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cph:gen
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Genmab A/S
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Nov 23rd, 2021 12:00AM
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Jul 5th, 2013 12:00AM
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https://www.uspto.gov?id=US11180572-20211123
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Dimeric protein with triple mutations
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The present invention relates to dimeric proteins comprising amino acids at three different positions which are different from those present in a human IgG1. Six of said dimeric proteins are capable of forming a hexameric structure in solution.
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11180572
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1. A dimeric protein comprising a first Fc polypeptide and a second Fc polypeptide, each Fc polypeptide comprising at least CH2 and CH3 regions of a human IgG1, IgG2, IgG3, or IgG4, wherein in one or both Fc polypeptides
(a) the amino acid in the position corresponding to E345 in a human IgG1 heavy chain is R or K,
(b) the amino acid in the position corresponding to E430 in a human IgG1 heavy chain is G or S, and
(c) the amino acid in the position corresponding to S440 in a human IgG1 heavy chain is Y or W,
wherein the numbering is according to the EU Index as set forth in Kabat.
2. The dimeric protein of claim 1, wherein in one or both Fc polypeptides, the amino acids at the positions corresponding to E345, E430, and S440 are R, G, and Y, respectively; K, G, and Y, respectively; R, S, and Y, respectively; or R, G, and W, respectively.
3. The dimeric protein of claim 1, wherein one or both Fc polypeptides further comprises a region capable of covalent binding between said first and second Fc polypeptides.
4. The dimeric protein of claim 1, wherein one or both Fc polypeptides further comprises a hinge region of an immunoglobulin heavy chain.
5. The dimeric protein of claim 3, wherein said first and second Fc polypeptides are interconnected via hinge region disulphide bonds.
6. The dimeric protein of claim 1, which is an antibody.
7. The dimeric protein of claim 6, wherein one or both Fc polypeptides comprise a full-length heavy chain constant region.
8. The dimeric protein of claim 1, further comprising a drug, toxin, radiolabel, radioopaque agent, paramagnetic agent, fluorescent agent, phosphorescent agent, ultrasound enhancing agent, or polyethyleneglycol (PEG).
9. The dimeric protein of claim 1, which is a homodimer or a heterodimer.
10. The dimeric protein of claim 1, which is a heterodimer.
11. The dimeric protein of claim 1, which is predominantly in oligomeric form in a phosphate buffer at a pH of about 6.8.
12. The dimeric protein of claim 1, which is predominantly in monomeric form at a pH of less than 6.0.
13. An oligomer comprising at least two non-covalently associated dimeric proteins, each according to claim 1.
14. A hexamer comprising six non-covalently associated dimeric proteins, each according to claim 1.
15. The hexamer of claim 14, wherein at least one dimeric protein of the hexamer is an antibody.
16. A hexamer comprising six non-covalently associated molecules, at least one of which is a dimeric protein according to claim 1, and at least one of which is an antibody comprising an Fc domain comprising at least CH2, CH3 and hinge regions.
17. The hexamer of claim 16, wherein the antibody is a monoclonal or polyclonal antibody.
18. A composition comprising the dimeric protein of claim 1, and a pharmaceutically acceptable carrier.
19. A composition comprising the dimeric protein of claim 1, one or more antibodies, and a pharmaceutically acceptable carrier.
20. A composition comprising first and second dimeric proteins according to claim 1, and a pharmaceutically acceptable carrier.
21. A composition comprising at least three dimeric proteins according to claim 1.
22. The composition of claim 20, wherein in one or both of said first and second Fc polypeptides of said first and second dimeric proteins, the amino acids at the positions corresponding to E345, E430, and S440 in a human IgG1 heavy chain are R, G, and Y, respectively.
23. The composition of claim 20, wherein at least one of the first and second dimeric proteins is an antibody.
24. The composition of claim 23, wherein both the first and the second dimeric proteins are antibodies.
25. The composition of claim 24, wherein the first and second antibodies bind to the same epitope of the same antigen.
26. The composition of claim 25, wherein the first and second antibodies comprise the same variable heavy and light chain region sequences.
27. The composition of claim 20, wherein the pharmaceutically acceptable carrier is an aqueous buffered solution, wherein the pH is at least about 6.5.
28. The composition of claim 20, wherein the pharmaceutically acceptable carrier is an aqueous buffered solution, wherein the pH is less than pH 6.5.
29. The composition of claim 28, comprising an acetate, histidine, glycine, citrate, nicotinate, lactate, or succinate buffer system.
30. A kit-of-parts comprising at least one dimeric protein according to claim 1.
31. The dimeric protein of claim 1, wherein in one or both Fc polypeptides, the amino acids in the positions corresponding to E345, E430, and S440 are R, G, and Y, respectively.
32. The dimeric protein of claim 1, wherein in one or both Fc polypeptides, the amino acids in the positions corresponding to E345, E430, and S440 are K, G, and Y, respectively.
33. The dimeric protein of claim 1, wherein in one or both Fc polypeptides, the amino acids in the positions corresponding to E345, E430, and S440 are R, S, and Y, respectively.
34. The dimeric protein of claim 1, wherein in one or both Fc polypeptides, the amino acids in the positions corresponding to E345, E430, and S440 are R, G, and W, respectively.
35. A polypeptide comprising an Fc region of a human IgG1, IgG2, IgG3, or IgG4, wherein:
(a) the amino acid in the position corresponding to E345 in a human IgG1 heavy chain is selected from the group consisting of R and K,
(b) the amino acid in the position corresponding to E430 in a human IgG1 heavy chain is selected from the group consisting of G and T, and
(c) the amino acid in the position corresponding to S440 in a human IgG1 heavy chain is selected from the group consisting of Y and W,
wherein the numbering is according to the EU Index as set forth in Kabat.
36. The dimeric protein of claim 1, wherein one or both Fc polypeptides comprise at least CH2 and CH3 regions of human IgG1.
37. A dimeric protein comprising a first Fc polypeptide and a second Fc polypeptide, each Fc polypeptide comprising at least CH2 and CH3 regions of a human IgG1, IgG2, IgG3, or IgG4, wherein in one or both Fc polypeptides, the amino acids at positions corresponding to E345, E430, and S440 in a human IgG1 heavy chain are
(a) R, G, and Y, respectively;
(b) K, G, and Y, respectively;
(c) R, S, and Y, respectively; or
(d) R, G, and W, respectively.
38. An antibody comprising a first Fc polypeptide and a second Fc polypeptide, each Fc polypeptide comprising at least CH2 and CH3 regions of a human IgG1, IgG2, IgG3, or IgG4, wherein in one or both Fc polypeptides
(a) the amino acid in the position corresponding to E345 in a human IgG1 heavy chain is selected from the group consisting of R and K,
(b) the amino acid in the position corresponding to E430 in a human IgG1 heavy chain is selected from the group consisting of G, and S, and
(c) the amino acid in the position corresponding to S440 in a human IgG1 heavy chain is selected from the group consisting of Y and W,
wherein the numbering is according to the EU Index as set forth in Kabat.
39. The antibody of claim 38, wherein in one or both Fc polypeptides, the amino acids at positions corresponding to E345, E430, and S440 in a human IgG1 heavy chain are
(a) R, G, and Y, respectively;
(b) K, G, and Y, respectively;
(c) R, S, and Y, respectively; or
(d) R, G, and W, respectively.
40. The antibody of claim 38, wherein each Fc polypeptide comprises at least the CH2 and CH3 regions of a human IgG1.
41. The antibody of claim 38, which is predominantly in monomeric form at a pH of less than 6.0.
42. The antibody of claim 38, which is predominantly in oligomeric form in a phosphate buffer at a pH of about 6.8.
43. The composition of claim 20, wherein
in one or both of said first and second Fc polypeptides of said first dimeric protein the amino acids at position corresponding to E345, E430 and S440 in a human IgG1 heavy chain are
(a) R, and Y, respectively;
(b) K, G, and Y, respectively;
(c) R, S, and Y, respectively; or
(d) R, G, and W, respectively.
44. The composition of claim 18, wherein
(a) in both Fc polypeptides of the dimeric protein, the amino acids corresponding to E345, E430 and S440 in human IgG1 heavy chain are R, G and Y, respectively; and
(b) the composition further comprises a second dimeric protein comprising a first Fc polypeptide and a second Fc polypeptide, each Fc polypeptide comprising at least CH2 and CH3 regions of a human IgG1, IgG2, IgG3, or IgG4, wherein in both Fc polypeptides of the second dimeric protein the amino acids corresponding to E345, E430 and S440 in a human IgG1 heavy chain are R, G and K, respectively.
45. The composition of claim 18, wherein in one or both Fc polypeptides, the amino acids at the positions corresponding to E345, E430, and S440 are R, G, and Y, respectively; K, G, and Y, respectively; R, S, and Y, respectively; or R, G, and W, respectively.
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RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national stage filing of International Application PCT/EP2013/064330, filed on Jul. 5, 2013, which claims priority to International Application PCT/EP2012/063339, filed on Jul. 6, 2012, U.S. Patent Application No. 61/751,045, filed on Jan. 10, 2013, and Danish Patent Application PA201300019, filed on Jan. 10, 2013. The contents of the aforementioned applications are hereby incorporated by reference.
FIELD OF THE INVENTION
The present invention relates to dimeric proteins, e.g. antibodies, comprising at least three mutations as compared to a parent dimeric protein. More particularly, the present invention relates to such dimeric proteins which are capable of forming oligomeric, e.g hexameric structures in solution. The present invention also relates to uses of such dimeric proteins and compositions comprising such dimeric proteins.
BACKGROUND OF THE INVENTION
The effector functions mediated by the Fc region of an antibody allow for the destruction of foreign entities, such as the killing of pathogens and the clearance and degradation of antigens. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) is initiated by binding of the Fc region to Fc receptor (FcR)-bearing cells, whereas complement-dependent cytotoxicity (CDC) is initiated by binding of the Fc region to C1q, which initiates the classical route of complement activation.
Each IgG antibody contains two binding sites for C1q, one in each heavy chain constant (Fc) region. A single molecule of IgG in solution, however, does not activate complement as the affinity of monomeric IgG for C1q is quite weak (Kd˜10−4 M) (Sledge et al., 1973 J. Biol. Chem. 248, 2818-13; Hughes-Jones et al., 1979 Mol. Immunol. 16, 697-701). Antigen-driven association of IgG can lead to much tighter binding of the multivalent C1q molecule (Kd˜10−8 M) and complement activation (Burton et al., 1990 Mol. Immunol. 22, 161-206). In contrast, IgM exists naturally in covalently bound penta- or hexamers, and upon binding of cellular expressed or immobilized antigen IgM pentamers and hexamers can efficiently elicit CDC. Antigen-binding is a requirement to induce a conformational change in IgM to expose the C1q binding sites (Feinstein et al., 1986, Immunology Today, 169-174).
It has been suggested that also IgG can achieve complement activation by the formation of hexameric ring structures, through interaction of the CH2/CH3 domains of the Fc region (Burton et al., 1990 Trends in Biochem. Sci. 15, 64-69). Evidence supporting the existence of such hexameric IgG structures has been found in two dimensional (Reidler et al., 1986 I Handbook of Experimental Immunology 4th edit. (Weir, D. M. ed.), pp 17.1-17.5. Blackwell, Edinburgh; Pinteric et al., 1971 Immunochem. 8, 1041-5) and three dimensional crystals, as well as for IgG1, IgG2a and IgG4 and human Fc in solution (Kuznetsov et al., 2000 J Struct. Biol. 131, 108-115). A hexameric ring formation was also observed in the crystal structure of the b12 human IgG1κ antibody directed against HIV-1 gp120 (1HZH in PDB) (Saphire et al., Science 2001 Aug. 10; 293(5532), 1155-9). In the b12 hexamer ring, six accessible C1q binding sites were presented at the hexamer surface, one from each of the six antibodies, while the other six binding sites faced downwards.
C1q resembles a bunch of tulips with six globular heads, containing the antibody combining regions, tethered to six collagenous stalks [Perkins et al., 1985 Biochem J. 228, 13-26; Poon et al., 1983 J Mol Biol. 168, 563-77; Reid et al., 1983 Biochem Soc Trans 11, 1-12; Weiss et al., 1986 J. Mol. Biol. 189, 573-81]. C1q was found to fit onto the b12 hexameric assembly of the 1HZH crystal structure, so that each of the six globular heads were in contact with one of the six C1q binding sites (Parren, FASEB Summer Research Conference, Snowmass, Co., 5-10 Jul. 2010; “Crystal Structure of an intact human IgG: implications for HIV-1 neutralization and effector Function”, Thesis by Erica Ollmann Saphire, for the Scripps Research Institute, La Jolla, Calif. November 2000). Mutations in selected amino acids in the Fc interfaces observed between symmetry-related b12 antibodies in the crystal structure were observed to decrease the binding avidity of C1q, indicating the contribution of these amino acids to the intermolecular Fc:Fc interaction.
Mekhaiel D N A et al, Nature Scientific Reports, 1:124, 19 Oct. 2011, disclose polymeric human Fc-fusion proteins with modified effector functions.
WO0042072 disclose polypeptide variants with altered effector functions.
US20080089892 disclose Fc region variants.
WO2006105062 disclose altered antibody Fc regions and uses thereof.
The present invention relates to dimeric proteins comprising certain amino acid residues, wherein six of said dimeric proteins are capable of forming non-covalent hexameric forms in solution.
SUMMARY OF THE INVENTION
In one aspect the present invention relates to a dimeric protein comprising a first and a second polypeptide, each polypeptide comprising at least CH2 and CH3 regions of an immunoglobulin heavy chain, wherein in said first and/or second polypeptides
the amino acids in the positions corresponding to E345 and E430 in a human IgG1 heavy chain are not E and
the amino acid in at least one position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254 is Y, K, R, or W; not Y; not D or E; not T; not E; not N; not Q; not I; and not S, for each position, respectively.
In another aspect the present invention relates to an oligomer comprising at least two non-covalently associated dimeric proteins of the present invention.
In another aspect the present invention relates to a hexamer comprising six non-covalently associated dimeric of the present invention.
In another aspect the present invention relates to a composition comprising the dimeric protein of the present invention, one or more antibodies, and a pharmaceutically acceptable carrier.
In another aspect the present invention relates to a composition comprising a first dimeric protein according to the present invention, a second dimeric protein according to the present invention, and optionally a pharmaceutically acceptable carrier.
In another aspect the present invention relates to a method of increasing oligomerization in solution and/or an effector function of a dimeric protein comprising a first and second polypeptide, each comprising at least CH2 and CH3 regions of an immunoglobulin heavy chain, the method comprising introducing into said first and/or second polypeptide, amino acid substitutions in at least the positions corresponding to E345, E430 and in a position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254 in a human IgG1 heavy chain.
In another aspect the present invention relates to a variant dimeric protein prepared by the method of the present invention.
In another aspect the present invention relates to a kit-of-parts comprising a first dimeric protein according to the present invention and a second dimeric protein according to the present invention for simultaneous, separate or sequential use in imaging, diagnostics or therapy.
In another aspect the present invention relates to a method for imaging of at least a part of the body of a human or other mammal, comprising administering a dimeric protein, oligomer, hexamer, composition or kit-of-parts according to the present invention.
In another aspect the present invention relates to a method for treating a bacterial, viral or parasitic infection, for imaging of at least a part of the body of human or other mammal, or for modulating clearance of a target molecule from the body of a human or other mammal, comprising administering a dimeric protein, oligomer, hexamer, composition or kit-of-parts according to the present invention.
In another aspect the present invention relates to a method for preventing or treating a disease, such as cancer, auto-immune diseases, organ transplant rejections, and C1q depletion in the humoral system, comprising administration of a dimeric protein, oligomer, hexamer, composition, kit-or-parts according to the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: (A) Schematic representation of IgG molecules in hexamer formation.
FIG. 2: Sequence alignment of the human IgG1, IgG1f, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE and IgM Fc segments corresponding to residues P247 to K447 in the IgG1 heavy chain, using Clustal 2.1 software, as numbered by the Eu index as set forth in Kabat. The sequence of the shown IgG1 (SEQ ID NO:6) represents residues 130 to 330 of the human IgG1 heavy chain constant region (SEQ ID NO:1; UniProt accession No. P01857) and the sequence of the shown IgG1m(f) (SEQ ID NO:7) represents residues 130 to 330 of the allotypic variant IgG1m(f) (SEQ ID NO:5); the sequence of the shown IgG2 (SEQ ID NO:8) represents residues 126 to 326 of the IgG2 heavy chain constant region (SEQ ID NO:2; UniProt accession No. P01859); and the sequence of the shown IgG3 (SEQ ID NO:9) represents residues 177 to 377 of the IgG3 heavy chain constant region (SEQ ID NO:3; UniProt accession No. P01860); and the sequence of the shown IgG4 (SEQ ID NO:10) represents residues 127 to 327 of the IgG4 heavy chain constant region (SEQ ID NO:4; UniProt accession No. P01861); and the sequence of the shown IgE (SEQ ID NO:11) represents residues 225-428 of the IgE constant region (Uniprot accession No. P01854); and the sequence of the shown IgA1 (SEQ ID NO:12) represents residues 133-353 of the IgA1 constant region (Uniprot accession No. P01876); and the sequence of the shown IgA2 (SEQ ID NO:13) represents residues 120-340 of the IgA2 constant region (SEQ ID NO:8; Uniprot accession No. P01877); and the sequence of the shown IgM (SEQ ID NO:14) represents residues 230-452 of the IgM constant region (Uniprot accession No. P01871); and the sequence of the shown IgD (SEQ ID NO:15) represents residues 176-384 of the IgD constant region (Uniprot accession No. P01880).
FIGS. 3A and 3B: Sequence alignment of anti-EGFr antibody 2F8 in an IgG1 (SEQ ID NO:3), IgG4 (SEQ ID NO:5) and (partial) IgG3 (SEQ ID NO:6) backbone. Amino acid numbering according to Kabat and according to the Eu-index are depicted (both described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
FIG. 4: Detailed view of the K439/S440 interactions between the Fc of adjacent molecules (Fc and Fc′, respectively) in an oligomeric (e.g., hexameric) arrangement, illustrating the interaction between wild-type, unmodified Fc and Fc′ molecules.
FIGS. 5A-5D: CDC mediated by mutants of CD38 antibody HuMAb 005 on CD38-positive cells. (FIG. 5A) CDC efficacy on Daudi cells by a concentration series of 005 mutants. (FIG. 5B) CDC efficacy on Raji cells by a concentration series of HuMAb 005 mutants. (FIG. 5C) CDC efficacy of E345R mutant of HuMAb 005 with either 20% or 50% NHS on Wien133 cells. (FIG. 5D) CDC efficacy of E345R mutants of HuMAb 005 and 7D8 with either 20% or 50% NHS on Raji cells. Unpurified antibody samples isolated from transient transfections were tested. As a negative control, supernatant of mock-transfected cells was used.
FIG. 6: CDC by wild type and E345R mutants of CD38 antibody HuMAb 005 in a competition experiment with an Fc-binding peptide. Cell lysis was measured after CDC on antibody-opsonized Daudi-cells incubated with a concentration series of the Fc-binding DCAWHLGELVWCT peptide (SEQ ID NO:7). Unpurified antibody samples isolated from transient transfections were used. As a negative control, supernatant of mock-transfected cells was used.
FIGS. 7A-7D: CDC on CD20- and CD38-positive Wien133 cells by CD20 antibody 7D8 mutants (FIG. 7A), CD38 antibody 005 mutants (FIG. 7B), mixtures of CD38 antibody 005 mutants and CD20 antibody 7D8 mutants (FIG. 7C) and (FIG. 7D).
FIGS. 8A and 8B: Evaluation of the in vivo efficacy of IgG1-005-E345R in a subcutaneous xenograft model with Raji-luc #2D1 cells.
FIG. 9: CDC on CD38-positive, EGFR-negative Wien133 cells by CD38/EGFR bispecific antibody with the E345R mutation.
FIGS. 10A and 10B: CDC on CD20-positive, CD38-negative Wien133 cells or Raji cells by CD20/CD38 bispecific antibody with and without the E345R mutation.
FIG. 11: CDC on EGFR-positive A431 cells by EGFR antibody 2F8 with the E345R mutation.
FIGS. 12A and 12B: CDC mediated by E345R mutant antibodies.
FIG. 13: Colocalization analysis of TF antibodies (FITC) with lysosomal marker LAMP1 (APC).
FIGS. 14A-14D: Introduction of E345R resulted in enhanced CDC-mediated killing compared to wild type rituximab tested on different B cell lines.
FIG. 14E: Introduction of E345R resulted in increased maximal CDC-mediated killing compared to wild type rituximab, independent of the expression levels of the complement regulatory proteins CD46 (A), CD55 (B) or CD59 (C) in different B cell lines with comparable CD20 expression levels.
FIGS. 15A-15D: CDC kinetics. E345R antibodies result in more rapid and more substantial target cell lysis by CDC compared to wild type antibodies.
FIG. 16: CDC kinetics. Introduction of the E345R mutation in the bispecific CD38×CD20 antibody results in more rapid and more substantial CDC-mediated target cell lysis.
FIG. 17: CDC kinetics. Introduction of the E345R mutation in bispecific antibody EGFRxCD38 that binds monovalently to the EGFR-negative Raji cells, results in more rapid and more substantial CDC-mediated target cell lysis than the bispecific EGFRxCD38 without E345R mutation.
FIGS. 18A-18F: CDC on Wien133 cells by a combination of a wild type antibody with a mutant antibody containing (FIGS. 18A-18C) E345R and Q386K or (FIGS. 18D-18F) E345R, E430G and Q386K. IgG1-b12 mutants do not bind Wien133 cells and were used as negative control antibodies.
