising the amino acid sequence set forth in SEQ ID NO: 12, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:12, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 15, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:15.
3. A chimeric antigen receptor (CAR), comprising:
a) an extracellular portion comprising an anti-CD19 antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising:
a heavy chain variable (VH) region comprising a CDR-H1, a CDR-H2, and a CDR-H3, contained within the VH region amino acid sequence set forth in SEQ ID NO: 11; and
a light chain variable (VL) region comprising a CDR-L1, a CDR-L2, and a CDR-L3, contained within the VL region amino acid sequence set forth in SEQ ID NO: 13, 14, 15, 16 or 17 and
b) an intracellular signaling domain, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).
4. The chimeric antigen receptor of claim 1, wherein the antibody or antigen binding fragment thereof comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 11, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:11, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 13, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:13.
5. The chimeric antigen receptor of claim 1, wherein the antibody or antigen binding fragment thereof comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 11, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:11, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 14, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:14.
6. The chimeric antigen receptor of claim 1, wherein the antibody or antigen binding fragment thereof comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 11, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:11, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 16, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:16.
7. The chimeric antigen receptor of claim 1, wherein:
the VH and VL regions of the antibody or fragment comprise the amino acid sequences set forth in SEQ ID NOs: 12 and 17, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences set forth in SEQ ID NOs: 12 and 15, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences set forth in SEQ ID NOs: 11 and 13, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences set forth in SEQ ID NOs: 11 and 14, respectively; or
the VH and VL regions of the antibody or fragment comprise the amino acid sequences set forth in SEQ ID NOs: 11 and 16, respectively.
8. The chimeric antigen receptor of claim 1, wherein the anti-CD19 antibody or antigen-binding fragment specifically binds to human CD19.
9. The chimeric antigen receptor of claim 1, wherein the antibody or antigen-binding fragment is human.
10. The chimeric antigen receptor of claim 1, wherein the antibody or antigen-binding fragment is a single chain molecule.
11. The chimeric antigen receptor of claim 10, wherein the single chain molecule comprises the antibody variable regions joined by a flexible linker.
12. The chimeric antigen receptor of claim 10, wherein the single chain molecule comprises an scFv.
13. The chimeric antigen receptor of claim 12, wherein the scFv comprises a linker comprising the sequence set forth SEQ ID NO: 34.
14. The chimeric antigen receptor of claim 12, wherein the scFv comprises a sequence that exhibits at least 95% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8, and 10.
15. The chimeric antigen receptor of claim 12, wherein the scFv comprises the amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8 and 10.
16. The chimeric antigen receptor of claim 1, wherein the intracellular signaling domain comprises a signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain.
17. The chimeric antigen receptor of claim 16, wherein the intracellular signaling domain further comprises an intracellular signaling region of a T cell costimulatory molecule.
18. The chimeric antigen receptor of claim 3, wherein the intracellular signaling domain comprises a signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain.
19. The chimeric antigen receptor of claim 18, wherein the intracellular signaling domain further comprises an intracellular signaling region of a T cell costimulatory molecule.
20. A conjugate, comprising:
a) an anti-CD19 antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment comprises:
a heavy chain variable (VH) region, wherein the VH region comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO: 18; a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 81; and a CDR-H3 comprising the amino acid sequence set forth as SEQ ID NO: 20; and
and a light chain variable (VL) region, wherein the VL region comprises:
a light chain complementarity determining region 1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO: 21, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 23; or
a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 24; or
a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 26, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 27; or
a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 30; or
a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 33; and
b) and a heterologous molecule or moiety.
21. The conjugate of claim 20, wherein the conjugate is a chimeric antigen receptor (CAR).
22. The chimeric antigen receptor of claim 1, wherein the CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 19 (GISWNSGRIGYADSVKG).
23. The chimeric antigen receptor of claim 3, wherein the anti-CD19 antibody or antigen-binding fragment specifically binds to human CD19.
24. The chimeric antigen receptor of claim 1, wherein the intracellular signaling domain further comprises an intracellular signaling region of a T cell costimulatory molecule.
25. The chimeric antigen receptor of claim 3, wherein the intracellular signaling domain further comprises an intracellular signaling region of a T cell costimulatory molecule.
26. The conjugate of claim 20, wherein the CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 19 (GISWNSGRIGYADSVKG).
27. The chimeric antigen receptor of claim 1, wherein the antibody or antigen binding fragment thereof comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 12, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:12, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 17, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:17.
28. The chimeric antigen receptor of claim 1, wherein the antibody or antigen binding fragment thereof comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 12, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:12, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 15, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:15.
29. The chimeric antigen receptor of claim 16, wherein the antibody or antigen binding fragment thereof comprises:
a VH region comprising the amino acid sequence set forth in SEQ ID NO: 11, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:11, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 13, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:13;
a VH region comprising the amino acid sequence set forth in SEQ ID NO: 11, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:11, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 14, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:14;
a VH region comprising the amino acid sequence set forth in SEQ ID NO: 11, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:11, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 16, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:16;
a VH region comprising the amino acid sequence set forth in SEQ ID NO: 12, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:12, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 17, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:17; or
a VH region comprising the amino acid sequence set forth in SEQ ID NO: 12, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:12, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 15, or a sequence having at least 90% amino acid sequence identity to SEQ ID NO:15.
30. The chimeric antigen receptor of claim 29, wherein the intracellular signaling domain further comprises an intracellular signaling region of a T cell costimulatory molecule.
31. The chimeric antigen receptor of claim 29, wherein:
the VH and VL regions of the antibody or fragment comprise the amino acid sequences set forth in SEQ ID NOs: 11 and 13, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences set forth in SEQ ID NOs: 11 and 14, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences set forth in SEQ ID NOs: 11 and 16, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences set forth in SEQ ID NOs: 12 and 15, respectively; or
the VH and VL regions of the antibody or fragment comprise the amino acid sequences set forth in SEQ ID NOs: 12 and 17, respectively.
32. The chimeric antigen receptor of claim 29, wherein the antibody or antigen binding fragment comprises an scFv.
33. The chimeric antigen receptor of claim 32, wherein the scFv comprises a sequence that exhibits at least 95% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8, or 10.
34. The chimeric antigen receptor of claim 1, wherein the VH region comprises a CDRH1, a CDRH2 and a CDRH3 set forth in SEQ ID NOS: 18, 19 and 20, respectively and the VL region comprises a CDRL1, a CDRL2 and a CDRL3 set forth in SEQ ID NOS: 21, 22 and 23, respectively.
35. The chimeric antigen receptor of claim 34, wherein the intracellular signaling domain comprises a signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain.
36. The chimeric antigen receptor of claim 35, wherein the intracellular signaling domain further comprises an intracellular signaling region of a T cell costimulatory molecule.
37. The chimeric antigen receptor of claim 1, wherein the VH region comprises a CDRH1, a CDRH2 and a CDRH3 set forth in SEQ ID NOS: 18, 19 and 20, respectively and the VL region comprises a CDRL1, a CDRL2 and a CDRL3 set forth in SEQ ID NOS: 21, 22 and 24, respectively.
38. The chimeric antigen receptor of claim 37, wherein the intracellular signaling domain comprises a signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain.
39. The chimeric antigen receptor of claim 38, wherein the intracellular signaling domain further comprises an intracellular signaling region of a T cell costimulatory molecule.
40. The chimeric antigen receptor of claim 1, wherein the VH region comprises a CDRH1, a CDRH2 and a CDRH3 set forth in SEQ ID NOS: 18, 19 and 20, respectively and the VL region comprises a CDRL1, a CDRL2 and a CDRL3 set forth in SEQ ID NOS: 25, 26 and 27, respectively.
41. The chimeric antigen receptor of claim 40, wherein the intracellular signaling domain comprises a signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain.
42. The chimeric antigen receptor of claim 41, wherein the intracellular signaling domain further comprises an intracellular signaling region of a T cell costimulatory molecule.
43. The chimeric antigen receptor of claim 1, wherein the VH region comprises a CDRH1, a CDRH2 and a CDRH3 set forth in SEQ ID NOS: 18, 19 and 20, respectively and the VL region comprises a CDRL1, a CDRL2 and a CDRL3 set forth in SEQ ID NOS: 28, 29 and 30, respectively.
44. The chimeric antigen receptor of claim 43, wherein the intracellular signaling domain comprises a signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain.
45. The chimeric antigen receptor of claim 44, wherein the intracellular signaling domain further comprises an intracellular signaling region of a T cell costimulatory molecule.
46. The chimeric antigen receptor of claim 1, wherein the VH region comprises a CDRH1, a CDRH2 and a CDRH3 set forth in SEQ ID NOS: 18, 19 and 20, respectively and the VL region comprises a CDRL1, a CDRL2 and a CDRL3 set forth in SEQ ID NOS: 31, 32 and 33, respectively.
47. The chimeric antigen receptor of claim 46, wherein the intracellular signaling domain comprises a signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain.
48. The chimeric antigen receptor of claim 47, wherein the intracellular signaling domain further comprises an intracellular signaling region of a T cell costimulatory molecule.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S. provisional application No. 62/043,273 filed Aug. 28, 2014, entitled “Antibodies and Chimeric Antigen Receptors Specific for CD19,” and U.S. provisional application No. 62/078,942 filed Nov. 12, 2014, entitled “Antibodies and Chimeric Antigen Receptors Specific for CD19,” the contents of which are incorporated by reference in their entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042000700SubSeqList.txt, created Feb. 9, 2016, which is 219,307 bytes in size. The information in electronic format of the Sequence Listing is incorporated by reference in its entirety.
FIELD
The present disclosure relates in some aspects to CD19 binding molecules, in particular, to anti-CD19 antibodies, including antibody fragments. The present disclosure further relates to recombinant receptors containing such antibodies, including chimeric antigen receptors (CARs), which contain such antibodies. The disclosure further relates to genetically engineered cells expressing such receptors and antibodies, and use thereof in adoptive cell therapy.
BACKGROUND
CD19 is expressed on normal B cells and by cells and tissues of various diseases and conditions, including most B cell malignancies. Most patients with B cell malignancies are not cured by available therapies, including therapies targeting CD19 and/or other B cell markers. Various CD19-binding molecules, including anti-CD19 antibodies, and chimeric antigen receptors containing anti-CD19 antibody portions, and cells expressing such chimeric receptors, are available. Improved CD19-binding molecules and engineered CD19-targeting cells are needed. For example, there is a need for molecules and cells with reduced immunogenicity and/or human antibodies, including antibody fragments that specifically bind to CD19 and chimeric receptors expressing such human antibodies for use in adoptive cell therapy. Provided are embodiments that meet such needs.
SUMMARY
Provided are CD19-binding molecules, including polypeptides, such as anti-CD19 antibodies, including antigen-binding antibody fragments such as single-chain fragments including scFv fragments, and polypeptides containing such antibodies, including fusion proteins, receptors, e.g., recombinant receptors, including chimeric receptors such as chimeric antigen receptors (CARs) containing the antibody as an antigen-recognition component. In particular embodiments, the antibodies are human antibodies, such as human single-chain fragments including scFvs.
Provided are antibodies or antigen-binding fragments thereof, including those that specifically bind to CD19. In some embodiments, the antibodies contain particular complementarity determining regions (CDRs), including heavy chain CDRs (CDR-Hs) and light chain CDRs (CDR-Ls). In some embodiments, the CDRs have or include amino acid sequences of CDRs of a reference antibody or chain or sequence thereof.
In some embodiments, the antibody or antigen-binding fragment thereof includes a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the antibody, e.g., the VH region thereof, includes a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence set forth as SEQ ID NO: 20. In some embodiments, the VH region comprises at least at or about 90% sequence identity to the VH region amino acid sequence set forth in SEQ ID NO: 11, 12, 60, 61, 63, or 62, e.g., at least at or about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the antibody or fragment includes a CDR-H1 of SEQ ID NO: 18 and a CDR-H3 of SEQ ID NO: 20. In some embodiments, the antibody or fragment further includes a CDR-H2 sequence comprising SEQ ID NO: 81, 82, 19 or 72.
In some embodiments, the antibody has a CDR-H1, a CDR-H2, and/or a CDR-H3 that respectively include the amino acid sequences of CDR 1, 2, and 3 sequences contained within the heavy chain variable (VH) region of a reference antibody. In some embodiments, the VH region of the reference antibody has the amino acid sequence set forth in SEQ ID NO: 11 or 12. In some embodiments, it has the amino acid sequence set forth in SEQ ID NO: 11, 12, 60, 61, 63, or 62.
In some embodiments, the antibody has, e.g., further includes, a CDR-L1, a CDR-L2, and/or a CDR-L3, respectively comprising the amino acid sequences of CDR 1, 2, and 3 sequences contained within the light chain variable (VL) region of a reference antibody. In some embodiments, the VL of the reference antibody has the amino acid sequence set forth in SEQ ID NO: 13, 14, 15, 16, or 17. In some embodiments, the VL of the reference antibody has the amino acid sequence set forth in SEQ ID NO: 13, 14, 15, 16, 17, 71, 65, 64, 66, 70, 69, 67, 90 or 91.
In some embodiments, the CDR within the reference antibody, VH, or VL refers to the CDR as defined by any numbering scheme, e.g., those defined herein. In some embodiments, the CDR in the reference antibody or VH or VL refers to the CDR as defined by Kabat numbering scheme as described herein, the CDR as defined by the Chothia scheme as described herein, or the Contact scheme as described herein.
In some embodiments, the antibody contains a VH chain that includes a CDR-H1, CDR-H2 and/or CDR-H3 in which the CDR-H1 comprises the amino acid sequence of DYAMH (SEQ ID NO: 18) or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 18; the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 81 or 82 or 19 or 72 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 81 or to SEQ ID NO: 82 or to SEQ ID NO: 19 or to SEQ ID NO: 72; and/or the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 20 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 20.
In some embodiments, the antibody comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 81 or 82, and a CDR-H3 comprising the amino acid sequence set forth as SEQ ID NO: 20.
In some embodiments, the antibody has a CDR-1 comprising the amino acid sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 110), wherein X1 is T, W, S or R; X2 is G or A; X3 is I, T, D or S; X4 is S, R, T or Q; X5 is null or S; X6 is null, D, N or G; X7 is null, V or L; X8 is X or null; X9 is X or null; X10 is X; X11 is X; X12 is Y, F, D or W; X13 is V, A or L and X14 is S, N or A. For example, in some embodiments, the antibody has a CDR-L1 comprising the amino acid sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 111), wherein X1 is T, Q, S, or R; X2 is G or A; X3 is I, T, D, or S; X4 is S, R, T, or Q; X5 is null or S; X6 is G, D, N, or null; X7 is null, V, or L; X8 is D, G, I, L, S, or null; X9 is S, G, A, I, R, or null; X10 is H, Y, F, S, or N; X11 is R, N, D, H, or Y; X12 is Y, F, D, or W; X13 is V, A, or L; and X14 is S, N, or A; and/or
a CDR-L2 comprising the amino acid sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 112), wherein X1 is D or S; X2 is F, V, N, K, or A; X3 is S, T, D, or N; X4 is K, V, N, Q, or R; X5 is R, V, or L; X6 is P, K, A, or E; and X7 is S, P, A, or T, and/or
a CDR-L3 comprising the amino acid sequence of X1X2X3X4X5X6X7X8X9X10X11X12 (SEQ ID NO: 115), wherein X1 is X; X2 is S, Q, A, or T; X3 is Y, S, W, R; X4 is A, D, R, T, or Y; X5 is X; X6 is X; X7 is S, P, L, Y, G; X8 is X or null; X9 is X or null; X10 is L or null; X11 is X; and X12 is V, T, or L. For example, in some embodiments, the antibody has a CDR-L3 comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12 (SEQ ID NO: 114), wherein X1 is S, G, T, A, Q, C or N; X2 is S, Q, A, or T; X3 is Y, S, W, R; X4 is A, D, R, T, or Y; X5 is A, S, P, G, N or D; X6 is I, S, G, T, A, L, H, R, N; X7 is S, P, L, Y, G; X8 is P, T, S, Q, M, R, N or null; X9 is S, L, N, A, M or null; X10 is L or null; X11 is Y, W, F, V, A or L; and X12 is V, T, or L.
In some such embodiments, in said CDR-L1, X3 is I, T, or S; X4 is S, T, or Q; X8 is D, G, I, S, or null; X9 is S, G, I, or null; X10 is H, Y, S, or N; X11 is R, N, D, or H; X12 is Y or D; and X13 is V or L; and/or in said CDR-L2, X1 is D; X4 is K, V, N, Q, or R; X6 is P, K, or A; and X7 is S, A, or T; and/or in said CDR-L3, X1 is S, G, T, A, Q, C, or N; X5 is A, S, P, G, N, or D; X6 is I, S, G, T, A, L, H, R, or N; X8 is P, T, S, Q, M, R, N, or null; X9 is S, L, N, A, M or null; and X11 is Y, W, F, V, A, or L. In some embodiments, in said CDR-L3, X1 is S, G, Q, or N; X2 is S, Q, or T; X4 is A, D, T, or Y; X5 is A, S, or G; and X6 is I, S, N, R, A, H, or T.
In some embodiments, the CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 19 (GISWNSGRIGYADSVKG); or the CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 72 (GISWNSGSIGYADSVKG).
In some embodiments, the CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 80, 77, 74, 73, 75, 79, 78, 76, 21, 25, 28, or 31. In some embodiments, the CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 80, 77, 74, 73, 78, 21, or 28.
In some embodiments, the CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 100, 97, 94, 93, 95, 99, 98, 96, 22, 26, 29, or 32. In some embodiments, the CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 100, 97, 94, 93, 98, 22, or 29.
In some embodiments, the CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 109, 106, 103, 101, 104, 108, 107, 105, 102, 23, 24, 27, 30, or 33. In some embodiments, the CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 109, 106, 103, 101, 107, 24 or 30.
In some embodiments, the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 21, 22, and 23, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 21, 22, and 24 or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 25, 26, and 27, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 28, 29 and 30, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 31, 32, and 33, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 80, 100, and 109, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs:77, 97, and 106, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 74, 94, and 103, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 73, 93, and 101, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs:75, 95, and 104, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 79, 99, and 108, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 78, 98, and 107, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 76, 96, and 105, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 73, 93, and 102, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto; or the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 77, 97, and 106, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto.
In some embodiments, the CDR-L3 comprises the amino acid sequence set forth as SEQ ID NO: 116, 117, 118, 119, 120, or 121, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto.
In some embodiments, the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 81, and 20, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 19, and 20, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 82, and 20, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto; or the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 72, and 20, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, respectively, thereto.
In some embodiments, the antibody has a CDR-L1 comprising the amino acid sequence X1GX3X4X5X6X7X8X9X10X11X12X13S (SEQ ID NO: 36), wherein X1 is T, S, or Q, X3 is T, S, or D, X4 is T or S, X5 is null or S, X6 is null, D, or N, X7 is null or V, X8 is null, G, or I, X9 is null, G, or R, X10 is S, Y, or N, X11 is D or N, X12 is D or Y, X13 is V or A; the CDR-L2 comprises the amino acid sequence X1X2X3X4RPS (SEQ ID NO: 37), wherein X1 is D or S, X2 is V, N, or K, X3 is S, N, or D, and X4 is K, Q, or N; and/or the CDR-L3 comprises the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12 (SEQ ID NO: 113), wherein X1 is C, S, A, G, or N; X2 is S, A, or T; X3 is Y, W, or R; X4 is A or D; X5 is G, D, or S; X6 is R, S, or N; X7 is Y, L, or G; X8 is N or S; X9 is S, N, or null; X10 is null; X11 is V, A, or W; and X12 is L or V.
In some embodiments, the antibody has a CDR-L1 comprising the amino acid sequence X1GX3X4X5X6X7X8X9X10X11X12X13S (SEQ ID NO: 36), wherein X1 is T, S, or Q, X3 is T, S, or D, X4 is T or S, X5 is null or S, X6 is null, D, or N, X7 is null or V, X8 is null, G, or I, X9 is null, G, or R, X10 is S, Y, or N, X11 is D or N, X12 is D or Y, X13 is V or A; the CDR-L2 comprises the amino acid sequence X1X2X3X4RPS (SEQ ID NO: 37), wherein X1 is D or S, X2 is V, N, or K, X3 is S, N, or D, and X4 is K, Q, or N; and/or the CDR-L3 comprises the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12 (SEQ ID NO: 38), wherein X1 is C, S, A, G, or N; X2 is S, A, or T; X3 is Y, W, or R; X4 is A or D; X5 is G, D, or S; X6 is R, S, or N; X7 is Y, L, or G; X8 is N or S; X9 is S or null; X10 is V, A or N; X11 is W or null; and X12 is L or V.
In some such embodiments, in the CDR-L1, X1 is T or S, X3 is T or S, X11 is D or N, and X13 is V; and/or in the CDR-L2, X2 is V or N and X4 is K or Q.
In some embodiments, the CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 19 (GISWNSGRIGYADSVKG) or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 19.
In some embodiments, the CDR-L1 comprises the sequence set forth in SEQ ID NO: 21, 25, 28, or 31 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto; and/or the CDR-L2 comprises the sequence set forth in SEQ ID NO: 22, 26, 29, or 32 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto; and/or the CDR-L3 comprises the sequence set forth in SEQ ID NO: 23, 24, 27, 30, or 33 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In some embodiments, the CDR-L1, CDR-L2, and/or CDR-L3 comprise the sequences of SEQ ID NOs: 21, 22, and/or 23, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively.
In some embodiments, the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 21, 22, and 24, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively.
In some embodiments, the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 25, 26, and 27, respectively or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively.
In some embodiments, the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 28, 29, and 30, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively. In some embodiments, the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 31, 32, and 33, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In some embodiments, the heavy and light chain CDRs are any combination of the aforementioned CDR-L and CDR-H sequences, including sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO: 11 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO: 12 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 13 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 14 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 15 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 16 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 17 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO: 63 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO: 60 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO: 61 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO: 63 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO: 62 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 71 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 90 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 91 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 68 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 65 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 64 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 66 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 70 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 69 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the antibody or fragment comprises a VL region comprising the amino acid sequence of SEQ ID NO: 67 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In particular embodiments, the VH region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 11, 60, 63, or 62 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto; and/or the VL region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 14, 16, 71, 90, 65, 64, or 69 or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In some embodiments, the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 17, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 15, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 13, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 14, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 16, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 71, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 62 and 68, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 65, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 60 and 64, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 61 and 66, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 70, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 62 and 69, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 67, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 91, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively; or
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 90, respectively, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, respectively.
In some embodiments, the VH region comprises SEQ ID NO: 11 and the VL region comprises SEQ ID NO: 13; in some embodiments, the VH region comprises SEQ ID NO: 11 and the VL region comprises SEQ ID NO: 14; in some embodiments, the VH region comprises SEQ ID NO: 11 and the VL region comprises SEQ ID NO: 15; in some embodiments, the VH region comprises SEQ ID NO: 11 and the VL region comprises SEQ ID NO: 16, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto; in some embodiments, the VH region comprises SEQ ID NO: 11 and the VL region comprises SEQ ID NO: 17, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In some embodiments, the VH region comprises SEQ ID NO: 12 and the VL region comprises SEQ ID NO: 13; in some embodiments, the VH region comprises SEQ ID NO: 12 and the VL region comprises SEQ ID NO: 14; in some embodiments, the VH region comprises SEQ ID NO: 12 and the VL region comprises SEQ ID NO: 15; in some embodiments, the VH region comprises SEQ ID NO: 12 and the VL region comprises SEQ ID NO: 16, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto; in some embodiments, the VH region comprises SEQ ID NO: 12 and the VL region comprises SEQ ID NO: 16, or sequences having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In some embodiments, the antibody is a single chain fragment, such as one with two or more variable regions joined by one or more flexible immunoglobulin linker. In some embodiments, the antibody is an scFv. In some embodiments, the scFv comprises a linker that is rich in serine and/or glycine, such as a linker comprising GGGS (SEQ ID NO: 122) or GGGGS (SEQ ID NO:123) repeats, such as one comprising the sequence set forth SEQ ID NO: 34. In some embodiments the linker comprises a sequence of SEQ ID NO: 43.
In some embodiments, the antibody fragment, e.g., scFv, contains a VH region or portion thereof, followed by a linker, followed by a VL or portions thereof. In some embodiments, the antibody fragment, e.g., scFv, contains a VL region or portion thereof followed by a linker, followed by a VH region or portion thereof.
In some embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10, or a sequence having at least or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.
In some embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 45, 47, 49, 51, 53, 55, 57, 59, 87, or 89, or has a sequence at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such a sequence.
In some embodiments, the antibody or fragment specifically binds to the same, similar, and/or an overlapping epitope of CD19 as the epitope specifically bound by a reference antibody, and/or the antibody competes for binding to CD19 with the reference antibody. In some aspects, the reference antibody is a murine or chimeric or human or humanized anti-CD19 antibody, FMC63, SJ25C1, an antibody having a variable region sequence of SEQ ID NO: 39 and/or 40, or an antibody having a variable region sequence of SEQ ID NO: 41 and/or 42. In some aspect, the reference antibody is an antibody including a sequence as described herein, including sequence(s) of any of the aforementioned embodiments. For example, in some embodiments, the reference antibody can be an scFv that contains the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 45, 47, 49, 51, 53, 55, 57, 59, 87, or 89. In some embodiments, the provided antibody or fragment contains one or more or all CDRs that are distinct from those in the reference antibody. For example, in some embodiments, the provided antibody or fragment contains one or more or all CDRs that are distinct from the corresponding CDRs in the antibody designated FMC63 or SJ25C1.
For example, provided are human antibody and antigen-binding fragments that specifically bind to the same or an overlapping epitope of CD19 as the epitope specifically bound by the reference antibody, such as FMC63, SJ25C1, an antibody having a variable region sequence of SEQ ID NO: 39 and/or 40, or an antibody having a variable region sequence of SEQ ID NO: 41 and/or 42, and comprising heavy and light chain CDRs that are distinct from the CDRs present in the reference antibody.
In some embodiments, the antibody competes for binding with the reference antibody to at least the same degree as the reference antibody competes for binding with itself to CD19, or a degree of competition that is no more than 1.5-fold or 2-fold lower, 3-fold lower, 4-fold lower, 5-fold lower, or 10-fold lower than the competition by the reference antibody, and/or a measured IC50 that is no more than 1.5-fold or 2-fold or 3-fold or 4-fold or 5-fold or 10-fold higher than the IC50 of the reference antibody competing for binding with itself, for example, as measured in the same assay.
In some embodiments, the antibody has a binding affinity that is at least as high or substantially as high as the binding affinity for CD19 of the reference antibody. In some aspects, the antibody has a binding affinity of an EC50 that is about the same or lower than the EC50 reference antibody or no more than about 1.5-fold or no more than about 2-fold greater, no more than 3-fold greater, and/or no more than 10-fold greater, than the EC50 of the reference antibody. In some embodiments, binding affinity of the antibody is compared to the corresponding form of the reference antibody. Comparison is generally by the same or a similar assay.
In some of any such embodiments, CD19 is a human CD19. In some of any such embodiments, the antibody or fragment specifically binds, exhibits binding affinity and/or competes for binding to human CD19.
In some embodiments, the antibody is human. In some embodiments, the antibody is recombinant. In some embodiments, the antibody is monoclonal. In some embodiments, the antibody is isolated.
In some embodiments, the antibody or fragment further includes at least a portion of an immunoglobulin constant region. The constant region may include any one or more of CH1, CH2, CH3, and/or CH4, and/or CL, of a human or other antibody, and be of any class, including IgG, IgM, IgA, IgE, and IgD, for example, including human IgG, e.g., IgG1 or IgG4, constant region domains. In some embodiments, the constant region comprises or is an Fc region, such as a human IgG Fc region.
Also provided are molecules such as chimeric and/or fusion molecules, including receptors, such as recombinant receptors, that include the antibody of any of the embodiments (e.g., contained in or part of an extracellular domain) and additional domains, such as intracellular signaling domains, spacers, linkers, and/or transmembrane domains. In some embodiments, the receptor is a chimeric antigen receptor, comprising an extracellular portion comprising the antibody or fragment of any of the embodiments and an intracellular signaling domain.