FIGS. 19A and 19B: CDC efficacy of IgG1, IgG2, IgG3 and IgG4 isotype antibodies containing the E345R mutation.
FIGS. 20A and 20B: Introduction of the Fc-Fc stabilizing E345R mutation in wild type CD38 antibody 005 results in enhanced killing of primary CLL cells in an ex vivo CDC assay (average±standard error of the mean).
FIG. 21: PAGE analysis of antibody variant IgG1-005-E345R/E430G/S440Y. Left panel: SDS-PAGE, non-reducing conditions. Middle panel: SDS-PAGE, reducing conditions. Right panel: native PAGE. Note: lane 1: IgG1-b12 control antibody. Lane 2: IgG1-005-E345R/E430G. Lane 3: IgG1-005-E345R/E430G/S440Y.
FIG. 22: HP-SEC analysis of wild type IgG1-005 antibody. Fraction monomer (i.e. single antibodies) was estimated at >99%.
FIG. 23: HP-SEC analysis of antibody variant IgG1-005-E345R/E430G/S440Y. Fraction oligomer was estimated at approximately 79%.
FIG. 24: Overlay of the HP-SEC profiles of wild type IgG1-005 antibody (dashed line) and IgG1-005-E345R/E430G/S440Y (solid line).
FIG. 25: C1q binding ELISA with IgG1-005, IgG1-005-E345R/E430G/S440Y and IgG1-005-E345R. Concentration series of the indicated antibodies were coated to the wells and incubated with a fixed concentration C1q.
FIG. 26: CDC efficacy by a concentration series of IgG1-005-WT, IgG1-005-E345R/E430G/S440Y and IgG1-005-E345R on CD38-positive Ramos cells.
FIG. 27: ADCC Reporter assay using CD38-positive Raji cells and a concentration series of IgG1-005-WT, IgG1-005-E345R/E430G/S440Y and IgG1-005-E345R
FIG. 28: Plasma human IgG concentrations in SCID mice in time as determined by total human IgG ELISA. Black circles: wild type IgG1-005; black triangles: IgG1-005-E345R/E430G/S440Y.
FIG. 29: Clearance rate of administered human IgG in SCID mice as determined by the anti-CD38 ELISA. Black circles: wild type IgG1-005; black triangles: IgG1-005-E345R/E430G/S440Y.
FIG. 30: HP-SEC profile of IgG1-005 in 0.1 M Na2SO4/0.1 M sodium phosphate pH 6.8.
FIG. 31: HP-SEC profile of IgG1-005-E345R/E430G/S440Y in 0.1 M Na2SO4/0.1 M sodium phosphate pH 6.8.
FIG. 32: Overlay of HP-SEC profiles of IgG1-005 in 0.15 M NaCl/0.1 M citrate pH 6.8 (dashed line) and pH 5.0 (solid line).
FIG. 33: HP-SEC profile of IgG1-005-E345R/E430G/S440Y in 0.15 M NaCl/0.1 M citrate pH 6.8 (dashed line) and pH 5.0 (solid line).
FIGS. 34A-34D: HP-SEC analysis of triple mutant antibodies IgG1-005-RGY, IgG-7D8-RGY, IgG1-ritux-RGY, IgG1-2F8-RGY and IgG1-M1-RGY.
FIGS. 35A and 35B: E345R/E430G/S440Y triple mutant CD20 antibodies show enhanced killing of primary CD20-positive CLL cells in an ex vivo assay with 7D8-derived antibodies (FIG. 35A) and rituximab-derived antibodies (FIG. 35B).
FIGS. 36A-36D: HP-SEC analysis of IgG1-005-RGY (FIG. 36A), IgG2-005-RGY (FIG. 36B), IgG3-005-RGY (FIG. 36C) and IgG4-005-RGY (FIG. 36D). Percentages indicate total multimers as fraction of total peak area.
FIGS. 37A and 37B: CDC efficacy by a concentration series of 005 antibody variants in different IgG isotype backbones on CD38-positive Daudi (FIG. 37A) and Wien133 (FIG. 37B) cells.
FIGS. 38A-38D: HP-SEC analysis of IgG1-005-KGY (FIG. 38A), IgG1-005-RSY (FIG. 38B), IgG1-005-RGW (FIG. 38C) and IgG1-005-RGI (FIG. 38D).
FIGS. 39A and 39B: CDC efficacy by a concentration series of IgG1-005-RGY, IgG1-005-KGY, IgG1-005-RSY, IgG1-005-RGW, and IgG1-005-RGI on CD38-positive Wien133 (FIG. 39A) and Ramos (FIG. 39B) cells.
FIG. 40: HP-SEC analysis of IGG1-005-RGE, IgG1-005-RGK and a mixture of IgG1-005-RGE+IgG1-005-RGK (AU indicates “arbitrary units”).
FIG. 41: HP-SEC analysis of IgG1-005-RGE, IgG1-005-RGIK and a mixture of IgG1-005-RGE+IgG1-005-RGIK (AU indicates “arbitrary units”).
FIG. 42: Overlay of HP-SEC traces of mixture IgG1-005-RGE+IgG1-005-RGK and mixture IgG1-005-RGE+IgG1-005-RGIK (AU indicates “arbitrary units”).
FIG. 43: HP-SEC analysis of triple mutant Fc fragment (Fc-RGY).
FIGS. 44A and 44B: FACS analysis of A431 (FIG. 44A) and Daudi (FIG. 44B) cells incubated with mixtures of Fc-RGY-647 with full length IgG1-RGY antibodies.
FIGS. 45A and 45B: HP-SEC analysis of IgG1-005-RGY at different pH levels. Percentages indicate total oligomers as fraction of total peak area.
FIG. 46: Programmed cell death is induced in different isotypic variants of IgG antibodies by introduction of the triple mutation RGY.
FIG. 47: HP-SEC analysis of IgG1-005-RGY (solid line) and hexameric IgM-005 (dashed line).
FIGS. 48A and 48B: CDC efficacy by a concentration series of IgG1-005, IgG1-005-RGY, IgM-005 on CD38-positive Daudi (FIG. 48A) and Wien133 (FIG. 48B) cells.
FIGS. 49A and 49B: In vitro CDC assay with IgG1-2F8-RGY on solid tumor cell lines A431 cells (FIG. 49A) and Difi (FIG. 49B).
FIG. 50: C4d produced by antibodies in normal human serum as a measure for complement activation in solution.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “immunoglobulin” refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four potentially inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. See for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region typically is comprised of three domains, CH1, CH2, and CH3. The heavy chains are inter-connected via disulfide bonds in the so-called “hinge region”. Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)). Unless otherwise stated or contradicted by context, the amino acids of the constant region sequences are herein numbered according to the Eu-index or numbering (described in Kabat, E. A. et al., Sequences of proteins of immunological interest. 5th Edition—US Department of Health and Human Services, NIH publication No. 91-3242, pp 662,680,689 (1991)).
The term “hinge region” as used herein is intended to refer to the hinge region of an immunoglobulin heavy chain. Thus, for example the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to the Eu numbering as set forth in Kabat.
The term “CH2 region” or “CH2 domain” as used herein is intended to refer the CH2 region of an immunoglobulin heavy chain. Thus, for example the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the Eu numbering system. However, the CH2 region may also be any of the other subtypes as described herein.
The term “CH3 region” or “CH3 domain” as used herein is intended to refer the CH3 region of an immunoglobulin heavy chain. Thus for example the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the Eu numbering system. However, the CH3 region may also be any of the other subtypes as described herein.
“Fc region”, “Fc fragment” or “Fc domain”, which may be used interchangeably herein, refers to an antibody region comprising, in the direction from the N- to C-terminal, at least a hinge region, a CH2 domain and a CH3 domain. An Fc region of an IgG1 antibody can, for example, be generated by digestion of an IgG1 antibody with papain.
The term “Fab fragment” in the context of the present invention, refers to a fragment of an immunoglobulin molecule, which comprises the variable regions of the heavy chain and light chain as well as the constant region of the light chain and the CH1 region of an immunoglobulin. The “CH1 region” refers e.g. to the region of a human IgG1 antibody corresponding to amino acids 118-215 according to the Eu numbering system. Thus, the Fab fragment comprises the binding region of an immunoglobulin.
The term “antibody” (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions with a half life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about eight hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about three, four, five, six, seven or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity). The antibody of the present invention comprises an Fc-domain of an immunoglobulin and an antigen-binding region. An antibody generally contains two CH2-CH3 regions and a connecting region, e.g. a hinge region, e.g. at least an Fc-domain. Thus the antibody of the present invention may comprise an Fc region and an antigen-binding region. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant or “Fc” regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q, the first component in the classical pathway of complement activation. An antibody may also be a multispecific antibody, such as a bispecific antibody or similar molecule. The term “bispecific antibody” refers to an antibody having specificities for at least two different, typically non-overlapping, epitopes. Such epitopes may be on the same or different targets. If the epitopes are on different targets, such targets may be on the same cell or different cells or cell types. As indicated above, unless otherwise stated or clearly contradicted by the context, the term antibody herein includes fragments of an antibody which comprise at least a portion of an Fc-region and which retain the ability to specifically bind to the antigen. Such fragments may be provided by any known technique, such as enzymatic cleavage, peptide synthesis and recombinant expression techniques. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “Ab” or “antibody” include, without limitation, monovalent antibodies (described in WO2007059782 by Genmab); heavy-chain antibodies, consisting only of two heavy chains and naturally occurring in e.g. camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMabs (Roche, WO2011069104), strand-exchange engineered domain (SEED or Seed-body) which are asymmetric and bispecific antibody-like molecules (Merck, WO2007110205); Triomab (Fresenius, Lindhofer et al. (1995 J Immunol 155:219); FcΔAdp (Regeneron, WO2010151792), Azymetric Scaffold (Zymeworks/Merck, WO2012/058768), mAb-Fv (Xencor, WO2011/028952), Dual variable domain immunoglobulin (Abbott, DVD-Ig, U.S. Pat. No. 7,612,181); Dual domain double head antibodies (Unilever; Sanofi Aventis, WO20100226923), Di-diabody (ImClone/Eli Lilly), Knobs-into-holes antibody formats (Genentech, WO9850431); DuoBody (Genmab, WO 2011/131746); Electrostatic steering antibody formats (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545), CrossMAbs (Roche, WO2011117329), LUZ-Y (Genentech), Biclonic (Merus), Dual Targeting domain antibodies (GSK/Domantis), Two-in-one Antibodies recognizing two targets (Genentech, NovImmune), Cross-linked Mabs (Karmanos Cancer Center), CovX-body (CovX/Pfizer), IgG-like Bispecific (ImClone/Eli Lilly, Shen, J., et al. J Immunol Methods, 2007. 318(1-2): p. 65-74), and DIG-body and PIG-body (Pharmabcine), and Dual-affinity retargeting molecules (Fc-DART or Ig-DART, by Macrogenics, WO/2008/157379, WO/2010/080538), Zybodies (Zyngenia), approaches with common light chain (Crucell/Merus, U.S. Pat. No. 7,262,028) or common heavy chains (κλBodies by NovImmune), as well as fusion proteins comprising a polypeptide sequence fused to an antibody fragment containing an Fc-domain like scFv-fusions, like BsAb by ZymoGenetics/BMS), HERCULES by Biogen Idec (U.S. Ser. No. 00/795,1918), SCORPIONS by Emergent BioSolutions/Trubion, Ts2Ab (MedImmune/AZ (Dimasi, N., et al. J Mol Biol, 2009. 393(3): p. 672-92), scFv fusion by Novartis, scFv fusion by Changzhou Adam Biotech Inc (CN 102250246), TvAb by Roche (WO 2012025525, WO 2012025530), mAb2 by f-Star (WO2008/003116), and dual scFv-fusions. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures (recombinant polyclonals) for instance generated by technologies exploited by Symphogen and Merus (Oligoclonics), and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies. An antibody as generated can potentially possess any isotype.
The term “full-length antibody” when used herein, refers to an antibody (e.g., a parent or variant antibody) which contains all heavy and light chain constant and variable domains corresponding to those that are normally found in a wild-type antibody of that isotype.
The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The terms “monoclonal antibody”, “monoclonal Ab”, “monoclonal antibody composition”, “mAb”, or the like, as used herein refer to a preparation of Ab molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term “human monoclonal antibody” refers to Abs displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs may be generated by a hybridoma which includes a B cell obtained from a transgenic or transchromosomal nonhuman animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene repertoire and a light chain transgene repertoire, rearranged to produce a functional human antibody and fused to an immortalized cell.
As used herein, “isotype” refers to the immunoglobulin class (for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM or any allotypes thereof such as IgGlm(za) and IgGlm(f)) that is encoded by heavy chain constant region genes. Further, each heavy chain isotype can be combined with either a kappa (κ) or lambda (λ) light chain.
The term “monovalent antibody” means in the context of the present invention that an antibody molecule is only capable of binding with one binding domain of the antibody to an antigen, e.g. has a single antigen-antibody interaction, and thus is not able of antigen crosslinking.
A “binding region” as used herein may be a polypeptide sequence, such as a protein, protein ligand, receptor, an antigen-binding region, or a ligand-binding region capable of binding to a target associated with a cell, bacterium, virion, or the like. A binding region may, for example, comprise part of a receptor, receptor ligand or antigen-binding region of an immunoglobulin or antibody.
As used herein, the term “target” is in the context of the present invention to be understood as a molecule to which the binding region of the polypeptide comprising a CH2, CH3, and optionally a hinge region, and a binding region binds. When used in the context of the binding of an antibody includes any antigen towards which the raised antibody is directed. The term “antigen” and “target” may in relation to an antibody be used interchangeably and constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention.
As used herein, the term “binding” in the context of the binding of an antibody to a predetermined antigen typically is a binding with an affinity corresponding to a KD of about 10−6 M or less, e.g. 10−7 M or less, such as about 10−8 M or less, such as about 10−9 M or less, about 10−19 M or less, or about 10−11 M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1,000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The amount with which the affinity is lower is dependent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is highly specific), then the amount with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000 fold. The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.
A “variant” of the present invention denotes a molecule, e.g. dimeric protein or which comprises one or more mutations as compared to a “parent molecule”, e.g. “parent dimeric protein”, such as a “parent antibody”. For an antibody variant, exemplary parent antibody formats include, without limitation, a wild-type antibody, a full-length antibody or Fc-containing antibody fragment, a bispecific antibody, a human antibody, or any combination thereof. Exemplary mutations include amino acid deletions, insertions, and substitutions of amino acids in the parent amino acid sequence. Amino acid substitutions may exchange a native amino acid for another naturally-occurring amino acid, or for a non-naturally-occurring amino acid derivative. The amino acid substitution may be conservative or non-conservative. In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected in one or more of the following three tables:
Amino Acid Residue Classes for Conservative Substitutions
Acidic Residues
Asp (D) and Glu (E)
Basic Residues
Lys (K), Arg (R), and His (H)
Hydrophilic Uncharged Residues
Ser (S), Thr (T), Asn (N), and
Gln (Q)
Aliphatic Uncharged Residues
Gly (G), Ala (A), Val (V), Leu (L),
and Ile (I)
Non-polar Uncharged Residues
Cys (C), Met (M), and Pro (P)
Aromatic Residues
Phe (F), Tyr (Y), and Trp (W)
Alternative Conservative Amino Acid Residue Substitution Classes
1
A
S
T
2
D
E
3
N
Q
4
R
K
5
I
L
M
6
F
Y
W
Alternative Physical and Functional Classifications of Amino Acid Residues
Alcohol group-containing residues
S and T
Aliphatic residues
I, L, V, and M
Cycloalkenyl-associated residues
F, H, W, and Y
Hydrophobic residues
A, C, F, G, H, I, L, M, R, T, V,
W, and Y
Negatively charged residues
D and E
Polar residues
C, D, E, H, K, N, Q, R, S, and T
Positively charged residues
H, K, and R
Small residues
A, C, D, G, N, P, S, T, and V
Very small residues
A, G, and S
Residues involved in turn formation
A, C, D, E, G, H, K, N, Q, R, S,
P, and T
Flexible residues
Q, T, K, S, G, P, D, E, and R
In the context of the present invention, a substitution in a variant is indicated as:
Original amino acid—position—substituted amino acid;
Referring to the well-recognized nomenclature for amino acids, the three letter code, or one letter code, are used, including the codes Xaa and X to indicate amino acid residue. Accordingly, the notation “E345R” or “Glu345Arg” means, that the variant comprises a substitution of Glutamic acid with Arginine in the variant amino acid position corresponding to the amino acid in position 345 in the parent antibody, when the two are aligned as indicated below.
Where a position as such is not present in an antibody, but the variant comprises an insertion of an amino acid, for example:
Position—substituted amino acid; the notation, e.g., “448E” is used.
Such notation is particular relevant in connection with modification(s) in a series of homologous polypeptides or antibodies.
Similarly when the identity of the substitution amino acid residues(s) is immaterial:
Original amino acid—position; or “E345”.
For a modification where the original amino acid(s) and/or substituted amino acid(s) may comprise more than one, but not all amino acid(s), the substitution of Glutamic acid for Arginine, Lysine or Tryptophan in position 345:
“Glu345Arg,Lys,Trp” or “E345R,K,W” or “E345R/K/W” or “E345 to R, K or W” may be used interchangeably in the context of the invention.
Furthermore, the term “a substitution” embraces a substitution into any one of the other nineteen natural amino acids, or into other amino acids, such as non-natural amino acids. For example, a substitution of amino acid E in position 345 includes each of the following substitutions: 345A, 345C, 345D, 345G, 345H, 345F, 345I, 345K, 345L, 345M, 345N, 345Q, 345R, 345S, 345T, 345V, 345W, 345P, and 345Y. This is, by the way, equivalent to the designation 345X, wherein the X designates any amino acid other than the original amino acid. These substitutions can also be designated E345A, E345C, etc, or E345A,C, etc, or E345A/C/etc. The same applies by analogy to each and every position mentioned herein, to specifically include herein any one of such substitutions.
The terms “amino acid” and “amino acid residue” may be used interchangeably.
The reference to “D/E356” refers in the present context to allotypic variants in the sequence of human IgG1. In the IgG1m(za) allotype of human IgG1 the amino acid in position 356 is D, while in the IgG1m(f) allotype of human IgG1 the amino acid in position 356 is E.
Unless otherwise stated or contradicted by the context, reference to an amino acid position number refers to the amino acid position number in a human IgG1 heavy chain.
An amino acid or segment in one sequence that “corresponds to” an amino acid or segment in another sequence is one that (i) aligns with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW or similar, typically at default settings and (ii) has a sequence identity to SEQ ID NO:1 of at least 50%, at least 80%, at least 90%, or at least 95%. For example, the sequence alignments shown in FIGS. 2 and 3 can be used to identify any amino acid in the shown immunoglobulin Fc sequences that corresponds to a particular amino acid in the IgG1 Fc sequence.
For purposes of the present invention, an amino acid at a position in an amino acid sequence which corresponds to a specific position in another, reference amino acid sequence, as well as the degree of identity between two amino acid or nucleotide sequences, can determined by alignment of the two sequences. Herein, unless otherwise indicated or contradicted by context, the reference amino acid sequence is the amino acid sequence of the human IgG1 heavy chain. The program “Align” which is a Needleman-Wunsch alignment (i.e. a global alignment) can be used for alignment of polypeptide, as well as nucleotide sequences. The default scoring matrix BLOSUM50 or BLOSUM62 can be used for polypeptide alignments, and the default identity matrix can be used for nucleotide alignments, the penalty of the first residue of a gap is −12 for polypeptides and −16 for nucleotides. The penalties for further residues of a gap are −2 for polypeptides, and −4 for nucleotides. “Align” is part of the FASTA package version v20u6 (see W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448, and W. R. Pearson (1990) “Rapid and Sensitive Sequence Comparison with FASTP and FASTA”, Methods in Enzymology 183:63-98). FASTA protein alignments use the Smith-Waterman algorithm with no limitation on gap size (see “Smith-Waterman algorithm”, T. F. Smith and M. S. Waterman (1981) J. Mol. Biolo. 147:195-197). Representative alignments between Fc regions of immunoglobulin heavy chains are shown in FIGS. 2 and 3.
The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of inducing transcription of a nucleic acid segment ligated into the vector. One type of vector is a “plasmid”, which is in the form of a circular double stranded DNA loop. Another type of vector is a viral vector, wherein the nucleic acid segment may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (for instance bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (such as non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (such as replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK-293 cells, PER.C6, NS0 cells, and lymphocytic cells, and prokaryotic cells such as E. coli and other eukaryotic hosts such as plant cells and fungi.
The term “transfectoma”, as used herein, includes recombinant eukaryotic host cells expressing the Ab or a target antigen, such as CHO cells, PER.C6, NS0 cells, HEK-293 cells, plant cells, or fungi, including yeast cells.
As used herein, the term “effector cell” refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, for instance lymphocytes (such as B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils. Some effector cells express Fc receptors (FcRs) or complement receptors and carry out specific immune functions. In some embodiments, an effector cell such as, e.g., a natural killer cell, is capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells and Kupffer cells which express FcRs, are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. In some embodiments the ADCC can be further enhanced by antibody driven classical complement activation resulting in the deposition of activated C3 fragments on the target cell. C3 cleavage products are ligands to complement receptors (CRs), such as CR3, expressoid on myeloid cells. The recognition of complement fragments by CRs on effector cells may promote enhanced Fc receptor-mediated ADCC. In some embodiments antibody driven classical complement activation leads to C3 fragments on the target cell. These C3 cleavage products may promote direct complement-dependent cellular cytotoxicity (CDCC). In some embodiments, an effector cell may phagocytose a target antigen, target particle or target cell. The expression of a particular FcR or complement receptor on an effector cell may be regulated by humoral factors such as cytokines. For example, expression of FcγRI has been found to be up-regulated by interferon γ (IFN γ) and/or G-CSF. This enhanced expression increases the cytotoxic activity of FcγRI-bearing cells against targets. An effector cell can phagocytose a target antigen or phagocytose or lyse a target cell. In some embodiments antibody driven classical complement activation leads to C3 fragments on the target cell. These C3 cleavage products may promote direct phagocytosis by effector cells or indirectly by enhancing antibody mediated phagocytosis.
As used herein, the term “effector functions” refers to functions that are a consequence of binding a dimeric protein, such as an antibody, to its target, such as an antigen, optionally on a cell, on a cell membrane, on a virion, or on another particle. Examples of effector functions include (i) C1q-binding, (ii) complement activation, (iii) complement-dependent cytotoxicity (CDC), (iv) oligomer formation, (v) oligomer stability, (vi) antibody-dependent cell-mediated cytotoxity (ADCC), (vii) FcRn-binding, (viii) Fc-gamma receptor-binding, (ix) antibody-dependent cellular phagocytosis (ADCP), (x) complement-dependent cellular cytotoxicity (CDCC), (xi) complement-enhanced cytotoxicity, (xii) binding to complement receptor of an opsonized antibody mediated by the antibody, (xiii) internalization, (xiv) downmodulation, (xv) induction of apoptosis, (xvi) opsonisation, (xvii) proliferation modulation, such as proliferation reduction, inhibition or stimulation, and (xii) a combination of any of (i) to (xvi).
As used herein, the term “affinity” is the strength of binding of one molecule, e.g. an antibody, to another, e.g. a target or antigen, at a single site, such as the monovalent binding of an individual antigen binding site of an antibody to an antigen.
As used herein, the term “avidity” refers to the combined strength of multiple binding sites between two structures, such as between multiple antigen binding sites of antibodies simultaneously interacting with a target or e.g. between antibody and C1q. When more than one binding interactions are present, the two structures will only dissociate when all binding sites dissociate, and thus, the dissociation rate will be slower than for the individual binding sites, and thereby providing a greater effective total binding strength (avidity) compared to the strength of binding of the individual binding sites (affinity).
As used herein, the term “oligomer” refers to a structure that consists of more than one but a limited number of units of a specific type of molecule (such as, e.g., antibody or other dimeric protein molecules according to the invention) in contrast to a polymer which, at least in principle, consists of an unlimited number of units. Thus, an oligomer according to the invention consists of a limited number of dimeric proteins according to any aspect or embodiment of the present invention. Exemplary oligomers are dimers, trimers, tetramers, pentamers, hexamers, and dodecamers. Greek prefixes are often used to designate the number of monomer units in the oligomer, for example a tetramer being composed of four units and a hexamer of six units. Likewise, the term “oligomerization”, as used herein, is intended to refer to a process that converts molecules to a finite degree of polymerization. Herein, it is observed, that antibodies and/or other dimeric proteins according to the invention can form oligomers, such as hexamers, via non-covalent association of Fc-domains in solution under certain pH conditions, as described in Example 31, or, in the case of dimeric proteins comprising target-binding regions, after target binding, e.g., at a cell surface. Oligomerization in solution can be evaluated, e.g., as described in Example 20. In a particular embodiment, the oligomerization in solution may be determined by performing HP-SEC (high pressure size exclusion chromatography) fractionation using a suitable size exclusion chromatography resin with a porse size capable of separating molecules in the range of 50 kDa to 1000 kDa, connected to an absorbance detector; separating into 50 μL samples containing 1.25 μg/mL protein at 1 mL/min in 0.1 M Na2SO4/0.1 M sodium phosphate buffered at pH 6.8; using a suitable software to process results; and expressing per peak as percentage of total peak area. The oligomerization of antibodies after antigen-binding can be evaluated (e.g. using a complement dependent cytotoxicity as described in Examples 3, 6, and 21). In a particular embodiment, CDC may be determined by pre-incubating suspension cells at a concentration of 1×106 cells/mL in round-bottom 96-well plates with an antibody at a final concentration ranging from 0.0003 to 30.0 μg/mL in a total volume of 100 μL for 15 min on a shaker at room temperature; adding normal human serum at a final concentration of 20%, 30% or 50%; incubating at 37° C. for 45 min; putting the plates on ice; adding 10 μL propidium iodide; and determining cell lysis by FACS analysis.
The term “C1q binding”, as used herein, is intended to refer to the binding of C1q in the context of the binding of C1q to an antibody bound to its antigen. The antibody bound to its antigen is to be understood as happening both in vivo and in vitro in the context described herein. C1q binding can be evaluated for example by using immobilized antibody on artificial surface (e.g. plastic in plates for ELISA, as described in example 21). In a particular embodiment, C1q binding may be determined by coating 96-well ELISA plates overnight at 4° C. with antibody in PBS at a concentration ranging from 0.007 to 25.0 μg/mL; washing the plates; blocking with 0.5×PBS/0.025% Tween 20/0.1% gelatin; sequentially incubating for 1 h at 37° C. plates with 3% pooled human serum, rabbit anti-human C1q, swine anti-rabbit IgG-HRP, by in-between washing; developing the plates for about 30 min with 1 mg/mL 2,2′-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid; adding 100 μL 2% oxalic acid; and measuring the absorbance at 405 nm in a microplate reader. The binding of C1q to an antibody oligomer is to be understood herein as a multivalent interaction resulting in high avidity binding.
As used herein, the term “complement activation” refers to the activation of the classical complement pathway, which is triggered by the binding of complement component C1q to an antibody bound to its antigen. C1q is the first protein in the early events of the classical complement cascade that involves a series of cleavage reactions that culminate in the formation of an enzymatic activity called C3 convertase, which cleaves complement component C3 into C3b and C3a. C3b binds covalently to C5 on the membrane to form C5b that in turn triggers the late events of complement activation in which terminal complement components C5b, C6, C7, C8 and C9 assemble into the membrane attack complex (MAC). The complement cascade results in the creation of pores due to which causes cell lysis, also known as CDC. Complement activation can be evaluated by using, CDC kinetics (as described in example 14, 15 and 16), CDC assays (as described in examples 3 and 21) or by the method Cellular deposition of C3b and C4b described in Beurskens et al Apr. 1, 2012 vol. 188 no. 7 3532-3541.
The term “complement-dependent cytotoxicity” (“CDC”), as used herein, is intended to refer to the process of antibody-mediated complement activation leading to lysis of a cell or virion as a result of pores in the membrane that are created by MAC assembly, when the antibody is bound to its target on said cell or virion. CDC can be evaluated by in vitro assays such as a CDC assay in which normal human serum is used as a complement source, as described above, e.g. in example 3 and 21.
The term “antibody-dependent cell-mediated cytotoxicity” (“ADCC”) as used herein, is intended to refer to a mechanism of killing of antibody-coated target cells or virions by cells expressing Fc receptors that recognize the constant region of the bound antibody. ADCC can be determined using methods such as, e.g., the ADCC assay described in example 21. In a particular embodiment, ADCC may be determined by incubating cells with antibody at a concentration ranging from 0.5 to 250 ng/mL; and quantifying ADCC activity with a ADCC bioluminescent reporter assay kit.
The term “antibody-dependent cellular phagocytosis” (“ADCP”) as used herein is intended to refer to a mechanism of elimination of antibody-coated target cells or virions by internalization by phagocytes. The internalized antibody-coated target cell or virion is contained in a vesicle called a phagosome, which then fuses with one or more lysosomes to form a phagolysosome. ADCP may be evaluated by using an in vitro cytotoxicity assay with marcophages as effector cells and video microscopy as described by van Bij et al. in Journal of Hepatology Volume 53, Issue 4, October 2010, Pages 677-685 or as described in example 24 for e.g. S. aureus phagocytos by PMN.
The term “complement-dependent cellular cytotoxicity” (“CDCC”) as used herein is intended to refer to a mechanism of killing of target cells or virions by cells expressing complement receptors that recognize complement 3 (C3) cleavage products that are covalently bound to the target cells or virions as a result of antibody-mediated complement activation. CDCC may be evaluated in a similar manner as described for ADCC, but in the presence of complement C5 depleted normal human serum.
The term “downmodulation”, as used herein, is intended to refer to a process that decreases the number of molecules, such as antigens or receptors, on a cellular surface, e.g. by binding of an antibody to a receptor.
The term “internalization”, as used herein, is intended to refer to any mechanism by which a dimeric protein of the present invention, e.g. an antibody or Fc-containing polypeptide, is internalized into a target-expressing cell from the cell-surface and/or from surrounding medium, e.g., via endocytosis. The internalization of an antibody can be evaluated using a direct assay measuring the amount of internalized antibody (such as, e.g., the lysosomal co-localization assay described in Example 12).
The term “programmed cell-death” or “PCD”, as used herein refers to the death of a cell in any form mediated by an intracellular signalling. Three forms of PCD are found; apoptosis, autophagy and necrosis/oncosis. In a particular embodiment, any of the three forms of programmed cell death may be determined by culturing 1.0×105 cells for 24 hours in 96-well U-bottom plates in the presence of antibody at a concentration ranging from 0.0025 to 10 μg/mL; staining dead cells with annexin V-FITC using a suitable annexin binding assay kit according to the manufacturer's instructions; and determining the amount of annexin V-FITC-positive cells using by FACS analysis.
The term “apoptosis”, as used herein, refers to the best-characterized type of programmed cell death because of its importance in development and homeostasis, and in the pathogenesis of different diseases, such as cancer. Apoptotic cells die in a controlled fashion in response to a variety of extrinsic or intrinsic signals (e.g., activation of tumor necrosis factor (TNF) receptors, DNA damage, mitochondrial pathways). Biochemical events lead to characteristic cell changes (morphology) and death. The hallmarks of apoptotic cell death include blebbing, exposure of phosphatidylserine on the extracellular face of the plasma membrane, activation of caspases, disruption of mitochondrial membrane potential, cell shrinkage, chromatin condensation, DNA fragmentation and DNA condensation. Binding of an antibody to a certain receptor may induce apoptosis.
The term “autophagy”, as used herein, refers to a selective degradation of intracellular molecules or structures, such as misfolded proteins and damaged organelles, and is an important homeostatic function. Autophagy performs in concert with the Ubiquitin-Proteasome System (UPS) to degrade aggregated/misfolded proteins that are ubiquitinated, labelling them for degradation by autophagy. The ubiquitinated cargo is carried to the phagophore and surrounds its cargo forming a double membrane vesicle, the autophagosome. The lyososome fuses to the autophagosome and the cargo is degraded inside the autolysosome.
The term “necrosis” or “oncosis”, as used herein, refers to an uncontrolled cell death characterized by cell swelling, as well as destruction of the plasma membrane and subcellular organelles, without nuclear fragmentation and condensation. Necrotic cell death is considered a heterogeneous phenomenon including both programmed and accidental cell death.
The term “proliferation”, as used herein refers to an increase in the number of cells as a result of cell growth and cell division.
The term “antibody-drug conjugate”, as used herein refers to a dimeric protein of the present invention, e.g. an antibody or Fc-containing polypeptide, having specificity for at least one type of malignant cell, a drug, and a linker coupling the drug to e.g. the antibody. The linker is cleavable or non-cleavable in the presence of the malignant cell; wherein the antibody-drug conjugate kills the malignant cell.
The term “antibody-drug conjugate uptake”, as used herein refers to the process in which antibody-drug conjugates are bound to a target on a cell followed by uptake/engulfment by the cell membrane and thereby is drawn into the cell. Antibody-drug conjugate uptake may be evaluated as “antibody-mediated internalization and cell killing by anti-TF ADC in an in vitro killing assay” as described in WO 2011/157741.
The term “FcRn”, as used herein is intended to refer to neonatal Fc receptor which is an Fc receptor. It was first discovered in rodents as a unique receptor capable of transporting IgG from mother's milk across the epithelium of newborn rodent's gut into the newborn's bloodstream. Further studies revealed a similar receptor in humans. In humans, however, it is found in the placenta to help facilitate transport of mother's IgG to the growing fetus and it has also been shown to play a role in monitoring IgG turnover. FcRn binds IgG at acidic pH of 6.0-6.5 but not at neutral or higher pH. Therefore, FcRn can bind IgG from the intestinal lumen (the inside of the gut) at a slightly acidic pH and ensure efficient unidirectional transport to the basolateral side (inside the body) where the pH is neutral to basic (pH 7.0-7.5). This receptor also plays a role in adult salvage of IgG through its occurrence in the pathway of endocytosis in endothelial cells. FcRn receptors in the acidic endosomes bind to IgG internalized through pinocytosis, recycling it to the cell surface, releasing it at the basic pH of blood, thereby preventing it from undergoing lysosomal degradation. This mechanism may provide an explanation for the greater half-life of IgG in the blood compared to other isotypes.
The term “Protein A”, as used herein is intended to refer to a 56 kDa MSCRAMM surface protein originally found in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topology, cellular osmolarity, and a two-component system called ArIS-ArIR. It has found use in biochemical research because of its ability to bind immunoglobulins. It is composed of five homologous Ig-binding domains that fold into a three-helix bundle. Each domain is able to bind proteins from many mammalian species, most notably IgGs. It binds the heavy chain Fc region of most immunoglobulins (overlapping the conserved binding site of FcRn receptors) and also interacts with the Fab region of the human VH3 family. Through these interactions in serum, IgG molecules bind the bacteria via their Fc region instead of solely via their Fab regions, by which the bacteria disrupts opsonization, complement activation and phagocytosis.
The term “Protein G”, as used herein is intended to refer to an immunoglobulin-binding protein expressed in group C and G Streptococcal bacteria much like Protein A but with differing specificities. It is a 65-kDa (G148 protein G) and a 58 kDa (C40 protein G) cell surface protein that has found application in purifying antibodies through its binding to the Fc region.
Dimeric Protein
The present invention relates in one aspect to a dimeric protein comprising a first and a second polypeptide, each polypeptide comprising at least CH2 and CH3 regions of an immunoglobulin heavy chain, wherein in said first and/or second polypeptide
the amino acids in the positions corresponding to E345 and E430 in a human IgG1 heavy chain are not E, and
the amino acid in at least one position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254 is Y, K, R or W; not Y; not D or E; not T; not E; not N; not Q; not I; and not S, for each position, respectively. Each of positions S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254 corresponds to the position in a human IgG1 heavy chain.
In one embodiment, in each of said first and second polypeptides
the amino acids in the positions corresponding to E345 and E430 in a human IgG1 heavy chain are not E, and
the amino acid in at least one position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254 is Y, K, R or W; not Y; not D or E; not T; not E; not N; not Q; not I; and not S, for each position, respectively. Each of positions S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254 corresponds to the position in a human IgG1 heavy chain.
The first and second polypeptides of the dimeric protein according to the invention dimerize by forming covalent or non-covalent interaction. Such an interaction may be found in any region of the polypeptides. Examples of covalent interaction are any CxxC peptide interaction, wherein the “x” represents any amino acid and the “C” represents cysteine residues. Another example is a TCRalpha chain constant domain and a TCRbeta chain constant domain. Examples on non-covalent interaction may be a leucine zipper such as described in Moll et al, Prot. Science, 2001, 10:649-655. In one embodiment, said first and/or second polypeptide may further comprise a region capable of covalent binding between said first and second polypeptide.
In one embodiment, the first and/or second polypeptides further comprise a hinge region.
For certain purposes of the present invention a part of the hinge region, such as amino acid positions corresponding to 226-230, suffices. Thus, in one embodiment, the first and/or second polypeptide may further comprise amino acids at positions corresponding to positions 226-230 in a human IgG1 heavy chain.
In one embodiment, in said first and second polypeptide the amino acid in the positions corresponding to E345 and E430 in a human IgG1 heavy chain, are not E, and the amino acid in at least one position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254 is Y or W; not Y; not D or E; not T; not E; not N; not Q; not I; and not S, for each position, respectively.
In one embodiment, in said first and second polypeptide the amino acid in the positions corresponding to E345 and E430 in a human IgG1 heavy chain, are not E, and the amino acid in at least one position selected from the group consisting of S440, Y436, E356, T359, E382, N434, Q438, I253 and S254 is Y, K, R or W; not Y; not E; not T; not E; not N; not Q; not I; and not S, for each position, respectively.
Thus, one embodiment the present invention relates to the dimeric protein comprising a first and a second polypeptide, each polypeptide comprising at least CH2, CH3 and hinge regions of an immunoglobulin heavy chain, wherein
the amino acids in the positions corresponding to E345 and E430 in a human IgG1 heavy chain are not E and
the amino acid in at least one position selected from the group consisting of S440, Y436, E356, T359, E382, N434, Q438, I253 and S254 is Y or W; not Y; not E; not T; not E; not N; not Q; not I; and not S, for each position, respectively.
In one embodiment, the first and second polypeptides are interconnected via hinge region disulphide binds.
Unless otherwise stated or contradicted by context, the amino acid positions mentioned refer to an amino acid position in a human IgG1 heavy chain in any aspect or embodiment of the present invention.
Furthermore, unless otherwise stated or contradicted by context, the amino acid numbering is according to Eu numbering as set forth in Kabat, as described above.
The dimeric protein may be prepared from a parent dimeric protein, and thereby be regarded as a variant dimeric protein, by introducing mutations in the positions corresponding to E345 and E430 in a human IgG1 heavy chain, and in at least one position selected from the group consisting of the positions corresponding to S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254 in a human IgG1 heavy chain, wherein the amino acid introduced in the position corresponding to S440 is Y, K, R or W. At the other amino acid positions any amino acid may be introduced, e.g. any naturally occurring amino acid.
In one embodiment, the amino acid at the position corresponding to E345 is, for one or both, such as each, of said first and second polypeptides of the dimeric protein, selected, e.g. separately, from the group consisting of R, Q, N, K, Y, A, C, D, F, G, H, I, L, M, P, S, T, V and W, such as from the group consisting of R, Q, N, K and Y.
In a further embodiment, the amino acid at the position corresponding to E345 is, for one or both, such as each, of said first and/or second polypeptides of the dimeric protein, selected, e.g. separately from the group consisting of R, Q, K and Y.
In a further embodiment, the amino acid at the position corresponding to E345 is, for one or both, such as each, of said first and/or second polypeptides of the dimeric protein, R.
In one embodiment, the amino acid at the position corresponding to E430 is, for one or both, such as each, of said first and/or second polypeptides of the dimeric protein, selected, e.g. separately from the group consisting of G, T, S, F, H, A, C, D, I, K, L, M, N, P, Q, R, V, W and Y, such as from the group consisting of G, T, S, F and H.
In a further embodiment, the amino acid at the position corresponding to E430 is, for one or both, such as each, of said first and/or second polypeptides of the dimeric protein, selected, e.g. separately from the group consisting of G, T, S and F.
In a further embodiment, the amino acid at the position corresponding to E430 is, for one or both, such as each, of said first and/or second polypeptides of the dimeric protein, G.
In one embodiment the amino acid in the position corresponding to S440 is, for one or both, such as each, of said first and/or second polypeptides of the dimeric protein, selected, e.g. separately from the group consisting of Y or W.
In one embodiment the amino acid in the position corresponding to S440 is, for one or both, such as each, of said first and/or second polypeptides of the dimeric protein, W.
In one embodiment the amino acid in the position corresponding to S440 is, for one or both, such as each, of said first and/or second polypeptides of the dimeric protein, Y.
In one embodiment, the amino acid in a position selected from the group consisting of Y436, D/E356, T359, E382, N434, Q438, I253 and S254 is, in one or both, such as each, of said first and/or second polypeptides of the dimeric protein,
(a) I, N, Q, S, T, R, A, E, F, H, K, L, M or V, such as I, N, Q or S;
(b) R, G, T, I, L, M, K, H, S, V, Y, Q, N, W, F, A, or C;
(c) R;
(d) V, L, M, D, Q, K, R, N, H, S, T, W or Y, such as V, L or M;
(e) W, H, K, Q, R, D, E, S, T, or Y, such as W, H, K, Q or R;
(f) N, S, T, A, E, G, H, K, Q, R, W or Y, such as N, S or T;
(g) V, L, N, Q, E, S or T, such as V, L, N or Q;
(h) L, G, I or V;
for each position, respectively.
The amino acid at the positions corresponding to E345 and E430 in a human IgG1 heavy chain, and at a position corresponding to a position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254, may in one embodiment be the same in the first and second polypeptide; or they may be different. The amino acids at said positions may be different, e.g. if the dimeric protein is a heterodimeric protein, such as a bispecific antibody described herein.