In some embodiments, the antibody or fragment comprises an scFv. In some embodiments, the intracellular signaling domain comprises an ITAM and/or signaling domain capable of delivering a signal approximating that of natural ligation of an ITAM-containing molecule or receptor complex such as a TCR receptor complex. In some aspects, the intracellular signaling domain comprises a signaling domain of a zeta chain of a CD3-zeta (CD3) chain.
In some embodiments, the receptor further includes one or more domains, such as a transmembrane domain, linking the antibody transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some aspects, the transmembrane domain comprises a transmembrane portion of a costimulatory molecule, such as a T cell costimulatory molecule, e.g., CD28 and/or 41BB. In some embodiments, the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB, and in some embodiments, the receptor includes signaling domains from CD28 and 41BB.
Also provided are nucleic acids encoding the antibody (including fragments) of any of embodiments or the receptor, e.g., chimeric antigen receptor of any of the embodiments, vectors including such nucleic acids, and cells containing the vectors and/or nucleic acids, for example, for expression of the antibodies and/or molecules.
Thus, also provided are cells and vectors for producing and expressing the molecules, including the antibodies and molecules such as receptors, e.g., chimeric antigen receptors (CARs). For example, provided are engineered cells expressing the chimeric antigen receptor of any of the embodiments. In some aspects, the cell is a T cell. In some aspects, the cell is an NK cell. In some aspects, the cell is a stem cell.
Also provided are compositions comprising the antibodies, receptors, molecules, and/or cells, including pharmaceutical compositions, e.g., further including pharmaceutically acceptable substances such as carriers.
Also provided are methods of administration, including methods of treatment, carried out by administering the cell, antibody, receptor, composition, or other molecule, of any of the embodiments, to a subject, for example, in an effective, e.g., therapeutically effective, amount. In some embodiments, the subject has or is suspected of having a disease or disorder associated with CD19, such as a B cell malignancy, such as B cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), pro-lymphocytic leukemias, hairy cell leukemias, common acute lymphocytic leukemias, Null-acute lymphoblastic leukemias, non-Hodgkin lymphomas, diffuse large B cell lymphomas (DLBCLs), multiple myelomas, follicular lymphoma, splenic, marginal zone lymphoma, mantle cell lymphoma, indolent B cell lymphoma, or Hodgkin lymphoma, or an autoimmune or inflammatory disease in which B cells are implicated.
In some embodiments, administration of the antibody or receptor is associated with a lower degree of immunogenicity as compared to administration of a reference antibody (or receptor containing the reference antibody) that competes for binding with the antibody or binds to an overlapping epitope. In some aspects, the reference antibody is a humanized, chimeric, or non-human antibody.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B show results from a binding assay comparing binding of exemplary human scFvs to CD19-expressing HEK293 cells as compared to binding to non-CD19-expressing HEK293 cells. MFI=mean fluorescence intensity.
FIG. 2 shows an SDS gel assessing purification of exemplary anti-CD19 antibodies (scFv fragments).
FIGS. 3A, 3B, and 3C show results from studies assessing binding affinities of various exemplary scFv antibodies (scFv fragments), including anti-CD19 antibodies. MFI=mean fluorescence intensity.
FIG. 4 show results from studies assessing binding affinities of various exemplary scFv antibodies, including anti-CD19 scFv antibody fragments. MFI=mean fluorescence intensity.
FIGS. 5A and 5B show results from competitive binding assays, assessing binding of respective labeled antibody in the presence of varying concentrations of competing antibodies. MFI=mean fluorescence intensity.
FIG. 6 shows results from competitive binding assays, assessing binding of a labeled reference scFv antibody in the presence of varying concentrations of competing scFv antibodies. MFI=mean fluorescence intensity.
FIG. 7A shows results from size-exclusion chromatography; a column was calibrated, standard proteins injected, and fractions collected to generate references. FIG. 7B shows results following injection of an anti-CD19 scFv (clone 18B) into the same column and collection of fraction under the same conditions.
FIG. 8A show results from a binding assay assessing binding of exemplary human scFv clones to CD19-expressing cells in order from left to right as follows: cells only, mock supernatant (Moc. Supe.) negative control antibody (Neg. Ctrl.), Clone 18, Clones 200 to 287, cells only, Moc. Supe, Neg. Ctrl. and Clone 18. Exemplary hits that show CD19-specific binding (indicated by an asterisk) are (in order from left to right): Clone 213, Clone 227, Clone 241, Clone 255, Clone 272, Clone 278, Clone 283 and Clone 285. MFI=mean fluorescence intensity.
FIG. 8B shows results from a binding assay assessing binding of exemplary human scFv clones to CD19-expressing cells in order from left to right as follows: cells only, mock supernatant (Moc. Supe.) negative control antibody (Neg. Ctrl.), Clone 18B, Clones 300-387, cells only, Moc. Supe., Neg. Ctrl. and Clone 18B. Exemplary hits that show CD19-specific binding (indicated by an asterisk) are (in order from left to right): Clone 302, Clone 305, Clone 313, Clone 314, Clone 318, Clone 324, Clone 327, Clone 328, Clone 336, Clone 339, Clone 377, Clone 379 and Clone 382. MFI=mean fluorescence intensity.
FIG. 8C shows results from a binding assay assessing binding of exemplary human scFv clones to CD19-expressing cells in order from left to right as follows: cells only, mock supernatant (Moc. Supe.) negative control antibody (Neg. Ctrl.), Clone 18B, Clones 400-487, cells only, Moc. Supe., Neg. Ctrl. and Clone 18B. Exemplary hits that show CD19-specific binding (indicated by an asterisk) are (in order from left to right): Clone 440 and Clone 448.
FIG. 8D shows results from a binding assay comparing binding of exemplary human scFvs to CD19-expressing K562 cells as compared to non-CD19-expressing K562 cells. MFI=mean fluorescence intensity.
FIG. 9 shows an SDS gel assessing purification of exemplary anti-CD19 antibodies (scFv fragments).
FIGS. 10A-E show results from separate binding assays assessing binding affinities of various exemplary scFv antibodies, including anti-CD19 scFv antibody fragments. MFI=mean fluorescence intensity.
FIG. 11 shows results from competitive binding assays, assessing binding of a labeled reference scFv antibody in the presence of varying concentrations of competing scFv antibodies. MFI=mean fluorescence intensity.
FIG. 12A shows cell surface expression of the various CARs, in either VH-VL (HL) orientation HL; dark line) or VL-VH orientation (LH; grey line), in transduced CD8+ T cells as measured by expression of EGFRt for cells before enrichment (pre) and after enrichment following sorting with an anti-EGFR antibody and expansion by stimulation with CD19+ B-LCL (post).
FIG. 12B shows an SDS gel assessing expression of exemplary human anti-CD19 CARs in transduced primary human T cells.
FIGS. 13A and 13B show cytolytic activity of primary human CD8+ T cells expressing various anti-CD19 specific CARs against CD19-expressing cells. C is EGFRt alone (negative control); FM is FMC63 scFv CAR, 18 is Clone 18 scFv CAR, 17 is Clone 17 scFv CAR, 76 is Clone 76 scFv CAR, 5 is Clone 5 scFv CAR and 18B is Clone 18B scFv CAR.
FIGS. 14A and 14B show cytokine secretion of primary human CD8+ T cells expressing various anti-CD19 specific CARs after co-culture with CD19-expressing cells. C is EGFRt alone (negative control); FM is FMC63 scFv CAR, 18 is Clone 18 scFv CAR, 17 is Clone 17 scFv CAR, 76 is Clone 76 scFv CAR, 5 is Clone 5 scFv CAR and 18B is Clone 18B scFv CAR.
FIG. 15 shows cytokine secretion of primary human CD4+ T cells expressing various anti-CD19 specific CARs after co-culture with CD19-expressing cells. C is EGFRt alone (negative control); FM is FMC63 scFv CAR, 18 is Clone 18 scFv CAR, 17 is Clone 17 scFv CAR, 76 is Clone 76 scFv CAR, 5 is Clone 5 scFv CAR and 18B is Clone 18B scFv CAR.
FIGS. 16A and 16B show proliferation of primary human CD8+ T cells or CD4+ T cells, respectively, expressing various anti-CD19 specific CARs against CD19-expressing cells after co-culture with CD19-expressing cells.
FIG. 17A shows the antitumor activity of primary human CD8+ T cells expressing various anti-CD19 specific CARs following administration to NSG mice engrafted with Raji cells that express firefly luciferase. FIG. 17B shows antitumor activity of primary human CD4+ and CD8+ T cells expressing various anti-CD19 specific CARs and administered at a 1:1 ratio to NSG mice engrafted with Raji cells.
FIG. 18A shows the amino acid sequence of a 74-residue or 75-residue membrane-proximal region for each of the three different chimeric CD19 molecules. Below all three sequences shown in FIG. 18A, each aligned position of the depicted region at which the human and rhesus sequences contain an identical amino acid is marked with an asterisk (“*”). Positions at which the rhesus sequence contains a non-identical but conservative amino acid substitution compared to the human sequence are marked with a “:”. Positions at which the rhesus sequence contains a non-identical but semi-conservative amino acid substitution compared to the human sequences are marked with a “.”. Positions at which the rhesus sequence contains an insertion or a non-identical, non-conservative/semi-conservative substitution compared with the human sequence are not marked with a symbol. FIG. 18B show cytokine secretion of primary human CD8+ T cells expressing various anti-CD19 specific CARs after co-culture with cells expressing human CD19, rhesus CD19 or chimeric rhesus/human CD19 molecules (V1, V2 or V3). C is EGFRt alone (negative control); FM is FMC63 scFv CAR, 18 is Clone 18 scFv CAR, 17 is Clone 17 scFv CAR, 76 is Clone 76 scFv CAR, 5 is Clone 5 scFv CAR and 18B is Clone 18B scFv CAR.
DETAILED DESCRIPTION
Provided are CD19-binding molecules, including antibodies (including antigen-binding antibody fragments, such as single chain fragments, including scFvs) and recombinant receptors, including chimeric receptors containing such antibodies and fragments, nucleic acids encoding such antibodies and fragments, and cells, such as recombinant cells, expressing and for production of these antibodies and fragments. Also provided are methods of making and using the antibodies and fragments as well as cells expressing or containing the antibodies and fragments.
I. CD19 BINDING MOLECULES
Provided in some aspects are CD19 binding molecules, such as CD19-binding polypeptides. Such binding molecules include antibodies that specifically bind to CD19, such as a human CD19 molecule, including antigen-binding fragments thereof. Also among the binding molecules are recombinant receptors such as chimeric antigen receptors containing such antibodies.
A. CD19 Antibodies
Provided are anti-CD19 antibodies, including functional antibody fragments, including those comprising a variable heavy chain and a variable light chain, such as scFvs. Also provided are molecules containing such antibodies, e.g., fusion proteins and/or recombinant receptors such as chimeric receptors, including antigen receptors. Among the provided anti-CD19 antibodies are human antibodies. In some embodiments, the antibodies, such as the human antibodies, specifically bind to a particular epitope or region of CD19, generally an extracellular epitope or region. In some embodiments, the antibodies bind to the same or a similar epitope or region of CD19 as bound by another antibody, such as one or more of the mouse antibodies, FMC63 or SJ25C1. In some embodiments, the antibodies bind to an overlapping epitope of CD19 as bound by one of these known antibodies and/or compete for binding with such an antibody. The antibodies include isolated antibodies. The molecules include isolated molecules.
The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab′)2 fragments, Fab′ fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
The terms “complementarity determining region,” and “CDR,” synonymous with “hypervariable region” or “HVR,” are known in the art to refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and/or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). “Framework regions” and “FR” are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).
The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (“Contact” numbering scheme), Lefranc M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme), and Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun. 8; 309(3):657-70, (“Aho” numbering scheme).
The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
Table 1, below, lists exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FR-L1 located between CDR-L1 and CDR-L2, and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.
TABLE 1
CDR
Kabat
Chothia
Contact
CDR-L1
L24-L34
L24-L34
L30-L36
CDR-L2
L50-L56
L50-L56
L46-L55
CDR-L3
L89-L97
L89-L97
L89-L96
CDR-H1
H31-H35B
H26-H32 . . . 34
H30-H35B
(Kabat Numbering1)
CDR-H1
H31-H35
H26-H32
H30-H35
(Chothia Numbering2)
CDR-H2
H50-H65
H52-H56
H47-H58
CDR-H3
H95-H102
H95-H102
H93-H101
1Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD
2Al-Lazikani et al., (1997) JMB 273, 927-948
Thus, unless otherwise specified, a “CDR” or “complementary determining region,” or individual specified CDRs (e.g., “CDR-H1, CDR-H2), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the aforementioned schemes. For example, where it is stated that a particular CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given VH or VL amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the aforementioned schemes. In some embodiments, specified CDR sequences are specified.
Likewise, unless otherwise specified, a FR or individual specified FR(s) (e.g., FR-H1, FR-H2), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) framework region as defined by any of the known schemes. In some instances, the scheme for identification of a particular CDR, FR, or FRs or CDRs is specified, such as the CDR as defined by the Kabat, Chothia, or Contact method. In other cases, the particular amino acid sequence of a CDR or FR is given.
The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
Among the provided antibodies are antibody fragments. An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody.
Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some aspects, the antibody fragments are scFvs.
A “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
Among the provided anti-CD19 antibodies are human antibodies. A “human antibody” is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences, including human antibody libraries. The term excludes humanized forms of non-human antibodies comprising non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human.
Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated. Human antibodies also may be derived from human antibody libraries, including phage display and cell-free libraries, containing antibody-encoding sequences derived from a human repertoire.
Among the provided antibodies are monoclonal antibodies, including monoclonal antibody fragments. The term “monoclonal antibody” as used herein refers to an antibody obtained from or within a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible variants containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. The term is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be made by a variety of techniques, including but not limited to generation from a hybridoma, recombinant DNA methods, phage-display and other antibody display methods.
The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains and other peptides, e.g., linkers and CD19-binding peptides, may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
Exemplary Anti-CD19 Antibodies
In some embodiments, the anti-CD19 antibody, e.g., antigen-binding antibody fragment, contains particular heavy and/or light chain CDR sequences and/or heavy and/or light chain variable (VH or VL) region sequences. Also among the provided antibodies are those having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such a sequence.
In some embodiments, the antibody, e.g., antigen-binding fragment thereof, includes a heavy chain complementarity determining region 3 (CDR-H3) comprising an amino acid sequence of a CDR-H3 present in a reference antibody, such as one present in a reference antibody having a VH region with the amino acid sequence set forth in set forth in SEQ ID NO: 11, 12, 60, 61, 63 62, 167 or 185, such as set forth in SEQ ID NO: 11, 12, 60, 61, 63, or 62. In some embodiments, the CDR-H3 comprises SEQ ID NO: 20. In some embodiments, the antibody, e.g., antigen-binding fragment thereof, has a VH region having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to (or 100% identity thereto) the VH region amino acid sequence of the reference antibody, such as to the VH region amino acid sequence set forth in SEQ ID NO: 11, 12, 60, 61, 63 62, 167 or 185, such as set forth in SEQ ID NO: 11, 12, 60, 61, 63, or 62.
In some embodiments, the CDR-H1 contains the amino acid sequence DYAMH (SEQ ID NO: 18), the CDR-H2 contains the amino acid sequence GISWNSGRIG (SEQ ID NO: 81), GISWNSGSIG (SEQ ID NO: 82), the amino acid sequence set forth in SEQ ID NO: 19 (GISWNSGRIGYADSVKG), or the amino acid sequence set forth in SEQ ID NO: 72 (GISWNSGSIGYADSVKG), and/or the CDR-H3 contains the amino acid sequence of SEQ ID NO: 20.
In some embodiments, the provided antibody contains a CDR-H3 having the amino acid sequence of SEQ ID NO: 20.
In some embodiments, the antibody contains a VH having the amino acid sequence set forth in SEQ ID NO: 11 or 12, or has a sequence at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such a sequence. In some embodiments, the antibody, e.g., antigen-binding fragment thereof, contains a VH region having the amino acid sequence set forth in SEQ ID NO: 11, 12, 60, 61, 63, or 62, or a sequence at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such a sequence. In some embodiments, the antibody, e.g., antigen-binding fragment thereof, contains a VH region having the amino acid sequence set forth in SEQ ID NO: 11, 12, 60, 61, 63, 62, 167 or 185, or a sequence at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such a sequence.
In some embodiments, the antibody contains the sequence of residues 1-119 of SEQ ID NO: 11, 12, 60, 61, 63, 62, 167 or 185 or a sequence comprising the portion of SEQ ID NO: 11, 12, 60, 61, 63, 62, 167 or 185 including the first three framework regions and the three heavy chain CDRs. In some embodiments, the antibody contains the sequence of residues 1-119 of SEQ ID NO: 11, 12, 60, 61, 63 or 62 or a sequence comprising the portion of SEQ ID NO: 11, 12, 60, 61, 63, or 62 including the first three framework regions and the three heavy chain CDRs.
In some embodiments, the anti-CD19 antibody includes light chain complementarity determining regions 1, 2, and/or 3 (CDR-L1, CDR-L2, and/or CDR-L3), respectively, having the amino acid sequences of CDR 1, 2, and/or 3 sequences contained within the light chain variable (VL) region amino acid sequence set forth in SEQ ID NO: 13, 14, 15, 16, 17, 71, 65, 64, 66, 70, 69, 67, 90, 91 or 187-205, such as set forth in SEQ ID NO: 13, 14, 15, 16, or 17, or in SEQ ID NO: 13, 14, 15, 16, 17, 71, 90, 91, 68, 65, 64, 66, 70, 69, or 67.
In some embodiments, the anti-CD19 antibody includes a CDR-L1, CDR-L2, and/or CDR-L3 in which:
In some embodiments, the CDR-L1 contains the amino acid sequence: X1X2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 110), wherein X1 is T, W, S or R; X2 is G or A; X3 is I, T, D or S; X4 is S, R, T or Q; X5 is null or S; X6 is null, D, N or G; X7 is null, V or L; X8 is X or null; X9 is X or null; X10 is X; X11 is X; X12 is Y, F, D or W; X13 is V, A or L and X14 is S, N or A. For example, in some embodiments, the CDR-L1 contains the amino acid sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 226), wherein X1 is T, Q, S, or R; X2 is G, A or E; X3 is I, T, A, D, or S; X4 is S, R, T Q, G or I; X5 is null, S, R or T; X6 is G, D, N, or null; X7 is null, V, L or I; X8 is D, G, I, L, S, or null; X9 is S, G, A, I, D, R, or null; X10 is H, Y, F, S, or N; X11 is R, N, D, H, Y or T; X12 is Y, F, D, W, H, T or S; X13 is V, A, or L; and X14 is S, N, or A. In some embodiments, the CDR-L1 contains the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 111), wherein X1 is T, Q, S, or R; X2 is G or A; X3 is I, T, D, or S; X4 is S, R, T, or Q; X5 is null or S; X6 is G, D, N, or null; X7 is null, V, or L; X8 is D, G, I, L, S, or null; X9 is S, G, A, I, R, or null; X10 is H, Y, F, S, or N; X11 is R, N, D, H, or Y; X12 is Y, F, D, or W; X13 is V, A, or L; and X14 is S, N, or A.
In some embodiments, the CDR-L2 contains the amino acid sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 227), wherein X1 is D, S or G; X2 is F, V, N, K, or A; X3 is S, T, D, or N; X4 is K, V, N, Q, or R; X5 is R, V, or L; X6 is P, K, A, or E; and X7 is S, P, A, or T. In some embodiments, the CDR-L2 contains the amino acid sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 112), wherein X1 is D or S; X2 is F, V, N, K, or A; X3 is S, T, D, or N; X4 is K, V, N, Q, or R; X5 is R, V, or L; X6 is P, K, A, or E; and X7 is S, P, A, or T.
In some embodiments, the CDR-L3 contains the amino acid sequence of X1X2X3X4X5X6X7X8X9X10X11X12 (SEQ ID NO: 228), wherein X1 is S, G, T, A, Q, C, or N; X2 is S, Q, A, or T; X3 is Y, S, W, R; X4 is A, D, R, T, or Y; X5 is A, S, P, G, N, or D; X6 is I, S, G, T, A, L, H, R, or N; X7 is S, P, L, Y, G; X8 is P, T, S, Q, M, R, N or null; X9 is S, L, N, A, M, R or null; X10 is L, D or null; X11 is Y, W, F, V, A, or L; and X12 is V, T, P or L. In some embodiments, the CDR-L3 contains the amino acid sequence of X1X2X3X4X5X6X7X8X9X10X11X12 (SEQ ID NO: 115), wherein X1 is X; X2 is S, Q, A, or T; X3 is Y, S, W, R; X4 is A, D, R, T, or Y; X5 is X; X6 is X; X7 is S, P, L, Y, G; X8 is X or null; X9 is X or null; X10 is L or null; X11 is X; and X12 is V, T, or L. For example, in some embodiments, the antibody has a CDR-L3 comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12 (SEQ ID NO: 114), wherein X1 is S, G, T, A, Q, C or N; X2 is S, Q, A, or T; X3 is Y, S, W, R; X4 is A, D, R, T, or Y; X5 is A, S, P, G, N or D; X6 is L S, G, T, A, L, H, R, N; X7 is S, P, L, Y, G; X8 is P, T, S, Q, M, R, N or null; X9 is S, L, N, A, M or null; X10 is L or null; X11 is Y, W, F, V, A or L; and X12 is V, T, or L
In some embodiments, in the CDR-L1, such as set forth in SEQ ID NO:110, 226 or 111, X3 is I, T, or S; X4 is S, T, or Q; X8 is D, G, I, S, or null; X9 is S, G, I, or null; X10 is H, Y, S, or N; X11 is R, N, D, or H; X12 is Y or D; and X13 is V or L; and/or in the CDR-L2, such as set forth in SEQ ID NO:227 or 112, X1 is D; X4 is K, V, N, Q, or R; X6 is P, K, or A; and X7 is S, A, or T; and/or in the CDR-L3, such as set forth in SEQ ID NO:228, 114 or 115, X1 is S, G, T, A, Q, C, or N; X5 is A, S, P, G, N, or D; X6 is I, S, G, T, A, L, H, R, or N; X8 is P, T, S, Q, M, R, N or null; X9 is S, L, N, A, M or null; and X11 is Y, W, F, V, A, or L. In some embodiments, in the CDR-L3, X1 is S, G, Q, or N; X2 is S, Q, or T; X4 is A, D, T, or Y; X5 is A, S, or G; and X6 is I, S, N, R, A, H, or T.
In some embodiments, the antibody includes a sequence of amino acids that contains a CDR-L1 set forth in SEQ ID NO:83, a CDR-L2 set forth in SEQ ID NO:84 and/or a CDR-L3 set forth in SEQ ID NO:85.
In some embodiments, the antibody, e.g., the antibody fragment contains a CDR-L1 that contains the amino acid sequence set forth in SEQ ID NO: 21, 25, 28, or 31. In some embodiments, the antibody or fragment contains a CDR-L1 that contains the amino acid sequence set forth in SEQ ID NO: 80, 77, 74, 73, 75, 79, 78, 76, 21, 25, 28, 31 or 146 to 152, such as contains the amino acid sequence set forth in SEQ ID NO: 80, 77, 74, 73, 75, 79, 78, 76, 21, 25, 28, or 31. In some embodiments, the antibody or fragment contains a CDR-L1 that contains the amino acid sequence set forth in SEQ ID NO: 80, 77, 74, 73, 78, 21, or 28.
In some embodiments, the antibody or fragment contains a CDR-L2 that contains the amino acid sequence set forth in SEQ ID NO: 22, 26, 29, or 32. In some embodiments, the antibody or fragment contains a CDR-L2 that contains the amino acid sequence SEQ ID NO: 100, 97, 94, 93, 95, 99, 98, 96, 22, 26, 29, 32 or 153 to 157, such as contains the amino acid sequence set forth in SEQ ID NO: 100, 97, 94, 93, 95, 99, 98, 96, 22, 26, 29, or 32. In some embodiments, the antibody or fragment contains a CDR-L2 that contains the amino acid sequence set forth in SEQ ID NO: 100, 97, 94, 93, 98, 22, or 29.
In some embodiments, the antibody or fragment contains a CDR-L3 that includes the sequence set forth in SEQ ID NO: 23, 24, 27, 30, or 33. In some embodiments, the antibody or fragment contains a CDR-L3 that includes the sequence set forth in SEQ ID NO: 109, 106, 103, 101, 104, 108, 107, 105, 102, 23, 24, 27, 30, 33, 158 or 159, such as contains the amino acid sequence set forth in SEQ ID NO: 109, 106, 103, 101, 104, 108, 107, 105, 102, 23, 24, 27, 30, or 33. In some embodiments, the antibody or fragment contains a CDR-L3 that includes the sequence set forth in SEQ ID NO: 109, 106, 103, 101, 107, 24 or 30.
In some embodiments, the CDR-L1, CDR-L2, and CDR-L3 contain the sequences of SEQ ID NOs: 21, 22, and 23, respectively; the CDR-L1, CDR-L2, and CDR-L3 include the sequences of SEQ ID NOs: 21, 22, and 24, respectively; the CDR-L1, CDR-L2, and CDR-L3 include the sequences of SEQ ID NOs: 25, 26, and 27, respectively; the CDR-L1, CDR-L2, and CDR-L3 contain the sequences of SEQ ID NOs: 28, 29, and 30, respectively; or the CDR-L1, CDR-L2, and CDR-L3 contain the sequences of SEQ ID NOs: 31, 32, and 33, respectively.
In some embodiments, the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 21, 22, and 23, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 21, 22, and 24, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 25, 26, and 27, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 28, 29, and 30, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 31, 32, and 33, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 80, 100, and 109, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs:77, 97, and 106, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 74, 94, and 103, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 73, 93, and 101, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs:75, 95, and 104, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 79, 99, and 108, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 78, 98, and 107, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 76, 96, and 105, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 73, 93, and 102, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 77, 97, and 106, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 163, 164, and 165, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 80, 100, and 109, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 146, 97, and 106, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 28, 153 and 158, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 74, 94, and 103, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 147, 154 and 121, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 148, 94 and 103, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 75, 95 and 104, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 149, 155 and 119, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 150, 22, and 120, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 21, 22 and 159, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 151, 26 and 118, respectively; the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 28, 156 and 116, respectively; or the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 152, 157 and 117, respectively.
Also provided are antibodies having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 81, and 20, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 19, and 20, respectively; the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 82, and 20, respectively; or the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 72, and 20, respectively.
Also provided are antibodies having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the VH region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 11, 12, 60, 61, 6362, 167 or 185, such as SEQ ID NO: 11, 12, 60, 61, 63, or 62; and/or the VL region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, 71, 90, 91, 68, 65, 64, 66, 70, 69, 67 or 187 to 205, such as SEQ ID NO: 13, 14, 15, 16, 17, 71, 90, 91, 68, 65, 64, 66, 70, 69, or 67. In some embodiments, the VH region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 11, 60, 63, or 62; and/or the VL region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 14, 16, 71, 90, 65, 64, or 69.
Also provided are antibodies having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 17, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 15, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 13, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 14, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 16, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 71, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 62 and 68, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 65, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 60 and 64, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 61 and 66, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 70, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 62 and 69, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 67, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 91, respectively; or the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 90, respectively. In some embodiments, the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 14, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 16, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 71, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 65, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 60 and 64, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 62 and 69, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 90, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 167 and 207, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 168 or 63 and 208, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 169 or 11 and 209, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 170 or 61 and 210, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 171 or 61 and 211, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 172 and 212, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 173 or 11 and 213, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 174 or 11 and 214, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 175 or 11 and 215, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 176 or 61 and 216, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 177 or 61 and 217, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 178 or 61 and 218, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 179 or 61 and 219, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 180 or 12 and 220, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 181 or 12 and 221, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 182 or 11 and 222, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 183 or 60 and 223, respectively; the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 184 or 11 and 224, respectively; or the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 185 and 225, respectively.
Also provided are antibodies having sequences at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such sequences.
In some embodiments, the antibody or fragment contains a VH region including the amino acid sequence of SEQ ID NO: 11 or 12 or residues 1-119 of such a sequence or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such a sequence.
In some embodiments, the antibodies include or further include a VL region including the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, or a sequence having at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such a sequence.
In some embodiments, the antibody is a single-chain antibody fragment, such as an scFv or diabody. In some embodiments, the single-chain antibody includes one or more linkers joining two antibody domains or regions, such as a variable heavy chain (VH) region and a variable light chain (VL). The linker typically is a peptide linker, e.g., a flexible and/or soluble peptide linker. Among the linkers are those rich in glycine and serine and/or in some cases threonine. In some embodiments, the linkers further include charged residues such as lysine and/or glutamate, which can improve solubility. In some embodiments, the linkers further include one or more proline.