The amino acids at the positions corresponding to E345, E430 and S440 may, for one or both, such as each, of said first and/or second polypeptides of the dimeric protein be one of the following non-limiting examples; E345R/E430G/S440Y, E345R/E430G/S440W, E345K/E430G/S440Y, E345K/E430G/S440W, E345Q/E430G/S440Y, E345Q/E430G/S440W, E345N/E430G/S440Y, E345N/E430G/S440W, E345Y/E430G/S440Y, E345Y/E430G/S440W, E345R/E430T/S440Y, E345R/E430T/S440W, E345K/E430T/S440Y, E345K/E430T/S440W, E345Q/E430T/S440Y, E345Q/E430T/S440W, E345N/E430T/S440Y, E345N/E430T/S440W, E345Y/E430T/S440Y, E345Y/E430T/S440W, E345R/E430S/S440Y, E345R/E430S/S440W, E345K/E430S/S440Y, E345K/E430S/S440W, E345Q/E430S/S440Y, E345Q/E430S/S440W, E345N/E430S/S440Y, E345N/E430S/S440W, E345Y/E430S/S440Y, E345Y/E430S/S440W, E345R/E430F/S440Y, E345R/E430F/S440W, E345K/E430F/S440Y, E345K/E430F/S440W, E345Q/E430F/S440Y, E345Q/E430F/S440W, E345N/E430F/S440Y, E345N/E430F/S440W, E345Y/E430F/S440Y, E345Y/E430F/S440W, E345R/E430G/S440K, E345R/E430G/S440R, E345K/E430G/S440K, E345K/E430G/S440R, E345Q/E430G/S440K, E345Q/E430G/S440R, E345N/E430G/S440K, E345N/E430G/S440R, E345Y/E430G/S440K, E345Y/E430G/S440R, E345R/E430T/S440K, E345R/E430T/S440R, E345K/E430T/S440K, E345K/E430T/S440R, E345Q/E430T/S440K, E345Q/E430T/S440R, E345N/E430T/S440K, E345N/E430T/S440R, E345Y/E430T/S440K, E345Y/E430T/S440R, E345R/E430S/S440K, E345R/E430S/S440R, E345K/E430S/S440K, E345K/E430S/S440R, E345Q/E430S/S440K, E345Q/E430S/S440R, E345N/E430S/S440K, E345N/E430S/S440R, E345Y/E430S/S440K, E345Y/E430S/S440R, E345R/E430F/S440K, E345R/E430F/S440R, E345K/E430F/S440K, E345K/E430F/S440R, E345Q/E430F/S440K, E345Q/E430F/S440R, E345N/E430F/S440K, E345N/E430F/S440R, E345Y/E430F/S440K, and E345Y/E430F/S440R.
In one embodiment, the amino acids in the positions corresponding to E345, E430 and S440 are, for one or both, such as each, of said first and/or second polypeptides of the dimeric protein, R, G and Y, respectively.
In an alternative embodiment, the amino acids in the positions corresponding to E345, E430 and S440 are, for said first and/or second polypeptides of the dimeric protein, K, G, and Y, respectively.
In an alternative embodiment, the amino acids in the positions corresponding to E345, E430, and S440 are, for said first and/or second polypeptides of the dimeric protein, R, S, and Y, respectively.
In an alternative embodiment, the amino acids in the positions corresponding to E345, E430 and S440 are, for said first and/or second polypeptides of the dimeric protein, R, G, and W, respectively.
In an alternative embodiment, the amino acids in the positions corresponding to E345, E430 and Y436 are, for said first and/or second polypeptides of the dimeric protein, R, G, and I, respectively.
In an alternative embodiment, the amino acids in the positions corresponding to E345, E430, Y436 and S440 are, for said first and/or second polypeptides of the dimeric protein, R, G, I, and K, respectively.
In one embodiment, the amino acids in the positions corresponding to E345, E430 and S440 are, for said first and/second polypeptides of the dimeric proteins, R, G, and K, respectively.
As described herein, the present invention inter alia relates to dimeric proteins comprising amino acids at three positions which are different from those naturally present in the CH2/CH3 region of a human IgG1 heavy chain.
In one embodiment, said first and second polypeptides of the dimeric protein are interconnected via hinge region disulphide bonds.
In one embodiment the isotype of the immunoglobulin heavy chain is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE and IgM. The isotype of the first and second polypeptide may be different, but in a particular embodiment, they are the same. In one embodiment, the isotype of the first and second polypeptide is different, such as the isotype of said first polypeptide may be an IgG1 immunoglobulin heavy chain and the isotype of said second polypeptide may be an IgG4 immunoglobulin heavy chain. The example is not to be understood limiting and thus, other combinations of isotypes is considered comprised in the present invention.
Any amino acid position, or mutation in an amino acid position, described herein as corresponding to an amino acid position in a human IgG1 heavy chain, can be identified or introduced at its equivalent position in IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgD and IgM as defined by the alignment in FIG. 2 to obtain a dimeric protein according to the invention.
In a particular embodiment the isotype of the immunoglobulin heavy chain is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, such as IgG1.
In another embodiment, the isotype of the immunoglobulin heavy chain is selected from the group consisting of IgA1 and IgA2.
In another embodiment, the isotype of the immunoglobulin heavy chain is selected from the group consisting of IgE, IgD and IgM.
In one embodiment, the immunoglobulin heavy chain is of mammalian origin.
In one embodiment the immunoglobulin heavy chain is of primate or murine origin, such as human origin.
In one embodiment, at least one of the polypeptides of the dimeric protein comprises a binding region which specifically binds to a target.
In a further embodiment, both the first and second polypeptide comprises a binding region, such as an antigen-binding region, specifically binding to a target. The binding region, such as an antigen-binding region, of the first and second polypeptide may bind to the same target, optionally to different epitopes of the same target, or they may bind to different targets.
The binding regions of the first and/or second polypeptide may be antigen binding regions.
Said binding region may bind any target, wherein the target may e.g. be a molecule present on a cell, bacterium, parasite or virion.
In a particular embodiment the target may be an antigen.
In a further embodiment said antigen may be expressed on the surface of a cell, such as a human tumor cell.
In one embodiment, said antigen is associated with a cell membrane.
In another embodiment, said antigen is associated with a virion, optionally wherein the antigen is comprised in the protein coat or a lipid envelope of the virion.
In a further embodiment, the target to which a binding regions binds may be an antigen expressed on the surface of a bacterial cell or a virion.
In another embodiment, the bacterial cell is selected from the group consisting of S. aureus, S. epidermidis, S. pneumonia, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia trachomatis, E. coli, Salmonella, Shigella, Yersinia, S. typhimurium, Neisseria meningitides, and Mycobacterium tuberculosis.
Examples of targets or antigens include but are not limited to: 5T4; ADAM-10; ADAM-12; ADAM17; AFP; alpha/beta T cell receptor (TCR); AXL; ANGPT2 anthrax antigen; antidrug antibody (ADA) BSG; CAIX; CAXII; CA 72-4; carcinoma associated antigen CTAA16.88; CCL11; CCL2; CCR4; CCR5; CCR6; CD2; CD3E; CD4; CD5; CD6; CD15; CD18; CD19; CD20; CD22; CD24; CD25; CD29; CD30; CD32B; CD33; CD37; CD38; CD40; CD40LG; CD44; CD47; CD52; CD55SC1; CD56; CD66E; CD72; CD74; CD79a; CD79b; CD80; CD86; CD98; CD137; CD147; CD138; CD168; CD200; CD248; CD254; CD257; CDH3; CEA; CEACAM5; CEACAM6; CEACAM8; Claudin4; CS-1; CSF2RA; CSPG-4; CTLA4; CRF-1; Cripto; DLL4; Death receptor 4; Death receptor 5; ED-B; EFNA2; EGFR; Endothelin B receptor; ENPP3; EPCAM; ERBB2; ERBB3; FAP alpha; FAS (aka APO-1, CD95); Fc gamma RI; FCER2; FGFR3; fibrin II beta chain; FLT1; FOLH1; FOLR1; FRP-1; G-28 glycolipid; GD1a; GD-2; GM-1; GD3 ganglioside; GM3; GDF2; GLP1R; Glypican-3; GPNMB; GRP78; Haemophilus influenza; HBV (hepatitis B virus); HCMV (human cytomegalovirus); heat shock protein 90 homolog [Candida albicans]; herpes simplex virus gD glycoprotein; HGF; HIV-1; HIV-1 IIIB gp120 V3 loop; HLA-DRB (HLA-DR beta); human anti-human antibodies (HAHA); human anti-murine antibodies (HAMA); human respiratory syncytial virus, glycoprotein F; ICAM1; IFNA1; IFNA1; IFNB1 bispecific; IgE, IgE Fc; IGF1R; IGHE connecting region; IL12B; IL13; IL15; IL17A; IL1A; IL1B; IL2RA; IL4; IL5; IL5RA; IL6; IL6R; IL9; interleukin-2 receptor beta subunit; ITGA2; ITGA2B ITGB3; ITGA4 ITGB7; ITGA5; ITGAL; ITGAV_ITGB3; ITGB2; KDR; L1CAM; Lewis-x; Lewis-y; lipid A, domain of lipopolyaccharide LPS; LTA; lipid A; Mannan (Candida albicans); MET; microbial proteases such as Staphylococcus aureus gluV8 and Streptococcus pyogenes IdeS; MMP14; MMp15; MST1R; MSTN; MUC1; MUC4; MUC16; MUC5AC; myelin; NCA-90 granulocyte cell antigen; Nectin 4; Neisseria meningitides, NGF; non-POU domain-containing octomer binding protein (NONO); NRP; NY-ESO-1; O-glycan; OX40L; PLAC-1; PLGF; PDGFRA; PD1; PDL1; PSCA; phosphatidylserine; PTK-7; Pseudomonas aeruginosa serotype IATS 011; RSV (human respiratory syncytial virus, glycoprotein F); ROR1; RTN4; SELL; SELP; STEAP1; Shiga-like toxin II B subunit [Escherichia coli]; SLAM7; SLC44A4; SOST; Staphylococcus epidermidis lipoteichoic acid; Streptococcus pneumonia; TAF-15; T cell receptor alpha_beta; Tissue Factor (TF); TGFB1; TGFB2; TMEFF2; TNC; TNF; TNFRSF10A; TNFRSF10B; TNFRSF12A; TNFSF13; TNFSF14; TNFSF2; TNFSF7; TRAILR2; TROP2; TYRP1; VAP-1; Vimentin; erbB1 (EGFR); erbB2 (HER2); erbB3; erbB4; MUC-1; CXCR5; c-Met; HERV-envelop protein; periostin; Bigh3; SPARC; BCR; and MRP3.
In a further embodiment, the antigens may be selected from CD20, EGFr and CD38, optionally, the dimeric protein of the present invention may be selected from 7D8, 2F8, 003 and 005 as described herein comprising the amino acid positions as defined by the present invention. Thus, 7D8, 2F8, 003, and 005, may be used as a parent dimeric protein according to the present invention.
In one embodiment, at least one of the polypeptides, of the dimeric protein, comprises an immunoglobulin heavy chain variable region.
In one embodiment, the dimeric protein of the present invention is an antibody.
In a further embodiment, wherein the dimeric protein is an antibody, one or both, such as each, of the first and second polypeptides comprises immunoglobulin heavy and light chain variable regions to form a first and a second antigen-binding region, optionally binding the same antigen.
In another embodiment, wherein the dimeric protein is an antibody, one or both, such as each, of the first and second polypeptides comprises an immunoglobulin heavy-chain variable region associated with an immunoglobulin light chain sequence comprising light chain variable and constant regions to form a first and a second antigen-binding region, optionally binding the same antigen. In such an embodiment, it is understood that said first and second polypeptides comprising immunoglobulin heavy-chain variable and constant regions are associated with an immunoglobulin light chain sequence comprising light chain variable and constant regions by interchain disulfide bonds between the constant domains of said heavy chain and said light chain, and thereby forming a first and a second antigen-binding region, optionally binding the same antigen.
In a further embodiment, wherein the dimeric protein is an antibody, one or both polypeptides comprise a full-length heavy chain constant region, such as a full-length human IgG1 heavy chain constant region.
The CH2 and CH3 regions of the first and/or second polypeptides may except for the amino acid positions defined by the present invention, comprise amino acids 114-223 and 224-330, respectively, of SEQ ID NO:1; amino acids 111-219 and 220-326, respectively, of SEQ ID NO:2; amino acids 161-270 and 271-377, respectively, of SEQ ID NO:3; amino acids 111-220 and 221-327, respectively, of SEQ ID NO:4; or amino acids 114-223 and 224-330, respectively, of SEQ ID NO:5.
Said first and/or second polypeptides may further comprise a hinge region, wherein said hinge region comprise amino acids 99-113 of SEQ ID NO:1; amino acids 99-110 of SEQ ID NO:2; amino acids 99-160 of SEQ ID NO:3; amino acids 99-110 of SEQ ID NO:4; or amino acids 99-113 of SEQ ID NO:5.
The first and/or second polypeptide may except for the amino acid positions defined by the present invention comprise a sequence according to any of SEQ ID NOs: 1, 2, 3, 4, and 5.
The dimeric protein of the present invention may in a particular embodiment as described above be an antibody. Furthermore, the dimeric protein, e.g. an antibody, may also be prepared by introducing mutations into a parent dimeric protein, e.g. a parent antibody, in the amino acids in the positions corresponding to E345 and E430 in a human IgG1 heavy chain, and an amino acid in at least one position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254. The examples of antibodies may thus refer to “an antibody of the present invention” or “a variant antibody of the present invention”, and a “parent antibody”.
Examples of suitable antibodies include but are not limited to monovalent antibodies heavy-chain antibodies, consisting only of two heavy chains and naturally occurring in e.g. camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMabs (Roche, WO2011069104), strand-exchange engineered domain (SEED or Seed-body) which are asymmetric and bispecific antibody-like molecules (Merck, WO2007110205); Triomab (Fresenius, Lindhofer et al. (1995 J Immunol 155:219); FcΔAdp (Regeneron, WO2010151792), Azymetric Scaffold (Zymeworks/Merck, WO2012/058768), mAb-Fv (Xencor, WO2011/028952), Dual variable domain immunoglobulin (Abbott, DVD-Ig, U.S. Pat. No. 7,612,181); Dual domain double head antibodies (Unilever; Sanofi Aventis, WO20100226923), Di-diabody (ImClone/Eli Lilly), Knobs-into-holes antibody formats (Genentech, WO9850431); DuoBody (Genmab, WO 2011/131746); Electrostatic steering antibody formats (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545), CrossMAbs (Roche, WO2011117329), LUZ-Y (Genentech), Biclonic (Merus), Dual Targeting domain antibodies (GSK/Domantis), Two-in-one Antibodies recognizing two targets (Genentech, NovImmune), Cross-linked Mabs (Karmanos Cancer Center), CovX-body (CovX/Pfizer), IgG-like Bispecific (ImClone/Eli Lilly, Shen, J., et al. J Immunol Methods, 2007. 318(1-2): p. 65-74), and DIG-body and PIG-body (Pharmabcine), and Dual-affinity retargeting molecules (Fc-DART or Ig-DART, by Macrogenics, WO/2008/157379, WO/2010/080538), Zybodies (Zyngenia), approaches with common light chain (Crucell/Merus, U.S. Pat. No. 7,262,028) or common heavy chains (κλBodies by NovImmune), as well as fusion proteins comprising a polypeptide sequence fused to an antibody fragment containing an Fc-domain like scFv-fusions, like BsAb by ZymoGenetics/BMS), HERCULES by Biogen Idec (U.S. Ser. No. 00/795,1918), SCORPIONS by Emergent BioSolutions/Trubion, Ts2Ab (MedImmune/AZ (Dimasi, N., et al. J Mol Biol, 2009. 393(3): p. 672-92), scFv fusion by Novartis, scFv fusion by Changzhou Adam Biotech Inc (CN 102250246), TvAb by Roche (WO 2012025525, WO 2012025530), mAb2 by f-Star (WO2008/003116) dual scFv-fusions, a mini-antibody, and a Dual Targeting (DT)-Ig antibody. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures (recombinant polyclonals) for instance generated by technologies exploited by Symphogen and Merus (Oligoclonics), and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies. An antibody as generated can potentially possess any isotype.
The parent antibody or an antibody of the present invention may be prepared from wild-type antibodies or non-naturally occurring antibody formats as any of those described herein, e.g. heterodimeric proteins, which are used as starting material into which the relevant modifications according to the present invention are introduced. The antibodies of the present invention may e.g. be produced by the hybridoma method first described by Kohler et al., Nature 256, 495 (1975), or may be produced by recombinant DNA methods. Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352, 624 628 (1991) and Marks et al., J. Mol. Biol. 222, 581 597 (1991). Monoclonal antibodies may be obtained from any suitable source. Thus, for example, monoclonal antibodies may be obtained from hybridomas prepared from murine splenic B cells obtained from mice immunized with an antigen of interest, for instance in form of cells expressing the antigen on the surface, or a nucleic acid encoding an antigen of interest. Monoclonal antibodies may also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or non-human mammals such as rabbits, rats, dogs, primates, etc.
The antibody may be e.g. a chimeric or humanized antibody. In another embodiment, the antibody is a human antibody. Human monoclonal antibodies may be generated using transgenic or transchromosomal mice, e.g. HuMAb mice, carrying parts of the human immune system rather than the mouse system. The HuMAb mouse contains a human immunoglobulin gene minilocus that encodes unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature 368, 856 859 (1994)). Accordingly, the mice exhibit reduced expression of mouse IgM or κ and in response to immunization, the introduced human heavy and light chain transgenes, undergo class switching and somatic mutation to generate high affinity human IgG,κ monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook of Experimental Pharmacology 113, 49 101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65 93 (1995) and Harding, F. and Lonberg, N. Ann. N.Y. Acad. Sci 764 536 546 (1995)). The preparation of HuMAb mice is described in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287 6295 (1992), Chen, J. et al., International Immunology 5, 647 656 (1993), Tuaillon et al., J. Immunol. 152, 2912 2920 (1994), Taylor, L. et al., International Immunology 6, 579 591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845 851 (1996). See also U.S. Pat. Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, 5,770,429, 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187. Splenocytes from these transgenic mice may be used to generate hybridomas that secrete human monoclonal antibodies according to well known techniques.
Further, human antibodies of the present invention or antibodies of the present invention from other species may be identified through display-type technologies, including, without limitation, phage display, retroviral display, ribosomal display, mammalian display, yeast display and other techniques known in the art, and the resulting molecules may be subjected to additional maturation, such as affinity maturation, as such techniques are well known in the art.
The antibody is not limited to antibodies which have a natural, e.g. a human Fc domain but it may also be an antibody having other mutations than those of the present invention, such as e.g. mutations that affect glycosylation, C1q binding, Fc receptor binding, or enables the antibody to be a bispecific antibody. By the term “natural antibody” is meant any antibody which does not comprise any genetically introduced mutations. An antibody which comprises naturally occurring variations, e.g. different allotypes, is thus to be understood as a “natural antibody” in the sense of the present invention. Such antibodies may serve as a template or starting material, e.g. parent antibody, for introducing the mutations according to the present invention, and thereby providing the antibodies of the invention. An example of an antibody comprising other mutations than those of the present invention is a bispecific antibody as described in WO2011/131746 (Genmab), utilizing reducing conditions to promote half-molecule exchange of two antibodies comprising IgG4-like matched CH3 regions, thus forming bispecific antibodies without concomitant formation of aggregates. Other examples of antibodies include but are not limited to bispecific antibodies such as heterodimeric bispecifics: Triomabs (Fresenius); bispecific IgG1 and IgG2 (Rinat Neurosciences Corporation); FcΔAdp (Regeneron); Knobs-into-holes (Genentech); Electrostatic steering (Amgen, Chugai, Oncomed); SEEDbodies (Merck); Azymetric scaffold (Zymeworks); mAb-Fv (Xencor); and LUZ-Y (Genentch). Other exemplary antibody formats include, without limitation, a wild-type antibody, a full-length antibody or Fc-containing antibody fragment, a human antibody, or any combination thereof.
Monoclonal antibodies for use in the present invention, may be produced, e.g., by the hybridoma method first described by Kohler et al., Nature 256, 495 (1975), or may be produced by recombinant DNA methods. Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies may be obtained from any suitable source. Thus, for example, monoclonal antibodies may be obtained from hybridomas prepared from murine splenic B cells obtained from mice immunized with an antigen of interest, for instance in form of cells expressing the antigen on the surface, or a nucleic acid encoding an antigen of interest. Monoclonal antibodies may also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or non-human mammals such as rats, dogs, primates, etc.
In one embodiment, the antibody is a human antibody. Human monoclonal antibodies directed against any antigen may be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. Such transgenic and transchromosomic mice include mice referred to herein as HuMAb® mice and KM mice, respectively, and are collectively referred to herein as “transgenic mice”.
The HuMAb® mouse contains a human immunoglobulin gene miniloci that encodes unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature 368, 856-859 (1994)). Accordingly, the mice exhibit reduced expression of mouse IgM or κ and in response to immunization, the introduced human heavy and light chain transgenes, undergo class switching and somatic mutation to generate high affinity human IgG,κ monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook of Experimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65-93 (1995) and Harding, F. and Lonberg, N. Ann. N.Y. Acad. Sci 764 536-546 (1995)). The preparation of HuMAb® mice is described in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994), Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845-851 (1996). See also U.S. Pat. Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, 5,770,429, 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.
The HCo7, HCo12, HCo17 and HCo20 mice have a JKD disruption in their endogenous light chain (kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)), a CMD disruption in their endogenous heavy chain genes (as described in Example 1 of WO 01/14424), and a KCo5 human kappa light chain transgene (as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)). Additionally, the Hco7 mice have a HCo7 human heavy chain transgene (as described in U.S. Pat. No. 5,770,429), the HCo12 mice have a HCo12 human heavy chain transgene (as described in Example 2 of WO 01/14424), the HCo17 mice have a HCo17 human heavy chain transgene (as described in Example 2 of WO 01/09187) and the HCo20 mice have a HCo20 human heavy chain transgene. The resulting mice express human immunoglobulin heavy and kappa light chain transgenes in a background homozygous for disruption of the endogenous mouse heavy and kappa light chain loci.