Accordingly, also provided are single-chain antibody fragments, such as scFvs and diabodies, particularly human single-chain fragments, typically comprising linker(s) joining two antibody domains or regions, such VH and VL domains. The linker typically is a peptide linker, e.g., a flexible and/or soluble peptide linker, such as one rich in glycine and serine.
In some aspects, the linkers rich in glycine and serine (and/or threonine) include at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% such amino acid(s). In some embodiments, they include at least at or about 50%, 55%, 60%, 70%, or 75%, glycine, serine, and/or threonine. In some embodiments, the linker is comprised substantially entirely of glycine, serine, and/or threonine. The linkers generally are between about 5 and about 50 amino acids in length, typically between at or about 10 and at or about 30, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and in some examples between 10 and 25 amino acids in length. Exemplary linkers include linkers having various numbers of repeats of the sequence GGGGS (4GS; SEQ ID NO:123) or GGGS (3GS; SEQ ID NO:122), such as between 2, 3, 4, and 5 repeats of such a sequence. Exemplary linkers include those having or consisting of a sequence set forth in SEQ ID NO: 34 (GGGGSGGGGSGGGGS). Exemplary linkers further include those having or consisting of the sequence set forth in SEQ ID NO: 43 (GSTSGSGKPGSGEGSTKG).
Accordingly, in some embodiments, also provided are single-chain fragments, e.g., scFvs, comprising one or more of the aforementioned linkers, such as glycine/serine rich linkers, including linkers having repeats of GGGS (SEQ ID NO:122) or GGGGS (SEQ ID NO:123), such as the linker set forth as SEQ ID NO: 34. In some embodiments, the linker has an amino acid sequence containing the sequence set forth SEQ ID NO: 34.
The fragment, e.g., scFv, may include a VH region or portion thereof, followed by the linker, followed by a VL or portions thereof. The fragment, e.g., the scFv, may include the VL, followed by the linker, followed by the VH.
In some aspects, the scFv has the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10, or has a sequence at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such a sequence.
In some aspects, the scFv has the amino acid sequence set forth set forth in SEQ ID NO: 2, 4, 6, 8, 10, 45, 47, 49, 51, 53, 55, 57, 59, 87, or 89, or has a sequence at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such a sequence.
In some aspects, the scFv has the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 45, 47, 49, 51, 53, 55, 57, 59, 87, 89, or 207 to 225 or has a sequence at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such a sequence.
In some aspects, the scFv contains the VH, linker and VL as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 45, 47, 49, 51, 53, 55, 57, 59, 87 89 or 207 to 225, or a sequence at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to such a sequence, but in which the VH and VL are configured in the opposite orientation, i.e. VL-VH, as compared to such sequence.
The antibody, e.g., antibody fragment, may contain at least a portion of an immunoglobulin constant region, such as one or more constant region domain. In some embodiments, the constant regions include a light chain constant region and/or a heavy chain constant region 1 (CH1). In some embodiments, the antibody includes a CH2 and/or CH3 domain, such as an Fc region. In some embodiments, the Fc region is an Fc region of a human IgG, such as an IgG1 or IgG4.
In some embodiments, any of the above antibodies, e.g., antibody fragments is human. For example, provided herein are human anti-CD19 antibodies that specifically bind CD19, such as specifically bind human CD19.
In some embodiments of a provided human anti-CD19 antibody, the human antibody contains a VH region that contains a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain V segment, a portion with at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence encoded by a germline nucleotide human heavy chain D segment, and/or a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence encoded by a germline nucleotide human heavy chain J segment; and/or contains a VL region that contains a portion with at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain V segment, and/or a portion with at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain J segment. In some embodiments, the portion of the VH region corresponds to the CDR-H1, CDR-H2 and/or CDR-H3. In some embodiments, the portion of the VH region corresponds to the framework region 1 (FR1), FR2, FR2 and/or FR4. In some embodiments, the portion of the VL region corresponds to the CDR-L1, CDR-L2 and/or CDR-L3. In some embodiments, the portion of the VL region corresponds to the FR1, FR2, FR2 and/or FR4.
In some embodiments, the human antibody contains a CDR-H1 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-H1 region within a sequence encoded by a germline nucleotide human heavy chain V segment. For example, the human antibody in some embodiments contains a CDR-H1 having a sequence that is 100% identical or with no more than one, two or three amino acid differences as compared to the corresponding CDR-H1 region within a sequence encoded by a germline nucleotide human heavy chain V segment.
In some embodiments, the human antibody contains a CDR-H2 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-H2 region within a sequence encoded by a germline nucleotide human heavy chain V segment. For example, the human antibody in some embodiments contains a CDR-H2 having a sequence that is 100% identical or with no more than one, two or three amino acid difference as compared to the corresponding CDR-H2 region within a sequence encoded by a germline nucleotide human heavy chain V segment.
In some embodiments, the human antibody contains a CDR-H3 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-H3 region within a sequence encoded by a germline nucleotide human heavy chain V segment, D segment and J segment. For example, the human antibody in some embodiments contains a CDR-H3 having a sequence that is 100% identical or with no more than one, two or three amino acid differences as compared to the corresponding CDR-H3 region within a sequence encoded by a germline nucleotide human heavy chain V segment, D segment and J segment.
In some embodiments, the human antibody contains a CDR-L1 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-L1 region within a sequence encoded by a germline nucleotide human light chain V segment. For example, the human antibody in some embodiments contains a CDR-L1 having a sequence that is 100% identical or with no more than one, two or three amino acid differences as compared to the corresponding CDR-L1 region within a sequence encoded by a germline nucleotide human light chain V segment.
In some embodiments, the human antibody contains a CDR-L2 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-L2 region within a sequence encoded by a germline nucleotide human light chain V segment. For example, the human antibody in some embodiments contains a CDR-L2 having a sequence that is 100% identical or with no more than one, two or three amino acid difference as compared to the corresponding CDR-L2 region within a sequence encoded by a germline nucleotide human light chain V segment.
In some embodiments, the human antibody contains a CDR-L3 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of the corresponding CDR-L3 region within a sequence encoded by a germline nucleotide human light chain V segment and J segment. For example, the human antibody in some embodiments contains a CDR-L3 having a sequence that is 100% identical or with no more than one, two or three amino acid differences as compared to the corresponding CDR-L3 region within a sequence encoded by a germline nucleotide human light chain V segment and J segment.
In some embodiments, the human antibody contains a framework region that contains human germline gene segment sequences. For example, in some embodiments, the human antibody contains a VH region in which the framework region, e.g. FR1, FR2, FR3 and FR4, has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a framework region encoded by a human germline antibody segment, such as a V and/or J segment. In some embodiments, the human antibody contains a VL region in which the framework region e.g. FR1, FR2, FR3 and FR4, has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a framework region encoded by a human germline antibody segment, such as a V and/or segment. For example, in some such embodiments, the framework sequence of the VH and/or VL sequence differs by no more than 10 amino acids, such as no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid, compared to the framework region encoded by a human germline antibody segment.
The antibody, e.g., antibody fragment, may contain at least a portion of an immunoglobulin constant region, such as one or more constant region domain. In some embodiments, the constant regions include a light chain constant region and/or a heavy chain constant region 1 (CH1). In some embodiments, the antibody includes a CH2 and/or CH3 domain, such as an Fc region. In some embodiments, the Fc region is an Fc region of a human IgG, such as an IgG1 or IgG4.
Also provided are nucleic acids encoding the antibodies and/or portions, e.g., chains, thereof. Among the provided nucleic acids are those encoding the anti-CD19 antibodies described herein. The nucleic acids may include those encompassing natural and/or non-naturally occurring nucleotides and bases, e.g., including those with backbone modifications. The terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide” may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide. Exemplary nucleic acids and vectors are those having the sequences set forth as SEQ ID NOs: 1, 3, 5, 7, 9, 44, 46, 48, 50, 52, 54, 56, 58, 86, and 88, and CDR-encoding portions thereof, as well as sequences containing at least at or about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity thereto. The nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody).
Also provided are vectors containing the nucleic acids, host cells containing the vectors, e.g., for producing the antibodies. Also provided are methods for producing the antibodies. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In some embodiments, a method of making the anti-CD19 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
Also provided are methods of making the anti-CD19 antibodies (including antigen-binding fragments). For recombinant production of the anti-CD19 antibody, nucleic acid encoding an antibody, e.g., as described above, may be isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been modified to mimic or approximate those in human cells, resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lec13 CHO cells, and FUT8 CHO cells; PER.C6® cells; and NSO cells. In some embodiments, the antibody heavy chains and/or light chains may be expressed in yeast. See, e.g., U.S. Publication No. US 2006/0270045 A1. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and/or light chains. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.
In some embodiments, the antibody is produced in a cell-free system. Exemplary cell-free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713 (2003).
The provided embodiments further include vectors and host cells and other expression systems for expressing and producing the antibodies and other binding proteins, including eukaryotic and prokaryotic host cells, including bacteria, filamentous fungi, and yeast, as well as mammalian cells such as human cells, as well as cell-free expression systems.
Exemplary Features
In some aspects, the provided antibodies, including antigen-binding fragments, have one or more specified functional features, such as binding properties, including binding to particular epitopes, such as epitopes that are similar to or overlap with those of other antibodies, the ability to compete for binding with other antibodies, and/or particular binding affinities.
In some embodiments, the antibodies specifically bind to CD19 protein. In some aspects of any of the embodiments herein, CD19 refers to human CD19. Generally, the observation that an antibody or other binding molecule binds to CD19 or specifically binds to CD19 does not necessarily mean that it binds to CD19 of every species. For example, in some embodiments, features of binding to CD19, such as the ability to specifically bind thereto and/or to compete for binding thereto with a reference antibody, and/or to bind with a particular affinity or compete to a particular degree, in some embodiments, refers to the ability with respect to human CD19 protein and the antibody many not have this feature with respect to a CD19 of another species, such as monkey or mouse.
In some embodiments, the provided antibodies, including antigen-binding fragments, bind to human CD19, such as to an epitope or region of human CD19, such as to human CD19 set forth in 92 (Accession No. P15391), or an allelic variant or splice variant thereof. In certain embodiments, the anti-CD19 antibody binds to an epitope of CD19 that is conserved among CD19 from different species. In some embodiments, the anti-CD19 antibody binds to an epitope of CD19 that is not conserved or not entirely conserved among CD19 from different species, such as among human and Macaca mulatta (rhesus macaque (rhesus)) CD19.
In some embodiments, the antibody binds to an epitope containing one or more amino acids within (or is entirely within) an extracellular domain of a CD19 and/or within (or is entirely within) a membrane-proximal region of the extracellular portion of CD19. In some embodiments, the antibody binds to an epitope containing one or more amino acids within, or is entirely within, the Ig-like domain 2 of CD19, a portion encoded by the fourth exon of the CD19, a portion corresponding to positions 176-277 of the human CD19 sequence set forth in SEQ ID NO: 92, and/or the membrane-proximal-most 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 44, 43, 43, 41, or 40 amino acid portion of the extracellular portion of the CD19. In some embodiments, such a portion or domain is required for binding of the antibody to CD19. In some embodiments, the epitope contains (or further contains) one or more amino acids that is within, or is entirely within, the Ig-like domain 1 of CD19, a portion encoded by the second exon of the CD19 and/or a portion corresponding to positions 20-117 of the human CD19 sequence set forth in SEQ ID NO: 92. In some embodiments, such a portion or domain is required for binding of the antibody to CD19. In some embodiments, the antibody specifically binds to a peptide comprising or consisting of or consisting essentially of the sequence of such a portion, and not containing the entire sequence of full-length CD19.
In some embodiments, the epitope contains one or more amino acids within, is within, or includes a portion of CD19 corresponding to residues 218-249 of the human CD19 sequence set forth in SEQ ID NO: 92, such as a portion having the sequence set forth in SEQ ID NO: 143.
In some embodiments, the epitope includes an amino acid at a position corresponding to one or more of the positions of CD19 corresponding to the following amino acids at the following positions of the human CD19 sequence set forth in SEQ ID NO: 92: the histidine (H) at position 218, the alanine (A) at position 236, the methionine (M) at position 242, the glutamate (E) at position 243, the proline (P) at position 249, and/or the lysine (K) and/or serine (S) at positions 223 and 224. In some embodiments, an amino acid at one or more such a position is important or necessary for binding of the antibody to CD19. In some embodiments, the amino acid in the epitope at such one or more position corresponds to the amino acid at the respective position in the human CD19 sequence set forth in SEQ ID NO: 92.
In some embodiments, the epitope includes an amino acid (such as a histidine) at a position of CD19 corresponding to the histidine at position 218 of the human CD19 sequence set forth in SEQ ID NO: 92; in some embodiments, such amino acid is important for binding of the antibody to CD19.
In some embodiments, the epitope includes an amino acid (such as an alanine) at a position of CD19 corresponding to the alanine at position 236 of the human CD19 sequence set forth in SEQ ID NO: 92; in some embodiments, such amino acid is important for binding of the antibody to CD19.
In some embodiments, the epitope includes an amino acid (such as a methionine) at a position of CD19 corresponding to the methionine at position 242 of the human CD19 sequence set forth in SEQ ID NO: 92; in some embodiments, such amino acid is important for binding of the antibody to CD19.
In some embodiments, the epitope includes an amino acid (such as a glutamate) at a position of CD19 corresponding to the glutamate at position 243 of the human CD19 sequence set forth in SEQ ID NO: 92; in some embodiments, such amino acid is important for binding of the antibody to CD19.
In some embodiments, the epitope includes an amino acid (such as a proline) at a position of CD19 corresponding to the proline at position 249 of the human CD19 sequence set forth in SEQ ID NO: 92; in some embodiments, such amino acid is important for binding of the antibody to CD19.
In some embodiments, the epitope contains amino acid(s) (such as lysine and/or serine) at one or two positions corresponding to the lysine and/or serine at positions 223 and 224 of the human CD19 sequence set forth in SEQ ID NO: 92; in some embodiments, such amino acid(s) are important for binding of the antibody to CD19.
In some embodiments, the epitope is the same as, similar to, overlapping with, or contains one or more of the same amino acids as an epitope that is specifically bound to by a reference antibody, such as FMC63 or SJ25C1. In some embodiments, the same one or more amino acids is important for the binding of the provided antibody and the reference antibody.
In some embodiments, the extent of binding of an anti-CD19 antibody to an unrelated, non-CD19 protein, such as non-human CD19 or other non-CD19 protein, is less than about 40% of the binding of the antibody to human CD19 as measured, for example, by a radioimmunoassay (RIA). In some embodiments, among provided antibodies are antibodies in which binding to a non-human CD19 or other non-CD19 protein is less than or about 30%, less than or about 20% or less than or about 10% of the binding of the antibody to human CD19.
In some embodiments, such properties of provided antibodies, including antigen-binding fragments, are described in relation to properties observed for another antibody, e.g., a reference antibody. In some embodiments, the reference antibody is a non-human anti-CD19 antibody, such as a murine or chimeric or humanized anti-CD19 antibody. In some aspects, the reference antibody is the antibody designated FMC63 or the antibody designated SJ25C1 (see, e.g., Zola H et al., Immunol Cell Biol. 1991 December; 69 (Pt 6):411-22; U.S. Pat. No. 7,446,190), and/or a fragment derived therefrom such as an scFv fragment thereof, and/or an antibody containing the VH and VL sequences of such an antibody and/or the heavy and light chain CDRs of such an antibody.
For example, in some embodiments, the reference antibody has a VH region containing the sequence set forth in SEQ ID NO: 39 or 41, or comprises CDR1, CDR2, and/or CDR3 within such a sequence, and/or has a VL containing the sequence set forth in SEQ ID NO: 40 or 42, or comprises CDR1, CDR2, and/or CDR3 within such a sequence. Thus, in some embodiments, the antibody competes for binding with, and/or binds to the same or an overlapping epitope of CD19 as, FMC63 or SJ25C1 or an antigen-binding fragment thereof.
In some embodiments, the reference antibody has a sequence present in an antibody or portion thereof as described herein. For example, in some embodiments, the reference antibody has a light chain variable (VL) region amino acid sequence set forth in SEQ ID NO: 13, 14, 15, 16, or 17 and/or set forth in SEQ ID NO: 13, 14, 15, 16, 17, 71, 90, 91, 68, 65, 64, 66, 70, 69, or 67, and/or has a heavy chain variable (VH) region set forth in SEQ ID NO: 11, 12, 60, 61, 63, or 62. In some embodiments, the antibody has heavy and/or light chain CDRs 1, 2, and/or 3 as present in such an antibody. In some embodiments, the reference antibody can be an scFv that contains the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 45, 47, 49, 51, 53, 55, 57, 59, 87 or 89.
In some embodiments, the antibody nonetheless contains heavy and light chain CDRs that are distinct from the CDRs present in the reference antibody or antibodies, such as FMC63 and SJ25C1. For example, among the provided antibodies are those that compete for binding with and/or bind to the same or overlapping epitopes of CD19 as those bound by a reference antibody or antibody, but nonetheless contain distinct CDRs, e.g., distinct heavy and/or light chain CDR1, CDR2, and CDR3. In some embodiments, the provided antibody contains heavy and light chain CDRs that are distinct from the CDRs present in the antibody designated FMC63, such as present in the VH region set forth in SEQ ID NO:39 and/or the VL region set forth in SEQ ID NO:40. In some embodiments, the provided antibody contains heavy and light chain CDRs that are distinct from the CDRs present in the antibody designated SJ25C1, such as present in the VH region set forth in SEQ ID NO:41 and/or the VL region set forth in SEQ ID NO:42.
For example, in some embodiments, the antibody specifically binds to an epitope that overlaps with the epitope of CD19 bound by a reference antibody, such as antibodies that bind to the same or a similar epitope as the reference antibody. In some embodiments, the antibody competes for binding to CD19 with the reference antibody.
In some embodiments, the antibodies display a binding preference for CD19-expressing cells as compared to CD19-negative cells, such as particular cells known in the art and/or described herein. In some embodiments, the binding preference is observed where a significantly greater degree of binding is measured to the CD19-expressing, as compared to the non-expressing, cells. In some embodiments, the fold change in degree of binding detected, for example, as measured by mean fluorescence intensity in a flow cytometry-based assay and/or dissociation constant or EC50, to the CD19-expressing cells as compared to the non-CD19-expressing cells, is at least at or about 1.5, 2, 3, 4, 5, 6, or more, and/or is about as great, about the same, at least as great or at least about as great, or greater, than the fold change observed for the reference antibody, such as the corresponding form of the reference antibody. In some cases, the total degree of observed binding to CD19 or to the CD19-expressing cells is approximately the same, at least as great, or greater than that observed for the reference antibody. In any of the provided embodiments, comparison of binding properties, such as affinities or competition, may be via measurement by the same or similar assay.
An antibody “competes for binding” to CD19 with a reference antibody if it competitively inhibits binding of the reference antibody to CD19, and/or if the reference antibody competitively inhibits binding of the antibody to CD19. An antibody competitively inhibits binding of a reference antibody to an antigen if the presence of the antibody in excess detectably inhibits (blocks) binding of the other antibody to its antigen. A particular degree of inhibition may be specified.
In some embodiments, addition of the provided antibody in excess, e.g., 1-, 2-, 5-, 10-, 50- or 100-fold excess, as compared to the amount or concentration of the reference antibody, inhibits binding to the antigen by the reference antibody (or vice versa). In some embodiments, the inhibition of binding is by at least 50%, and in some embodiments by at least 75%, 90% or 99%. In some aspects, the competitive inhibition is as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).
In some embodiments, where the reference antibody is present at a concentration of 10 nM, the provided antibody inhibits binding of the reference antibody with an IC50 of less than at or about 100, 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nM, or less than at or about 9, 8, 7, 6, or 5 nM. In some embodiments, where the provided antibody is present at a concentration of 10 nM, the reference antibody inhibits binding of the provided antibody with an IC50 of less than at or about 100, 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nM, or less than at or about 9, 8, 7, 6, or 5 nM.
In some embodiments, competitive inhibition of the reference antibody's binding by the provided antibody (or vice versa) is at or about or least at or about the same degree as the degree of competitive inhibition of the reference antibody's binding by the reference antibody itself, e.g., unlabeled reference antibody. In some embodiments, the provided antibody inhibits binding of the reference antibody, such as binding of FMC63 or SJ25C1, to human CD19 by at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Competitive inhibition assays are known and include ELISA-based, flow cytometry-based assays, and RIA-based assays. In some aspects, competitive inhibition assays are carried out by incorporating an excess of an unlabeled form of one of the antibodies and assessing its ability to block binding of the other antibody, which is labeled with a detectable marker, such that degree of binding and reduction thereof can be assessed by detection of the label or marker.
In some embodiments, two antibodies specifically bind to the same epitope if all or essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody. In some embodiments, two antibodies specifically bind to an overlapping epitope if at least some of the amino acid mutations in the antigen that reduce binding or eliminate binding to the antigen by one antibody also reduce or eliminate binding to the antigen by the other antibody.
In some embodiments, the provided antibodies are capable of binding CD19, such as human CD19, with at least a certain affinity, as measured by any of a number of known methods. In some embodiments, the affinity is represented by a dissociation constant (Kd); in some embodiments, the affinity is represented by EC50. In certain embodiments, the binding affinity (EC50) and/or the dissociation constant of the antibody to CD19 is at or about or less than at or about 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nM, such as between at or about 1 nM and at or about 15 nM, e.g., between at or about 5 and at or about 10 nM. In one embodiment, the extent of binding of an anti-CD19 antibody to an unrelated, non-CD19 protein is less than at or about 10% of the binding of the antibody to CD19 as measured, e.g., by a radioimmunoassay (RIA).
In some aspects, the affinity is at or about the same degree or substantially the same degree of affinity compared to the reference antibody, such as murine CD19 antibody, for example FMC63 or SJ25C1. In some aspects, the affinity is at least 80, 85, 90, 95, or 99% the same as that of the reference antibody. In some embodiments, binding affinity is compared with respect to the corresponding form of the reference antibody.
In some embodiments, the antibody has an affinity, e.g., EC50 or Kd, about the same as or lower than that of the reference antibody, such as of the corresponding form of the reference antibody, e.g., no more than about 1.5-fold or no more than about 2-fold greater, no more than 3-fold greater, and/or no more than 10-fold greater, than the EC50 of the reference antibody, e.g., as measured in the same or similar assay.
Anti-CD19 antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various known assays. In one aspect, the antibody is tested for its antigen binding activity, e.g., by known methods such as ELISA, Western blotting, and/or flow cytometric assays, including cell-based binding assays, for example, assessing binding of the antibody (e.g., conjugated to a fluorescent marker or tagged) to a cell expressing the target antigen, e.g., CD19, in some cases compared to results using cells that do not express the target antigen, e.g., CD19. Binding affinity may be measured as Kd or EC50.
Competition assays may be used to identify an antibody that competes with any of the antibodies described herein. Assays for mapping epitopes bound by the antibodies and reference antibodies also may be used and are known.
Immunoconjugates
In some embodiments, the antibody is or is part of an immunoconjugate, in which the antibody is conjugated to one or more heterologous molecule(s), such as, but not limited to, a cytotoxic agent, an imaging agent, a detectable moiety a multimerization domain or other heterologous molecule. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins. In some embodiments, the antibody is conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
Among the immunoconjugates are antibody-drug conjugates (ADCs), in which an antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC 1065.
Also among the immunoconjugates are those in which the antibody is conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
Also among the immunoconjugates are those in which the antibody is conjugated to a radioactive atom to form a radioconjugate. Exemplary radioactive isotopes include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu.
Conjugates of an antibody and cytotoxic agent may be made using any of a number of known protein coupling agents, e.g., linkers, (see Vitetta et al., Science 238:1098 (1987)), WO94/11026. The linker may be a “cleavable linker” facilitating release of a cytotoxic drug in the cell, such as acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, and disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020).
Conjugates may also include fusion proteins such as Fc-fusions and chimeric molecules.
Multispecific Antibodies
In certain embodiments, the CD19-binding molecules, e.g., antibodies are multispecific. Among the multispecific binding molecules are multispecific antibodies, including, e.g. bispecific. Multispecific binding partners, e.g., antibodies, have binding specificities for at least two different sites, which may be in the same or different antigens. In certain embodiments, one of the binding specificities is for CD19 and the other is for another antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of CD19. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express CD19. Bispecific antibodies can be prepared as full length antibodies or antibody fragments. Among the bispecific antibodies are multispecific single-chain antibodies, e.g., diabodies, triabodies, and tetrabodies, tandem di-scFvs, and tandem tri-scFvs. Also provided are multispecific chimeric receptors, such as multispecific CARs, containing the antibodies.
Exemplary additional antigens include other B cell specific antigens and antigens expressed on T cells. Exemplary antigens include CD4, CD5, CD8, CD14, CD15, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138, B7, MUC-1, Ia, HM1.24, HLA-DR, tenascin, an angiogenesis factor, VEGF, PIGF, ED-B fibronectin, an oncogene, an oncogene product, CD66a-d, necrosis antigens, Ii, IL-2, T101, TAC, IL-6, TRAIL-R1 (DR4) and TRAIL-R2 (DR5).
Variants
In certain embodiments, the antibodies include one or more amino acid variations, e.g., substitutions, deletions, insertions, and/or mutations, compared to the sequence of an antibody described herein. Exemplary variants include those designed to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
In certain embodiments, the antibodies include one or more amino acid substitutions, e.g., as compared to an antibody sequence described herein and/or compared to a sequence of a natural repertoire, e.g., human repertoire. Sites of interest for substitutional mutagenesis include the CDRs and FRs. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, improved half-life, and/or improved effector function, such as the ability to promote antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In some embodiments, the variant antibody exhibits retained or improved binding to CD19.
In some embodiments, one or more residues within a CDR of a parent antibody (e.g. a humanized or human antibody) is/are substituted. In some embodiments, the substitution is made to revert a sequence or position in the sequence to a germline sequence, such as an antibody sequence found in the germline (e.g., human germline), for example, to reduce the likelihood of immunogenicity, e.g., upon administration to a human subject.
In some embodiments, alterations are made in CDR “hotspots,” residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. Such alterations may, for example, be outside of antigen contacting residues in the CDRs. In certain embodiments of the variant VH and VL sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.
Modifications
In certain embodiments, the antibody is altered to increase or decrease the extent to which the antibody is glycosylated, for example, by removing or inserting one or more glycosylation sites by altering the amino acid sequence and/or by modifying the oligosaccharide(s) attached to the glycosylation sites, e.g., using certain cell lines. Glycosylation sites include asparagine 297 of the heavy chain (according to Kabat numbering).
Exemplary modifications, variants, and cell lines are described, e.g., in Patent Publication Nos. US 2003/0157108, US 2004/0093621, US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107); WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.); WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
Among the modified antibodies are those having one or more amino acid modifications in the Fc region, such as those having a human Fc region sequence or other portion of a constant region (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
Such modifications can be made, e.g., to improve half-life, alter binding to one or more types of Fc receptors, and/or alter effector functions.
Also among the variants are cysteine engineered antibodies such as “thioMAbs” and other cysteine engineered variants, in which one or more residues of an antibody are substituted with cysteine residues, in order to generate reactive thiol groups at accessible sites, e.g., for use in conjugation of agents and linker-agents, to produce immunoconjugates. Cysteine engineered antibodies are described, e.g., in U.S. Pat. Nos. 7,855,275 and 7,521,541.
In some embodiments, the antibodies are modified to contain additional nonproteinaceous moieties, including water soluble polymers. Exemplary polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
B. Recombinant Receptors
Among the provided CD19 binding molecules are recombinant receptors, such as antigen receptors and other chimeric receptors, that specifically bind to CD19, such as receptors containing the provided anti-CD19 antibodies, e.g., antibody fragments. Among the antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Also provided are cells expressing the recombinant receptors and uses thereof in adoptive cell therapy, such as treatment of diseases and disorders associated with CD19 expression.
Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in international patent application publication numbers WO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, WO2013/123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the antigen receptors include a CAR as described in U.S. Pat. No. 7,446,190, and those described in International Patent Application Publication No.: WO/2014055668 A1. Exemplary of the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190, 8,389,282, e.g., and in which the antigen-binding portion, e.g., scFv, is replaced by an antibody, e.g., as provided herein.
Among the chimeric receptors are chimeric antigen receptors (CARs). The chimeric receptors, such as CARs, generally include an extracellular antigen binding domain that includes, is, or is comprised within, one of the provided anti-CD19 antibodies. Thus, the chimeric receptors, e.g., CARs, typically include in their extracellular portions one or more CD19-binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable domains, and/or antibody molecules, such as those described herein. In some embodiments, the CAR includes a CD19-binding portion or portions of the antibody molecule, such as a variable heavy (VH) chain region and/or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment.
CD19-targeting CARs are described, for example, by Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190, and 8,389,282.
In some embodiments, the recombinant receptor, such as a CAR, such as the antibody portion thereof, further includes a spacer, which may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region, e.g., an IgG4 hinge region, and/or a CH1/CL and/or Fc region. In some aspects, the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. In some examples, the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, international patent application publication number WO2014031687, U.S. Pat. No. 8,822,647 or published app. No. US2014/0271635.
In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgG1. In some embodiments, the spacer has the sequence ESKYGPPCPPCP (set forth in SEQ ID NO: 124), and is encoded by the sequence set forth in SEQ ID NO: 125. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 126. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 127. In some embodiments, the constant region or portion is of IgD. In some embodiments, the spacer has the sequence set forth in SEQ ID NO:128. In some embodiments, the spacer has a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 124, 126, 127 or 128.
The antigen recognition domain generally is linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and/or signal via another cell surface receptor. Thus, in some embodiments, the CD19-specific binding component (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154, and/or transmembrane regions containing functional variants thereof such as those retaining a substantial portion of the structural, e.g., transmembrane, properties thereof. In some embodiments, the transmembrane domain is a transmembrane domain derived from CD4, CD28, or CD8, e.g., CD8alpha, or functional variant thereof. In some embodiments the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and/or transmembrane domain(s).
Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
The receptor, e.g., the CAR, generally includes at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the CD19-binding antibody is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25 or CD 16.
In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling domain of the CAR activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functional capability.
In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.
T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.
In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD8, CD22, CD79a, CD79b, and CD66d. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.
In some embodiments, the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10, or ICOS, or CD27. In some aspects, the same CAR includes both the activating and costimulatory components.
In some embodiments, the activating domain (e.g. CD3 zeta) is included within one CAR, whereas the costimulatory component (e.g. CD28 or 4-1BB) is provided by another CAR recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see WO2014/055668). In some aspects, the CD19-targeting CAR is the stimulatory or activating CAR; in other aspects, it is the costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than CD19, whereby an activating signal delivered through the CD19-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.
In some embodiments, the intracellular signaling component of the recombinant receptor, such as CAR, comprises a CD3 zeta intracellular domain and a costimulatory signaling region. In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and/or CD 137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.
In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.
In some embodiments, the CAR or other antigen receptor further includes a marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor, such as a truncated version of a cell surface receptor, such as truncated EGFR (tEGFR). In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor (e.g., tEGFR) or a functional variant thereof. In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. For example, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence. An exemplary polypeptide for a truncated EGFR (e.g. tEGFR) comprises the sequence of amino acids set forth in SEQ ID NO: 138 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:138. An exemplary T2A linker sequence comprises the sequence of amino acids set forth in SEQ ID NO:137 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:137.
In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.
In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self” by the immune system of the host into which the cells will be adoptively transferred.
In some embodiments, the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive transfer and encounter with ligand.
In some cases, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR in some aspects is one that includes multiple costimulatory domains of different costimulatory receptors.
In some embodiments, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment includes an scFv and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain includes a signaling domain of a zeta chain of a CD3-zeta (CD3ζ) chain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some aspects, the transmembrane domain contains a transmembrane portion of CD28. The extracellular domain and transmembrane domain can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein. In some embodiments, the receptor contains extracellular portion of the molecule from which the transmembrane domain is derived, such as a CD28 extracellular portion. In some embodiments, the chimeric antigen receptor contains an intracellular domain derived from a T cell costimulatory molecule or a functional variant thereof, such as between the transmembrane domain and intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.
For example, in some embodiments, the CAR contains an antibody, e.g., an antibody fragment, as provided herein, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some embodiments, the CAR contains an antibody, e.g., antibody fragment, as provided herein, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4-1BB or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some such embodiments, the receptor further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4 hinge, such as a hinge-only spacer.
In some embodiments, the transmembrane domain of the recombinant receptor, e.g., the CAR, is or includes a transmembrane domain of human CD28 (e.g. Accession No. P01747.1) or variant thereof, such as a transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 129 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:129; in some embodiments, the transmembrane-domain containing portion of the recombinant receptor comprises the sequence of amino acids set forth in SEQ ID NO: 130 or a sequence of amino acids having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
In some embodiments, the intracellular signaling component(s) of the recombinant receptor, e.g. the CAR, contains an intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as a domain with an LL to GG substitution at positions 186-187 of a native CD28 protein. For example, the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO:131 or 132 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:131 or 132. In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of 4-1BB (e.g. (Accession No. Q07011.1) or functional variant or portion thereof, such as the sequence of amino acids set forth in SEQ ID NO:133 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 133.
In some embodiments, the intracellular signaling domain of the recombinant receptor, e.g. the CAR, comprises a human CD3 zeta stimulatory signaling domain or functional variant thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human CD3 (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. Nos. 7,446,190 or 8,911,993. For example, in some embodiments, the intracellular signaling domain comprises the sequence of amino acids 134, 135 or 136 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:134, 135 or 136.
In some aspects, the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgG1, such as the hinge only spacer set forth in SEQ ID NO:124. In other embodiments, the spacer is or contains an Ig hinge, e.g., an IgG4-derived hinge, optionally linked to a CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains, such as set forth in SEQ ID NO:127. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a CH3 domain only, such as set forth in SEQ ID NO:126. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers.
For example, in some embodiments, the CAR includes an anti-CD19 antibody such as an anti-CD19 antibody fragment, such as any of the provided human anti-CD19 antibodies, e.g., single-chain antibodies including scFvs, described herein, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR includes an anti-CD19 antibody or fragment, such as any of the human anti-CD19 antibodies, including scFvs described herein, a spacer such as any of the Ig-hinge containing spacers, a CD28-derived transmembrane domain, a 4-1BB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.
In some embodiments, such CAR constructs further includes a T2A ribosomal skip element and/or a tEGFR sequence, e.g., downstream of the CAR, such as set forth in SEQ ID NO:137 and/or 138, respectively, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 137 or 138
C. Engineered Cells
Also provided are cells, cell populations, and compositions containing the cells, e.g., the engineered cells, e.g. that contain an engineered antigen receptor, e.g., that contains an extracellular domain including the anti-CD19 antibody or fragment, described herein. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.
Thus also provided are genetically engineered cells expressing the recombinant receptors containing the antibodies, e.g., cells containing the CARs. The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, as described herein, and re-introducing them into the same patient, before or after cryopreservation.
Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naïve T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
In some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.
Vectors and Methods for Genetic Engineering
Also provided are methods, nucleic acids, compositions, and kits, for expressing the binding molecules, including receptors comprising the antibodies, and for producing the genetically engineered cells expressing such binding molecules. The genetic engineering generally involves introduction of a nucleic acid encoding the recombinant or engineered component into the cell, such as by retroviral transduction, transfection, or transformation.
In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.
In some contexts, overexpression of a stimulatory factor (for example, a lymphokine or a cytokine) may be toxic to a subject. Thus, in some contexts, the engineered cells include gene segments that cause the cells to be susceptible to negative selection in vivo, such as upon administration in adoptive immunotherapy. For example in some aspects, the cells are engineered so that they can be eliminated as a result of a change in the in vivo condition of the patient to which they are administered. The negative selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Negative selectable genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell II:223, 1977) which confers ganciclovir sensitivity; the cellular hypoxanthine phosphribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase, (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).
In some aspects, the cells further are engineered to promote expression of cytokines or other factors. Various methods for the introduction of genetically engineered components, e.g., antigen receptors, e.g., CARs, are well known and may be used with the provided methods and compositions. Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.
In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 Nov. 29(11): 550-557.
In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
Methods of lentiviral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.
In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).
Other approaches and vectors for transfer of the nucleic acids encoding the recombinant products are those described, e.g., in international patent application, Publication No.: WO2014055668, and U.S. Pat. No. 7,446,190.
Among additional nucleic acids, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.
Preparation of Cells for Engineering
In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for introduction of the CD19-binding molecule, e.g., CAR, may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig.
In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.
In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++/Mg++ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.
In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques.
For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (markerhigh) on the positively or negatively selected cells, respectively.
In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.
In embodiments, memory T cells are present in both CD62L+ and CD62L− subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L−CD8+ and/or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.
In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.
CD4+ T helper cells are sorted into naïve, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L− and CD45RO−.
In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher© Humana Press Inc., Totowa, N.J.).
In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads or MACS beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.
In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.
The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.
In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.
In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.
In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.
In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Number WO2009/072003, or US 20110003380 A1.
In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.
In some aspects, the separation and/or other steps is carried out using CliniMACS system (Miltenyi Biotic), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.
The CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.
In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.
In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.
In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are then frozen to −80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.
In some embodiments, the provided methods include cultivation, incubation, culture, and/or genetic engineering steps. For example, in some embodiments, provided are methods for incubating and/or engineering the depleted cell populations and culture-initiating compositions.
Thus, in some embodiments, the cell populations are incubated in a culture-initiating composition. The incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.
In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agents include IL-2 and/or IL-15, for example, an IL-2 concentration of at least about 10 units/mL.
In some aspects, incubation is carried out in accordance with techniques such as those described in U.S. Pat. No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.
In some embodiments, the T cells are expanded by adding to the culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.
In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.
In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigen-specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.
II. COMPOSITIONS, METHODS AND USES
Also provided are compositions including the CD19 binding molecules and engineered cells, including pharmaceutical compositions and formulations, and methods of using and uses of the molecules and compositions, such as in the treatment of diseases, conditions, and disorders in which CD19 is expressed, and/or detection, diagnostic, and prognostic methods.
A. Pharmaceutical Compositions and Formulations
Provided are pharmaceutical formulations including the CD19-binding molecule, e.g., antibody or chimeric receptor, and/or the engineered cells expressing the molecules. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.
The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
In some aspects, the choice of carrier is determined in part by the particular cell, binding molecule, and/or antibody, and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).
Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
Formulations of the antibodies can include lyophilized formulations and aqueous solutions.
The formulation or composition may also contain more than one active ingredients useful for the particular indication, disease, or condition being treated with the binding molecules or cells, preferably those with activities complementary to the binding molecule or cell, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the cells or antibodies are administered in the form of a salt, e.g., a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p-toluenesulphonic acid.
Active ingredients may be entrapped in microcapsules, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. In certain embodiments, the pharmaceutical composition is formulated as an inclusion complex, such as cyclodextrin inclusion complex, or as a liposome. Liposomes can serve to target the host cells (e.g., T-cells or NK cells) to a particular tissue. Many methods are available for preparing liposomes, such as those described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
The pharmaceutical composition in some aspects can employ time-released, delayed release, and sustained release delivery systems such that the delivery of the composition occurs prior to, and with sufficient time to cause, sensitization of the site to be treated. Many types of release delivery systems are available and known. Such systems can avoid repeated administrations of the composition, thereby increasing convenience to the subject and the physician.
The pharmaceutical composition in some embodiments contains the binding molecules and/or cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
In certain embodiments, in the context of genetically engineered cells containing the binding molecules, a subject is administered the range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges, and/or such a number of cells per kilogram of body weight of the subject.
The may be administered using standard administration techniques, formulations, and/or devices. Provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. Administration of the cells can be autologous or heterologous. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cell populations are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyoi (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
Sterile injectable solutions can be prepared by incorporating the binding molecule in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.
Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules.
The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
B. Therapeutic and Prophylactic Methods and Uses
Also provided are methods for using and uses of the CD19 binding molecules, including the anti-CD19 antibodies, e.g., antibody fragments, and/or engineered cells expressing the recombinant receptors. Such methods and uses include therapeutic methods and uses, for example, involving administration of the molecules, cells, or compositions containing the same, to a subject having a disease, condition, or disorder expressing or associated with CD19 expression, and/or in which cells or tissues express CD19. In some embodiments, the molecule, cell, and/or composition is administered in an effective amount to effect treatment of the disease or disorder. Uses include uses of the antibodies and cells in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods are carried out by administering the antibodies or cells, or compositions comprising the same, to the subject having or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject.
As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.
As used herein, “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.
“Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided molecules and compositions are used to delay development of a disease or to slow the progression of a disease.
As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, an antibody or composition or cell which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody or composition or cell.
An “effective amount” of an agent, e.g., a pharmaceutical formulation, binding molecule, antibody, or cells, or composition, in the context of administration, refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.
A “therapeutically effective amount” of an agent, e.g., a pharmaceutical formulation, antibody, or cells, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered. In some embodiments, the provided methods involve administering the molecules, cells, and/or compositions at effective amounts, e.g., therapeutically effective amounts.
A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human. The diseases and disorders include B cell malignancies, such as B cell leukemias and lymphomas, including B cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), pro-lymphocytic leukemias, hairy cell leukemias, common acute lymphocytic leukemias, Null-acute lymphoblastic leukemias, non-Hodgkin lymphomas, diffuse large B cell lymphomas (DLBCLs), multiple myelomas, follicular lymphoma, splenic, marginal zone lymphoma, mantle cell lymphoma, indolent B cell lymphoma, Hodgkin lymphoma. Also among the diseases and conditions are autoimmune and inflammatory diseases, including those associated with inappropriate or enhanced B cell numbers and/or activation. Exemplary diseases and conditions include multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus (SLE).
In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another CD19-specific antibody and/or cells expressing a CD19-targeting chimeric receptor and/or other therapy, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another CD19-targeted therapy. In some embodiments, the subject has not relapsed but is determined to be at risk for relapse, such as at a high risk of relapse, and thus the compound or composition is administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.
In some embodiments, the treatment does not induce an immune response by the subject to the therapy, and/or does not induce such a response to a degree that prevents effective treatment of the disease or condition. In some aspects, the degree of immunogenicity and/or graft versus host response is less than that observed with a different but comparable treatment. For example, in the case of adoptive cell therapy using cells expressing CARs including the provided anti-CD19 antibodies, the degree of immunogenicity is reduced compared to CARs including a different antibody that binds to a similar, e.g., overlapping epitope and/or that competes for binding to CD19 with the provided antibody, such as a mouse antibody.
In some embodiments, the methods include adoptive cell therapy, whereby genetically engineered cells expressing the provided anti-CD19-containing receptors (e.g., CD19-targeted CARs) are administered to subjects. Such administration can promote activation of the cells (e.g., T cell activation) in a CD19-targeted manner, such that the cells of the disease or disorder are targeted for destruction.
Thus, the provided methods and uses include methods and uses for adoptive cell therapy. In some embodiments, the methods include administration of the cells or a composition containing the cells to a subject, tissue, or cell, such as one having, at risk for, or suspected of having the disease, condition or disorder. In some embodiments, the cells, populations, and compositions are administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells or compositions are administered to the subject, such as a subject having or at risk for the disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptom of the disease or condition, such as by lessening tumor burden in a CD19-expressing cancer.
Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.
In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.
In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.
In some embodiments, the subject, to whom the cells, cell populations, or compositions are administered is a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent. In some examples, the patient or subject is a validated animal model for disease, adoptive cell therapy, and/or for assessing toxic outcomes such as cytokine release syndrome (CRS).
The CD19-binding molecules, such as antibodies and chimeric receptors containing the antibodies and cells expressing the same, can be administered by any suitable means, for example, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing and administration may depend in part on whether the administration is brief or chronic. Various dosing schedules include but are not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion.
For the prevention or treatment of disease, the appropriate dosage of the binding molecule or cell may depend on the type of disease to be treated, the type of binding molecule, the severity and course of the disease, whether the binding molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the binding molecule, and the discretion of the attending physician. The compositions and molecules and cells are in some embodiments suitably administered to the patient at one time or over a series of treatments.
Depending on the type and severity of the disease, dosages of antibodies may include about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg), about 1 μg/kg to 100 mg/kg or more, about 0.05 mg/kg to about 10 mg/kg, 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg. Multiple doses may be administered intermittently, e.g. every week or every three weeks. An initial higher loading dose, followed by one or more lower doses may be administered.
In certain embodiments, in the context of genetically engineered cells containing the binding molecules, a subject is administered the range of about one million to about 100 billion cells and/or that amount of cells per kilogram of body weight, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges and/or per kilogram of body weight. Again, dosages may vary depending on attributes particular to the disease or disorder and/or patient and/or other treatments.
In some embodiments, the cells or antibodies are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as another antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.
The cells or antibodies in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells or antibodies are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells or antibodies are administered after to the one or more additional therapeutic agents.
Once the cells are administered to a mammal (e.g., a human), the biological activity of the engineered cell populations and/or antibodies in some aspects is measured by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells also can be measured by assaying expression and/or secretion of certain cytokines, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
In certain embodiments, engineered cells are modified in any number of ways, such that their therapeutic or prophylactic efficacy is increased. For example, the engineered CAR or TCR expressed by the population can be conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating compounds, e.g., the CAR or TCR, to targeting moieties is known in the art. See, for instance, Wadwa et al., J. Drug Targeting 3: 1 1 1 (1995), and U.S. Pat. No. 5,087,616.
C. Diagnostic and Detection Methods
Also provided are methods involving use of the provided binding molecules, e.g., antibodies, including antibody fragments, and molecules (such as conjugates and complexes) containing one or more of such antibodies, for detection, prognosis, diagnosis, staging, determining binding of a particular treatment to one or more tissues or cell types, and/or informing treatment decisions in a subject, such as by the detection of CD19 and/or the presence of an epitope thereof recognized by the antibody. In some embodiments, the methods are diagnostic and/or prognostic methods in association with a CD19-expressing disease or condition. The methods in some embodiments include incubating and/or probing a biological sample with the antibody and/or administering the antibody to a subject. In certain embodiments, a biological sample includes a cell or tissue or portion thereof, such as tumor or cancer tissue or biopsy or section thereof. In certain embodiments, the contacting is under conditions permissive for binding of the anti-CD19 antibody to CD19 present in the sample. In some embodiments, the methods further include detecting whether a complex is formed between the anti-CD19 antibody and CD19 in the sample, such as detecting the presence or absence or level of such binding. Such a method may be an in vitro or in vivo method. In one embodiment, an anti-CD19 antibody is used to select subjects eligible for therapy with an anti-CD19 antibody or engineered antigen receptor, e.g. where CD19 is a biomarker for selection of patients.
In some embodiments, a sample, such as a cell, tissue sample, lysate, composition, or other sample derived therefrom is contacted with the anti-CD19 antibody and binding or formation of a complex between the antibody and the sample (e.g., CD19 in the sample) is determined or detected. When binding in the test sample is demonstrated or detected as compared to a reference cell of the same tissue type, it may indicate the presence of an associated disease or condition, and/or that a therapeutic containing the antibody (e.g., antibody fragment) will specifically bind to a tissue or cell that is the same as or is of the same type as the tissue or cell or other biological material from which the sample is derived. In some embodiments, the sample is from human tissues and may be from diseased and/or normal tissue, e.g., from a subject having the disease or condition to be treated and/or from a subject of the same species as such subject but that does not have the disease or condition to be treated. In some cases, the normal tissue or cell is from a subject having the disease or condition to be treated but is not itself a diseased cell or tissue, such as a normal tissue from the same or a different organ than a cancer that is present in a given subject.
Various methods known in the art for detecting specific antibody-antigen binding can be used. Exemplary immunoassays include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). An indicator moiety, or label group, can be attached to the subject antibodies and is selected so as to meet the needs of various uses of the method which are often dictated by the availability of assay equipment and compatible immunoassay procedures. Exemplary labels include radionuclides (e.g. 125I, 131I, 35S, 3H, or 32P and/or chromium (51Cr), cobalt (57Co), fluorine (18F), gadolinium (153Gd, 159Gd), germanium (68Ge), holmium (166Ho), indium (115In, 113In, 112In, 111In), iodine (125I, 123I, 121I), lanthanium (140La), lutetium (177Lu), manganese (54Mn), molybdenum (99Mo), palladium (103Pd), phosphorous (32P), praseodymium (142Pr), promethium (149Pm), rhenium (186Re, 188Re), rhodium (105Rh), rutheroium (97Ru), samarium (153Sm), scandium (47Sc), selenium (75Se), (85Sr), sulphur (35S), technetium (99Tc), thallium (201Ti) tin (113Sn, 117Sn), tritium (3H), xenon (133Xe), ytterbium (169Yb, 175Yb), yttrium (90Y),), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or β-glactosidase), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot™ nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, Calif.). Various general techniques to be used in performing the various immunoassays noted above are known.
For purposes of diagnosis, the antibodies can be labeled with a detectable moiety including but not limited to radioisotopes, fluorescent labels, and various enzyme-substrate labels know in the art. Methods of conjugating labels to an antibody are known in the art.
In some embodiments, antibodies need not be labeled, and the presence thereof can be detected using a labeled antibody which binds to any of the antibodies.
The antibodies provided herein can be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).
The antibodies and polypeptides can also be used for in vivo diagnostic assays, such as in vivo imaging. Generally, the antibody is labeled with a radionuclide (such as 111In, 99Tc, 14C, 131I, 125I, or 3H) so that the cells or tissue of interest can be localized in vivo following administration to a subject.
The antibody may also be used as staining reagent in pathology, e.g., using known techniques.
III. ARTICLES OF MANUFACTURE
Also provided are articles of manufacture containing the provided binding molecules, e.g., antibodies and CARs and/or genetically engineered cells, and/or compositions. The articles of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container in some embodiments holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition. In some embodiments, the container has a sterile access port. Exemplary containers include an intravenous solution bags, vials, including those with stoppers pierceable by a needle for injection. The label or package insert may indicate that the composition is used for treating the CD19-expressing or -associated disease or condition. The article of manufacture may include (a) a first container with a composition contained therein, wherein the composition includes the antibody or engineered antigen receptor; and (b) a second container with a composition contained therein, wherein the composition includes a further agent, such as a cytotoxic or otherwise therapeutic agent. The article of manufacture may further include a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further include another or the same container comprising a pharmaceutically-acceptable buffer. It may further include other materials such as other buffers, diluents, filters, needles, and/or syringes.
As used herein, reference to a “corresponding form” of an antibody means that when comparing a property or activity of two antibodies, the property is compared using the same form of the antibody. For example, if it is stated that an antibody has greater activity compared to the activity of the corresponding form of a first antibody, that means that a particular form, such as a scFv of that antibody, has greater activity compared to the scFv form of the first antibody.
As used herein, recitation that nucleotides or amino acid positions “correspond to” nucleotides or amino acid positions in a disclosed sequence, such as set forth in the Sequence listing, refers to nucleotides or amino acid positions identified upon alignment with the disclosed sequence to maximize identity using a standard alignment algorithm, such as the GAP algorithm. For example, in some embodiments, exemplary corresponding residues of a CD19 protein, such as a human CD19 protein, can be identified by alignment of a sequence with an exemplary Vpx sequence set forth in SEQ ID NO:92. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides. In general, to identify corresponding positions, the sequences of amino acids are aligned so that the highest order match is obtained (see, e.g.: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New. Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carrillo et al. (1988) SIAM J Applied Math 48: 1073).
“Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.
The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).
An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
“Isolated nucleic acid encoding an anti-CD19 antibody” refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
As used herein, “percent (%) amino acid sequence identity” and “percent identity” when used with respect to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence (e.g., the subject antibody or fragment) that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
An amino acid substitution may include replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1. Amino acid substitutions may be introduced into a binding molecule, e.g., antibody, of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
Amino acids generally can be grouped according to the following common side-chain properties:
(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
(3) acidic: Asp, Glu;
(4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro;
(6) aromatic: Trp, Tyr, Phe.
Non-conservative amino acid substitutions will involve exchanging a member of one of these classes for another class.
The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include “consisting” and/or “consisting essentially of” aspects and variations.
Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.
The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.
As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
As used herein, a statement that a cell or population of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.
As used herein, a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.
Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
IV. EXEMPLARY EMBODIMENTS
Among the embodiments provided herein are:
1. An anti-CD19 antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein:
said VH region comprises a heavy chain complementarity determining region 3 (CDR-H3) comprising the amino acid sequence set forth as SEQ ID NO: 20 or
said VH region comprises at least 90% sequence identity to the VH region amino acid sequence set forth in SEQ ID NO: 11, 12, 60, 61, 63, or 62.
2. An antibody or antigen-binding fragment thereof comprising:
a CDR-H1, a CDR-H2, and a CDR-H3, respectively comprising the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 sequences contained within the VH region amino acid sequence set forth in SEQ ID NO: 11, 12, 60, 61, 63, or 62; and/or
light chain complementarity determining regions 1, 2, and 3 (CDR-L1, CDR-L2, and CDR-L3), respectively comprising the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 sequences contained within the light chain variable (VL) region amino acid sequence set forth in SEQ ID NO: 13, 14, 15, 16, 17, 71, 65, 64, 66, 70, 69, 67, 90 or 91.
3. An antibody or antigen-binding fragment thereof comprising:
a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 81 or 82, and a CDR-H3 comprising the amino acid sequence set forth as SEQ ID NO: 20; and/or
a CDR-L1 comprising the amino acid sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14 (SEQ ID NO: 111), wherein X1 is T, Q, S, or R; X2 is G or A; X3 is I, T, D, or S; X4 is S, R, T, or Q; X5 is null or S; X6 is G, D, N, or null; X7 is null, V, or L; X8 is D, G, I, L, S, or null; X9 is S, G, A, I, R, or null; X10 is H, Y, F, S, or N; X11 is R, N, D, H, or Y; X12 is Y, F, D, or W; X13 is V, A, or L; and X14 is S, N, or A;
a CDR-L2 comprising the amino acid sequence of X1X2X3X4X5X6X7 (SEQ ID NO: 112), wherein X1 is D or S; X2 is F, V, N, K, or A; X3 is S, T, D, or N; X4 is K, V, N, Q, or R; X5 is R, V, or L; X6 is P, K, A, or E; and X7 is S, P, A, or T, and
a CDR-L3 comprising the amino acid sequence of X1X2X3X4X5X6X7X8X9X10X11X12 (SEQ ID NO: 115), wherein X1 is X; X2 is S, Q, A, or T; X3 is Y, S, W, R; X4 is A, D, R, T, or Y; X5 is X; X6 is X; X7 is S, P, L, Y, G; X8 is X or null; X9 is X or null; X10 is L or null; X11 is X; and X12 is V, T, or L.
4. The antibody or antigen-binding fragment thereof of embodiment 3, wherein:
in said CDR-L1, X3 is I, T, or S; X4 is S, T, or Q; X8 is D, G, I, S, or null; X9 is S, G, I, or null; X10 is H, Y, S, or N; X11 is R, N, D, or H; X12 is Y or D; and X13 is V or L; and/or
in said CDR-L2, X1 is D; X4 is K, V, N, Q, or R; X6 is P, K, or A; and X7 is S, A, or T; and/or
in said CDR-L3, X1 is S, G, T, A, Q, C, or N; X5 is A, S, P, G, N, or D; X6 is I, S, G, T, A, L, H, R, or N; X8 is P, T, S, Q, M, R, N or null; X9 is S, L, N, A, M or null; and X11 is Y, W, F, V, A, or L.
5. The antibody or antigen binding fragment of embodiment 3 or embodiment 4, wherein, in said CDR-L3, X1 is S, G, Q, or N; X2 is S, Q, or T; X4 is A, D, T, or Y; X5 is A, S, or G; and X6 is I, S, N, R, A, H, or T.
6. The antibody or fragment of any of embodiments 1-5, wherein:
the CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 19 (GISWNSGRIGYADSVKG); or
the CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 72 (GISWNSGSIGYADSVKG).
7. The antibody or fragment of any of embodiments 1-6, wherein the CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 80, 77, 74, 73, 75, 79, 78, 76, 21, 25, 28, or 31.
8. The antibody or fragment of embodiment 7, wherein said CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO: 80, 77, 74, 73, 78, 21, or 28.
9. The antibody or fragment of any of embodiments 1-8, wherein the CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 100, 97, 94, 93, 95, 99, 98, 96, 22, 26, 29, or 32.