In the KM mouse strain, the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al., EMBO J. 12, 811-820 (1993) and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of WO 01/09187. This mouse strain carries a human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also carries a human heavy chain transchromosome composed of chromosome 14 fragment hCF (SC20) as described in WO 02/43478. HCo12-Balb/C mice can be generated by crossing HCo12 to KCo5[J/K](Balb) as described in WO/2009/097006. Splenocytes from these transgenic mice may be used to generate hybridomas that secrete human monoclonal antibodies according to well-known techniques.
Further, any antigen-binding regions of the present invention may be obtained from human antibodies or antibodies from other species identified through display-type technologies, including, without limitation, phage display, retroviral display, ribosomal display, and other techniques, using techniques well known in the art and the resulting molecules may be subjected to additional maturation, such as affinity maturation, as such techniques are well known in the art (see for instance Hoogenboom et al., J. Mol. Biol. 227, 381 (1991) (phage display), Vaughan et al., Nature Biotech 14, 309 (1996) (phage display), Hanes and Plucthau, PNAS USA 94, 4937-4942 (1997) (ribosomal display), Parmley and Smith, Gene 73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245 (1992), Cwirla et al., PNAS USA 87, 6378-6382 (1990), Russel et al., Nucl. Acids Research 21, 1081-1085 (1993), Hogenboom et al., Immunol. Reviews 130, 43-68 (1992), Chiswell and McCafferty TIBTECH 10, 80-84 (1992), and U.S. Pat. No. 5,733,743). If display technologies are utilized to produce antibodies that are not human, such antibodies may be humanized.
The dimeric protein, e.g. antibody of the present invention may comprise human IgG1 heavy chain comprising except for the mutations described herein the sequence of SEQ ID NO: 1 (UniProt accession No. P01857), such as comprising the relevant segment, I253 to K447, e.g. P247 to K447, corresponding to the underlined residues 136 to 330, e.g. 130 to 330, of the human IgG1 heavy chain constant region; SEQ ID NO:1:
1 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs
wnsgaltsgv
51 htfpavlqss glyslssvvt vpssslgtqt yicnvnhkps
ntkvdkkvep
101 kscdkthtcp pcpapellgg psvflfppkp kdtlmisrtp
evtcvvvdvs
151 hedpevkfnw yvdgvevhna ktkpreeqyn styrvvsvlt
vlhqdwlngk
201 eykckvsnka lpapiektis kakgqprepq vytlppsrde
ltknqvsltc
251 lvkgfypsdi avewesngqp ennykttppv ldsdgsffly
skltvdksrw
301 qqgnvfscsv mhealhnhyt qkslslspgk
The allotype of the above-referenced IgG1 is IgG1m(za). The CH1 domain, hinge region, CH2 domain, and CH3 domain in SEQ ID NO:1 are for the present invention amino acids 1-98, 99-113, 114-223 and 224-330, respectively. The CH1 domain, hinge region, CH2 domain, and CH3 domain when numbered according to Eu numbering as set forth in Kabat are numbered as amino acids 118-215, 216-230, 231-340, and 340-447, respectively.
The dimeric protein, e.g. antibody of the present invention can also comprise a human IgG2 heavy chain comprising except for the mutations described herein the sequence of SEQ ID NO:2. Amino acid residues I253 to K447, e.g. P247 to K447, of the IgG1 heavy chain correspond to the underlined residues 132 to 326, e.g. 126 to 326, of the IgG2 heavy chain constant region (accession number P01859; SEQ ID NO:2)
1 astkgpsvfp lapcsrstse staalgclvk dyfpepvtvs
wnsgaltsgv
51 htfpavlqss glyslssvvt vpssnfgtqt ytcnvdhkps
ntkvdktver
101 kccvecppcp appvagpsvf lfppkpkdtl misrtpevtc
vvvdvshedp
151 evqfnwyvdg vevhnaktkp reeqfnstfr vvsvltvvhq
dwlngkeykc
201 kvsnkglpap iektisktkg gpregavytl ppsreemtkn
qvsltclvkg
251 fypsdiavew esngqpenny kttppmldsd gsfflysklt
vdksrwqqgn
301 vfscsvmhea lhnhytqksl slspgk
The CH1 domain, hinge region, CH2 domain, and CH3 domain in SEQ ID NO:2 are for the present invention amino acids 1-98, 99-110, 111-219 and 220-326, respectively.
The dimeric protein, e.g. antibody, of the present invention can also comprise a human IgG3 heavy chain comprising except for the mutations described herein the sequence of SEQ ID NO:3. Amino acid residues I253 to K447, e.g. P247 to K447, of the IgG1 heavy chain correspond to residues 183 to 377, e.g. 177 to 377, of the IgG3 heavy chain constant region (UniProt accession No. P01860, SEQ ID NO:3), underlined in the following:
1 astkgpsvfp lapcsrstsg gtaalgclvk dyfpepvtvs
wnsgaltsgv
51 htfpavlqss glyslssvvt vpssslgtqt ytcnvnhkps
ntkvdkrvel
101 ktplgdttht cprcpepksc dtpppcprcp epkscdtppp
cprcpepksc
151 dtpppcprcp apellggpsv flfppkpkdt lmisrtpevt
cvvvdvshed
201 pevqfkwyvd gvevhnaktk preeqynstf rvvsvltvlh
qdwlngkeyk
251 ckvsnkalpa piektisktk gqprepqvyt lppsreemtk
nqvsltclvk
301 gfypsdiave wessgqpenn ynttppmlds dgsfflyskl
tvdksrwqqg
351 nifscsvmhe alhnrftqks lslspgk
The CH1 domain, hinge region, CH2 domain, and CH3 domain in SEQ ID NO:3 are for the present invention amino acids 1-98, 99-160, 161-270 and 271-377, respectively.
The dimeric protein, e.g. antibody, of the present invention can also comprise a human IgG4 heavy chain comprising except for the mutations described herein the sequence of SEQ ID NO:4. Amino acid residues I253 to K447, e.g. P247 to K447, of the IgG1 heavy chain correspond to the underlined residues 133 to 327, e.g. 127 to 327, of the IgG4 heavy chain constant region (accession number P01859, SEQ ID NO:4)
1 astkgpsvfp lapcsrstse staalgclvk dyfpepvtvs
wnsgaltsgv
51 htfpavlqss glyslssvvt vpssslgtkt ytcnvdhkps
ntkvdkrves
101 kygppcpscp apeflggpsv flfppkpkdt lmisrtpevt
cvvvdvsqed
151 pevqfnwyvd gvevhnaktk preeqfnsty rvvsvltvlh
qdwlngkeyk
201 ckvsnkglps siektiskak gqprepqvyt lppsqeemtk
nqvsltclvk
251 gfvpsdiave wesngqpenn ykttppvlds dgsfflysrl
tvdksrwqeg
301 nvfscsvmhe alhnhytqks lslslgk
The CH1 domain, hinge region, CH2 domain, and CH3 domain in SEQ ID NO:4 are for the present invention amino acids 1-98, 99-110, 111-220 and 221-327, respectively.
The dimeric protein, e.g. antibody, of the present invention can also comprise a human IgG1m(f) allotype heavy chain comprising except for the mutations described herein the sequence of SEQ ID NO:5. Amino acid residues I253 to K447 of the IgG1m(f) allotype heavy chain correspond to the underlined residues 136-330 of SEQ ID NO:5
1 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs
wnsgaltsgv
51 htfpavlqss glyslssvvt vpssslgtqt yicnvnhkps
ntkvdkrvep
101 kscdkthtcp pcpapellgg psvflfppkp kdtlmisrtp
evtcvvvdvs
151 hedpevkfnw yvdgvevhna ktkpreeqyn styrvvsvlt
vlhqdwlngk
201 eykckvsnka lpapiektis kakgqprepq vytlppsree
mtknqvsltc
251 lvkgfypsdi avewesngqp ennykttppv ldsdgsffly
skltvdksrw
301 qqgnvfscsv mhealhnhyt qkslslspgk
The CH1 domain, hinge region, CH2 domain, and CH3 domain in SEQ ID NO:5 are for the present invention amino acids 1-98, 99-113, 114-223 and 224-330, respectively.
The dimeric protein of the present invention may be of another allotype of human IgG1 immunoglobulins, such as IgG1m(a), IgG1m(z), and IgG1m(x). Such allotypes have been described to contain different amino acid in one or more positions corresponding to positions 214, 356, 358, and 431 according to Eu numbering as set forth in Kabat.
An alignment of the respective segments of the IgG1, IgG2, IgG3, IgG4, IgG1m(f), IgA1, IgA2, IgE, IgD and IgM constant regions is shown in FIG. 2. Accordingly, any amino acid position, or mutation in an amino acid position, described herein as corresponding to an amino acid position in a human IgG1 heavy chain, can be identified or introduced at its equivalent position in IgG2, IgG3, IgG4, IgG1m(f), IgA1, IgA2, IgE, IgD and IgM as defined by the alignment in FIG. 2 to obtain a dimeric protein according to the invention.
In any aspect or embodiment of a dimeric protein of the present invention, the first and/or second polypeptide of the dimeric protein may comprise the sequence of residues 130 to 330 of SEQ ID NO:1, residues 126 to 326 of SEQ ID NO:2, residues 177 to 377 of SEQ ID NO:3, residues 127 to 327 of SEQ ID NO:4, or residues 130 to 330 of SEQ ID NO:5.
In one embodiment, the first and/or second polypeptide of the dimeric protein comprises a sequence selected from SEQ ID No.: 1-5, such as SEQ ID No.:1, SEQ ID No.:2, SEQ ID No.:3, SEQ ID No.:4, or SEQ ID No.:5.
In one embodiment the antibody is a human full-length antibody, such as a human full-length IgG1 antibody.
In one embodiment, the antibody is a human IgG1 antibody, e.g. the IgG1m(za) or IgG1m(f) allotype, optionally wherein the first and/or second polypeptide, e.g. both polypeptides, comprises except for the mutations described herein, SEQ ID NO:1 or 5, In one embodiment, the antibody is a human antibody which may be any of the allotypes known within that isotype.
In one embodiment, the antibody is a human IgG2 antibody, optionally wherein the first and/or second polypeptide, e.g. both polypeptides, comprises SEQ ID NO:2.
In one embodiment, the antibody is a human IgG3 antibody, optionally wherein the first and/or second polypeptide, e.g. both polypeptides, comprises SEQ ID NO:3.
In one embodiment, the antibody is a human IgG4 antibody, optionally wherein the first and/or second polypeptide, e.g. both polypeptides, comprises SEQ ID NO:4.
In particular embodiments of any dimeric protein of the present invention, the first and/or second polypeptide, e.g. both polypeptides, of the dimeric protein comprises an amino acid sequence which has a degree of identity to amino acids P247 to K447, e.g. I253 to K447, of SEQ ID Nos: 1, 2, 3, 4, and 5 of at least 70%, 72%, 74%, 76%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or of at least about 99%, except for the amino acid positions defined by the present invention.
Thus, the first and/or second polypeptide, e.g. both polypeptides, of the dimeric protein may comprise a sequence according to SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No: 4, or SEQ ID No:5 except for any amino acid residues of the present invention and defined herein.
The inventors of the present invention have found that six antibodies in which the amino acids E, E and S at the positions corresponding to E345, E430 and S440 of a human IgG1 heavy chain, have been substituted with the amino acids R, G and Y, respectively, are capable of forming non-covalent hexameric structures in solution.
Hence, in one embodiment, the dimeric protein of the present invention is predominantly in oligomeric form, such as hexameric form, in a phosphate buffer at a pH of about 6.8.
Hence, in one embodiment a dimeric protein of the present invention is capable of forming a non-covalent hexameric structure under the conditions described in Example 20 or 23, e.g. in a solution of 12.6 mM sodium phosphate, 140 mM NaCl at pH 7.4, or in a solution of 0.1 M Na2SO4, 0.1 M sodium phosphate at pH 6.8, or in a solution of 0.15 M NaCl, 0.1M citrate buffer at pH 6.8. In the context of the present invention the term “capable of forming non-covalent hexameric structure” means that more than 30%, such as more than 40%, or more than 50%, or more than 60%, or more than 70%, or more than 80%, or more than 85%, or more than 90%, or more than 95% of the dimeric proteins, e.g. antibodies are in a non-covalent hexameric structure when determined as described in Example 20. In a further embodiment the dimeric protein of the present invention is not capable of forming non-covalent structure in a solution of 0.15 M NaCl, 0.1M citrate buffer at pH 5.0. The term “is not able to form non-covalent hexameric structure” means in the context of the present invention that less than 10%, such as less than 8%, or less than 7%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or less than 0.5% of the dimeric proteins, e.g. antibodies, are in a non-covalent hexameric structure when determined as described in Example 20. Thus, in one embodiment, the hexameric structure may be determined by performing HP-SEC (high pressure size exclusion chromatography) fractionation using a suitable size exclusion chromatography resin with a pore size capable of separating molecules in the range of 50 kDa to 1000 kDa, connected to an absorbance detector; separating in 50 μL samples containing 1.25 μg/mL protein at 1 mL/min in 0.1 M Na2SO4/0.1 M sodium phosphate buffered at pH 6.8; using suitable software to process results; and expressing per peak as percentage of total peak area.
The ability of a dimeric protein of the present invention of forming an oligomeric, e.g. a hexameric structure makes the dimeric protein suitable for binding targets not only present on a cell but also soluble targets. Thus, the dimeric protein of the present invention may e.g. be used to remove soluble factors, e.g. bacterial toxins, or other unwanted factors, from the blood stream, such as complement components, e.g. C1q.
Phenotyping of erythrocytes, such as determination of the Rhesus D status, is important in case of blood transfusions and to determine the risk of hemolytic disease of a newborn. For the phenotyping of erythrocytes currently monoclonal human IgG antibodies are used in a laboratory test, e.g. Coombs test. However, many of the IgGs used in these assays induce poor agglutination. In stead of IgG, oligomeric structures, e.g. hexameric structures, of the dimeric proteins of the present invention may be used as reagent in phenotyping assays. Using a stable oligomeric, such as a hexameric, structure of the dimeric proteins of the present invention for erythrocyte phenotyping may have several advantages, such as the oligomeric structure could by itself induce crosslinking of cells bypassing need for a secondary antibody, could improve the sensitivity of the assay, and two or more dimeric proteins having different binding regions could be used for phenotyping multiple erythrocyte antigens simultaneously. Thus, in one embodiment, the dimeric protein of the present invention may be used for phenotyping erythrocytes. In one embodiment, two or more, such as three, four, five or six, dimeric proteins of the present invention having different binding regions may be used for phenotyping of multiple erythrocyte antigens.
In a further embodiment the dimeric protein of the present invention has an increased effector function compared to a parent dimeric protein. The dimeric protein of the present invention may be regarded as a variant of a parent dimeric protein wherein the variant comprises amino acid mutations in the positions 345, 430, and an amino acid mutation in at least one position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254, compared to the parent dimeric protein. A parent dimeric protein is in this context a dimeric protein in which the amino acids of said first and/or second polypeptides correspond to those of a human IgG1 heavy chain at positions E345, E430 and amino acid in at least one position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254, wherein the amino acid position selected from the group consisting of S440, Y436, E356, T359, E382, N434, Q438, I253 and S254, corresponds to those of a human IgG1 heavy chain at that position. As described above, the parent dimeric protein may be any isotype.
For an antibody, typically, the efficacy of the antibody may be expressed by the EC50 value, which is the concentration of the antibody necessary to obtain 50% of the maximal effect. This similarly applies to a dimeric protein of the present invention.
Maximal effect is the effect obtained when a saturating amount of the antibody is used, in which saturating is intended to refer to the amount of antibody at which all antigens for the antibody are bound by the antibody. This similarly applies to a dimeric protein of the present invention.
The term “increasing an effector function” or “improving an effector function” refers in the context of the present invention to a decrease in the EC50 value of the dimeric protein of the present invention compared to the parent dimeric protein. The decrease in the EC50 value may e.g. be at least or about 2-fold, such as at least or about 3-fold, or at least or about 5-fold, or at least or about 10-fold. Alternatively, “increasing an effector function” or “improving an effector function” means that there is an increase in the maximal amount of cells lysed (where the total amount of cells is set at 100%) by e.g. from 10% to 100% of all cells, such as by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100% under conditions where the parent dimeric protein lyses less than 100% of all cells.
A dimeric protein could be tested for increased or improved effector function by cloning the variable domain of the IgG1-005 or IgG1-7D8 heavy chain into the dimeric protein and test its efficacy in CDC assays, such as described for Daudi (Example 3) and Wien (Example 6). In one embodiment, CDC efficacy may be determined by pre-incubating suspension cells at a concentration of 1×106 cells/mL in round-bottom 96-well plates with an antibody in the range from 0.0003 to 30.0 μg/mL final concentration in a total volume of 100 μL for 15 min on a shaker at room temperature, adding normal human serum at a concentration of 20%, 30% or 50% final concentration, incubating at 37° C. for 45 min, putting the plates on ice, adding 10 μL propidium iodide, and determining cell lysis by FACS analysis.
Using an IgG1-7D8 HC variable domain and Daudi cells, an increase would be defined by a more than 2 fold lower EC50 than the EC50 of IgG1-7D8 under the studied condition, such as about 2-fold, about 3-fold, about 5-fold, about 10-fold or a more than 10-fold lower EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-005 HC variable domain and Daudi cells, an increase would be defined by a more than 2 fold lower EC50 than the EC50 of IgG1-005 under the studied condition, such as about 2-fold, about 3-fold, about 5-fold, about 10-fold or a more than 10-fold lower EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-7D8 HC variable domain and Wien133 cells, an increase would be defined by a more than 2 fold lower EC50 than the EC50 of IgG1-7D8 under the studied condition, such as about 2-fold, about 3-fold, about 5-fold, about 10-fold or a more than 10-fold lower EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-005 HC variable domain and Wien133 cells, an increase would be defined by an increase in the maximal lysis ranging from 10% to 100% of all cells, such as by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%. An increase in CDC efficacy could also be defined by a more than 2-fold lower EC50 than the EC50 of IgG1-005 under the studied condition, such as about 2-fold, about 3-fold, about 5-fold, about 10-fold or a more than 10-fold lower EC50 value, the concentration at which half-maximal lysis is observed under conditions where lysis of Wien133 cells is detectable.
The inventors of the present invention have found that an antibody wherein the amino acids in positions corresponding to E345, E430 and S440 in a human IgG1 heavy chain are not E, E and S, respectively have a lower EC50 value for binding to C1q and a lower EC50 value for CDC (see example 21) compared to the same antibody wherein the amino acids at said positions are E, E and S, respectively.
In a further embodiment, an effector function, such as CDC, of the dimeric protein of the present invention may be increased when the dimeric protein is bound to its target on a cell or virion where the target is present on the virion or cell membrane, as compared to the parent dimeric protein.
Other Amino Acid Position(s)
The first and/or second polypeptide of the dimeric protein of the present invention may further comprise other specific amino acids at indicated position(s). As described herein, a dimeric protein of the present invention, e.g. an antibody, may be prepared by introducing mutations at the amino acid positions as specified by the present invention.
Examples of such further other amino acids positions, or mutations, include amino acids positions where the specific amino acid affects one or more effector functions of the dimeric protein. Examples of such amino acids include amino acid residues which are capable of enhancing CDC, C1q binding, Fc-gamma receptor binding or FcRn-binding and/or improving Fc-gamma receptor-mediated effector functions.
Examples of effector functions include (i) C1q-binding, (ii) complement activation, (iii) CDC, (iv) oligomer formation, (v) oligomer stability, (vi) antibody-dependent cell-mediated cytotoxity (ADCC), (vii) FcRn-binding, (viii) Fc-gamma receptor-binding, (ix) antibody-dependent cellular phagocytosis (ADCP), (x) complement-dependent cellular cytotoxicity (CDCC), (xi) complement-enhanced cytotoxicity, (xii) binding to complement receptor of an opsonized antibody mediated by the antibody, (xiii) internalization, (xiv) downmodulation, (xv) induction of apoptosis, (xvi) opsonisation, (xvii) proliferation modulation, such as proliferation reduction, inhibition, and stimulation, and (xvii) a combination of any of (i) to (xvi), of a dimeric protein, such as an antibody when e.g. bound to its antigen on a cell, on a cell membrane, on a virion, or on another particle.
In one embodiment, a dimeric protein according to the invention further comprises an amino acid residue, or a modification of an amino acid residue, which is known as enhancing CDC e.g., an exchange of segments between IgG isotypes to generate chimeric IgG molecules (Natsume et al., 2008 Cancer Res 68(10), 3863-72); one or more amino acid substitutions in the hinge region (Dall'Acqua et al., 2006 J Immunol 177, 1129-1138), and/or one or more amino acid substitutions in or near the C1q-binding site in the CH2 domain, centered around residues D270, K322, P329, and P331 (Idusogie et al., 2001 J Immunol 166, 2571-2575; Michaelsen et al., 2009 Scand J Immunol 70, 553-564; WO 99/51642; Moore et al., 2010 mAbs 2(2), 181-189). For example, in one embodiment, a dimeric protein according to the invention further comprises a combination of any of the amino acid substitutions S267E, H268F, S324T, S239D, G236A and I332E, providing enhanced effector function via CDC or ADCC (Moore et al., 2010 mAbs 2(2), 181-189 and WO 2011/091078 A2). Other Fc mutations affecting binding to Fc-receptors (described in WO 2006/105062, WO 00/42072, U.S. Pat. Nos. 6,737,056 and 7,083,784) or physical properties of the antibodies (described in WO 2007/005612 A1) can also be used in the variants of the invention.
Hence, in one embodiment, the amino acid in at least one position corresponding to S267, H268, S324, S239, G236 and I332, may be E, F, T, D, A and E, respectively.