10. The antibody or fragment of embodiment 9, wherein the CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO: 100, 97, 94, 93, 98, 22, or 29.
11. The antibody or fragment of any of embodiments 1-10, wherein the CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 109, 106, 103, 101, 104, 108, 107, 105, 102, 23, 24, 27, 30, or 33.
12. The antibody or fragment of embodiment 11, wherein the CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO: 109, 106, 103, 101, 107, 24 or 30.
13. The antibody or fragment of any of embodiments 1-12, wherein:
the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 21, 22, and 23, respectively;
the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 21, 22, and 24, respectively;
the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 25, 26, and 27, respectively;
the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 28, 29, and 30, respectively;
the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 31, 32, and 33, respectively;
the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 80, 100, and 109, respectively;
the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs:77, 97, and 106, respectively;
the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 74, 94, and 103, respectively;
the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 73, 93, and 101, respectively;
the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs:75, 95, and 104, respectively;
the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 79, 99, and 108, respectively;
the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 78, 98, and 107, respectively;
the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 76, 96, and 105, respectively;
the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 73, 93, and 102, respectively; or
the CDR-L1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 77, 97, and 106, respectively.
14. The antibody or antigen-binding fragment thereof of any of embodiments 1-13, wherein:
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 81, and 20, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 19, and 20, respectively;
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 82, and 20, respectively; or
the CDR-H1, CDR-H2, and CDR-H3 comprise the sequences of SEQ ID NOs: 18, 72, and 20, respectively.
15. The antibody or fragment of any of embodiments 1-14, wherein:
the VH region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 11, 12, 60, 61, 63, or 62; and/or
the VL region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, 71, 90, 91, 68, 65, 64, 66, 70, 69, or 67.
16. The antibody or fragment of embodiment 15, wherein:
the VH region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 11, 60, 63, or 62; and/or
the VL region of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 14, 16, 71, 90, 65, 64, or 69.
17. The antibody or fragment of any of embodiments 1-16, wherein:
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 17, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 15, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 13, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 14, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 16, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 71, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 62 and 68, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 65, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 60 and 64, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 61 and 66, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 70, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 62 and 69, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 67, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 12 and 91, respectively; or
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 90, respectively.
18. The antibody or fragment of embodiment 17, wherein:
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 14, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 16, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 71, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 11 and 65, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 60 and 64, respectively;
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 62 and 69, respectively; or
the VH and VL regions of the antibody or fragment comprise the amino acid sequences of SEQ ID NOs: 63 and 90, respectively.
19. The antibody or fragment of any of embodiments 1-18, wherein the antibody specifically binds to CD19.
20. The antibody or fragment of embodiment 19, wherein the antibody specifically binds to the same or an overlapping epitope of CD19 as the epitope specifically bound by a reference anti-CD19 antibody selected from the group consisting of FMC63 and SJ25C1.
21. The antibody or fragment of embodiment 19, wherein the antibody competes for binding to CD19 with an anti-CD19 antibody selected from the group consisting of FMC63 and SJ25C1.
22. A human antibody fragment that specifically binds to the same or an overlapping epitope of CD19 as the epitope specifically bound by a reference antibody, which is the antibody or fragment thereof of any of embodiments 1-21 or is an anti-CD19 antibody selected from the group consisting of FMC63 and SJ25C1, said human antibody fragment comprising heavy and light chain CDRs that are distinct from the CDRs present in FMC63 and SJ25C1.
23. A human antibody fragment that specifically binds to CD19 and competes for binding to CD19 with a reference antibody, which is the antibody or fragment of any of embodiments 1-21 or is an anti-CD19 antibody selected from the group consisting of FMC63 and SJ25C1, said antibody fragment comprising heavy and light chain CDRs that are distinct from the CDRs present in FMC63 and SJ25C1.
24. The antibody or fragment of embodiment 21 or 23, which competes for binding with the reference antibody to at least the same degree as the reference antibody competes for binding with itself to CD19, or a degree of competition that is no more than 1.5-fold or 2-fold lower than the competition by the reference antibody.
25. The antibody or fragment of any of embodiment 1-24, wherein the antibody has a binding affinity that is at least as high or substantially as high as the binding affinity for CD19 of a reference antibody selected from the group consisting of FMC63 and SJ25C1.
26. The antibody or fragment of embodiment 25, which has a binding affinity of an EC50 that is about the same or lower than the EC50 of the reference antibody or no more than about 1.5-fold or no more than about 2-fold greater, no more than 3-fold greater, and/or no more than 10-fold greater, than the EC50 of the reference antibody.
27. The antibody or fragment of any of embodiments 1-26, wherein the antibody or fragment is human.
28. The antibody or fragment of any of embodiments 1-27, wherein the antibody or fragment is recombinant.
29. The antibody or fragment of any of embodiments 1-28, which is monoclonal.
30. The antibody or fragment of any of any of embodiments 1-29, which is a single chain fragment.
31. The antibody or fragment of any of embodiments 1-30, which is a fragment comprising antibody variable regions joined by a flexible immunoglobulin linker.
32. The antibody or fragment of embodiment 30 or 31, wherein the fragment comprises an scFv.
33. The antibody or fragment of embodiment 32, wherein the scFv comprises a linker comprising the sequence set forth SEQ ID NO: 34.
34. The antibody or fragment of embodiment 32, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 45, 47, 49, 51, 53, 55, 57, 59, 87, or 89.
35. The antibody or fragment of any of embodiments 1-34, which further comprises at least a portion of an immunoglobulin constant region.
36. The antibody or fragment of embodiment 35, wherein the at least a portion of an immunoglobulin constant region comprises an Fc region.
37. The antibody or fragment of embodiment 36, wherein the Fc region is an Fc region of a human IgG.
38. The antibody or fragment of any of embodiments 1-37, wherein CD19 is human CD19.
39. A chimeric antigen receptor (CAR) comprising an extracellular portion comprising the antibody or fragment of any of embodiments 1-38 and an intracellular signaling domain.
40. The chimeric antigen receptor of embodiment 38, wherein the antibody or fragment comprises an scFv and the intracellular signaling domain comprises an ITAM.
41. The chimeric antigen receptor of embodiment 39 or 40, wherein the intracellular signaling domain comprises a signaling domain of a zeta chain of a CD3-zeta (CD3) chain.
42. The chimeric antigen receptor of any of embodiments 39-41, further comprising a transmembrane domain linking the extracellular domain and the intracellular signaling domain.
43. The chimeric antigen receptor of embodiment 42, wherein the transmembrane domain comprises a transmembrane portion of CD28.
44. The chimeric antigen receptor of any of embodiments 39-43, further comprising an intracellular signaling domain of a T cell costimulatory molecule.
45. The chimeric antigen receptor of embodiment 44, wherein the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB.
46. An engineered cell expressing the chimeric antigen receptor of any of embodiments 39-45.
47. The engineered cell of embodiment 46, which is a T cell.
48. A method of treatment, comprising administering the cell of embodiment 46 or 47 to a subject having a disease or disorder associated with CD19.
49. A method of treatment, comprising administering the antibody of any of embodiments 1-38 to a subject having a disease or disorder associated with CD19.
50. The method of embodiment 48 or 49, wherein the disease or disorder is a B cell malignancy.
51. The method of embodiment 50, wherein the B cell malignancy is selected from the group consisting of B cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), pro-lymphocytic leukemias, hairy cell leukemias, common acute lymphocytic leukemias, Null-acute lymphoblastic leukemias, non-Hodgkin lymphomas, diffuse large B cell lymphomas (DLBCLs), multiple myelomas, follicular lymphoma, splenic, marginal zone lymphoma, mantle cell lymphoma, indolent B cell lymphoma, and Hodgkin lymphoma.
52. A nucleic acid encoding the antibody or fragment thereof of any of embodiments 1-38 or the chimeric antigen receptor of any of embodiments 39-45.
53. A composition comprising the antibody or fragment thereof of any of embodiments 1-38, the CAR of any of embodiments 39-45, or the cell of embodiment 46 or 47.
54. A method of treatment, comprising administering the composition of embodiment 53 to a subject having a disease or disorder associated with CD19.
55. An antibody or antigen-binding fragment thereof comprising:
heavy chain complementarity determining regions 1, 2, and 3 (CDR-H1, CDR-H2, and CDR-H3), respectively comprising the amino acid sequences of CDR 1, 2, and 3 sequences contained within the heavy chain variable (VH) region amino acid sequence set forth in SEQ ID NO: 11 or 12; and
light chain complementarity determining regions 1, 2, and 3 (CDR-L1, CDR-L2, and CDR-L3), respectively comprising the amino acid sequences of CDR 1, 2, and 3 sequences contained within the light chain variable (VL) region amino acid sequence set forth in SEQ ID NO: 13, 14, 15, 16, or 17.
56. The antibody or fragment of embodiment 55, wherein:
the CDR-H1 comprises the amino acid sequence of DYAMH (SEQ ID NO: 18);
the CDR-H2 comprises the amino acid sequence GISWNSGRIGY (SEQ ID NO: 35);
the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 20;
the CDR-L1 comprises the amino acid sequence X1GX3X4X5X6X7X8X9X10X11X12X13S (SEQ ID NO: 36), wherein X1 is T, S, or Q, X3 is T, S, or D, X4 is T or S, X5 is null or S, X6 is null, D, or N, X7 is null or V, X8 is null, G, or I, X9 is null, G, or R, X10 is S, Y, or N, X11 is D or N, X12 is D or Y, X13 is V or A;
the CDR-L2 comprises the amino acid sequence X1X2X3X4RPS (SEQ ID NO: 37), wherein X1 is D or S, X2 is V, N, or K, X3 is S, N, or D, and X4 is K, Q, or N; and
the CDR-L3 comprises the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12 (SEQ ID NO: 38), wherein X1 is C, S, A, G, or N, X2 is S, A, or T, X3 is Y, W, or R, X4 is A or D, X5 is G, D, or S, X6 is R, S, or N, X7 is Y, L, or G, X8 is N or S, X9 is S or null, X10 is V, A, or N, X11 is W or null, and X12 is L or V.
57. The antibody or fragment of embodiment 56, wherein: in the CDR-L1, X1 is T or S, X3 is T or S, XII is D or N, and X13 is V; in the CDR-L2, X2 is V or N and X4 is K or Q; and/or in the CDR-L3, X1 is C, S, A, or G, X3 is Y or W, X5 is G or D, X7 is Y or L, X10 is V or A, and X11 is null.
58. The antibody or fragment of any of embodiments 55-57, wherein the CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO: 19 (GISWNSGRIGYADSVKG).
59. The antibody or fragment of any of embodiments 55-58, wherein the CDR-L1 comprises the sequence set forth in SEQ ID NO: 21, 25, 28, or 31.
60. The antibody or fragment of any of embodiments 55-59, wherein the CDR-L2 comprises the sequence set forth in SEQ ID NO: 22, 26, 29, or 32.
61. The antibody or fragment of any of embodiments 55-60, wherein the CDR-L3 comprises the sequence set forth in SEQ ID NO: 23, 24, 27, 30, or 33.
62. The antibody or fragment of any of embodiments 55-61, wherein:
the CDRL1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 21, 22, and 23, respectively;
the CDRL1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 21, 22, and 24, respectively;
the CDRL1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 25, 26, and 27, respectively;
the CDRL1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 28, 29, and 30, respectively; or
the CDRL1, CDR-L2, and CDR-L3 comprise the sequences of SEQ ID NOs: 31, 32, and 33, respectively.
63. The antibody or fragment of any of embodiments 55-62, wherein the antibody or fragment comprises:
a VH region comprising the amino acid sequence of SEQ ID NO: 11 or 12; and
a VL region comprising the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, or 17.
64. The antibody or fragment of embodiment 63, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 11.
65. The antibody or fragment of embodiment 63, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 12.
66. The antibody or fragment of any of embodiments 55-65, wherein the antibody specifically binds to CD19.
67. The antibody or fragment of embodiment 66, wherein the antibody specifically binds to the same or an overlapping epitope of CD19 as the epitope specifically bound by a reference anti-CD19 antibody selected from the group consisting of FMC63 and SJ25C1.
68. The antibody or fragment of embodiment 66, wherein the antibody competes for binding to CD19 with an anti-CD19 antibody selected from the group consisting of FMC63 and SJ25C1.
69. A human antibody fragment that specifically binds to the same or an overlapping epitope of CD19 as the epitope specifically bound by a reference antibody, which is the antibody or fragment thereof of any of embodiments 55-68 or by an anti-CD19 antibody selected from the group consisting of FMC63 and SJ25C1, said human antibody fragment comprising heavy and light chain CDRs that are distinct from the CDRs present in FMC63 and SJ25C1.
70. A human antibody fragment that specifically binds to CD19 and competes for binding to CD19 with a reference antibody, which is the antibody or fragment of any of embodiments 55-68 or an anti-CD19 antibody selected from the group consisting of FMC63 and SJ25C1, said antibody fragment comprising heavy and light chain CDRs that are distinct from the CDRs present in FMC63 and SJ25C1.
71. The antibody of embodiment 68 or 70, which competes for binding with the reference antibody to at least the same degree as the reference antibody competes for binding with itself to CD19, or a degree of competition that is no more than 1.5-fold or 2-fold lower than the competition by the reference antibody.
72. The antibody of any of embodiments 55-71, wherein the antibody has a binding affinity that is at least as high or substantially as high as the binding affinity for CD19 of a reference antibody selected from the group consisting of FMC63 and SJ25C1.
73. The antibody of embodiment 72, which has a binding affinity of an EC50 that is about the same or lower than the EC50 reference antibody or no more than about 1.5-fold or no more than about 2-fold greater, no more than 3-fold greater, and/or no more than 10-fold greater, than the EC50 of the reference antibody.
74. The antibody or fragment of any of embodiments 55-73, wherein the antibody is human.
75. The antibody or fragment of any of embodiments 55-74, wherein the antibody is recombinant.
76. The antibody or fragment of any of embodiments 55-75, which is monoclonal.
77. The antibody or fragment of any of any of embodiments 55-76, which is a single chain fragment.
78. The antibody or fragment of any of embodiments 55-77, which is a fragment comprising antibody variable regions joined by a flexible immunoglobulin linker.
79. The antibody or fragment of embodiment 77 or 78, wherein the fragment comprises an scFv.
80. The antibody or fragment of embodiment 79, wherein the scFv comprises a linker comprising the sequence set forth SEQ ID NO: 34.
81. The antibody or fragment of embodiment 80, wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10.
82. The antibody or fragment of any of embodiments 55-81, which further comprises at least a portion of an immunoglobulin constant region.
83. The antibody or fragment of embodiment 82, wherein the at least a portion of an immunoglobulin constant region comprises an Fc region.
84. The antibody or fragment of embodiment 83, wherein the Fc region is an Fc region of a human IgG.
85. The antibody or fragment of any of embodiments 55-84, wherein the CD19 is a human CD19.
86. A chimeric antigen receptor comprising an extracellular portion comprising the antibody or fragment of any of embodiments 55-85 and an intracellular signaling domain.
87. The chimeric antigen receptor of embodiment 86, wherein the antibody or fragment comprises an scFv and the intracellular signaling domain comprises an ITAM.
88. The chimeric antigen receptor of embodiment 87, wherein the intracellular signaling domain comprises a signaling domain of a zeta chain of a CD3-zeta (CD3) chain.
89. The chimeric antigen receptor of any of embodiments 86-88, further comprising a transmembrane domain linking the extracellular domain and the intracellular signaling domain.
90. The chimeric antigen receptor of embodiment 89, wherein the transmembrane domain comprises a transmembrane portion of CD28.
91. The chimeric antigen receptor of any of embodiments 86-90, further comprising an intracellular signaling domain of a T cell costimulatory molecule.
92. The chimeric antigen receptor of embodiment 91, wherein the T cell costimulatory molecule is selected from the group consisting of CD28 and 41BB.
93. An engineered cell expressing the chimeric antigen receptor of any of embodiments 86-92.
94. The engineered cell of embodiment 93, which is a T cell.
95. A method of treatment, comprising administering the cell of embodiment 93 or 94 to a subject having a disease or disorder associated with CD19.
96. A method of treatment, comprising administering the antibody of any of embodiments 55-85 to a subject having a disease or disorder associated with CD19.
97. The method of embodiment 95 or 96, wherein the disease or disorder is a B cell malignancy.
98. The method of embodiment 97, wherein the B cell malignancy is selected from the group consisting of B cell chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), pro-lymphocytic leukemias, hairy cell leukemias, common acute lymphocytic leukemias, Null-acute lymphoblastic leukemias, non-Hodgkin lymphomas, diffuse large B cell lymphomas (DLBCLs), multiple myelomas, follicular lymphoma, splenic, marginal zone lymphoma, mantle cell lymphoma, indolent B cell lymphoma, and Hodgkin lymphoma.
99. A nucleic acid encoding the antibody of any of embodiments 55-85 or the chimeric antigen receptor of any of embodiments 86-92.
100. A composition comprising the antibody of any of embodiments 55-85, the CAR of any of embodiments 86-92, or the cell of embodiment 93 or 94.
101. A method of treatment, comprising administering the composition of embodiment 100 to a subject having a disease or disorder associated with CD19.
102. The antibody or fragment of any of embodiments 1-38 or 55-85, the CAR of any of embodiments 39-45 or 86-92, the cell of any of embodiments 46, 47, 93, and 94, the method of any of claims 48-51, 54, 95-98, and 101, the nucleic acid of embodiment 52 or 99, or the composition of any of claims 53 and 100, wherein the antibody or fragment specifically binds to an epitope containing one or more amino acids within a region of the extracellular portion of a CD19.
103. The antibody or fragment of any of embodiments 1-38 or 55-85, the CAR of any of embodiments 39-45 or 86-92, the cell of any of embodiments 46, 47, 93, and 94, the method of any of claims 48-51, 54, 95-98, and 101, the nucleic acid of embodiment 52 or 99, or the composition of any of claims 53 and 100, wherein the antibody or fragment specifically binds to an epitope that is within a region of the extracellular portion of a CD19.
103. The antibody or fragment of any of embodiments 1-38 or 55-85, the CAR of any of embodiments 39-45 or 86-92, the cell of any of embodiments 46, 47, 93, and 94, the method of any of claims 48-51, 54, 95-98, and 101, the nucleic acid of embodiment 52 or 99, or the composition of any of claims 53 and 100, wherein the antibody or fragment specifically binds to a polypeptide consisting or consisting essentially of region of the extracellular portion of a CD19, or that comprises the region of the extracellular portion of the CD19 but not any, or substantially no, other portion of CD19.
104. The antibody, fragment, cell, method, nucleic acid, or composition of any of embodiments 102-103, wherein the region of the extracellular portion of the CD19 is a membrane-proximal region.
105. The antibody, fragment, cell, method, nucleic acid, or composition of any of embodiments 102-104, wherein the region of the extracellular portion of the CD19 is a portion encoded by the fourth exon of the CD19 or a portion corresponding to positions 176-277 of the human CD19 sequence set forth in SEQ ID NO: 92.
106. The antibody, fragment, cell, method, nucleic acid, or composition of any of embodiments 102-105, wherein the region of the extracellular portion of the CD19 consists of or comprises the membrane-proximal-most 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 44, 43, 43, 41, or 40 amino acid portion of the extracellular portion of the CD19.
107. The antibody, fragment, cell, method, nucleic acid, or composition of any of embodiments 102-106, wherein the region of the extracellular portion consists of or comprises the Ig-like domain 1 of CD19, a portion encoded by the second exon of the CD19 and/or a portion corresponding to positions 20-117 of the human CD19 sequence set forth in SEQ ID NO: 92
108. The antibody, fragment, cell, method, nucleic acid, or composition of any of embodiments 102-106, which optionally is any of the portions of the extracellular region described in any of embodiments 105-107.
109. The antibody or fragment of any of embodiments 1-38 or 55-85, the CAR of any of embodiments 39-45 or 86-92, the cell of any of embodiments 46, 47, 93, and 94, the method of any of claims 48-51, 54, 95-98, and 101, the nucleic acid of embodiment 52 or 99, or the composition of any of claims 53 and 100, wherein the antibody or fragment specifically binds to an epitope of CD19 that contains one or more amino acids within, is within, or includes a portion of CD19 corresponding to residues 218-249 of the human CD19 sequence set forth in SEQ ID NO: 92.
110. The antibody, fragment, cell, method, nucleic acid, or composition of embodiment 109, wherein the portion comprises the sequence set forth in SEQ ID NO: 143.
111. The antibody or fragment of any of embodiments 1-38 or 55-85, the CAR of any of embodiments 39-45 or 86-92, the cell of any of embodiments 46, 47, 93, and 94, the method of any of claims 48-51, 54, 95-98, and 101, the nucleic acid of embodiment 52 or 99, or the composition of any of claims 53 and 100, wherein the antibody or fragment specifically binds to an epitope of CD19 that contains an amino acid at a position corresponding to a position of the human CD19 sequence set forth in SEQ ID NO: 92 selected from the group consisting of: the histidine (H) at position 218, the alanine (A) at position 236, the methionine (M) at position 242, the glutamate (E) at position 243, the proline (P) at position 249, and/or the lysine (K) and/or serine (S) at positions 223 and 224, and combinations thereof.
112. The antibody or fragment of any of embodiments 1-38 or 55-85, the CAR of any of embodiments 39-45 or 86-92, the cell of any of embodiments 46, 47, 93, and 94, the method of any of claims 48-51, 54, 95-98, and 101, the nucleic acid of embodiment 52 or 99, or the composition of any of claims 53 and 100, wherein an amino acid at a position corresponding to a position of the human CD19 sequence set forth in SEQ ID NO: 92 selected from the group consisting of: the histidine (H) at position 218, the alanine (A) at position 236, the methionine (M) at position 242, the glutamate (E) at position 243, the proline (P) at position 249, and/or the lysine (K) and/or serine (S) at positions 223 and 224, and combinations thereof is necessary or important for binding of the antibody to human CD19.
113. The antibody, fragment, cell, method, nucleic acid, or composition of embodiment 111 or 112, wherein the amino acid is identical to the amino acid present at the corresponding position in SEQ ID NO: 92.
114. The antibody, fragment, cell, method, nucleic acid, or composition of any of embodiments 111-113, wherein the amino acid is or comprises an amino acid at the position corresponding to the histidine at position 218 of the human CD19, optionally wherein the amino acid at said position is a histidine.
115. The antibody, fragment, cell, method, nucleic acid, or composition of any of embodiments 111-114, wherein the amino acid is or comprises an amino acid at the position corresponding to the alanine at position 236 of the human CD19 sequence, optionally wherein the amino acid at said position is an alanine.
116. The antibody, fragment, cell, method, nucleic acid, or composition of any of embodiments 111-115, wherein the amino acid is or comprises an amino acid at the position corresponding to the methionine at position 242 of the human CD19, optionally wherein the amino acid at said position is a methionine.
117. The antibody, fragment, cell, method, nucleic acid, or composition of any of embodiments 111-116, wherein the amino acid is or comprises an amino acid at the position corresponding to the glutamate at position 243 of the human CD19, optionally wherein the amino acid at said position is a glutamate.
118. The antibody, fragment, cell, method, nucleic acid, or composition of any of embodiments 111-117, wherein the amino acid is or comprises an amino acid at the position corresponding to the proline at position 249 of the human CD19, optionally wherein the amino acid at said position is a proline.
119. The antibody, fragment, cell, method, nucleic acid, or composition of any of embodiments 111-118, wherein the amino acid is or comprises an amino acid(s) at one or both of the positions corresponding to the lysine and/or serine at positions 223 and 224 of the human CD19.
120. The antibody or fragment of any of embodiments 1-38 or 55-85, the CAR of any of embodiments 39-45 or 86-92, the cell of any of embodiments 46, 47, 93, and 94, the method of any of claims 48-51, 54, 95-98, and 101, the nucleic acid of embodiment 52 or 99, or the composition of any of claims 53 and 100, wherein the antibody or fragment specifically binds to an epitope that overlaps with or is identical to or comprises an epitope specifically bound by a reference antibody, wherein the overlapping portion comprises or is within a portion of CD19 (a) comprising SEQ ID NO: 143, (b) corresponding to residues 218-249 of the human CD19 sequence set forth in SEQ ID NO: 92, or (c) corresponding to a region of CD19 encoded by exon 4 of human CD19, or (d) within a portion corresponding to the 75-most or 80-most membrane proximal residues of human CD19.
121. The antibody, fragment, cell, method, nucleic acid, or composition of embodiment 120, wherein the reference antibody is FMC63, or is wherein the reference antibody is SJ25C1.
V. EXAMPLES
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1: Generation and Assessment of Anti-CD19 Antibodies
Exemplary anti-CD19 antibodies that specifically bind to CD19-expressing cells with similar binding properties to murine anti-CD19 reference antibodies, and/or compete for binding with murine anti-CD19 reference antibodies were generated and assessed.
1A. Library Selection, Antibody Generation
Exemplary anti-CD19 antibodies (scFvs) were generated through a series of selection steps carried out on dsDNA-encoded human naive antibody libraries displayed in a cell-free system. Members of a VH library were selected for binding to live cells through three successive rounds, enriching for members that bind specifically to stably-transfected CD19-expressing HEK293 cells, but not parental HEK293 cells and/or to CHOK1 cells that did not expresses CD19. At the end of each selection round, three separate elution pools were generated by (a) surface stripping to recover binders from target cells, (b) competitive elution using a murine anti-CD19 antibody, FMC63 IgG, and (c) competitive elution using another murine anti-CD19 antibody, SJ25C1 ((b) and (c) carried out to enrich for binders that compete with FMC63 and/or SJ25C1 for binding to CD19).
At end of 3 rounds of selections, these enriched VH libraries were then converted to scFv libraries by shuffling VH members of these respective pools and a naïve human VL library in VH-(G4S)3—VL format. The resulting scFv libraries were subjected to a fourth round, enriching for members that bound specifically to CD19-expressing HEK293 cells and not to parental cells, followed by surface stripping.
A fifth round was carried out to further enrich for members that bound to other CD19-expressing cells (CD19/K562). Selections were followed by the generation of separate elution pools using either (a) surface stripping, (b) FMC63 competitive elution, or (c) SJ25C1 competitive elution. In a sixth round, these three pools were individually further enriched by negative selection for members that did not bind parental cells (HEK293, twice, K562), followed by positive selection for members that bound CD19-expressing HEK293 cells and immunoprecipitation with an anti-Myc antibody that recognized a C-terminal tag on CD19 expressed on HEK293 cells.
In one study, forty-eight (48) clones from each of the three R6 scFv resulting pools were sequenced using forward and reverse primers to determine amino acid sequences. 130 of the determined scFv sequences showed full length reading. Convergence was observed among the sequences. Eighteen (18) replicates were identified among the 130 scFv sequences (representing forty-six (46) of the 130 clones). In this study, one VH portion sequence containing CDRs 1-3 and FRs 1-3 was detected fourteen (14) times in two of the different pools (10 copies from one and 4 copies from another), paired with 5 different VLs. Other replicates were identified between 2 and 5 times in different pools; others were single-copy sequences. In another study, additional CD19-binding clones were identified and sequenced. The same VH portion appeared among them, with different VL sequences.
1B. Specific Binding to CD19-Expressing Cells
Binding of the sequenced clones to CD19-expressing and control HEK293 cells, as compared to cells that did not express CD19, was assessed by flow cytometry either with in vitro translated crude cell lysate or with bacterially-produced supernatant. Briefly, RNA of each clone was normalized and in vitro translated as crude scFv with a C-terminal FLAG tag. CD19-expressing HEK293 and control (mock transfected) HEK293 cells were used in the assay. Binding of the individual scFvs to CD19 and control cells was measured with a secondary anti-FLAG-Alexa647 conjugate. Alternatively, scFv binding pools were cloned into E. coli expression vectors and were produced as HIS-tagged scFvs which were detected with anti-HIS-Alexa647 conjugate in flow-cytometric assays. Murine anti-CD19 antibodies (FMC63 scFv and FMC63 IgG) were used as positive controls; a control scFv also was used. Mean fluorescence intensity (MFI) was assessed by flow cytometry. The results are shown in FIGS. 1A and 1B, demonstrating binding of identified clones to CD19-expressing cells. Among the clones assessed were scFvs, including clones 5, 17, 18 (identified with in vitro translated lysates), and 76 (identified with bacterial supernatant), that displayed clear binding preference for CD19-expressing cells as compared to CD19-negative cells.
As shown in FIGS. 1A and 1B, for some clones, the fold change in degree of binding detected, in this case as measured by mean fluorescence intensity, to the CD19-expressing cells as compared to the non-CD19-expressing cells, was about as great, at least as great, or greater than the fold change observed for the positive control reference antibodies, murine anti-CD19 antibodies FMC63 scFv and/or FMC63 IgG. In some cases, the total degree of observed binding to the CD19-expressing cells was approximately the same, at least as great, or greater than that observed for one or more of the positive control reference antibody.
Four (4) scFv clones that displayed clear binding preference for CD19-expressing cells compared to non-CD19-expressing cells (“clone 18,” “clone 17,” “clone 5,” and “clone 76”) were further analyzed. Sequencing revealed that the clones shared common CDR sequences within their VH sequences, with different VL sequences and different CDR-Ls. Sequence identifiers corresponding to sequences, including exemplary scFv, VH, VL, and CDR (Kabat) amino acid sequences and encoding nucleotide scFv sequences, for the four clones are listed Table 2. A germline variant of clone 18 (deemed “clone 18B”) was generated by a cysteine (C) to serine (S) substitution at Kabat position 89; sequences for this clone also are listed in Table 2. Each of the clones had a VH3 chain sequence. Clone 18 included a light chain framework derived from a Vλ2 sequence (with clone 18B having the Vλ2 germline framework sequence); clones 17 and 76 had Vλ1 sequences, and clone 5 included a Vλ3 sequence. Clones 18 and 17 were derived from multiple branches and libraries, including VH—VL shuffling and scFv. Clone 76 was derived from VH—VL SJ25C1 competitive elution (Round 6); clone 5 was derived from VH—VL FMC63 competitive elution (Round 6).
TABLE 2
Sequences for Exemplary Clones (SEQ ID NO.)
Heavy Chain
Light Chain
CDR-H
CDR-L
Variable (VH)
Variable (VL)
ScFv Sequence
(1, 2, 3)
(1, 2, 3)
Region
Region
(Amino Acid,
(Kabat)
(Kabat)
Clone #
(Amino Acid)
(Amino Acid)
Nucleotide)
(Amino Acid)
(Amino Acid)
5
12
17
10
18, 19, 20
31, 32, 33
17
12
15
6
18, 19, 20
25, 26, 27
18
11
13
2
18, 19, 20
21, 22, 23
18B
11
14
4
18, 19, 20
21, 22, 24
76
11
16
8
18, 19, 20
28, 29, 30
1C. Binding Affinities, Competition with Reference Antibodies
Clones 5, 17, 18, 18B, and 76, were purified by single-step purification and purification assessed via SDS gel. A gel from an exemplary study is shown in FIG. 2 (lanes 1 and 2=clone 5, non-reduced, reduced; lanes 3 and 4=clone 17, non-reduced, reduced; lanes 5 and 6=clone 18, non-reduced and reduced; lanes 7 and 8=clone 76, non-reduced and reduced). In this study, isoelectric points were measured as 5.36, 5.32, 7.11, and 5.32, respectively for clones 5, 17, 18, and 76.
Melting temperature (Tm) measurements were made using BioTad CFX96 instrument to analyze sypro orange protein incorporation at incremental temperatures, revealing similar Tm values as those observed for the reference antibody FMC63 scFv. The results are presented in Table 3.
TABLE 3
Assessment of Tm
Clone, Condition
Tm (° C.)
5, Imidazole
53
5, pH 6
61
5, pH 7
57
5, pH 8
57
17
51
18
59
18B
59
FMC63 scFv
56
Clones were titrated, and their binding affinities (EC50) to CD19-expressing K562 cells assessed by flow cytometry, with a reference murine CD19 antibody, FMC63 scFv, used as a positive control. Results from three separate assays, each including and comparing other binding affinities to that for clone 18, are shown in FIGS. 3A-3C.
In the assay the results of which are shown in FIG. 3A, EC50 values for clone 18, clone 17, another clone identified by the study (deemed clone 192; see sequences in Table 6), and the reference antibody (FMC63 scFv) were measured as 3.79 nM, 14.86 nM, 12.80 nM, and 7.37 nM, respectively. In the assay the results of which are shown in FIG. 3B, EC50 values for clone 18, clone 18B, and clone 76 were measured as 7.1 nM and 9.3 nM, and 7.9 nM, respectively. In the assay the results of which are shown in FIG. 3C, EC50 values for clone 18 and clone 76 were measured as 4.1 nM and 8.8 nM respectively.
Thus, each of the clones assayed specifically bound to the CD19-expressing cells with affinities similar to that of the reference antibody, e.g., having EC50s about the same as or lower than that of the reference antibody, or no more than about 1.5-fold or no more than about 2-fold, or no more than about 3-fold greater than the EC50 of the reference antibody.
In another assay, clones 18, 5, 17, other clones identified (161, 170, 1 (see sequence information in Table 6)), and the positive control reference antibody FMC63 scFv (one plate) and clone 18, other clones identified (177, 184, 192, 198), and the positive control reference antibody FMC63 scFv (another plate) were assessed by the same assay. Results are presented in FIG. 4. EC50 values observed for the two plates are presented in Tables 4A and 4B. As shown, clones were observed to have comparable binding affinities with that of the reference antibody.
TABLE 4A
Exemplary
Clone
Additional Clones
FMC63
18
Clone 5
Clone 17
(clones 161, 170, 1)
scFv
EC50 (nM)
4.79
15.84
8.32
52.26, 96.68, 213.80
5.06
TABLE 4B
Exemplary Additional Clones
Clone 18
(clones 177, 184, 192, 198)
FMC63 scFv
EC50 (nM)
3.11
53.33, 113.90, 12.02, 13.21
5.83
Competition binding assays were performed to assess competition of various antibodies for binding to CD19-expressing cells. In one assay, binding of 0.5 nM (˜EC50) FITC-labeled SJ25C1 to Ramos cells was assessed in the presence or absence of various concentrations of unconjugated competitor FMC63 IgG or a control IgG; binding was assessed by flow cytometry (mean fluorescence intensity). The results are shown in FIG. 5A, indicating that FMC63 IgG competed for binding to CD19 with SJ25C1 IgG1 in this study, suggesting that SJ25C1 and FMC63 bound to overlapping epitopes e.g., a common epitope, of CD19. In another assay, CD19-expressing cells were incubated with labeled FMC63 IgG in the presence of various concentrations of (or absence of) clone 18 scFv, FMC63 scFv (positive control) and a control scFv (negative control). Results are shown in FIG. 5B. As shown, both the clone 18 scFv and FMC63 scFv (but not the negative control scFv) were observed to compete with the FMC63 IgG for binding to CD19-expressing cells, with comparable IC50 values (24.0 nM and 19.8 nM, respectively), indicating that clone 18 bound to an epitope of CD19 that overlaps with the epitope recognized by FMC63, and competed for binding with the reference antibody to a similar degree.
In another assay, 10 nM (EC50) Alexa647-labeled FMC63 scFv was incubated with CD19-expressing K562 cells in the presence or absence of varying concentrations of clone 18 scFv, clone 18B scFv, clone 17 scFv, clone 76 scFv, a reference antibody (FMC63 scFv) and a negative control antibody (R12). Results are presented in FIG. 6. The clones and reference antibody, but not the negative control antibody, displayed competition for binding to CD19 with the FMC63 scFv, and competition by the reference antibody with itself was similar to competition observed for the tested clones.
Collectively, in a number of studies, the following EC50 (binding affinity) and IC50 (competition) values were observed for the various clones, as listed in Table 5. As shown, among the identified human CD19 antibodies were those having similar degrees of binding affinity for CD19 and similar degrees of competitive inhibition for a murine anti-CD19 reference antibody, as compared to the reference antibody itself, for example, about the same, less than, or no more than 1.5-fold, 2-fold, or 3-fold greater EC50 and/or IC50.
TABLE 5
Summary of Data from Exemplary Binding Studies
EC50
IC50 (nM)
(CD19-Expressing Cells)
(competition for binding
Clone/Antibody
(nM)
with FMC63)
Clone 18
4.1 ± .57 (n = 7)
20.1 ± 9.8 (n = 3)
Clone 18B
5.4 ± 1.3 (n = 5)
28 (n = 1)
Clone 76
8.04 ± 0.3
18.2 ± 1.5 (n = 2)
Clone 17
11.7 ± 1.9
35.4 ± 3.9 (n = 2)
Clone 5
15.8 (n = 1)
50 (n = 1)
FMC63
6.1 ± 1.2 (n = 6)
20.5 ± 6.7 (n = 3)
1D. Size Exclusion Chromatography
Biophysical properties of clone 18B were assessed via size-exclusion chromatography. A HiLoad 16/600 Superdex 200 column was calibrated and Bio-Rad gel filtration standard 150-1901 kDa proteins were injected, and fractions collected at 1.5 mL/min to generate references. 770 ug of clone 18B scFv was injected into the column and fraction collected under the same conditions. The results are shown in FIGS. 7A and 7B (FIG. 7A=standard; FIG. 7B=Clone 18B). The results for clone 18B scFv revealed a single peak, with minimal large size aggregates observed.
Example 2: Generation and Assessment of Additional Anti-CD19 Antibodies
Additional exemplary anti-CD19 antibodies (scFv fragments) having similar binding properties to (and/or that compete for binding with) murine anti-CD19 reference antibodies were generated and assessed.
2A. Library Selection, Antibody Generation
Additional exemplary anti-CD19 scFvs were generated by two different selection approaches, each involving a series of selection steps carried out on dsDNA-encoded human antibody libraries displayed in a cell-free system.
In one approach (deemed “clone 18 CDR3 grafting”), a heavy chain CDR3 (CDR-H3) sequence present in clones identified in Example 1 (SEQ ID NO: 20, DQGYHYYDSAEHAFDI) was grafted into human naïve VH library frameworks. Members of the resulting CDR3-grafted VH library were shuffled with members of a naïve human VL library to generate an scFv library as VH-(G4S)3-VL format. The resulting scFv library was subjected to three rounds of selection, to enrich for members that bound specifically to CD19-expressing HEK293 cells and not to parental cells, followed by surface stripping for round (R1), immunoprecipitation and off-rate for round 2 (R2).
In another approach (deemed “FMC63 guided selection”), two initial scFv libraries were generated, respectively, by (a) shuffling members of a naïve VH library with the VL region of FMC63 and (b) shuffling members of a naïve VL library with the VH region of FMC63. After two and three rounds of selection, respectively, to enrich the library members from (a) and (b) for CD19-binding with the guidance of the parental FMC63 VH or VL. The binding molecules were eluted off by surface stripping from CD19/HEK293 cells (R1) and FMC63 elution from CD19/K562 cells (R2 and R3). A third scFv library was generated by shuffling the VH sequences from the selection in (a) with the VL sequences resulting from the selection in (b). Three further rounds of selection were carried out on CD19/HEK293 cells with surface stripping (R1), followed by CD19/K562 cells with FMC63 elution (R2) and CD19/HEK293 cells with immunoprecipitation (R3). Binding by the selected scFv clones to CD19-expressing cells was confirmed by flow cytometry using bacterially-produced supernatant. The selected scFv pools were cloned into E. coli expression vectors and produced as HIS-tagged scFvs. Binding of individual clones to CD19-transfected HEK293 cells was detected with anti-HIS-Alexa647 conjugate by flow cytometry. Clone 18 or Clone 18B were used as positive controls, along with various negative controls. The results are shown in FIGS. 8A-C (MFI=mean fluorescence intensity).
The results shown in FIG. 8D confirm CD19-specific binding by an exemplary twenty-three (23) of the hits (marked with asterisks in FIGS. 8A-C, representing 4 hits identified via the CDR3 grafting approach and 19 via FMC63-guided selection). Binding of in vitro-translated FLAG-tagged scFvs to CD19-expressing K562 cells, as compared to control (mock transfected) K562 cells, was assessed by flow cytometry as described in Example 1. As shown, the clones specifically bound to CD19-expressing cells.
These and additional CD19-specific scFv clones generated by the selection approaches in Examples 1 and 2 were further assessed. Sequencing revealed several CD19-specific binding antibodies (scFvs) with various different light chain sequences and sharing a common CDR-H3 sequence (SEQ ID NO: 20) also present in scFvs described in Example 1. Sequence identifiers corresponding to various sequences of additional CD19-binding scFvs are listed in Table 6, including for scFv, VH, VL, and CDR (Kabat) amino acid sequences (and the encoding scFv nucleotide sequences). Among the CD19-specific scFv clones were those having several different light chain variable and CDR sequences, some of which had CDR-H1, CDR-H2, and/or CDR-H3 present in SEQ ID NO: 11, CDR-H1, CDR-H2, and/or CDR-H3 having sequences of SEQ ID NOs: 18, 19, and/or 20, and/or CDR-H1, CDR-H2, and/or CDR-H3 having sequences of 18, 72, and 20. Each of the clones listed in Table 6 was derived from a human VH3 framework (with kappa and lambda gene V segments from which the clones are derived indicated).
TABLE 6
Sequences for Exemplary Clones (SEQ ID NO.)
Heavy Chain
Light Chain
ScFv
CDR-H
CDR-L
Variable (VH)
Variable (VL)
Sequence
(1, 2, 3)
(1, 2, 3)
Light Chain
Region
Region
(Amino Acid,
(Kabat)
(Kabat)
Framework
Clone #
(Amino Acid)
(Amino Acid)
Nucleotide)
(Amino Acid)
(Amino Acid)
Derived From
488
63
71
45, 44
18, 72, 20
80, 100, 109
Vκ3
1304
62
68
47, 46
18, 72, 20
77, 97, 106
Vκ1
285
11
65
49, 48
18, 19, 20
74, 94, 103
Vλ2
192B
60
64
51, 50
18, 19, 20
73, 93, 101
Vλ2
328
61
66
53, 52
18, 19, 20
75, 95, 104
Vλ2
227
63
70
55, 54
18, 72, 20
79, 99, 108
Vκ1
1300
62
69
57, 56
18, 72, 20
78, 98, 107
Vκ1
1
12
67
59, 58
18, 19, 20
76, 96, 105
Vλ1
192
12
91
87, 86
18, 19, 20
73, 93, 102
Vλ2
241
63
90
89, 88
18, 72, 20
77, 97, 106
Vκ1
2B. Purification and Assessment
Clones described above, including clones listed in Table 6 and/or described in Example 1, were purified by single-step purification and purification assessed via SDS gel. The results are presented in FIG. 9 (lane 1=MW marker; lanes 2, 9, and 10=clone 5 (1530, 2880, 1130 μg/mL); lane 3=clone 18B (660 μg/mL); lanes 4, 11, 12, and 13=clone 17 (300, 1060, 180, 1440 μg/mL); lane 5=clone 192B (1580 μg/mL); lanes 6 and 14=clone 76 (1340, 3220 μg/mL); lane 7=clone 835 (470 μg/mL); lane 8=clone 488 (340 μg/mL)). Melting temperature (Tm) measurements were made as described in Example 1, revealing similar Tm values as those observed for the reference antibody and clones in Example 1 (Table 7).
TABLE 7
Assessment of Tm
Clone
Tm (° C.)
5
58
18B
57
17
52
192B
64
76
51/59
488
63
285
68
227
60
Various clones were titrated and their binding affinity (EC50) to various CD19-expressing cells assessed by flow cytometry. The FMC63 scFv reference antibody was used as a positive control. The results from five separate assays assessing binding affinities for various CD19-specific scFv clones are shown in FIGS. 10A-10E. As shown, the selections resulted in several CD19-specific scFv clones with various binding affinities and a range of saturatable binding activity.
Competition binding assays were performed as described in Example 1 to assess the ability of various identified antibodies (scFv clones) to compete for binding with a murine reference antibody for binding to CD19-expressing cells. In one example, CD19-expressing cells were incubated with 10 nM labeled FMC63 scFv in the presence of various concentrations of the indicated scFv clones having different light chain sequences and sharing a common heavy chain CDR3 (or FMC63 scFv (positive control)). The results, shown in FIG. 11, demonstrated that the clones competed with FMC63 scFv for binding to CD19-expressing cells, with various IC50 values. Similar studies were carried out to assess properties of other clones identified in the screening approaches described in Examples 1 and 2. EC50 (binding affinity) and IC50 (competition) values observed for various CD19-binding antibodies (scFvs) are listed in Table 8. CDR-L3 sequences for clones 79, 835, 184, 505, 506, and 305 are set forth as SEQ ID NOs: 116, 117, 118, 119, 120, 121, and respectively.
TABLE 8
Results from Various Binding and Competition Assays
EC50 (CD19-Expressing
IC50 (nM) (competition
Clone/Antibody
Cells) (nM)
for binding with FMC63)
Clone 18B
4.9 ± 0.8 (n = 7)
32.9 ± 3.2 (n = 3)
Clone 17
11.6 ± 1.1 (n = 5)
35.4 ± 3.9 (n = 2)
Clone 76
7.0 ± 1.4 (n = 5)
18.2 ± 1.5 (n = 2)
Clone 5
15.8 (n = 1)
50 (n = 1)
Clone 192B
7.7 ± 1.4 (n = 3)
15.7 ± 2.5 (n = 3)
Clone 488
2.9 ± 0.4 (n = 4)
6.1 ± 0.7 (n = 6)
Clone 79
65.7 (n = 1)
102.5 (n = 1)
Clone 835
71.8 (n = 1)
>200
Clone 184
113.9 (n = 1)
N/A
Clone 505
138.9 (n = 1)
N/A
Clone 506
179.3 (n = 1)
N/A
Clone 1
213.8 (n = 1)
N/A
Clone241
5.2 ± 0.1 (n = 2)
14.6 ± 2.7 (n = 3)
1300
1.3 ± 0.1 (n = 3)
3.9 ± 0.5 (n = 2)
227
31.8 ± 5.3 (n = 3)
56.1 ± 3.9 (n = 2)
285
2.5 ± 0.5 (n = 4)
9.4 ± 1.4 (n = 3)
305
32.2 ± 6.9 (n = 2)
>500 (n = 2)
328
10.9 ± 4.6 (n = 4)
32.9 (n = 1)
FMC63
6.0 ± 0.8 (n = 9)
15.0 ± 2.8 (n = 10)
Among the identified human CD19 antibodies (scFv fragments), many demonstrated similar or greater degrees of binding affinity (e.g., similar or lower EC50 values) for CD19 as compared to a murine anti-CD19 reference antibody, FMC63. Many also demonstrated similar or greater degrees of competition (e.g., similar or lower IC50 values) with a murine anti-CD19 reference antibody for CD19 binding, as compared to the reference antibody's ability to compete with itself.
For example, clones were observed with EC50 values that were less than, about the same as, or no more than at or about 1.5-fold greater, 2-fold greater, or 3-fold greater than those for the reference antibody. Likewise, several of the identified anti-CD19 antibodies (scFvs) were observed to compete with labeled FMC63 scFv for binding to CD19-expressing cells with IC50 values that were lower than the IC50 values observed for FMC63 scFv, about the same as the IC50 values observed for FMC63, or no more than 1.5-fold or 2-fold or 3-fold higher (e.g., a degree of competition that is no more than 1.5-fold or 2-fold or 3-fold lower than the competition by the reference antibody). The results indicated that these studies identified a plurality of antibodies that bind to an epitope of CD19 that overlaps with the epitope specifically bound by FMC63.
Example 3: Generation of Chimeric Antigen Receptors (CARs) Against CD19 and Engineering of Cells Expressing Such CARs
Various exemplary chimeric antigen receptors (CARs) were generated, with antigen-binding regions containing human anti-CD19 scFvs as described in Example 1. Specifically, nucleic acid molecules were generated that encoded CARs with scFvs (in the VH-VL format) derived from the following clones and having the amino acid sequences set forth in the indicated sequence identifiers: Clone 18 (SEQ ID NO:2), Clone 18B (SEQ ID NO:4), Clone 17 (SEQ ID NO:6), Clone 76 (SEQ ID NO: 8), and Clone 5 (SEQ ID NO:10). Additionally, for each clone, constructs encoding a CAR having the same VH and VL sequences, but present in the reverse orientation (VL-VH), also were generated. A CAR containing a murine anti-CD19 scFv derived from FMC63 (in the VH-VL orientation) was used as a control. Each CAR further contained an Ig-derived spacer; a human CD28-derived transmembrane domain; a human 4-1BB-derived intracellular signaling domain; and a human CD3 zeta-derived signaling domain, a truncated EGFR (EGFRt) sequence, for use as a transduction marker, separated from the CAR sequence by a self-cleaving T2A sequence.
Primary human T cell populations expressing the various CARs were generated. Nucleic acid molecules encoding each CAR were individually cloned into a lentiviral vector, which was used to transduce CD4+ and CD8+ T cells in populations isolated from human PBMC samples obtained from healthy donors (essentially as described by Yam et al. (2002) Mol. Ther. 5:479; WO2015/095895).
After transduction and expansion, staining with anti-EGFR antibody was used to verify expression of the EGFRt transduction marker on the surface of CD4+ and CD8+ T cells by flow cytometry. FIG. 12A provides representative results for expression of the various CARs in CD8+ cells; similar results were observed for CD4+ cells. CAR protein expression was confirmed by western blotting using an anti-CD247 (CD3 zeta) antibody (which in each case detected a band at approximately 50 kD, representing the CAR, and a band at approximately 18 kDa, representing the endogenous CD3 zeta chain present in the cells) (FIG. 12B). The results demonstrated comparable degrees of transduction and CAR protein expression for each of the various human scFv-containing CAR constructs (including VH-VL and VL-VH orientations) and control (murine, FMC63-derived) CAR constructs in primary T cell populations. No EGFRt expression was detected in cells not subjected to transduction. Results from western blotting confirmed that the CAR derived from clone 76, in the VH-VL orientation, was present in different glycosylation forms.
As shown in FIG. 12A, T cell populations were successfully enriched for transduced cells (at or close to 100% EGFRt+ as confirmed by flow cytometry) by staining with an anti-EGFR antibody, sorting on a flow cytometer, and stimulation in the presence of irradiated (8,000 rad) cells from a CD19+ B-lymphoblastoid cell line (B-LCL) essentially as described by Yam et al. (2002) Mol. Ther. 5:479; WO2015/095895.
Example 4: Assessing Effector Functions of T Cells Engineered to Express Anti-CD19 Chimeric Antigen Receptor (CAR) In Vitro
Genetically engineered human T cells (either CD8+ or CD4+) expressing various CARs containing human anti-CD19 scFvs, produced as described in Example 3, were assessed for various responses following co-culture with CD19-expressing cells.
A. Cytolytic Activity
CD19-expressing target cells were incubated with CD8+ T cells expressing the various CARs and separately with cells transduced with EGFRt alone (negative control). Following incubation, lysis of target cells was monitored. Specifically, lysis of CD19-transduced K562 cells (K562/CD19), Raji (CD19+ B cell lymphoma line) cells, and non-transduced K562 control cells (negative control) (FIG. 13A) and primary human chronic lymphocytic leukemia cells (CLL; FIG. 13B) were tested.
The target cells (K562/CD19 Raji non-transduced K562 control cells or CLL) were labeled overnight with 51Cr. Labeled cells were washed and incubated in triplicate with effector T cells (CAR-expressing and negative control CD8+ cells) at an effector to target (E:T) ratio of 30:1. To measure spontaneous lysis, target cells were incubated with an equal volume of media but without effector cells and maximum lysis was determined following incubation of target cells with detergent to completely lyse the target cells. Supernatants were harvested for 7-counting after a 4 hour incubation. The percent specific lysis for the experimental conditions was calculated as:
[(Experimental Release−Spontaneous Release)/(Maximum Release−Spontaneous Release)]×100.
The results are set forth in FIGS. 13A and 13B. As shown in FIG. 13A, engineered CD8+ T cells expressing the various human anti-CD19 scFv-containing CARs exhibited antigen-specific cytolytic activity against CD19+ cells, to a comparable degree as cells expressing CARs containing the murine anti-CD19 (FMC63) scFv. This cytotoxic activity was not observed against control K562 cells not expressing CD19. The degree of cytolytic activity observed for cells expressing CARs with the human scFvs in the VH-VL orientation (HL) was observed to be comparable or greater than that observed for cells expressing the murine scFv-containing CAR. The degree of cytolytic activity observed for cells expressing a CAR with a given human scFv in the VH-VL (HL) orientation was generally greater than that observed for cells expressing a CAR with the corresponding scFv in the reverse VL-VH orientation (LH). As shown in FIG. 13B, the results also demonstrated antigen-specific cytolytic activity against the primary human CLL cells by the engineered CD8+ cells expressing the various human anti-CD19 scFv-containing CARs (VH-VL orientation) also was observed.
B. Cytokine Release
Cytokine release was assessed following incubation of the CAR-expressing cells with antigen-expressing and control target cells. Transduced CD8+ and CD4+ T cells were co-cultured in triplicate with target cells (K562, K562/CD19, Raji) at an effector to target (E:T) ratio of 2:1. Cytokine secretion following co-culture of transduced CD8+ cells with primary human chronic lymphocytic leukemia cells (CLL) also was similarly tested. The co-cultured cells were incubated for about 24 hours, and then supernatants were collected for measurement of IFN-γ (CD8+ cells) or IFN-γ, TNF-α, or IL-2 (CD4+ cells) using a multiplex cytokine immunoassay (Luminex®).
The results for CD8+ cells are set forth in FIGS. 14A and 14B. Engineered CD8+ T cells expressing the various human anti-CD19 scFv-containing CARs were observed to secrete IFN-γ in an antigen-specific manner following incubation with CD19+ cells, to a comparable degree as that observed for cells expressing CARs containing the murine anti-CD19 (FMC63) scFv. The cytokine secretion was not observed following incubation control K562 cells not expressing CD19. The levels of cytokine secretion observed for cells expressing CARs with the tested human anti-CD19 scFvs in the VH-VL orientation were comparable and in some cases greater than that observed for cells expressing the murine anti-CD19 scFv-containing CAR. The degree of IFNγ secretion observed for cells expressing a CAR with a given human scFv in the VH-VL orientation was generally greater that observed for cells expressing a CAR with the corresponding scFv in the reverse (VL-VH) orientation. As shown in FIG. 14B, antigen-specific cytokine secretion by CD8+ engineered T cells expressing the various human anti-CD19 scFv-containing CARs (VH-VL orientation) also was observed following co-culture with the CLL cells.
The results for CD4+ CAR-expressing T cells are set forth in FIG. 15. Engineered CD4+ T cells expressing the various human anti-CD19 scFv-containing CARs (VH-VL orientation) were observed to secrete cytokines in an antigen-specific manner following incubation with CD19+ target cells, at levels comparable to and in general greater than those observed for cells expressing the murine-scFv (FMC63)-containing CAR. The cytokine secretion was not observed following CD19-negative control cells.
C. T Cell Proliferation
Proliferation of the various CAR-expressing T cells following incubation with CD19-expressing target cells was assessed by flow cytometry. CD8+ or CD4+ CAR-expressing T cells were labeled with 0.2 μM carboxyfluorescein succinmidyl ester (CFSE). Cells were washed and incubated for 72 hours in triplicate with target cells (K562, K562/CD19 or Raji) in serum-containing medium without exogenous cytokines. Division of live T cells was indicated by CFSE dilution, as assessed by flow cytometry.
The results are set forth in FIGS. 16A and 16B for CD8+ CAR-expressing T cells and CD4+ CAR-expressing T cells, respectively. As shown in FIG. 16A, CD8+ T cells expressing each of the tested human anti-CD19 scFv-containing CAR constructs proliferated after co-culture with CD19-expressing K562/CD19 or Raji target cells, but generally not with K562 control cells. The degree of proliferation observed for T cells expressing CARs with the tested human anti-CD19 scFv was comparable to that observed for cells expressing the murine anti-CD19 scFv-containing CAR. The degree of proliferation of cells expressing a CAR with a given human scFv in the VH-VL orientation was generally observed to be greater than observed for cells expressing a CAR with the corresponding scFv in the reverse (VL-VH) orientation.
Antigen-specific proliferation of CAR-expressing T cells also was observed for CD4+ cells. As shown in FIG. 16B, CD4+ T cell expressing each of the tested human anti-CD19 scFv-containing CAR constructs proliferated after co-culture with CD19-expressing K562/CD19 or Raji target cells. The degree of proliferation observed for CD4+ T cells expressing CARs with the tested human anti-CD19 scFv was comparable to that observed for cells expressing the murine anti-CD19 scFv-containing CAR.