In one embodiment, a dimeric protein according to the invention further comprises modifications enhancing Fc-gamma receptor binding and/or Fc-gamma receptor-mediated effector function. Such modifications include (i) reducing the amount of fucose in the CH2 attached glycosylation (glyco-engineering) (Umana P, et al., Nat Biotechnol 1999; 17: 176-80; Niwa R, et al., Clin Cancer Res 2004; 10: 6248-55.)), and (ii) site-directed mutagenesis of amino acids in the hinge or CH2 regions of antibodies (protein-engineering) (Lazar G A, et al., Proc Natl Acad Sci USA 2006; 103: 4005-10).
In another embodiment such further mutations may be mutations which inhibit or reduce the effector functions of the dimeric protein. In clinical applications where engagement of the immune system is not required and may even cause unwanted side-effects the first and/or second polypeptide of the dimeric protein may then be further mutated in the CH2 domain to abolish C1q and/or FcGammaReceptor interactions.
Some amino acid residues in the Fc-domain that play a dominant role in the interactions with C1q and the FcGammaReceptors have been identified. Positions 234 and 235 were shown to have a strong modulating effect on human Fc binding to human CD64 (Canfield & Morrison, 1991; Chappel et al., 1991; Hezareh et al., 2001), CD32A (Hezareh et al., 2001; Armour et al., 2003), CD16 (Hezareh et al., 2001) and C1q (Xu et al., 2000; Hezareh et al., 2001). The CH2 position 331 was shown to be a major determinant for human IgG binding to human CD64 (Canfield & Morrison, 1991, J Exp Med.; 173:1483-91) and C1q (Tao et al., 1993; Idusogie et al., 2000) and a triple mutation L234F/L235E/P331S causes a profound decrease in binding to human CD64, CD32A, CD16 and C1q.
Based on this knowledge several variants were described to make Fc-domain inactive for interactions with Fcgamma receptors and C1q for therapeutic antibody development.
For IgG1 mutating L234A and L235A and P331S were described (Hezareh M, et al., J Virol 2001, 75:12161-12168, Xu D et al. Cell Immunol 2000, 200:16-26, Shields R L, et al. J Biol Chem 2001, 276:6591-6604) and L234A combined with L235A was used in the clinic (Herold K C, et al. Diabetes 2005, 54:1763-1769). Hence, in one embodiment, the amino acid in at least one position corresponding to L234, L235 and P331, may be A, A and S, respectively.
Also mutating these same positions to L234F and L235E was described to result in Fc-domains with abrogated interactions with FcGammaReceptors and C1q (Oganesyan Acta Cryst. (2008). D64, 700-704, Canfield & Morrison, 1991 J Exp Med.; 173:1483-91., Duncan, 1988 Nature 332:738-40). Hence, in one embodiment, the amino acids in the positions corresponding to L234 and L235, may be F and E, respectively.
Mutating position D265A showed decreased binding to all FcγReceptors and prevented ADCC (Shields R L et al. J Biol Chem 2001, 276:6591-6604). Hence, in one embodiment, the amino acid in a position corresponding to D265, may be A.
Binding to C1q could be abrogated by mutating positions D270, K322, P329, and P331 (Idusogie et al., J Immunol 2000, 164:4178-4184). Mutating these positions to either D270A or K322A or P329A or P331A made the antibody deficient in CDC activity. Hence, in one embodiment, the amino acids in at least one position corresponding to D270, K322, P329 and P331, may be A, A, A, and A, respectively.
An alternative approach to minimize the interaction of the Fc-domain with FcgammaReceptors and C1q is by removal of the glycosylation site of an antibody. Mutating position N297 to eg Q, A, and E removes a glycosylation site which is critical for IgG-Fcgamma receptor interactions (Tao and Morrison, J Immunol. 1989 Oct. 15; 143(8):2595-601, Bolt S et al., Eur J Immunol 1993, 23:403-411). Hence, in one embodiment, the amino acid in a position corresponding to N297, may be Q, A or E.
Alternatively, human IgG2 and IgG4 subclasses are naturally compromised in their interactions with C1q and FcgammaReceptors. However, residual interactions with FcγReceptors (FcgammaReceptors) have been described (Parren et al., J Clin Invest 1992, 90:1537-1546.). Mutations abrogating these residual interactions have been described for both isotypes and result in reduction of unwanted side-effects associated with FcR binding. For IgG2 mutating L234A and G237A was described (Cole M S et al. J Immunol 1997, 159:3613-3621 and for IgG4 L235E was described (Reddy M P et al., J Immunol 2000, 164:1925-1933). Hence, in one embodiment, the amino acid in a position corresponding to L234 and G237 in a human IgG2 heavy chain, may be A and A, respectively. In one embodiment, the amino acid in a position corresponding to L235 in a human IgG4 heavy chain, may be E.
Other approaches to further minimize the interaction with FcgammaReceptors and C1q IgG2 antibodies were described in WO 2011/066501 A1 (PCT/US2010/058188) and Lightle, S., et al.; Protein Science (19):753-62 (2010).
Alternatively, the hinge region of the antibody is of importance with respect of interactions with FcgammaReceptors and complement. Mutations in the hinge region have been described to influence effector functions of an antibody (Brekke et al., J Immunol 2006, 177:1129-1138, Dall'Acqua W F, et al. J Immunol 2006, 177:1129-1138. Either mutating or deleting the hinge region will affect Fc effector functions of an antibody.
Hence, in one embodiment the first and/or second polypeptide of the dimeric protein of the present invention may further comprise any of the above mentioned mutations which inhibit or reduce one or more effector functions of the dimeric protein.
Combining sets of mutations described above may result in an even more inert Fc-domain, for instance combining mutations L234F, L235E, D265A; or L234F, L235E, N297Q and D265A in an IgG1 Fc-domain or other variations generated by the information described above. Hence, in one embodiment, the amino acids in at least one or a combination of positions corresponding to L234, L235, D265; or L234, L235, N297 and D265, may be F, E, A, F, E, Q and A, respectively.
In one embodiment the first and/or second polypeptide, e.g. both polypeptides of the dimeric protein of the present invention may further comprise any combination of the above mentioned mutations which inhibit or reduce one or more effector functions of the dimeric protein.
Typically, the effect of an antibody on an effector function may be measured by the EC50 value, which is the concentration of the antibody necessary to obtain half the value of the maximal lysis. This similarly applies to a dimeric protein of the present invention.
Maximal lysis is the effect obtained when a saturating amount of the antibody is used in which saturating is intended to refer to the amount of antibody at which all antigens for the antibody are bound by the antibody. This similarly applies to a dimeric protein of the present invention.
Thus, in one embodiment, the first and/or second polypeptides of the dimeric protein may further comprise amino acid substitutions in the amino acid positions corresponding to L234, L235 and D265 in a human IgG1 heavy chain, which are F, E, and A, respectively.
The term “decreasing an effector function” refers in the context of the present invention that there is an increase in the EC50 value of the dimeric protein compared to the parent dimeric protein, wherein parent dimeric protein has the meaning as described above. The increase in the EC50 value may e.g. be at least or about 2-fold, such as at least or about 3-fold, or at least or about 5-fold, or at least or about 10-fold. Alternatively, “decreasing an effector function” means that there is an decrease in the maximal amount of cells lysed by e.g. from 10% to 100% of all cells, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100% under conditions where the parent dimeric protein lyses less than 100% of all cells.
A dimeric protein could be tested for decreased effector function by cloning the variable domain of the IgG1-005 or IgG1-7D8 heavy chain into the dimeric protein and test its efficacy in CDC assays, such as described for Daudi (Example 3) and Wien (Example 6). Using an IgG1-7D8 HC variable domain and Daudi cells, an decrease would be defined by a more than 2 fold higher EC50 than the EC50 of IgG1-7D8 under the studied condition, such as about 2-fold, about 3-fold, about 5-fold, about 10-fold or a more than 10-fold higher EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-005 HC variable domain and Daudi cells, a decrease would be defined by a more than 2-fold higher EC50 than the EC50 of IgG1-005 under the studied condition, such as about 2-fold, about 3-fold, about 5-fold, about 10-fold or a more than 10-fold higher EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-7D8 HC variable domain and Wien133 cells, an decrease would be defined by a more than 2 fold higher EC50 than the EC50 of IgG1-7D8 under the studied condition, such as about 2-fold, about 3-fold, about 5-fold, about 10-fold or a more than 10-fold higher EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-005 HC variable domain and Wien133 cells, a decrease would be defined by a decrease in the maximal lysis ranging from 10% to 100% of all cells, such as by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%. A decrease in CDC efficacy could also be defined by a more than 2-fold higher EC50 than the EC50 of IgG1-005 under the studied condition, such as about 2-fold, about 3-fold, about 5-fold, about 10-fold or a more than 10-fold higher EC50 value, the concentration at which half-maximal lysis is observed under conditions where lysis of Wien133 cells is detectable.
FcRn is a major histocompatibility complex class I-related receptor and plays a role in the passive delivery of immunoglobulin (Ig)Gs from mother to young and in the regulation of serum IgG levels by protecting IgG from intracellular degradation (Ghetie V et al., Annu Rev Immunol. 18, 739-66 (2000)). As FcRn is responsible for the extended persistence of IgG and other Fc-conjugated proteins in the serum, modulating the FcRn-Fc interaction will allow the deliberate control of the half-life of these agents in the circulation to various ends.
In one embodiment, the dimeric protein may comprise other amino acid residues or further, such as amino acid substitutions, which affect the pharmacokinetic profile, e.g. by affecting binding to FcRn.
In one embodiment, the plasma clearance of hexameric forms of dimeric proteins according to the present invention is decreased, for example to allow lower dosing and minimize adverse reactions caused by high doses, decrease frequency of injection, maximize transcytosis to specific tissue sites, enhance efficiency of trans-placental delivery, or decrease production costs.
In a further embodiment, the first and/or second polypeptide, e.g. both polypeptides, of the dimeric protein according to the present invention have been further modified e.g. in the CH2 and/or CH3 region, for example, to improve the pharmacokinetic profile, e.g. via improving the binding to FcRn, e.g. at pH 6.0. These modifications include, but are not limited to, mutations at any one or more of amino acid positions P238, T250, M252, I253, S254, R255, T256, D265, E272, N286, K288, V303, V305, T307, L309, H310, Q311, D312, K317, K340, D356, K360, Q362, D376, A378, E380, E382, Q386, E388, S400, D413, S415, S424, M428, H433, N434, H435, Y436, K439 or K447 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat (Shields, R. L., et al, J Biol Chem. 9, 6591-604 (2001), Dall'Acqua, W. F., et al, J Immunol. 9, 5171-80 (2002), Hinton, P., et al, J Biol Chem. 8, 6213-6 (2004), Dall'Acqua, W. F., et al, J Biol Chem. 33, 23514-24 (2006), Petkova, S. B., et al, Int Immunol. 12, 1759-69 (2006), Datta-Mannan, A., et al, J Biol Chem. 3, 1709-17 (2007), Yeung, Y. A., J Immunol. 12, 7663-71 (2009), Kabat, E. A. in US Department of Health and Human Services, NIH publication n° 91-3242, 5th edition 662, 680, 689 (1991)). Hence, in one embodiment, in the first and/or second, such as both, polypeptides of the dimeric protein, an amino acid in at least one position corresponding to a position selected from the group consisting of P238, T250, M252, I253, S254, R255, T256, D265, E272, N286, K288, V303, V305, T307, L309, H310, Q311, D312, K317, K340, D356, K360, Q362, D376, A378, E380, E382, Q386, E388, S400, D413, S415, S424, M428, H433, N434, H435, Y436, K439 or K447; is not P, not T, not M, not I, not S, not R, not T, not D, not E, not N, not K, not V, not V, not T, not L, not H, not Q, not D, not K, not K, not D, not K, not Q, not D, not A, not E, not E, not Q, not E, not S, not D, not S, not S, not M, not H, not N, not H, not Y, not K or not K, for each position, respectively.
In an even further embodiment, the first and/or second polypeptide, e.g. both polypeptides, of the dimeric protein according to the present invention have been further modified to improve the pharmacokinetic profile, via improving the binding to FcRn by the specific mutations N434A (Shields, R. L., et al, J Biol Chem. 9, 6591-604 (2001)), T307A/E380A/N434A (Shields, R. L., et al, J Biol Chem. 9, 6591-604 (2001)), T250Q/M428L (Hinton, P., et al, J Biol Chem. 8, 6213-6 (2004)) or M252Y/S254T/T256E (Dall'Acqua, W. F., et al, J Immunol. 9, 5171-80 (2002)), wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat (Kabat, E. A. in US Department of Health and Human Services, NIH publication n° 91-3242, 5th edition 662, 680, 689 (1991). Hence, in one embodiment, an amino acid in position corresponding to N434 may be A. In another embodiment, amino acids in positions corresponding to T307, E380 and N434 may be A, A and A, respectively. In another embodiment, amino acids in positions corresponding to positions T250 and M428 may be Q and L, respectively. In another embodiment, amino acids in positions in positions corresponding to M252, S254 and T256 may be Y, T and E, respectively. Thus, in one embodiment, said first and/or second polypeptides of the dimeric protein, may comprise amino acid substitutions in the amino acid positions corresponding to E345, E430, N434, and S440 in a human IgG1 heavy chain, which are not E; not E; A; and Y or W, respectively.
In one embodiment the dimeric protein may comprise a substitution of one or more of P238, T256, T307, Q311, D312, E380, E382, and N434 into an alanine residue improving FcRn binding (Shields R L, et al. J. Biol. Chem. 2001; 276:6591); or an amino acid substitution or combination of amino acid substitutions selected from M252Y/S254T/T256E, M252W, M252Y, M252Y/T256Q, M252F/T256D, V308T/L309P/Q311S, G385D/Q386P/N389S, G385R/Q386T/P387R/N389P, H433K/N434F/Y436H, N434F/Y436H, H433R/N434Y/Y436H, M252Y/S254T/T256E-H433K/N434F/Y436H or M252Y/S254T/T256E-G385R/Q386T/P387R/N389P in IgG1, increasing the affinity for FcRn (Dall'Acqua et al., supra). Hence, in one embodiment, one or more amino acids in position(s) corresponding to positions to those selected from the group consisting of P238, T256, T307, Q311, D312, E380, E382 and N434 may, for each polypeptide of the dimeric protein, be an A for each position, respectively. In another embodiment the amino acids in a positions corresponding to M252, S254 and T256, may be Y, T and E, respectively; or in position corresponding to M252 may be W; or in a position corresponding to M252 may be Y; or in positions corresponding to M252 and T256 may be Y and Q, respectively; or in positions corresponding to M252 and T256 may be F and D, respectively; or in positions corresponding to V308, L309 and Q311 may be T, P and S, respectively; or in positions corresponding to G385, Q386 and N389 may be D, P and S, respectively; or in positions corresponding to G385, Q386, P387 and N389 may be R, T, R and P, respectively; or in positions corresponding to H433, N434 and Y436 may be K, F and H, respectively; or in positions corresponding to N434 and Y436 may be F and H, respectively; or in positions corresponding to H433, N434 and Y436 may be R, Y and H, respectively; or in positions corresponding to M252, S254, T256, H433, N434 and Y436 may be Y, T, E, K, F and H, respectively; or in positions corresponding to M252, S254, T256, G385, Q386, P387 and N389 may be Y, T, E, R, T, R and P, respectively.
In one embodiment, the half-life of hexameric forms of the dimeric proteins according to the present invention is shortened, for example to ensure rapid clearance of dimeric proteins used for imaging and/or radioimmunotherapy, or promote clearance of pathogenic target molecules.
In a further embodiment, the first and/or second polypeptide, e.g. both polypeptides, of the dimeric protein according to the present invention have been further modified e.g. in the CH2 and/or CH3 region, for example, to modulate the pharmacokinetic profile, e.g. via reducing or abrogating the binding to FcRn. These modifications include, but are not limited to, mutations at any one or more of amino acid positions P238, T250, M252, I253, S254, R255, T256, D265, E272, N286, K288, V303, V305, T307, L309, H310, Q311, D312, K317, K340, D356, K360, Q362, D376, A378, E380, E382, Q386, E388, S400, D413, S415, S424, M428, H433, N434, H435, Y436, K439 or K447 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat (Shields, R. L., et al, J Biol Chem. 9, 6591-604 (2001), Dall'Acqua, W. F., et al, J Immunol. 9, 5171-80 (2002), Hinton, P., et al, J Biol Chem. 8, 6213-6 (2004), Dall'Acqua, W. F., et al, J Biol Chem. 33, 23514-24 (2006), Petkova, S. B., et al, Int Immunol. 12, 1759-69 (2006), Datta-Mannan, A., et al, J Biol Chem. 3, 1709-17 (2007), Yeung, Y. A., J Immunol. 12, 7663-71 (2009), Kabat, E. A. in US Department of Health and Human Services, NIH publication n° 91-3242, 5th edition 662, 680, 689 (1991)). Hence, in one embodiment an amino acid in at least one position corresponding to a position selected from the group consisting of P238, T250, M252, I253, S254, R255, T256, D265, E272, N286, K288, V303, V305, T307, L309, H310, Q311, D312, K317, K340, D356, K360, Q362, D376, A378, E380, E382, Q386, E388, S400, D413, S415, S424, M428, H433, N434, H435, Y436, K439 or K447; is not P; not T; not M; not I; not S; not R; not T; not D; not E; not N; not K; not V; not V; not T; not L; not H; not Q; not D; not K; not K; not D; not K; not Q; not D; not A; not E; not E; not Q; not E; not S; not D; not S; not S; not M; not H; not N; not H; not Y; not K or not K, for each position, respectively.
In an even further embodiment, the first and/or second polypeptide, e.g. both polypeptides, of the dimeric protein according to the present invention have been further modified to improve the pharmacokinetic profile, via reducing or abrogating the binding to FcRn by the specific mutations I253A, H310A, H433A, H435A, Y436A, mutations I253A/H310A/H435A (Kim, J. K. et al. Eur. J. Immunol. 29, 2819-2825 (1999), Shields, R. L., et al, J Biol Chem. 9, 6591-604 (2001)), wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat (Kabat, E. A. in US Department of Health and Human Services, NIH publication n° 91-3242, 5th edition 662, 680, 689 (1991)). Hence, in one embodiment, an amino acid in at least one position corresponding to a position selected from the group consisting of I253, H310, H433, H435 and Y436, may be A for each position, respectively. In another embodiment, the amino acids in positions corresponding to I253, H310A and H435A may each be A.
Another example of other amino acid positions where the specific amino acid may be relevant, e.g. where a parent dimeric protein is mutated to change the amino acid, may be amino acids which affect the interaction in the Fc-region between dimeric proteins, e.g. antibodies. Such mutations may be used to minimize the interaction of a therapeutic dimeric protein, e.g. therapeutic antibody, with antibodies naturally present in a patient to whom the therapeutic dimeric protein, e.g. therapeutic antibody, is administered.
Such amino acid residues or mutations have previously been described in PCT/EP12/063339, and include combinations of two amino acid residues or mutations which individually decrease an effector function but when used together, e.g. by combining two dimeric proteins each comprising one of said amino acid residues or mutations the effector function is similar to a parent dimeric protein where said amino acid residue is not mutated, thus it corresponds to that of a human IgG1 heavy chain. When such two dimeric proteins, each comprising one of such a pair of mutations, are used together, the specificity for interaction between said two dimeric proteins may be increased compared to the interaction between two dimeric proteins comprising only one of the mutations of such a pair of mutations. Similarly, the interaction between two dimeric proteins each comprising one of such a pair of mutations, may also be stronger than the interaction between a dimeric protein comprising only one of the mutations of such a pair with a dimeric protein not comprising any of the mutations of the pair.
Hence, in one embodiment the first and/or second polypeptide of the dimeric protein of the present invention may further comprise a mutation or amino acid residue according to this aspect.
Thus, without being bound by any theory, it is foreseen that by including such two mutations in a therapeutic dimeric protein, the induction of C1q binding in a patient will be limited to oligomeric complexes containing therapeutic dimeric protein, e.g. antibodies, comprising such a combination or pair of amino acids. This may allow a reduction of any potential side-effects caused by interaction of the therapeutic dimeric protein with the patients own antibodies which do not comprise such mutations.
In a particular embodiment, a dimeric protein may be used in combination with another dimeric protein, wherein each of the dimeric proteins comprises one of the amino acids of such a “pair” of amino acids. Thus, in one embodiment the present invention relates to a dimeric protein comprising one of the amino acids of such a “pair” in the first and/or second polypeptides. Such a dimeric protein, e.g. first dimeric protein, comprising one amino acid of such a “pair” may be used together with a second dimeric protein comprising in the first and/or second polypeptide the other amino acid of such a “pair”. Examples of such amino acids include those of Table 1. Thus, in a further embodiment, an amino acid in a position corresponding to K439, S440, K447, 448 and 449 may be, for each polypeptide of the dimeric protein of the present invention, as described in Table 1.
TABLE 1
Amino acid position
Preferred
(IgG1)
Exemplary substitutions
substitutions
K439
439DER
439E
S440
440DEKR
440K
K447
447DE
447E
K447/448
447KRH/448P
447/448P
K447
447DE
447E
K447/448/449
447KRH/448KRH/449P
447/448K/449P
Specific combinations of dimeric protein comprising such amino acids may be as described herein.
Thus, in a further aspect of the present invention the amino acid in the position corresponding to K439 is, for one or both, such as each, polypeptides of the dimeric protein, not K.
In a further embodiment the amino acid in the position corresponding to K439 is E or D.
In a further embodiment the amino acid in the position corresponding to K439 is E.
In a further embodiment the amino acid in the position corresponding to K439 is D.
In one embodiment, in the first or second polypeptide of the dimeric protein, the amino acid in positions corresponding to E345, E430, K439, and S440, in a human IgG1 heavy chain, are R, K, Q, N or Y; G, S, T, F, or H; E or D; and Y or W, respectively.
In one embodiment, in said first and/or second polypeptide, the amino acids in the positions corresponding to E345, E430, K439, and S440 in a human IgG1 heavy chain, are R; G; E; and Y, respectively.
In one embodiment, in said first and/or second polypeptide, the amino acid in the position corresponding to S440 in a human IgG1 heavy chain is K or R.
In one embodiment, the amino acid in the position corresponding to S440 is K.
In one embodiment, the amino acid in the position corresponding to S440 is R.
In one embodiment, in said first and/or second polypeptide the amino acid in the position corresponding to E345, E430, and S440 in a human IgG1 heavy chain are R; G; and K, respectively.
In a further embodiment of the present invention at least one amino acid in a position selected from the group consisting of Y436, D/E356, T359, E382, N434, Q438, I253 and S254 is, for one or both, such as each, polypeptides of the dimeric protein not Y; D or E; T; E; N; Q; I; and S, for each position, respectively, and the amino acid in the position corresponding to S440 is K or R.
In a further embodiment, the amino acid in the position corresponding to Y436 is I, and the amino acid corresponding to S440 is K.
In one embodiment, in said first and/or second polypeptide, the amino acids in the positions corresponding to E345, E430, Y436 and S440 in a human IgG1 heavy chain, are R; G; I; and K, respectively.
In one embodiment, the pair of K439D/E/R and S440D/E/K/R substitutions are used. Thus, in one embodiment, the first polypeptide comprises K439D/E/R, and the second polypeptide comprises S440D/E/K/R, or vice versa, and wherein in the first and/or second polypeptide the amino acids corresponding to the positions E345 and E430 are not E. In a further embodiment in the polypeptide comprising S440D/E/K/R one of the amino acids corresponding to the positions selected from the group consisting of Y436, D/E356, T359, E382, N434, Q438, I253 and S254 is not Y; D or E; T; E; N; Q; I; and S, for each position, respectively. Thus, in another aspect of the present invention, at least one amino acid in a position selected from the group consisting of Y436, D/E356, T359, E382, N434, Q438, I253 and S254 is, for one or both, such as each, polypeptides of the dimeric protein not Y; D or E; T; E; N; Q; I; and S, for each position, respectively, and the amino acid in the position corresponding to S440 is not S, Y or W.
In another embodiment the amino acid residue in the position corresponding to K447 is, for one or both, such as each, polypeptides of the dimeric protein D or E.
In a further embodiment, the amino acid in a position corresponding to K447 is D.
In a further embodiment, the amino acid in a position corresponding to K447 is E.
In another embodiment the amino acid residue in the position corresponding to K447 is, for one or both, such as each, of the polypeptides of the dimeric protein, K, R or H and the polypeptides comprise
(a) an amino acid residue in position 448 which is P; or
(b) an amino acid residue in position 448 which is K, R or H and an amino acid residue in position 449 which is P.
In a further embodiment, the amino acid in the position corresponding to K447 is K.
In a further embodiment, the amino acid in the position corresponding to K447 is R.
In a further embodiment, the amino acid in the position corresponding to K447 is H.
In a further embodiment, the amino acid in the position corresponding to 448 is K.
In a further embodiment, the amino acid in the position corresponding to 448 is R.
In a further embodiment, the amino acid in the position corresponding to 448 is H.
In a further embodiment, the amino acid in the position corresponding to Q386 is, for one or both, such as each, polypeptides in the dimeric protein, K.
As described in the Examples 3-5, antibody variants comprising only one of the K439E and S440K mutations had a drastically increased KD for C1q, reflecting a decreased complement activation and/or CDC capability. Surprisingly, it was found that antibody variants of HuMAb 7D8 or 005 comprising both mutations had a restored or increased C1q-binding or CDC. Without being bound by any specific theory, the underlying mechanism could perhaps be explained by the respective mutations sterically compensating for each other, as illustrated in FIG. 4.
In a further embodiment, an amino acid in at least one position corresponding to L234, L235, G236, G237, S239, P238, T250, M252, I253, S254, R255, T256, D265, S267, H268, D270, E272, N286, K288, N297, V303, V305, T307, V308, L309, H310, Q311, D312, K317, K322, S324, P329, P331, I332 K340, D356, K360, Q362, D376, A378, E380, E382, G385, Q386, P387, E388, N389, S400, D413, S415, S424, M428, H433, N434, H435, Y436, K439 or K447, is not, for one or both, such as each, polypeptides of the dimeric protein, L; not L; not G; not G; not S; not P; not T; not M; not I; not S; not R; not T; not D; not S; not H; not D; not E; not N; not K; not N; not V; not V; not T; not V; not L; not H; not Q; not D; not K; not K; not S; not P; not P; not I; not K; not D; not K; not Q; not D; not A; not E; not E; not G; not Q; not P; not E; not N; not S; not D; not S; not S; not M; not H; not N; not H; not Y; not K and not K, respectively.
In one embodiment, the dimeric protein of the present invention is a homodimer. Thus, in one embodiment, both the first and second polypeptides of the dimeric protein comprise the same or identical amino acid substitutions according to any aspect or embodiment of the present invention.
Heterodimeric Format
In another embodiment, the dimeric protein of the present invention is a heterodimer.
In a further embodiment, at least one of the polypeptides comprises a binding region that specifically binds to a target. In a further embodiment, each polypeptide of the heterodimeric protein comprises a binding region specifically binding to a target, optionally the same target.
The target may be any of those described herein.
In a further embodiment the binding regions of the first and second polypeptide of the heterodimeric protein may bind to different epitopes on the same target. In another embodiment the binding regions of the first and second polypeptide of the heterodimeric protein may bind to different targets.
In another embodiment, the binding regions of the first and second polypeptide of the heterodimeric protein may bind to different targets on different cells.
In a particular embodiment, the heterodimeric protein may be a bispecific antibody.
In a further embodiment the binding regions of the first and second polypeptide of said heterodimeric antibody may bind to different epitopes on the same target. In another embodiment the binding regions of the first and second polypeptide of said heterodimeric antibody may bind to different targets.
In another embodiment, the binding regions of the first and second polypeptide of the heterodimeric protein may bind to different targets on different cells.
If the dimeric protein is a heterodimeric protein, the amino acids at the positions corresponding to E345, E430 and at least one position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254 in a human IgG1 heavy chain, may in one embodiment be different in the first and second polypeptide, however, they may also in another embodiment be the same.
Said amino acids may for example be different if the heterodimeric protein is produced as described in WO2011/131746.
The bispecific antibody of the present invention is not limited to a particular format and it may be any of those described herein.
Exemplary bispecific antibody molecules which may be used in the present invention comprise (i) a single antibody that has two arms comprising different antigen-binding regions, (ii) a single chain antibody that has specificity to two different epitopes, e.g., via two scFvs linked in tandem by an extra peptide linker; (iii) a dual-variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (iv) a chemically-linked bispecific (Fab′)2 fragment; (v) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vi) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (vii) a so called “dock and lock” molecule, based on the “dimerization and docking domain” in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (viii) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fab-arm; and (ix) a diabody.
In one embodiment, the bispecific antibody of the present invention is a diabody, a cross-body, or a bispecific antibody obtained via a controlled Fab arm exchange (such as described in WO 11/131746) as those described in the present invention.
Examples of different classes of bispecific antibodies include but are not limited to
IgG-like molecules with complementary CH3 domains to force heterodimerisation
recombinant IgG-like dual targeting molecules, wherein the two sides of the molecule each contain the Fab fragment or part of the Fab fragment of at least two different antibodies;
IgG fusion molecules, wherein full length IgG antibodies are fused to extra Fab fragment or parts of Fab fragment;
Fc fusion molecules, wherein single chain Fv molecules or stabilized diabodies are fused to heavy-chain constant-domains, Fc-regions or parts thereof;
Fab fusion molecules, wherein different Fab-fragments are fused together, fused to heavy-chain constant-domains, Fc-regions or parts thereof;
ScFv- and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) wherein different single chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g. domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule fused to heavy-chain constant-domains, Fc-regions or parts thereof.
Examples of IgG-like molecules with complementary CH3 domains molecules include but are not limited to the Triomab/Quadroma (Trion Pharma/Fresenius Biotech), the Knobs-into-Holes (Genentech), CrossMAbs (Roche) and the electrostatically-matched (Amgen, Chugai, Oncomed), the LUZ-Y (Genentech), the Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), the Biclonics (Merus), FcΔAdp (Regeneron), bispecific IgG1 and IgG2 (Pfizer/Rinat), Azymetric scaffold (Zymeworks), mAb-Fv (Xencor), bivalent bispecific antibodies (Roche) and the DuoBody (Genmab A/S).
Examples of recombinant IgG-like dual targeting molecules include but are not limited to Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-one Antibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star) and CovX-body (CovX/Pfizer).
Examples of IgG fusion molecules include but are not limited to Dual Variable Domain (DVD)-Ig (Abbott), IgG-like Bispecific (ImClone/Eli Lilly), Ts2Ab (MedImmune/AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec) and TvAb (Roche).
Examples of Fc fusion molecules include but are not limited to ScFv/Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS), Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics) and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine—China).
Examples of Fab fusion bispecific antibodies include but are not limited to F(ab)2 (Medarex/AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech).
Examples of ScFv-, diabody-based and domain antibodies include but are not limited to Bispecific T Cell Engager (BITE) (Micromet, Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) and COMBODY (Epigen Biotech), dual targeting nanobodies (Ablynx), dual targeting heavy chain only domain antibodies.
In a particular embodiment, the bispecific antibody has the format described in WO 2011/131746.
Thus, in one embodiment the present invention relates to a heterodimeric protein according to the present invention, wherein
the amino acid in a position selected from K409, T366, L368, K370, D399, F405, and Y407 is not K, T, L, K, D, F and Y, respectively, in the first polypeptide, and
the amino acid in a position selected from F405, T366, L368, K370, D399, Y407, and K409 is not F, T, L, K, D, Y and K, respectively, in the second polypeptide.
In a particular embodiment of the heterodimeric protein, the amino acid in position K409 is R in the first polypeptide, and the amino acid in position F405 is L in the second polypeptide.
Accordingly, in one embodiment, the sequences of said first and second polypeptide contain asymmetrical amino acid residues or mutations, i.e. amino acid residues or mutations at different positions in the first and second polypeptide, e.g. a specific amino acid or mutation at position 405 in one of the polypeptides and a specific amino acid or mutation at position 409 in the other polypeptide. Reference to first and second polypeptide in this respect is not to be understood as limiting, as the amino acid residues or mutations may similarly be present in the opposite polypeptide. Thus, e.g. the amino acid in position F405 is L in said first polypeptide, and the amino acid in position K409 is R in said second polypeptide; or vice versa, the amino acid in position K409 is R in said first polypeptide, and the amino acid in position F405 is L in said second polypeptide.
In one embodiment, the first polypeptide has an amino acid other than Lys, Leu or Met at position 409, such as Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Phe, Tyr, Trp or Cys, and said second polypeptide has an amino acid in a position selected from the group consisting of: 366, 368, 370, 399, 405 and 407, wherein said amino acid is not T, L, K, D, F, and Y. In one such embodiment, said first polypeptide has an amino acid other than Lys, Leu or Met at position 409, e.g. Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Phe, Tyr, Trp or Cys, and said second polypeptide has an amino acid other than Phe at position 405, e.g. Lys, Leu, Met, Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Tyr, Trp or Cys. In a further embodiment hereof, said first polypeptide has an amino acid other than Lys, Leu or Met, e.g. Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Phe, Tyr, Trp or Cys, at position 409 and said second polypeptide has an amino acid other than Phe, Arg or Gly, e.g. e.g. Lys, Leu, Met, His, Asp, Glu, Ser, Thr, Asn, Gln, Pro, Ala, Val, Ile, Tyr, Trp or Cys, at position 405.
In another embodiment, said first polypeptide comprises a Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409, e.g. Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Phe, Tyr, Trp or Cys, and said second polypeptide comprises an amino acid other than Phe, e.g. Lys, Leu, Met, Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Tyr, Trp or Cys, at position 405 and a Lys at position 409. In a further embodiment hereof, said first polypeptide comprises a Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409, e.g. Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Phe, Tyr, Trp or Cys, and said second polypeptide comprises an amino acid other than Phe, Arg or Gly at position 405, e.g. Lys, Leu, Met, His, Asp, Glu, Ser, Thr, Asn, Gln, Pro, Ala, Val, Ile, Tyr, Trp or Cys, and a Lys at position 409.
In another embodiment, said first polypeptide comprises a Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409, e.g. Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Phe, Tyr, Trp or Cys, and said second polypeptide comprises a Leu at position 405 and a Lys at position 409. In a further embodiment hereof, said first polypeptide comprises a Phe at position 405 and an Arg at position 409 and said second polypeptide comprises an amino acid other than Phe, Arg or Gly, e.g. Lys, Leu, Met, His, Asp, Glu, Ser, Thr, Asn, Gln, Pro, Ala, Val, Ile, Tyr, Trp or Cys, at position 405 and a Lys at position 409. In another embodiment, said first polypeptide comprises Phe at position 405 and an Arg at position 409 and said second polypeptide comprises a Leu at position 405 and a Lys at position 409.
In a further embodiment, said first polypeptide comprises an amino acid other than Lys, Leu or Met at position 409, e.g. Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Phe, Tyr, Trp or Cys, and said second polypeptide comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405. In a further embodiment, said first polypeptide comprises an Arg at position 409 and said second polypeptide comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405.
In an even further embodiment, said first polypeptide comprises a Lys at position 370, a Phe at position 405 and an Arg at position 409 and said second polypeptide comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405.
In another embodiment, said first polypeptide comprises an amino acid other than Lys, Leu or Met at position 409, e.g. Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Phe, Tyr, Trp or Cys, and said second polypeptide comprises a Lys at position 409 and: a) an Ile at position 350 and a Leu at position 405, or b) a Thr at position 370 and a Leu at position 405.
In another embodiment, said first polypeptide comprises an Arg at position 409 and said second polypeptide comprises a Lys at position 409 and: a) an Ile at position 350 and a Leu at position 405, or b) a Thr at position 370 and a Leu at position 405.
In another embodiment, said first polypeptide comprises a Thr at position 350, a Lys at position 370, a Phe at position 405 and an Arg at position 409 and said second polypeptide comprises a Lys at position 409 and: a) an Ile at position 350 and a Leu at position 405, or b) a Thr at position 370 and a Leu at position 405.
In another embodiment, said first polypeptide comprises a Thr at position 350, a Lys at position 370, a Phe at position 405 and an Arg at position 409 and said second polypeptide comprises an Ile at position 350, a Thr at position 370, a Leu at position 405 and a Lys at position 409.
In another embodiment, said first polypeptide has an amino acid other than Lys, Leu or Met at position 409, e.g. Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Phe, Tyr, Trp or Cys, and said second polypeptide has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407, e.g. His, Asn, Gly, Pro, Ala, Val, Ile, Trp, Leu, Met or Cys. In another embodiment, said first polypeptide has an amino acid other than Lys, Leu or Met at position 409, e.g. Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Phe, Tyr, Trp or Cys, and said second polypeptide has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407.
In another embodiment, said first polypeptide has an amino acid other than Lys, Leu or Met at position 409, e.g. Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Phe, Tyr, Trp or Cys, and said second polypeptide has a Gly, Leu, Met, Asn or Trp at position 407.
In another embodiment, said first polypeptide has a Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409, e.g. Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Phe, Tyr, Trp or Cys, and said second polypeptide has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407, e.g. His, Asn, Gly, Pro, Ala, Val, Ile, Trp, Leu, Met or Cys, and a Lys at position 409.
In another embodiment, said first polypeptide has a Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409, e.g. Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Phe, Tyr, Trp or Cys, and said second polypeptide has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and a Lys at position 409.
In another embodiment, said first polypeptide has a Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409, e.g. Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Phe, Tyr, Trp or Cys, and said second polypeptide has a Gly, Leu, Met, Asn or Trp at position 407 and a Lys at position 409.
In another embodiment, said first polypeptide has a Tyr at position 407 and an Arg at position 409 and said second polypeptide has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407, e.g. His, Asn, Gly, Pro, Ala, Val, Ile, Trp, Leu, Met or Cys and a Lys at position 409.
In another embodiment, said first polypeptide has a Tyr at position 407 and an Arg at position 409 and said second polypeptide has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and a Lys at position 409.
In another embodiment, said first polypeptide has a Tyr at position 407 and an Arg at position 409 and said second polypeptide has a Gly, Leu, Met, Asn or Trp at position 407 and a Lys at position 409.
In one embodiment, the first polypeptide has an amino acid other than Lys, Leu or Met at position 409, e.g. Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Phe, Tyr, Trp or Cys, and the second polypeptide has
(i) an amino acid other than Phe, Leu and Met at position 368, e.g. Arg, His, Asp, Glu, Ser, Thr, Asn, Gln, Gly, Pro, Ala, Val, Ile, Lys, Tyr, Trp or Cys or
(ii) a Trp at position 370, or
(iii) an amino acid other than Asp, Cys, Pro, Glu or Gln at position 399, e.g. Arg, His, Ser, Thr, Asn, Gly, Ala, Val, Ile, Phe, Tyr, Trp, Lys, Leu, or Met, or
(iv) an amino acid other than Lys, Arg, Ser, Thr, or Trp at position 366, e.g. Leu, Met, His, Asp, Glu, Asn, Glu, Gly, Pro, Ala, Val, Ile, Phe, Tyr or Cys.
In one embodiment, the first polypeptide has an Arg, Ala, His or Gly at position 409, and the second polypeptide has
(i) a Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or
(ii) a Trp at position 370, or
(iii) an Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr at position 399 or
(iv) an Ala, Asp, Glu, His, Asn, Val, Gln, Phe, Gly, Ile, Leu, Met, or Tyr at position 366.
In one embodiment, the first polypeptide has an Arg at position 409, and the second polypeptide has
(i) an Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or
(ii) a Trp at position 370, or
(iii) a Phe, His, Lys, Arg or Tyr at position 399, or
(iv) an Ala, Asp, Glu, His, Asn, Val, Gln at position 366.
In addition to the above-specified amino-acid substitutions, said first and second polypeptide may contain further amino-acid substitutions, deletion or insertions relative to wild-type Fc sequences.
Such bispecific antibodies according to the invention can be generated as described in Example 8. Furthermore, the effect on CDC killing by the generated heterodimeric proteins can be tested by using an assay as used in Example 9. Thus, in a particular embodiment, CDC killing may be determined by pre-incubating suspension cells at a concentration of 1×106 cells/mL in round-bottom 96-well plates with an antibody in the range from 0.0003 to 30.0 μg/mL final concentration in a total volume of 100 μL for 15 min on a shaker at room temperature, adding normal human serum at a final concentration of 20%, 30% or 50%, incubating at 37° C. for 45 min, putting the plates on ice, adding 10 μL propidium iodide, and determining cell lysis by FACS analysis.
In a particular embodiment of the heterodimeric protein, the amino acid in a position corresponding to K409 is R in the first polypeptide, and the amino acid in a position corresponding to F405 is L in the second polypeptide, and wherein the amino acids of each of said first and second polypeptide in the positions corresponding to E345 and E430 in a human IgG1 heavy chain are not E and an amino acid in at least one position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254 is Y or W, not Y, not E, not T, not E, not N, not Q, not I and not S, for each position, respectively.
In a further particular embodiment of the heterodimeric protein, the amino acid in a position corresponding to K409 is R in the first polypeptide, and the amino acid in a position corresponding to F405 is L in the second polypeptide, or vice versa; and wherein the amino acids of the first and second polypeptide at the positions corresponding to E345, E430 and S440 are R, G and Y, respectively.
In a further particular embodiment of the heterodimeric protein, the amino acid in a position corresponding to K409 is R in the first polypeptide, and the amino acid in a position corresponding to F405 is L in the second polypeptide, or vice versa; and wherein the amino acids of the first and/or second polypeptide at the positions corresponding to E345, E430 and S440 are K, G and Y, respectively.
In a further particular embodiment of the heterodimeric protein, the amino acid in a position corresponding to K409 is R in the first polypeptide, and the amino acid in a position corresponding to F405 is L in the second polypeptide, or vice versa; and wherein the amino acids of the first and/or second polypeptide at the positions corresponding to E345, E430 and S440 are R, S and Y, respectively.
In a further particular embodiment of the heterodimeric protein, the amino acid in a position corresponding to K409 is R in the first polypeptide, and the amino acid in a position corresponding to F405 is L in the second polypeptide, or vice versa; and wherein the amino acids of the first and/or second polypeptide at the positions corresponding to E345, E430 and S440 are R, G and W, respectively.
In a further particular embodiment of the heterodimeric protein, the amino acid in a position corresponding to K409 is R in the first polypeptide, and the amino acid in a position corresponding to F405 is L in the second polypeptide, or vice versa; and wherein the amino acids of the first and/or second polypeptide at the positions corresponding to E345, E430 and Y436 are R, G and I, respectively.
In a further embodiment, any other amino acids in the heterodimeric protein may be as further described in the section “Other amino acid positions”.