Example 5: Anti-Tumor Effect of CAR-Expressing T Cells after Adoptive Transfer In Vivo
The anti-tumor effects of CAR-expressing engineered primary human T cells was assessed by monitoring tumors following adoptive transfer of cells to patient-derived xenograft (PDX) tumor model animal subjects. Six- to eight-week old female NOD.Cg.PrkdcscidIL2rgtmIWjl/SzJ (NSG) mice were injected intravenously (i.v.) with 0.5×106 Raji lymphoma tumor cells transfected with firefly luciferase (Raji-ffluc). Tumor engraftment was allowed to occur for 6 days and verified using bioluminescence imaging. On day 7, mice received a single intravenous (i.v.) injection of a sub-optimal dose (1×106 CAR-expressing T cells in this study) of the various engineered primary human T cells (CD8+ cells alone (FIG. 17A) or combined CD4+ and CD8+ cells at a 1:1 ratio (FIG. 17B)) described in Example 3. As a control, mice were administered cells that were transduced with EGFRt alone (negative control). The sub-optimal dose was used in order to better visualize differences in anti-tumor effects.
Anti-tumor activity of the adoptively transferred CAR-expressing cells was monitored by bioluminescence imaging on days 6, 9, 13, 20 27 and 34. For bioluminescence imaging, mice received intraperitoneal (i.p.) injections of luciferin substrate (CaliperLife Sciences, Hopkinton, Mass.) resuspended in PBS (15 μg/g body weight). Mice were anesthetized and imaged essentially as described in WO2015/095895. The average radiance (p/s/cm2/sr) was determined.
As shown in FIGS. 17A and 17B, tumors in control mice continued to grow over the course of the study following adoptive transfer of control T cells (CD8+ cells alone (FIG. 17A) or combination of CD4+ and CD8+ cells (FIG. 17B) transduced with EGFRt alone). Compared to the control mice, mice having been administered adoptive transfer of engineered T cells expressing each of the various tested anti-CD19 scFv-containing CARs were observed to have a lower degree of bioluminescence signal, indicating a reduction in tumor size over time and/or a lower degree of tumor growth in the treated animals. In general, as shown in FIG. 17A, adoptive transfer of CD8+ T cells expressing the tested human anti-CD19 scFv CARs alone led to a comparative reduction in tumor size to at least the same degree as adoptive transfer of cells expressing a CAR containing the mouse anti-CD19 scFv (FMC63). As shown in FIG. 17B, adoptive transfer of the combination of CD8+ and CD4+ T cells expressing the tested human anti-CD19 CARs was observed to reduce tumor size over time. Tumor size (as indicated by bioluminescence signal) following adoptive transfer of such human anti-CD19 CAR-expressing cells was observed to be comparatively lower than that detected following adoptive transfer of the mouse-scFv-derived CAR-expressing cells.
Example 6: Identification of Region in Human CD19 Recognized by Anti-CD19 Antibodies
CARs containing certain anti-CD19 antibodies (scFvs) described in Example 1, or the murine anti-CD19 (FMC63) scFv, were assessed for binding to various CD19 molecules. K562 cells were engineered to express (a) a human CD19 (having the amino acid sequence set forth in SEQ ID NO:92), (b) a Macaca mulatta (rhesus macaque (rhesus)) CD19 (having the amino acid sequence set forth in SEQ ID NO:139; Accession No. F7F486), or (c) one the three different human/rhesus chimeric CD19 molecules, V1, V2, and V3, which contained membrane-proximal regions having the sequences depicted in FIG. 18A. Aside from the region depicted in FIG. 18A, the remaining regions of each chimeric molecule were identical in sequence to the corresponding regions of the rhesus CD19.
Chimeric CD19 V1:
The 74-amino acid membrane-proximal region depicted in FIG. 18A of the chimeric molecule designated V1 had the amino acid sequence set forth in SEQ ID NO: 140, which was identical to the sequence of the corresponding region (residues 218 to 291) of the human CD19 molecule having the sequence set forth in SEQ ID NO: 92.
Chimeric CD19 V2:
The 75-amino acid membrane-proximal region depicted in FIG. 18A of the chimeric CD19 molecule designated V2 had the amino acid sequence set forth in SEQ ID NO: 141. Within this region, the 27-amino acid membrane-proximal portion was identical in sequence to the corresponding portion (residues 265 to 291) of human CD19. The remaining portion of the shown region was identical in sequence to the corresponding portion of the rhesus CD19 sequence set forth in SEQ ID NO: 139. Positions in this remaining portion having a substitution or an insertion compared to the corresponding human sequence are underlined.
Chimeric CD19 V3:
The 74-amino acid region depicted in FIG. 18A of the chimeric CD19 molecule designated V3 had the amino acid sequence set forth in SEQ ID NO: 142. Within this depicted region, a 47-amino acid portion was identical in sequence to the corresponding portion (residues 218-264) of the human CD19 sequence set forth in SEQ ID NO: 92. The remaining 27-amino acid membrane-proximal portion was identical in sequence to the corresponding portion of rhesus CD19 sequence set forth in SEQ ID NO: 139. Positions in this remaining 27-amino acid portion having a substitution compared to the corresponding human sequence are underlined.
Primary human T cells expressing various human anti-CD19 scFv-containing CARs or a murine anti-CD19 scFv (FMC63)-containing CAR were generated as described in Example 3 and co-cultured with the various K562 target cells transfected with nucleic acid molecules encoding the various CD19 molecules, at an effector to target (E:T) ratio of 2:1. The cells were incubated for 24 hours, and supernatants were collected for measurement of IFN-γ, using a cytokine immunoassay, as an indicator of functional binding by the anti-CD19 scFv-containing CARs to the respective CD19 molecules on the surface of the target cells. The results are shown in FIG. 18B.
Each of the tested anti-CD19 CARs exhibited detectable levels of cytokine following co-culture with cells expressing the human CD19 molecule (indicating functional binding thereto), but not following co-culture with cells expressing the rhesus CD19. For each of the tested anti-CD19 CARs, detectable levels of secretion were observed following co-culture with cells expressing the rhesus/human chimeric molecules designated V1 (entire membrane-proximal 74-amino acid region human-derived) and V3 (27-amino acid membrane-proximal portion rhesus-derived), but not to cells expressing the rhesus/human chimeric molecule designated V2 (27-amino acid membrane-proximal portion human-derived).
These results indicated that at least part of a 32-amino acid portion (SEQ ID NO: 143 (HPKGPKSLLSLELKDDRPARDMWVMETGLLLP) of the human CD19 molecule (corresponding to residues 218-249 of SEQ ID NO: 92), was important for functional binding to CD19 by each of the tested anti-CD19 CARs. Specifically, whereas each of V1 and V3 contained this 32-amino acid sequence (set forth in bold in FIG. 18A), the corresponding portion of V2 contained the 33-residue amino acid sequence set forth in SEQ ID NO: 144 (RPKGPKSSLLSLELKDDRPDRDMWVVDTGLLLT), which was identical in sequence to the corresponding portion of the rhesus CD19 molecule, but contained five amino acid substitutions (at positions 218, 236, 242, 243, and 249 of the human CD19 sequence of SEQ ID NO: 92) and one insertion (between positions 223 and 224 of the human CD19 sequence of SEQ ID NO: 92) compared with the corresponding human sequence, each underlined in FIG. 18A. Thus, the results indicate that the amino acid(s) present at at least one of these position(s) in the human sequence (positions 218, 236, 242, 243, 249 and/or 223-224 of SEQ ID NO: 92) was important for the ability of each CAR tested to specifically bind to human CD19. Thus, the results support a conclusion that each of the tested human scFv-containing CARs bound to a similar and/or overlapping epitope as compared to the CAR containing the mouse scFv, FMC63.
The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.
Sequences
TABLE 9
SEQ
ID
NO.
Sequence
Description
1
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTC
Clone 18 scFv
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCAT
(nt)
GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA
TTAGTTGGAATAGTGGTAGGATAGGCTATGCGGACTCTGTAAAGGGCCGA
TTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTTTCTGCAAATGAA
CAGTCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCAGG
GGTATCATTACTATGATAGTGCCGAACATGCTTTTGATATCTGGGGCCAAG
GGACAGTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGC
TCTGGCGGTGGCGGATCGCAGTCTGCCCTGACTCAGCCTCGCTCAGTGTCC
GGCTTTCCTGGACAATCAGTCACCATCTCCTGCACTGGAACCACCAGTGAT
GATGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCCAACTTATGCT
TTATGATGTCAGTAAGCGGCCCTCCGGGGTCCCTCATCGCTTCTCTGGCTC
CAGGTCTGGCAGAGCGGCCTCCCTGATCATCTCTGGGCTCCAGACTGAGG
ATGAGGCTGATTATTTCTGCTGCTCATATGCAGGCCGATACAACTCTGTCC
TTTTCGGCGGAGGGACCAAGCTGACCGTCCTA
2
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
Clone 18 scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
(aa)
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPRSVSGFP
GQSVTISCTGTTSDDVSWYQQHPGKAPQLMLYDVSKRPSGVPHRFSGSRSGR
AASLIISGLQTEDEADYFCCSYAGRYNSVLFGGGTKLTVL
3
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTC
Clone 18B
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCAT
scFv (nt)
GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA
TTAGTTGGAATAGTGGTAGGATAGGCTATGCGGACTCTGTAAAGGGCCGA
TTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTTTCTGCAAATGAA
CAGTCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCAGG
GGTATCATTACTATGATAGTGCCGAACATGCTTTTGATATCTGGGGCCAAG
GGACAGTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGC
TCTGGCGGTGGCGGATCGCAGTCTGCCCTGACTCAGCCTCGCTCAGTGTCC
GGCTTTCCTGGACAATCAGTCACCATCTCCTGCACTGGAACCACCAGTGAT
GATGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCCAACTTATGCT
TTATGATGTCAGTAAGCGGCCCTCCGGGGTCCCTCATCGCTTCTCTGGCTC
CAGGTCTGGCAGAGCGGCCTCCCTGATCATCTCTGGGCTCCAGACTGAGG
ATGAGGCTGATTATTTCTGCAGCTCATATGCAGGCCGATACAACTCTGTCC
TTTTCGGCGGAGGGACCAAGCTGACCGTCCTA
4
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
Clone 18B
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
scFv (aa)
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPRSVSGFP
GQSVTISCTGTTSDDVSWYQQHPGKAPQLMLYDVSKRPSGVPHRFSGSRSGR
AASLIISGLQTEDEADYFCSSYAGRYNSVLFGGGTKLTVL
5
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTC
Clone 17 scFv
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCAT
(nt)
GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA
TTAGTTGGAATAGTGGTAGGATAGGCTATGCGGACTCTGTAAAGGGCCGA
TTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTTTCTGCAAATGAA
CAGTCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCAGG
GGTATCATTACTATGATAGTGCCGAACATGCTTTTGATATCTGGGGCCAAG
GGACAATGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGC
TCTGGCGGTGGCGGATCGCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCT
GGGTCTCCTGGACAGTCGATCACCATCTTCTGCACTGGAACCAGCAGTGA
CGTTGGTGGTTATAACTATGTCTCCTGGTACCAGCAGCTCCCAGGAACGGC
CCCCAAACTCCTCATCTATAGTAATAATCAGCGGCCCTCAGGGGTCCCTG
ACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTG
GGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGAC
AGCCTGAGTGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTC
6
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
Clone 17 scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
(aa)
HYYDSAEHAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGS
PGQSITIFCTGTSSDVGGYNYVSWYQQLPGTAPKLLIYSNNQRPSGVPDRFSG
SKSGTSASLAISGLRSEDEADYYCAAWDDSLSVVFGGGTKLTVL
7
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTC
Clone 76 scFv
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCAT
(nt)
GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA
TTAGTTGGAATAGTGGTAGGATAGGCTATGCGGACTCTGTAAAGGGCCGA
TTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTTTCTGCAAATGAA
CAGTCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCAGG
GGTATCATTACTATGATAGTGCCGAACATGCTTTTGATATCTGGGGCCAAG
GGACAGTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGC
TCTGGCGGTGGCGGATCGCAGTCTGTGCTGACGCAGCCGCCCTCAGTGTC
TGCGGCCCCAGGACAGGAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCA
ACATTGGGAATAATTATGTATCCTGGTACCAGCAACTCCCAGGAACAGCC
CCCAAACTCCTCATTTATGACAATGATAAGCGACCCTCAGGGATTCCTGA
CCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCG
GACTCCAGACTGGGGACGAGGCCGATTATTACTGCGGAACATGGGATGGC
AATCTGAGTGCTGTATTCGGCGGAGGGACCAAGGTGACCGTCCTA
8
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
Clone 76 scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
(aa)
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSAA
PGQEVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNDKRPSGIPDRFSGSK
SGTSATLGITGLQTGDEADYYCGTWDGNLSAVFGGGTKVTVL
9
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTC
Clone 5 scFv
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCAT
(nt)
GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA
TTAGTTGGAATAGTGGTAGGATAGGCTATGCGGACTCTGTAAAGGGCCGA
TTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTTTCTGCAAATGAA
CAGTCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCAGG
GGTATCATTACTATGATAGTGCCGAACATGCTTTTGATATCTGGGGCCAAG
GGACAATGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGC
TCTGGCGGTGGCGGATCGTCCTATGAGCTGACTCAGGACCCTGCTGTGTCT
GTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAG
AAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTAC
TTGTCATCTATGATAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTC
TCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCA
GGCGGAAGATGAGGCTGACTACTACTGCAACTCCCGGGACAGCAGTGGTA
ACAATTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA
10
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
Clone 5 scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
(aa)
HYYDSAEHAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSSYELTQDPAVSVA
LGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYDKNNRPSGIPDRFSGSSS
GNTASLTITGAQAEDEADYYCNSRDSSGNNWVFGGGTKLTVL
11
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
(clones 18, 18B
HYYDSAEHAFDIWGQGTVVTVSS
reversion, 76,
285)
(aa)
12
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH (clones 17,
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
5, 1, 192)
HYYDSAEHAFDIWGQGTMVTVSS
(aa)
13
QSALTQPRSVSGFPGQSVTISCTGTTSDDVSWYQQHPGKAPQLMLYDVSKRP
VL Clone 18
SGVPHRFSGSRSGRAASLIISGLQTEDEADYFCCSYAGRYNSVLFGGGTKLTV
(aa)
L
14
QSALTQPRSVSGFPGQSVTISCTGTTSDDVSWYQQHPGKAPQLMLYDVSKRP
VL, Clone 18B
SGVPHRFSGSRSGRAASLIISGLQTEDEADYFCSSYAGRYNSVLFGGGTKLTV
(aa)
L
15
QSALTQPASVSGSPGQSITIFCTGTSSDVGGYNYVSWYQQLPGTAPKLLIYSN
VL, Clone 17
NQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSVVFGGGT
(aa)
KLTVL
16
QSVLTQPPSVSAAPGQEVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDND
VL, Clone 76
KRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDGNLSAVFGGGTK
(aa)
VTVL
17
SYELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYDKNN
VL, Clone 5
RPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNNWVFGGGTKL
(aa)
TVL
18
DYAMH
CDR-H1 (aa)
19
GISWNSGRIGYADSVKG
CDR-H2 (aa)
20
DQGYHYYDSAEHAFDI
CDR-H3 (aa)
21
TGTTSDDVS
Clones 18, 18B
CDR-L1 (aa)
22
DVSKRPS
Clones 18, 18B
CDR-L2 (aa)
23
CSYAGRYNSVL
Clone 18 CDR-
L3 (aa)
24
SSYAGRYNSVL
Clone 18B
CDR-L3 (aa)
25
TGTSSDVGGYNYVS
Clone 17 CDR-
L1 (aa)
26
SNNQRPS
Clone 17 CDR-
L2 (aa)
27
AAWDDSLSVV
Clone 17 CDR-
L3 (aa)
28
SGSSSNIGNNYVS
Clone 76 CDR-
L1 (aa)
29
DNDKRPS
Clone 76 CDR-
L2 (aa)
30
GTWDGNLSAV
Clone 76 CDR-
L3 (aa)
31
QGDSLRSYYAS
Clone 5 CDR-
L1 (aa)
32
DKNNRPS
Clone 5 CDR-
L2 (aa)
33
NSRDSSGNNWV
Clone 5 CDR-
L3 (aa)
34
GGGGSGGGGSGGGGS
Linker (aa)
35
GISWNSGRIGY
CDR-H2
36
X1GX3X4X5X6X7X8X9X10X11X12X13S
CDR-L1
X1 = T, S, or Q; X3 = T, S, or D; X4 = T or S;
consensus
X5 = null or S; X6 = null, D, or N; X7 = null or V;
X8 = null, G, or I; X9 = null, G, or R; X10 = S, Y,
or N; X11 = D or N; X12 = D or Y; X13 = V or A
37
X1X2X3X4RPS
CDR-L2
X1 = D or S; X2 = V, N, or K; X3 = S, N, or D;
consensus
X4 = K, Q, or N
38
X1X2X3X4X5X6X7X8X9X10X11X12
CDR-L3
X1 = C, S, A, G, or N; X2 = S, A, or T; X3 = Y, W,
or R; X4 = A or D; X5 = G, D, or S; X6 = R, S, or N;
X7 = Y, L, or G; X8 = N or S; X9 = S or null;
X10 = V, A, or N; X11 = W or null; X12 = L or V.
39
EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIW
FMC63 VH
GSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGS
YAMDYWGQGTSVTVSS
40
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSR
FMC63VL
LHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT
41
EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQI
SJ25C1VH
YPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISS
VVDFYFDYWGQGTTVTVSS
42
DIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSAT
SJ25C1 VL
YRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKL
EI
43
GSTSGSGKPGSGEGSTKG
Linker
44
GAAGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTC
Clone 488 scFv
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCAT
(nt)
GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA
TTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCGA
TTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAA
CAGTCTGAGAGCTGAGGACACCGCCGTGTATTACTGTGCGAGAGATCAGG
GGTATCATTACTATGATAGTGCCGAACATGCTTTTGATATCTGGGGCCAAG
GGACAGTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGC
TCTGGCGGTGGCGGATCGGAAATTGTGTTGACGCAGTCTCCAGCCACCCT
GTCTTTGTCTCCAGGGGAGACCGCCACCCTCTCCTGCAGGGCCAGTCAGA
GTATTAACCACTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCC
CGGCTCCTCATCTATGATGCCTCCAACAGGGCCACTGGCATCCCAGCCAG
GTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCC
TAGAGCCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTCACC
CTCGAATGTACACTTTTGGCCAGGGGACCAAACTGGATATCAAA
45
EVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
Clone 488 scFv
SWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDQGY
(aa)
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLS
PGETATLSCRASQSINHYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGS
GTDFTLTISSLEPEDFATYYCQQSYSHPRMYTFGQGTKLDIK
46
CAGATGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTC
Clone 1304
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCAT
scFv (nt)
GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA
TTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCGA
TTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAA
CAGTCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCAGG
GGTATCATTACTATGATAGTGCCGAACATGCTTTTGATATCTGGGGCCAAG
GGACAGTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGC
TCTGGCGGTGGCGGATCGGCCATCCGGATGACCCAGTCTCCATCCTCCCTG
TCTGCATCTGTAGGAGACAGAGTCACCGTCACTTGCCAGGCGAGTCAGGA
CATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGAAGAGCCCCTA
AGCTCCTGATCTACGATGCATCCAATGTGAAAGCAGGGGTCCCATCAAGG
TTCAGTGGGGGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTG
CAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCT
CAGGCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA
47
QMQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSG
Clone 1304
ISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDQGY
scFv (aa)
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSAIRMTQSPSSLSAS
VGDRVTVTCQASQDISNYLNWYQQKPGRAPKLLIYDASNVKAGVPSRFSGG
GSGTDFTLTISSLQPEDFATYYCQQSYSTPQAYTFGQGTKLEIK
48
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTC
Clone 285 scFv
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCAT
(nt)
GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA
TTAGTTGGAATAGTGGTAGGATAGGCTATGCGGACTCTGTAAAGGGCCGA
TTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTTTCTGCAAATGAA
CAGTCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCAGG
GGTATCATTACTATGATAGTGCCGAACATGCTTTTGATATCTGGGGCCAAG
GGACAGTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGC
TCTGGCGGTGGCGGATCGCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCT
GGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGA
CCTTGGTGGTTACAATTATGTCTCCTGGTATCAACACCGCCCAGGCAAAGC
CCCCAAACTCATCATTTATGATGTCACTGTTCGGCCCTCAGGGGTTTCTGA
TCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGG
GCTCCAGGCTGAGGACGAGGCTGATTATTACTGCGGCTCATATACAAGCA
GTAGCACTCTTCTTTGGGTGTTCGGCGGAGGGACCAAGCTCACCGTCCTA
49
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
Clone 285 scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
(aa)
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGS
PGQSITISCTGTSSDLGGYNYVSWYQHRPGKAPKLIIYDVTVRPSGVSDRFSGS
KSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGTKLTVL
50
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTC
Clone 192B
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCAT
scFv (nt)
GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA
TTAGTTGGAATAGTGGTAGGATAGGCTATGCGGACTCTGTAAAGGGCCGA
TTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTTTCTGCAAATGAA
CAGTCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCAGG
GGTATCATTACTATGATAGTGCCGAACATGCTTTTGATATCTGGGGCCAAG
GGACAATGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGC
TCTGGCGGTGGCGGATCGCAGGCTGTGCTGACTCAGCCTCGCTCAGTGTC
CGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAATCAGCAGTG
GTGTTGATAGTCATAGGTATGTCTCCTGGTACCAACACCACCCAGGCAAA
GCCCCCAAACTCATGATTTATGATTTCAGTAAGCGGCCCTCAGGGGTCCCT
GATCGTTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCT
GGGCTCCAGGCTGAGGATGAGGCTGATTACTATTGCAGCTCATATGCAGC
CATCTCCCCTAATTATGTCTTCGGAACTGGGACCAAGCTCACCGTCCTA
51
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
Clone 192B
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
scFv (aa)
HYYDSAEHAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQAVLTQPRSVSGS
PGQSVTISCTGISSGVDSHRYVSWYQHHPGKAPKLMIYDFSKRPSGVPDRFSG
SKSGNTASLTISGLQAEDEADYYCSSYAAISPNYVFGTGTKLTVL
52
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTC
Clone 328 scFv
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCAT
(nt)
GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA
TTAGTTGGAATAGTGGTAGGATAGGCTATGCGGACTCTGTAAAGGGCCGA
TTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTTTCTGCAAATGAA
CAGTCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCAGG
GGTATCATTACTATGATAGTGCCGAACATGCTTTTGATATCTGGGGCCAAG
GGACAGTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGC
TCTGGCGGTGGCGGATCGCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCT
GGGTCTCCTGGACATTCGATCACCATCTCCTGCACTGGAACCAGAAGTGA
CGTCGGTGGTTTTGATTATGTCTCCTGGTACCAGCATAACCCAGGCAAAGC
CCCCAAACTCATAATTTATGATGTCACTAAGCGGCCCTCAGGGGTCTCTAA
TCGCTTCTCTGGCGCCAAGTCTGGCATCACGGCCTCCCTGACCATCTCTGG
GCTCCAGGCTGAGGACGAGGCTGATTATTACTGCACCTCATATAGACCCG
GTCCAACATTTGTCTTCGGCACCGGGACCAAGCTCACCGTCCTA
53
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
Clone 328 scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
(aa)
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGS
PGHSITISCTGTRSDVGGFDYVSWYQHNPGKAPKLIIYDVTKRPSGVSNRFSG
AKSGITASLTISGLQAEDEADYYCTSYRPGPTFVFGTGTKLTVL
54
GAAGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTC
Clone 227 scFv
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCAT
(nt)
GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA
TTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCGA
TTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAA
CAGTCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCAGG
GGTATCATTACTATGATAGTGCCGAACATGCTTTTGATATCTGGGGCCAAG
GGACAGTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGC
TCTGGCGGTGGCGGATCGGACATCCAGTTGACCCAGTCTCCTTCCACCCTG
TCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAG
TATTAGTAGGTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTA
AGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCCAGG
TTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTG
CAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCT
CTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
55
EVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
Clone 227 scFv
SWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDQGY
(aa)
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSDIQLTQSPSTLSAS
VGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGS
GTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIK
56
CAGATGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTC
Clone 1300
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCAT
scFv (nt)
GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA
TTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCGA
TTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAA
CAGTCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCAGG
GGTATCATTACTATGATAGTGCCGAACATGCTTTTGATATCTGGGGCCAAG
GGACAGTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGC
TCTGGCGGTGGCGGATCGGCCATCCGGATGACCCAGTCTCCTTCCACCCTG
TCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAG
CATTAGTCACTACTTGGCCTGGTATCAACAGAAACCAGGGAAAGCCCCTA
AGCTCCTGATCTTTGATGCCTCCCGTTTGGCAAGTGGGGTCCCATCAAGGT
TCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGC
AACCTGAAGATTTTGCGACATACTACTGTCAACAGAGTTACGGTGCCCCT
ATGTTCACTTTCGGCCCTGGGACCAGAGTGGATCTCAAA
57
QMQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSG
Clone 1300
ISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDQGY
scFv (aa)
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSAIRMTQSPSTLSAS
VGDRVTITCRASQSISHYLAWYQQKPGKAPKLLIFDASRLASGVPSRFSGSGS
GTDFTLTISSLQPEDFATYYCQQSYGAPMFTFGPGTRVDLK
58
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTC
Clone 1 scFv
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCAT
(nt)
GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA
TTAGTTGGAATAGTGGTAGGATAGGCTATGCGGACTCTGTAAAGGGCCGA
TTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTTTCTGCAAATGAA
CAGTCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCAGG
GGTATCATTACTATGATAGTGCCGAACATGCTTTTGATATCTGGGGCCAAG
GGACAATGGTCACAGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGC
TCTGGCGGTGGCGGATCGCAGTCTGCCCTGACTCAGCCCGCCTCCGTGTCT
GGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGGACCAGCAGTGA
CGTTGGTGCTTATAACTTTGTCTCCTGGTACCAGCAGCTCCCAGGAACAGC
CCCCAAATTCCTCATTTATGACAATAATAAACGACCCCCAGGGATTCCTG
ACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACC
GGACTCCAGACTGGGGACGAGGCCGATTATTACTGCGCAACATGGGATAG
CGGCCTGAGTGCTGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA
59
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
Clone 1 scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
(aa)
HYYDSAEHAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGS
PGQSITISCTGTSSDVGAYNFVSWYQQLPGTAPKFLIYDNNKRPPGIPDRFSGS
KSGTSATLGITGLQTGDEADYYCATWDSGLSAVVFGGGTKLTVL
60
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 192B
HYYDSAEHAFDIWGQGTMVTVSS
(aa)
61
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 328
HYYDSAEHAFDIWGQGTVVTVSS
(aa)
62
QMQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSG
VH
ISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDQGY
Clone 1304
HYYDSAEHAFDIWGQGTVVTVSS
Clone 1300
(aa)
63
EVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDQGY
Clones 227,
HYYDSAEHAFDIWGQGTVVTVSS
488, 241
(aa)
64
QAVLTQPRSVSGSPGQSVTISCTGISSGVDSHRYVSWYQHHPGKAPKLMIYDF
VL
SKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCSSYAAISPNYVFGTGT
Clone 192B
KLTVL
(aa)
65
QSALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQHRPGKAPKLIIYDVT
VL
VRPSGVSDRFSGSKSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGT
Clone 285
KLTVL
(aa)
66
QSALTQPASVSGSPGHSITISCTGTRSDVGGFDYVSWYQHNPGKAPKLIIYDVT
VL
KRPSGVSNRFSGAKSGITASLTISGLQAEDEADYYCTSYRPGPTFVFGTGTKLT
Clone 328
VL
(aa)
67
QSALTQPASVSGSPGQSITISCTGTSSDVGAYNFVSWYQQLPGTAPKFLIYDN
VL
NKRPPGIPDRFSGSKSGTSATLGITGLQTGDEADYYCATWDSGLSAVVFGGGT
Clone 1
KLTVL
(aa)
68
AIRMTQSPSSLSASVGDRVTVTCQASQDISNYLNWYQQKPGRAPKLLIYDAS
VL
NVKAGVPSRFSGGGSGTDFTLTISSLQPEDFATYYCQQSYSTPQAYTFGQGTK
Clone 1304
LEIK
(aa)
69
AIRMTQSPSTLSASVGDRVTITCRASQSISHYLAWYQQKPGKAPKLLIFDASRL
VL
ASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYGAPMFTFGPGTRVDL
Clone 1300
K
(aa)
70
DIQLTQSPSTLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIYDASN
VL
LETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIK
Clone 227
(aa)
71
EIVLTQSPATLSLSPGETATLSCRASQSINHYLAWYQQKPGQAPRLLIYDASNR
VL
ATGIPARFSGSGSGTDFTLTISSLEPEDFATYYCQQSYSHPRMYTFGQGTKLDI
Clone 488
K
(aa)
72
GISWNSGSIGYADSVKG
CDR-H2
Clone 1304
Clone 1300
Clone 227
Clone 488
Clone 241
(aa)
73
TGISSGVDSHRYVS
CDR-L1
Clone 192B
Clone 192
(aa)
74
TGTSSDLGGYNYVS
CDR-L1
Clone 285
(aa)
75
TGTRSDVGGFDYVS
CDR-L1
Clone 328
(aa)
76
TGTSSDVGAYNFVS
CDR-L1
Clone 1
(aa)
77
QASQDISNYLN
CDR-L1
Clone 1304
Clone 241
(aa)
78
RASQSISHYLA
CDR-L1
Clone 1300
(aa)
79
RASQSISRWLA
CDR-L1
Clone 227
(aa)
80
RASQSINHYLA
CDR-L1
Clone 488
(aa)
81
GISWNSGRIG
CDR-H2
82
GISWNSGSIG
CDR-H2
83
X1X2X3X4X5X6X7X8X9X10X11X12X13X14
CDR-L1
X1 = T, Q, or R; X2 = G or A; X3 = I, T, or S;
Consensus
X4 = S, R, or Q; X5 = null or S; X6 = null, D, or G;
X7 = null, V, or L; X8 = D, G, or S; X9 = S, G, A, or
I; X10 = H, Y, F, S, or N; X11 = R, N, D, or H; X12 =
Y, F, or W; X13 = V or L; X14 = S, N, or A
84
DX2X3X4X5X6X7
CDR-L2
X2 = F, V, N, or A; X3 = S, T, or N; X4 = K, V, N, or
Consensus
R; X5 = R, V, or L; X6 = P, K, A, or E; X7 = S, P, A,
or T
85
X1X2X3X4X5X6X7X8X9X10X11X12X13X14
CDR-L3
X1 = S, G, T, A, or Q; X2 = S, T, or Q; X3 = Y, W, or
Consensus
S; X4 = A, T, R, D, or Y; X5 = A, S, P, G, or N; X6 =
I, S, G, T, L, A, or H; X7 = S, P, or L; X8 = P, T, S,
Q, M, R, or null; X9 = N, L, A, M, or null; X10 = L or
null; X11 = Y, W, F, V, or L; X12 = V or T
86
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTC
Clone 192 scFv
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCAT
(nt)
GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA
TTAGTTGGAATAGTGGTAGGATAGGCTATGCGGACTCTGTAAAGGGCCGA
TTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTTTCTGCAAATGAA
CAGTCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCAGG
GGTATCATTACTATGATAGTGCCGAACATGCTTTTGATATCTGGGGCCAAG
GGACAATGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGC
TCTGGCGGTGGCGGATCGCAGGCTGTGCTGACTCAGCCTCGCTCAGTGTC
CGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAATCAGCAGTG
GTGTTGATAGTCATAGGTATGTCTCCTGGTACCAACACCACCCAGGCAAA
GCCCCCAAACTCATGATTTATGATTTCAGTAAGCGGCCCTCAGGGGTCCCT
GATCGTTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCT
GGGCTCCAGGCTGAGGATGAGGCTGATTACTATTGCTGCTCATATGCAGC
CATCTCCCCTAATTATGTCTTCGGAACTGGGACCAAGCTGACCGTCCTA
87
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
Clone 192 scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
(aa)
HYYDSAEHAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQAVLTQPRSVSGS
PGQSVTISCTGISSGVDSHRYVSWYQHHPGKAPKLMIYDFSKRPSGVPDRFSG
SKSGNTASLTISGLQAEDEADYYCCSYAAISPNYVFGTGTKLTVL
88
GAAGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTC
Clone 241 scFv
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCAT
(nt)
GCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTA
TTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCGA
TTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAA
CAGTCTGAGAGCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATCAGG
GGTATCATTACTATGATAGTGCCGAACATGCTTTTGATATCTGGGGCCAAG
GGACAGTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGC
TCTGGCGGTGGCGGATCGGCCATCCGGATGACCCAGTCTCCATCCTCCCTG
TCTGCATCTGTAGGAGACAGAGTCACCGTCACTTGCCAGGCGAGTCAGGA
CATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAGAGCCCCTA
AGCTCCTGATCTACGATGCATCCAATGTGAAAGCAGGGGTCCCATCAAGG
TTCAGTGGGGGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTG
CAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCT
CAGGCGTACACTTTTGGCCAGGGGACCAAGCTGGATATCAAA
89
EVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
Clone 241 scFv
SWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDQGY
(aa)
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSAIRMTQSPSSLSAS
VGDRVTVTCQASQDISNYLNWYQQKPGRAPKLLIYDASNVKAGVPSRFSGG
GSGTDFTLTISSLQPEDFATYYCQQSYSTPQAYTFGQGTKLDIK
90
AIRMTQSPSSLSASVGDRVTVTCQASQDISNYLNWYQQKPGRAPKLLIYDAS
VL
NVKAGVPSRFSGGGSGTDFTLTISSLQPEDFATYYCQQSYSTPQAYTFGQGTK
Clone 241 (aa)
LDIK
91
QAVLTQPRSVSGSPGQSVTISCTGISSGVDSHRYVSWYQHHPGKAPKLMIYDF
VL Clone 192
SKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAAISPNYVFGTGT
KLTVL
92
MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTW
CD19
SRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKA
Accession No.
WQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLY
P15391
VWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSR
Homo
GPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKY
Sapiens
YCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGIL
HLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGR
AQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSE
EDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEEL
TQPVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGILYAAPQLRSIRGQP
GPNHEEDADSYENMDNPDGPDPAWGGGGRMGTWSTR
93
DFSKRPS
CDR-L2
Clone 192B,
Clone 192
94
DVTVRPS
CDR-L2
Clone 285
95
DVTKRPS
CDR-L2
Clone 328
96
DNNKRPP
CDR-L2
Clone 1
97
DASNVKA
CDR-L2
Clone 1304
Clone 241
98
DASRLAS
CDR-L2
Clone 1300
99
DASNLET
CDR-L2
Clone 227
100
DASNRAT
CDR-L2
Clone 488
101
SSYAAISPNYV
CDR-L3
Clone 192B
102
CSYAAISPNYV
CDR-L3
Clone 192
103
GSYTSSSTLLWV
CDR-L3
Clone 285
104
TSYRPGPTFV
CDR-L3
Clone 328
105
ATWDSGLSAVV
CDR-L3
Clone 1
106
QQSYSTPQAYT
CDR-L3
Clone 1304
Clone 241
107
QQSYGAPMFT
CDR-L3
Clone 1300
108
QQYDNLPLT
CDR-L3
Clone 227
109
QQSYSHPRMYT
CDR-L3
Clone 488
110
X1X2X3X4X5X6X7X8X9X10X11X12X13X14
CDR-L1
X1 = T, Q, S, or R;
consensus
X2 = G or A;
X3 = I, T, D, or S;
X4 = S, R, T, or Q;
X5 = null or S;
X6 = null, D, D, or G;
X7 = null, V, or L;
X8 = X or null;
X9 = X or null;
X10 = X;
X11 = X;
X12 = Y, F, D, or W;
X13 = V, A, or L;
X14 = S, N, or A
111
X1X2X3X4X5X6X7X8X9X10X11X12X13X14
CDR-L1
X1 = T, Q, S, or R;
consensus
X2 = G or A;
X3 = I, T, D, or S;
X4 = S, R, T, or Q;
X5 = null or S;
X6 = G, D, N, or null;
X7 = null, V, or L;
X8 = D, G, I, L, S, or null;
X9 = S, G, A, I, R, or null;
X10 = H, Y, F, S, or N;
X11 = R, N, D, H, or Y;
X12 = Y, F, D, or W;
X13 = V, A, or L;
X14 = S, N, or A
112
X1X2X3X4X5X6X7
CDR-L2
X1 = D or S;
Consensus
X2 = F, V, N, K, or A;
X3 = S, T, D, or N;
X4 = K, V, N, Q, or R;
X5 = R, V, or L;
X6 = P, K, A, or E;
X7 = S, P, A, or T
113
X1X2X3X4X5X6X7X8X9X10X11X12
CDR-L3
X1 = C, S, A, G, or N;
X2 = S, A, or T;
X3 = Y, W, or R;
X4 = A or D;
X5 = G, D, or S;
X6 = R, S, or N;
X7 = Y, L, or G;
X8 = N or S;
X9 = S, N, or null;
X10 = null;
X11 = V, A, or W;
X12 = L or V.
114
X1X2X3X4X5X6X7X8X9X10X11X12
CDR-L3
X1 = S, G, T, A, Q, C, or N;
consensus
X2 = S, Q, A, or T;
X3 = Y, S, W, R;
X4 = A, D, R, T, or Y;
X5 = A, S, P, G, N, or D;
X6 = I, S, G, T, A, L, H, R, N;
X7 = S, P, L, Y, G;
X8 = P, T, S, Q, M, R, N or null
X9 = S, L, N, A, M or null;
X10 = L or null;
X11 = Y, W, F, V, A, or L;
X12 = V, T, or L
115
X1X2X3X4X5X6X7X8X9X10X11X12
CDR-L3
X1 = X;
consensus
X2 = S, Q, A, or T;
X3 = Y, S, W, R;
X4 = A, D, R, T, or Y;
X5 = X
X6 = X
X7 = S, P, L, Y, G;
X8 = X or null
X9 = X or null
X10 = L or null;
X11 = X
X12 = V, T, or L
116
GTWDISLRFGV
CDR-L3
Clone 79
117
CSYEAPTHTYV
CDR-L3
Clone 835
118
AAWDDSLNVV
CDR-L3
Clone 184
119
CSYAGSYTFEV
CDR-L3
Clone 505
120
CSFAGYYTYWL
CDR-L3
Clone 506
121
SSXAGRKYV
CDR-L3
Clone 305
122
GGGS
Linker
artificial
123
GGGGS
Linker
artificial
124
ESKYGPPCPPCP
spacer
(IgG4hinge)
(aa)
homo sapien
125
GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT
spacer
(IgG4hinge)
(nt)
homo sapien
126
ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW
Hinge-CH3
ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH
spacer
NHYTQKSLSLSLGK
Homo sapien
127
ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE
Hinge-CH2-
VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK
CH3 spacer
VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS
Homo sapien
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
MHEALHNHYTQKSLSLSLGK
128
RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEE
IgD-hinge-Fc
QEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHL
Homo sapien
TWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSL
WNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWL
LCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAP
PSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH
129
FWVLVVVGGVLACYSLLVTVAFIIFWV
CD28 (amino
acids 153-179
of Accession
No. P10747)
Homo sapien
130
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP
CD28 (amino
FWVLVVVGGVLACYSLLVTVAFIIFWV
acids 114-179
of Accession
No. P10747)
Homo sapien
Homo sapien
131
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
CD28 (amino
acids 180-220
of P10747)
Homo sapien
132
RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
CD28 (LL to
GG)
Homo sapien
133
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
4-1BB (amino
acids 214-255
of Q07011.1)
Homo sapien
134
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
CD3 zeta
RKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY
Homo sapien
QGLSTATKDTYDALHMQALP PR
135
RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR
CD3 zeta
KNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY
Homo sapien
QGLSTATKDTYDALHMQALP PR
136
RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR
CD3 zeta
RKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY
Homo sapien
QGLSTATKDTYDALHMQALP PR
137
LEGGGEGRGSLLTCGDVEENPGPR
T2A
artificial
138
MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTS
tEGFR
ISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLH
artificial
AFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYA
NTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSC
RNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNC
IQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGP
GLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM
139
MPPPCLLFFLLFLTPMEVRPQEPLVVKVEEGDNAVLQCLEGTSDGPTQQLVW
Rhesus
CRDSPFEPFLNLSLGLPGMGIRMGPLGIWLLIFNVSNQTGGFYLCQPGLPSEKA
macaque CD19
WQPGWTVSVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLNSSQLY
Accession No.
VWAKDRPEMWEGEPVCGPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVS
F7F486
RGPLSWTHVRPKGPKSSLLSLELKDDRPDRDMWVVDTGLLLTRATAQDAGK
YYCHRGNWTKSFYLEITARPALWHWLLRIGGWKVPAVTLTYLIFCLCSLVGI
LQLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLG
RAQRWAAGLGGTAPSYGNPSSDVQVDGAVGSRSPPGAGPEEEEGEGYEEPD
SEEGSEFYENDSNFGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDE
ELTQPVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGLLYAAPQLRTIRG
QPGPNHEEDADSYENMDNPDGPDPAWGGGGRMGTWSAR
140
HPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLT
V1
MSFHLEITARPVLWHWLLRTGGWK
chimeric/rhesus
human
corresponding
to residues 218
to 291 of
human CD19
141
RPKGPKSSLLSLELKDDRPDRDMWVVDTGLLLTRATAQDAGKYYCHRGNLT
V2
MSFHLEITARPVLWHWLLRTGGWK
chimeric/rhesus
human
corresponding
to residues 218
to 291 of
human CD19
142
HPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNWT
V3
KSFYLEITARPALWHWLLRIGGWK
chimeric/rhesus
human
corresponding
to residues 218
to 291 of
human CD19
143
HPKGPKSLLSLELKDDRPARDMWVMETGLLLP
Artificial
144
RPKGPKSSLLSLELKDDRPDRDMWVVDTGLLLT
Artificial
145
DQGXHXYDSAEHAFXI
CDR-H3
clone 305
146
QASQDISNYLN
CDR-L1
Clone 255
147
TGTGRDIGAYDYVS
CDR-L1
Clone 305
148
TETSSDLGGYNYVS
CDR-L1
Clone 327
149
TGASTDVGGYNYVS
CDR-L1
Clone 505
150
TGASSDVGGYDHVS
CDR-L1
Clone 506
151
SGSSSNIGSNTVN
CDR-L1
Clone 184
152
TGPISGVGDYTSVS
CDR-L1
Clone 835
153
DNNKRPS
CDR-L2
Clone 272
154
GVNKRPS
CDR-L2
Clone 305
155
DVNKRPS
CDR-L2
Clone 505
156
DNNKRPS
CDR-L2
Clone 79
157
DVTQRPS
CDR-L2
Clone 835
158
GTWDSSLNRDWV
CDR-L3
Clone 272
159
CSYAGRYNSVP
CDR-L3
Clone 508
160
TSGVGVG
CDR-H1
Clone 1265
161
LIYWDDDKRYSPSLKS
CDR-H2
Clone 1265
162
IDYGSGSYSPRTSYYYYMSV
CDR-H3
Clone 1265
163
RASQGISSYLN
CDR-L1
Clone 1265
164
AASNLQS
CDR-L2
Clone 1265
165
QQGDAFPLT
CDR-L3
Clone 1265
166
QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLALIY
VH
WDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCAHIDYGSG
Clone 1265
SYSPRTSYYYYMSVWGKGTTVTVSS
167
QVQLVQSGGGVVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSG
VH
ISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDQGY
Clone 213
HYYDSAEHAFDIWGQGTVVTVSS
168
EVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDQGY
Clone 255
HYYDSAEHAFDIWGQGTVVTVSS
169
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 272
HYYDSAEHAFDIWGQGTVVTVSS
170
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 283
HYYDSAEHAFDIWGQGTVVTVSS
171
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 302
HYYDSAEHAFDIWGQGTVVTVSS
172
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSXXGRXXISRDNAKNSLFLQMNSLRAEDTAXYYCAXDQGX
Clone 305
HXYDSAEHAFXIWGQGTVVTVSS
173
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 314
HYYDSAEHAFDIWGQGTVVTVSS
174
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 379
HYYDSAEHAFDIWGQGTVVTVSS
175
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 324
HYYDSAEHAFDIWGQGTVVTVSS
176
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAXNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 327
HYYDSAEHAFDIWGQGTVVTVSS
177
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 336
HYYDSAEHAFDIWGQGTVVTVSS
178
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 440
HYYDSAEHAFDIWGQGTVVTVSS
179
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 448
HYYDSAEHAFDIWGQGTVVTVSS
180
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 505
HYYDSAEHAFDIWGQGTMVTVSS
181
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 506
HYYDSAEHAFDIWGQGTMVTVSS
182
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 508
HYYDSAEHAFDIWGQGTVVTVSS
183
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 184
HYYDSAEHAFDIWGQGTMVTVSS
184
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 79
HYYDSAEHAFDIWGQGTVVTVSS
185
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRLAPGKGLEWVSGI
VH
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAKDTAVYYCARDQGY
Clone 835
HYYDSAEHAFDIWGQGTMVTVSS
186
AIQLTQSPSFLSASVGDRVTITCRASQGISSYLNWYQQRAGKAPELLIYAASNL
VL
QSGVPSRFSGSGSGTDFTLTITSVQPEDFATYFCQQGDAFPLTFGPGTKVTIR
Clone 1265
187
EIVLTQSPATLSLSPGETATLSCRASQSINHYLAWYQQKPGQAPRLLIYDASNR
VL
ATGIPARFSGSGSGTDFTLTISSLEPEDFATYYCQQSYSHPRMYTFGQGTKLEI
Clone 213
K
188
AIRMTQSPSSLSASVGDRVTVTCQASQDISNYLNWYQQKPGRAPKLLIYDAS
VL
NVKAGVPSRFSGGGSGTDFTLTISSLQPEDFATYYCQQSYSTPQAYTFGQGTK
Clone 255
LDIK
189
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNN
VL
KRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLNRDWVFGGG
Clone 272
TKLTVL
190
QSALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQHRPGKAPKLIIYDVT
VL
VRPSGVSDRFSGSKSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGT
Clone 283
KLTVL
191
QSALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQHRPGKAPKLIIYDVT
VL
VRPSGVSDRFSGSKSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGT
Clone 302
KLTVL
192
QSVLXXPXXASGSPGQSVTVSCTGTGRDIGAYDYVSWYQQHPGKAPKLLIYG
VL
VNKRPSGVPDRFSGSKSDNTASLTVSGLQVEDEADYYCSSXAGRKYVFGTGX
Clone 305
KVTVL
193
QSALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQHRPGKAPKLIIYDVT
VL
VRPSGVSDRFSGSKSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGT
Clone 314
KLTVL
194
QSALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQHRPGKAPKLIIYDVT
VL
VRPSGVSDRFSGSKSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGT
Clone 379
KLTVL
195
QSALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQHRPGKAPKLIIYDVT
VL
VRPSGVSDRFSGSKSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGT
Clone 324
KLTVL
196
QSALTQPASVSGSPGQSITISCTETSSDLGGYNYVSWYQHRPGKAPKLIIYDVT
VL
VRPSGVXDRFSGSKSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGT
Clone 327
KLTVL
197
QSALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQHRPGKAPKLIIYDVT
VL
VRPSGVSDRFSGSKSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGT
Clone 336
KLTVL
198
QSALTQPASVSGSPGHSITISCTGTRSDVGGFDYVSWYQHNPGKAPKLIIYDVT
VL
KRPSGVSNRFSGAKSGITASLTISGLQAEDEADYYCTSYRPGPTFVFGTGTKL
Clone 440
DIK
199
QSALTQPASVSGSPGQSITISCTGTSSDLGGYNYVSWYQHRPGKAPKLIIYDVT
VL
VRPSGVSDRFSGSKSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGT
Clone 448
KLDIK
200
QSVLTQPRSLSGSPGQSVTIACTGASTDVGGYNYVSWYQQHPGKAPKLMIYD
VL
VNKRPSGVPDRFSGSKSGNTAFLTISGLQAEDEADYYCCSYAGSYTFEVFGG
Clone 505
GTKLTVL
201
QLVLTQPPSVSGSPGQSVTFSCTGASSDVGGYDHVSWYQHHPGKGPKLLIYD
VL
VSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSFAGYYTYWLFGG
Clone 506
GTKVTVL
202
QSALTQPRSVSGFPGQSVTISCTGTTSDDVSWYQQHPGKAPQLMLYDVSKRP
VL
SGVPHRFSGSRSGRAASLIISGLQTEDEADYFCCSYAGRYNSVPFGGGTKLTV
Clone 508
L
203
SYVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQFPGTAPKLLIYSNNQ
VL
RPSGVPDRFSGSKSGTSASLAISGLQSEDEAEYYCAAWDDSLNVV
Clone 184
204
QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNN
VL
KRPSGIPDRFSGSKSGTSATLGITGLQTGDEGDYYCGTWDISLRFGVFGGGTK
Clone 79
VTVL
205
QSVLTQPRSVSGSPGQSVTISCTGPISGVGDYTSVSWYQHYPGKTPKLIIYDVT
VL
QRPSGVPNRFSGSKSGNTASLTISGLQADDEADYYCCSYEAPTHTYVFGTGTK
Clone 835
LTVL
206
QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLALIY
scFv
WDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCAHIDYGSG
Clone 1265
SYSPRTSYYYYMSVWGKGTTVTVSSGGGGSGGGGSGGGGSAIQLTQSPSFLS
ASVGDRVTITCRASQGISSYLNWYQQRAGKAPELLIYAASNLQSGVPSRFSGS
GSGTDFTLTITSVQPEDFATYFCQQGDAFPLTFGPGTKVTIR
207
QVQLVQSGGGVVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSG
scFv
ISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDQGY
Clone 213
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLS
PGETATLSCRASQSINHYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGS
GTDFTLTISSLEPEDFATYYCQQSYSHPRMYTFGQGTKLEIK
208
EVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
scFv
SWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDQGY
Clone 255
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSAIRMTQSPSSLSAS
VGDRVTVTCQASQDISNYLNWYQQKPGRAPKLLIYDASNVKAGVPSRFSGG
GSGTDFTLTISSLQPEDFATYYCQQSYSTPQAYTFGQGTKLDIK
209
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 272
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSAA
PGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSK
SGTSATLGITGLQTGDEADYYCGTWDSSLNRDWVFGGGTKLTVL
210
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 283
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGS
PGQSITISCTGTSSDLGGYNYVSWYQHRPGKAPKLIIYDVTVRPSGVSDRFSGS
KSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGTKLTVL
211
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 302
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGS
PGQSITISCTGTSSDLGGYNYVSWYQHRPGKAPKLIIYDVTVRPSGVSDRFSGS
KSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGTKLTVL
212
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
scFv
SWNSGRIGYADSXXGRXXISRDNAKNSLFLQMNSLRAEDTAXYYCAXDQGX
Clone 305
HXYDSAEHAFXIWGQGTVVTVSSGGGGSGGGGSGGGGSQSVLXXPXXASGS
PGQSVTVSCTGTGRDIGAYDYVSWYQQHPGKAPKLLIYGVNKRPSGVPDRFS
GSKSDNTASLTVSGLQVEDEADYYCSSXAGRKYVFGTGXKVTVL
213
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 314
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGS
PGQSITISCTGTSSDLGGYNYVSWYQHRPGKAPKLIIYDVTVRPSGVSDRFSGS
KSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGTKLTVL
214
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 379
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGS
PGQSITISCTGTSSDLGGYNYVSWYQHRPGKAPKLIIYDVTVRPSGVSDRFSGS
KSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGTKLTVL
215
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 324
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGS
PGQSITISCTGTSSDLGGYNYVSWYQHRPGKAPKLIIYDVTVRPSGVSDRFSGS
KSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGTKLTVL
216
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
scFv
SWNSGRIGYADSVKGRFTISRDNAXNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 327
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGS
PGQSITISCTETSSDLGGYNYVSWYQHRPGKAPKLIIYDVTVRPSGVXDRFSGS
KSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGTKLTVL
217
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 336
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGS
PGQSITISCTGTSSDLGGYNYVSWYQHRPGKAPKLIIYDVTVRPSGVSDRFSGS
KSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGTKLTVL
218
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 440
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGS
PGHSITISCTGTRSDVGGFDYVSWYQHNPGKAPKLIIYDVTKRPSGVSNRFSG
AKSGITASLTISGLQAEDEADYYCTSYRPGPTFVFGTGTKLDIK
219
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 448
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGS
PGQSITISCTGTSSDLGGYNYVSWYQHRPGKAPKLIIYDVTVRPSGVSDRFSGS
KSGNTASLTISGLQAEDEADYYCGSYTSSSTLLWVFGGGTKLDIK
220
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 505
HYYDSAEHAFDIWGQGTMVTVSSQSVLTQPRSLSGSPGQSVTIACTGASTDV
GGYNYVSWYQQHPGKAPKLMIYDVNKRPSGVPDRFSGSKSGNTAFLTISGLQ
AEDEADYYCCSYAGSYTFEVFGGGTKLTVL
221
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 506
HYYDSAEHAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQLVLTQPPSVSGS
PGQSVTFSCTGASSDVGGYDHVSWYQHHPGKGPKLLIYDVSKRPSGVPDRFS
GSKSGNTASLTISGLQAEDEADYYCCSFAGYYTYWLFGGGTKVTVL
222
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 508
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSALTQPRSVSGFP
GQSVTISCTGTTSDDVSWYQQHPGKAPQLMLYDVSKRPSGVPHRFSGSRSGR
AASLIISGLQTEDEADYFCCSYAGRYNSVPFGGGTKLTVL
223
QVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 184
HYYDSAEHAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSSYVLTQPPSASGT
PGQRVTISCSGSSSNIGSNTVNWYQQFPGTAPKLLIYSNNQRPSGVPDRFSGSK
SGTSASLAISGLQSEDEAEYYCAAWDDSLNVV
224
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGI
scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARDQGY
Clone 79
HYYDSAEHAFDIWGQGTVVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSAA
PGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSK
SGTSATLGITGLQTGDEGDYYCGTWDISLRFGVFGGGTKVTVL
225
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRLAPGKGLEWVSGI
scFv
SWNSGRIGYADSVKGRFTISRDNAKNSLFLQMNSLRAKDTAVYYCARDQGY
Clone 835
HYYDSAEHAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPRSVSGS
PGQSVTISCTGPISGVGDYTSVSWYQHYPGKTPKLIIYDVTQRPSGVPNRFSGS
KSGNTASLTISGLQADDEADYYCCSYEAPTHTYVFGTGTKLTVL
226
X1X2X3X4X5X6X7X8X9X10X11X12X13X14
CDR-L1
X1 = T, Q, R, or S;
consensus
X2 = G, A, or E;
X3 = I, T, S, D, A, or P;
X4 = S, R, Q, G, or I;
X5 = null, S, R, or T;
X6 = G, D, N, or null;
X7 = V, L, null, or I;
X8 = D, G, S, I, L, or null;
X9 = S, G, A, I, null, or D;
X10 = H, Y, F, S, or N;
X11 = R, N, D, H, Y, or T;
X12 = Y, F, W, D, H, T, or S;
X13 = V, A, or L;
X14 = S, N, or A
227
X1X2X3X4X5X6X7
CDR-L2
X1 = D, S, or G;
consensus
X2 = F, V, N, K, or A;
X3 = S, T, N, or D;
X4 = K, V, N, R, or Q;
X5 = R, V, or L;
X6 = P, K, A, or E;
X7 = S, P, A, or T
228
X1X2X3X4X5X6X7X8X9X10X11X12
CDR-L3
X1 = S, G, T, A, Q, C, or N;
consensus
X2 = S, Q, A, or T;
X3 = Y, S, W, or R;
X4 = A, D, R, T, or Y;
X5 = A, S, P, G, N, or D;
X6 = I, S, G, T, A, L, H, R, or N;
X7 = S, P, L, Y, or G;
X8 = P, T, S, Q, M, R, or N;
X9 = S, L, N, A, M, null, or R;
X10 = L, null, or D;
X11 = Y, W, F, V, A, or L;
X12 = V, T, L, or P
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14839911
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juno therapeutics, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Mar 30th, 2022 06:04PM
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Mar 30th, 2022 06:04PM
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Bristol-Myers Squibb
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Health Care
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Pharmaceuticals & Biotechnology
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