Example 11, shows that introducing the E345R mutation to a bispecific CD20×EGFR antibody enhances the CDC efficacy. Thus, in one embodiment, CDC efficacy may be determined by pre-incubating suspension cells of a concentration of 1×106 cells/mL in round-bottom 96-well plates with an antibody at a final concentration ranging from 0.0003 to 30.0 μg/mL in a total volume of 100 μL for 15 min on a shaker at room temperature, adding normal human serum at a final concentration of 20%, 30% or 50%, incubating at 37° C. for 45 min, putting the plates on ice, adding 10 μL propidium iodide, and determining cell lysis by FACS analysis.
Examples 9, 15 and 16 also describe some of the different bispecific antibodies.
The bispecific antibody may, for example, comprise an antigen-binding region of a CD20 antibody and an antigen-binding region of a CD38 antibody, and the amino acids according to the present invention. Exemplary CD20-binding regions include those of ofatumumab (2F2), 7D8 and 11B8, described in WO2004/035607, which is hereby incorporated by reference in its entirety, and rituximab (WO 2005/103081). Exemplary CD38-binding regions include those of 003 and daratumumab (005), described in WO2006/099875, which is hereby incorporated by reference in its entirety.
In one embodiment, the bispecific antibody binds different epitopes on the same or different target. Thus, the binding region of the first and the second polypeptide may in one embodiment bind to the same target, but different epitopes. In another embodiment the binding region of the first and second polypeptide may bind to different targets.
In another embodiment, the binding region of the first and second polypeptide may bind to different targets on different cells.
In one embodiment, the amino acids in the first and second polypeptide in the positions corresponding to E345, E430 and corresponding to a position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254 in a human IgG1 heavy chain, are not D or E; E; S; Y; E; T; E; N; Q; I; and S, respectively, may be the same or different.
In a further embodiment, one or more further amino acids may be as described herein. In a particular embodiment, the amino acid in a position corresponding to K439 is D or E, in each of the polypeptides of the heterodimeric protein. In another particular embodiment, the amino acid in a position corresponding to S440 is K or R. Thus, in a particular embodiment, in said first polypeptide the amino acid in positions corresponding to E345 and E430 in a human IgG1 heavy chain, is not E, the amino acid in at least one position corresponding to a position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254 in a human IgG1 heavy chain, is Y, K, R, or W; not Y; not D or E; not T; not E; not N; not Q; not I; and not S, for each position, respectively, and the amino acid in the position corresponding to K439 in a human IgG1 heavy chain is D or E, and in said second polypeptide the amino acid in positions corresponding to E345 and E430 in a human IgG1 heavy chain, is not E, the amino acid in the position S440 in a human IgG1 heavy chain, is K or R.
Fc-Fusion Proteins
In one aspect of the present invention, the dimeric protein according to any aspect or embodiment of the invention is part of a fusion protein. A fusion protein according to the invention may refer to a protein consisting of two or more covalently linked protein fragments which are not naturally expressed as a single protein. Fusion proteins may e.g. be produced by recombinant cloning and expression technologies commonly known in the art, or the method of creating fusion proteins may be post-production. Examples of such processes are intein, protein ligase, or other enzymatic processes commonly known in the art. Thus, a fusion protein according to the present invention is understood to be said dimeric protein comprising a first and a second polypeptide, each comprise at least a CH2 and CH3 region of an immunoglobulin heavy chain, wherein said first and/or second polypeptide may further comprise a binding region.
Thus, the first and/or second polypeptides of the dimeric protein according to the invention may further comprise a binding region. A binding region according to the invention is understood to be a polypeptide sequence which is capable of binding to a target. Thus, the binding region may be a protein, protein ligand, receptor, an antigen-binding region, or a ligand-binding region capable of binding to a target associated with a cell, bacterium, virion, or the like. A binding region may, for example, comprise part of a receptor, receptor ligand, ligand, cytokine, hormone, or antigen-binding region of an immunoglobulin or antibody.
In one embodiment, the binding region is a cytokine which is selected from the group consisting of IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα, IFNβ, IFNγ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFα.
In one embodiment, the binding region is an antigen-binding region. In some embodiments, said first and/or second polypeptides of said dimeric protein comprise, in addition to the Fc region, one or more or all of the other regions of an antibody, i.e. a CH1 region, a VH region, a CL region and/or a VL region. Thus, in one embodiment, said first polypeptide is a full-length antibody. In another embodiment, said second polypeptide is a full-length antibody.
In another embodiment, the binding region is a toxin, such as a naturally occurring toxin.
Conjugates
In one aspect, the dimeric protein the present invention, further comprises a drug, toxin, radiolabel, radioopaque agent, paramagnetic agent, fluorescent agent, phosphorescent agent, ultrasound enhancing agent, sialylation, or polyethyleneglycol (PEG), optionally conjugated to at least one of the polypeptides via a linker.
In one embodiment said dimeric protein is part of a fusion protein.
In one embodiment, the dimeric protein of the invention comprises a radiolabel.
In one embodiment, the dimeric protein of the invention comprises a radiopaque agent.
In one embodiment, the dimeric protein of the invention comprises a paramagnetic agent.
In one embodiment, the dimeric protein of the invention comprises a fluorescent agent.
In one embodiment, the dimeric protein of the invention comprises a phosphorescent agent.
In one embodiment, the dimeric protein of the invention comprises an ultrasound enhancing agent.
In one embodiment, the dimeric protein of the invention comprises a polyethyleneglycol (PEG).
In another aspect, the dimeric protein of the invention is not conjugated at the C-terminus to another molecule, such as a toxin or label. In one embodiment, the dimeric protein is conjugated to another molecule at another site, typically at a site which does not interfere with oligomer formation. For example, the dimeric protein may, at the other site, be linked to a compound selected from the group consisting of a toxin (including a radioisotope) a prodrug or a drug. Such a compound may make killing of target cells more effective, e.g. in cancer therapy. The resulting dimeric protein is thus an immunoconjugate.
Thus, in a further aspect, the present invention provides a dimeric protein, such as an antibody linked or conjugated to one or more therapeutic moieties, such as a cytotoxin, a chemotherapeutic drug, a cytokine, an immunosuppressant, and/or a radioisotope. Such conjugates are referred to herein as “immunoconjugates” or “drug conjugates”. Immunoconjugates which include one or more cytotoxins are referred to as “immuno-toxins”.
A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Suitable therapeutic agents for forming immunoconjugates of the present invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, maytansine or an analog or derivative thereof, enediyene antitumor antibiotics including neocarzinostatin, calicheamycins, esperamicins, dynemicins, lidamycin, kedarcidin or analogs or derivatives thereof, anthracyclins, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin; as well as duocarmycin A, duocarmycin SA, CC-1065 (a.k.a. rachelmycin), or analogs or derivatives of CC-1065), dolastatin, pyrrolo[2,1-c][1,4] benzodiazepins (PDBs) or analogues thereof, antibiotics (such as dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), anti-mitotic agents (e.g., tubulin-inhibitors) such as monomethyl auristatin E, monomethyl auristatin F, or other analogs or derivatives of dolastatin 10; Histone deacetylase inhibitors such as the hydroxamic acids trichostatin A, vorinostat (SAHA), belinostat, LAQ824, and panobinostat as well as the benzamides, entinostat, CI994, mocetinostat and aliphatic acid compounds such as phenylbutyrate and valproic acid, proteasome inhibitors such as Danoprevir, bortezomib, amatoxins such as alpha-amantin, diphtheria toxin and related molecules (such as diphtheria A chain and active fragments thereof and hybrid molecules); ricin toxin (such as ricin A or a deglycosylated ricin A chain toxin), cholera toxin, a Shiga-like toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins. Other suitable conjugated molecules include antimicrobial/lytic peptides such as CLIP, Magainin 2, mellitin, Cecropin, and P18; ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, diphtherin toxin, and Pseudomonas endotoxin. See, for example, Pastan et al., Cell 47, 641 (1986) and Goldenberg, Calif. A Cancer Journal for Clinicians 44, 43 (1994). Therapeutic agents that may be administered in combination with a dimeric protein of the present invention as described elsewhere herein, such as, e.g., anti-cancer cytokines or chemokines, are also candidates for therapeutic moieties useful for conjugation to a dimeric protein of the present invention.
In one embodiment, the drug conjugates of the present invention comprise a dimeric protein as disclosed herein conjugated to auristatins or auristatin peptide analogs and derivates (U.S. Pat. Nos. 5,635,483; 5,780,588). Auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis and nuclear and cellular division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584) and have anti-cancer (U.S. Pat. No. 5,663,149) and anti-fungal activity (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42:2961-2965. The auristatin drug moiety may be attached to the dimeric protein via a linker, through the N (amino) terminus or the C (carboxy) terminus of the peptidic drug moiety.
Exemplary auristatin embodiments include the N-terminus-linked monomethyl auristatin drug moieties DE and DF, disclosed in Senter et al., Proceedings of the American Association for Cancer Research. Volume 45, abstract number 623, presented Mar. 28, 2004 and described in US 2005/0238649).
An exemplary auristatin embodiment is MMAE (monomethyl auristatin E). Another exemplary auristatin embodiment is MMAF (monomethyl auristatin F).
In one embodiment, a dimeric protein of the present invention comprises a conjugated nucleic acid or nucleic acid-associated molecule. In one such embodiment, the conjugated nucleic acid is a cytotoxic ribonuclease, an antisense nucleic acid, an inhibitory RNA molecule (e.g., a siRNA molecule) or an immunostimulatory nucleic acid (e.g., an immunostimulatory CpG motif-containing DNA molecule). In another embodiment, a dimeric protein of the present invention is conjugated to an aptamer or a ribozyme.
In one embodiment, dimeric proteins comprising one or more radiolabeled amino acids are provided. A radiolabeled dimeric protein may be used for both diagnostic and therapeutic purposes (conjugation to radiolabeled molecules is another possible feature). Non-limiting examples of labels for polypeptides include 3H, 14C, 15N, 35S, 90Y, 99Tc, and 125I, 131I, and 186Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art, (see, for instance Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2nd Ed., Chafner and Longo, eds., Lippincott Raven (1996)) and U.S. Pat. Nos. 4,681,581; 4,735,210; 5,101,827; 5,102,990 (U.S. RE35,500), U.S. Pat. Nos. 5,648,471 and 5,697,902. For example, a radioisotope may be conjugated by the chloramine-T method.
In one embodiment, the dimeric protein of the present invention is conjugated to a radioisotope or to a radioisotope-containing chelate. For example, the dimeric protein can be conjugated to a chelator linker, e.g. DOTA, DTPA or tiuxetan, which allows for the dimeric protein to be complexed with a radioisotope. The dimeric protein may also or alternatively comprise or be conjugated to one or more radiolabeled amino acids or other radiolabeled molecule. A radiolabeled dimeric protein may be used for both diagnostic and therapeutic purposes. In one embodiment the dimeric protein of the present invention is conjugated to an alpha-emitter. Non-limiting examples of radioisotopes include 3H, 14C, 15N, 35S, 90Y, 99Tc, 125I, 111In, 131I, 186Re, 213Bs, 225Ac and 227Th.
In one embodiment the dimeric protein of the present invention may be conjugated to a cytokine selected from the group consisting of IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα, IFNβ, IFNγ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFα.
Dimeric proteins of the present invention may also be chemically modified by covalent conjugation to a polymer to for instance increase their circulating half-life. Exemplary polymers, and methods to attach them to peptides, are illustrated in for instance U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285 and 4,609,546. Additional polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., a PEG with a molecular weight of between about 1,000 and about 40,000, such as between about 2,000 and about 20,000).
Any method known in the art for conjugating the dimeric protein of the present invention to the conjugated molecule(s), such as those described above, may be employed, including the methods described by Hunter et al., Nature 144, 945 (1962), David et al., Biochemistry 13, 1014 (1974), Pain et al., J. Immunol. Meth. 40, 219 (1981) and Nygren, J. Histochem. and Cytochem. 30, 407 (1982). Such dimeric proteins may be produced by chemically conjugating the other moiety to the N-terminal side or C-terminal side of the dimeric protein or fragment thereof (e.g., an antibody H or L chain) (see, e.g., Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). Such conjugated dimeric protein derivatives may also be generated by conjugation at internal residues or sugars, where appropriate.
The agents may be coupled either directly or indirectly to a dimeric protein of the present invention. One example of indirect coupling of a second agent is coupling via a spacer or linker moiety to cysteine or lysine residues in a bispecific antibody. In one embodiment, a dimeric protein is conjugated to a prodrug molecule that can be activated in vivo to a therapeutic drug via a spacer or linker. In some embodiments, the linker is cleavable under intracellular conditions, such that the cleavage of the linker releases the drug unit from the dimeric protein in the intracellular environment. In some embodiments, the linker is cleavable by a cleavable agent that is present in the intracellular environment (e. g. within a lysosome or endosome or caveola). For example, the spacers or linkers may be cleavable by tumor-cell associated enzymes or other tumor-specific conditions, by which the active drug is formed. Examples of such prodrug technologies and linkers are described in WO02083180, WO2004043493, WO2007018431, WO2007089149, WO2009017394 and WO201062171 by Syntarga B V, et al. Suitable antibody-prodrug technology and duocarmycin analogs can also be found in U.S. Pat. No. 6,989,452 (Medarex), incorporated herein by reference. The linker can also or alternatively be, e.g. a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleaving agents can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside the target cells (see e. g. Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit (valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine) linker (see e.g. U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the Val-Cit linker and different examples of Phe-Lys linkers). Examples of the structures of a Val-Cit and a Phe-Lys linker include but are not limited to MC-vc-PAB described below, MC-vc-GABA, MC-Phe-Lys-PAB or MC-Phe-Lys-GABA, wherein MC is an abbreviation for maleimido caproyl, vc is an abbreviation for Val-Cit, PAB is an abbreviation for p-aminobenzylcarbamate and GABA is an abbreviation for γ-aminobutyric acid. An advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high.
In yet another embodiment, the linker unit is not cleavable and the drug is released by dimeric protein or antibody degradation (see US 2005/0238649). Typically, such a linker is not substantially sensitive to the extracellular environment. As used herein, “not substantially sensitive to the extracellular environment” in the context of a linker means that no more than 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linkers, in a sample of dimeric protein drug conjugate compound, are cleaved when the dimeric protein drug conjugate compound is present in an extracellular environment (e.g. plasma). Whether a linker is not substantially sensitive to the extracellular environment can be determined for example by incubating the dimeric protein drug conjugate compound with plasma for a predetermined time period (e.g. 2, 4, 8, 16 or 24 hours) and then quantitating the amount of free drug present in the plasma. Exemplary embodiments comprising MMAE or MMAF and various linker components have the following structures (wherein Ab means antibody and p, representing the drug-loading (or average number of cytostatic or cytotoxic drugs per antibody molecule), is 1 to about 8, e.g. p may be from 4-6, such as from 3-5, or p may be 1, 2, 3, 4, 5, 6, 7 or 8).
Examples where a cleavable linker is combined with an auristatin include MC-vc-PAB-MMAF (also designated as vcMMAF) and MC-vc-PAB-MMAE (also designated as vcMMAE), wherein MC is an abbreviation for maleimido caproyl, vc is an abbreviation for the Val-Cit (valine-citruline) based linker, and PAB is an abbreviation for p-aminobenzylcarbamate.
Other examples include auristatins combined with a non-cleavable linker, such as mcMMAF (mc (MC is the same as mc in this context) is an abbreviation of maleimido caproyl).
In one embodiment, the drug linker moiety is vcMMAE. The vcMMAE drug linker moiety and conjugation methods are disclosed in WO2004010957, U.S. Pat. Nos. 7,659,241, 7,829,531, 7,851,437 and U.S. Ser. No. 11/833,028 (Seattle Genetics, Inc.), (which are incorporated herein by reference), and the vcMMAE drug linker moiety is bound to the dimeric proteins at the cysteines using a method similar to those disclosed therein.
In one embodiment, the drug linker moiety is mcMMAF. The mcMMAF drug linker moiety and conjugation methods are disclosed in U.S. Pat. No. 7,498,298, U.S. Ser. No. 11/833,954, and WO2005081711 (Seattle Genetics, Inc.), (which are incorporated herein by reference), and the mcMMAF drug linker moiety is bound to the dimeric protein at the cysteines using a method similar to those disclosed in therein.
In one embodiment, the dimeric protein of the present invention is attached to a chelator linker, e.g. tiuxetan, which allows for e.g. a bispecific antibody to be conjugated to a radioisotope.
In one embodiment, the dimeric protein is conjugated to toxins or payloads, such as drugs, which have optimal function at a lower pH than neutral pH.
In one embodiment, both the first and second polypeptide of the dimeric protein is coupled directly or indirectly to the same one or more therapeutic moieties.
In one embodiment, only the first or second polypeptide of the dimeric protein is coupled directly or indirectly to one or more therapeutic moieties.
In one embodiment, the first and second polypeptide of the dimeric protein is coupled directly or indirectly to different therapeutic moieties. For example, in embodiments where the dimeric protein is a bispecific antibody and is prepared by controlled Fab-arm exchange of two different monospecific antibodies, e.g. a first and second antibody such bispecific antibodies can be obtained by using monospecific antibodies which are conjugated or associated with different therapeutic moieties.
Oligomer
The present invention is based, in part, on the discovery that dimeric proteins comprising at least the CH2 and CH3 regions, and optionally a hinge region, of immunoglobulin heavy chains can form oligomers such as hexamers not only when bound to a target molecule but also in solution. The oligomerization occurs via non-covalent association of adjacent Fc-regions, and has in particular been observed for antibodies having mutations in E345, E430 and S440, as described in the Examples.
In one aspect the present invention relates to an oligomer comprising at least two non-covalently associated dimeric proteins, each according to any aspect or embodiment herein described.
In one embodiment, the invention provides a hexamer comprising six non-covalently associated dimeric proteins, each according to any one of the preceding aspects or embodiments. In one embodiment, at least one, such as at least two, at least three, at least four, at least five or six dimeric proteins of the hexamer are antibodies.
In one embodiment, the invention provides an oligomer comprising six non-covalently associated dimeric proteins, at least one of which is a dimeric protein according to any aspect or embodiment of the invention and at least one of which is an antibody comprising an Fc domain comprising at least CH2 and CH3 regions and optionally a hinge region.
In one embodiment, the invention provides a hexamer comprising six non-covalently associated molecules, such as dimeric proteins, at least one of which is of a dimeric protein according to any preceding aspect or embodiment and at least one of which is an antibody comprising an Fc domain comprising at least CH2, CH3 and hinge regions in which at least one of the positions corresponding to E345, E430 and S440 in a human IgG1 heavy chain, is E, E and S, respectively. In one embodiment, the antibody is a monoclonal or polyclonal antibody, the monoclonal antibody optionally selected from the known antibodies denoted “second antibodies” in the section below.
Compositions
The present invention also relates to a composition comprising one or more dimeric proteins of the present invention, optionally in the form of oligomers, such as hexamers according to any preceding aspect or embodiment. The composition of the present invention may be a pharmaceutical composition comprising a dimeric protein of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical compositions may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995.
The composition of the present invention may be a pharmaceutical composition comprising a dimeric protein according to any aspect or embodiments of the present invention, one or more antibodies, and a pharmaceutically acceptable carrier.
In a particular embodiment, the composition comprises a first dimeric protein according to any aspect or embodiment of the invention and a second dimeric protein according to any aspect or embodiment of the invention, and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients should be suitable for the dimeric protein of the present invention and the chosen mode of administration. Suitability for carriers and other components of pharmaceutical compositions is determined based on the lack of significant negative impact on the desired biological properties of the dimeric protein or pharmaceutical composition of the present invention (e.g., less than a substantial impact (10% or less relative inhibition, 5% or less relative inhibition, etc.)) on antigen binding.
A pharmaceutical composition of the present invention may also include diluents, fillers, salts, buffers, detergents (e.g., a nonionic detergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars, sugar alcohols such as sorbitol and mannitol, or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and/or other materials suitable for inclusion in a pharmaceutical composition.
Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible with a dimeric protein of the present invention.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethyl cellulose colloidal solutions, tragacanth gum and injectable organic esters, such as ethyl oleate, and/or various buffers. Other carriers are well known in the pharmaceutical arts.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the dimeric protein, use thereof in the pharmaceutical compositions of the present invention is contemplated.
Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
Pharmaceutical compositions of the present invention may also comprise pharmaceutically acceptable antioxidants for instance (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Pharmaceutical compositions of the present invention may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the compositions.
The pharmaceutical compositions of the present invention may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the pharmaceutical composition. The dimeric protein of the present invention may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid alone or with a wax, or other materials well known in the art. Methods for the preparation of such formulations are generally known to those skilled in the art. See e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
In one embodiment, the dimeric protein of the present invention may be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the dimeric protein, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active compounds may also be incorporated into the compositions.
Pharmaceutical compositions for injection must typically be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier may be an aqueous or nonaqueous solvent or dispersion medium containing for instance water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions may be prepared by incorporating the dimeric protein in the required amount in an appropriate solvent with one or a combination of ingredients e.g. as enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the dimeric protein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients e.g. from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Sterile injectable solutions may be prepared by incorporating the dimeric protein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the dimeric protein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
pH
The compositions of the invention may comprise a suitable buffer system to control the pH and thereby the oligomerization state of the dimeric protein(s) present. For example, at a pH of 6.4 or below, such as at pH 5.0, a dimeric protein according to the invention is typically predominantly in monomeric form, i.e. a single dimeric protein, whereas at above pH 6.4, such as at pH 6.8, a dimeric protein is predominantly in oligomeric form, such as hexamer form. The hexameric form of the dimeric protein, is composed of six dimeric proteins which non-covalently associate with each other to form a hexameric form. The term “monomeric form” in the context of dimeric protein according to the present invention refers to a single, individual dimeric protein, which is composed of dimeric proteins that do not associate non-covalently with each other. Example 31 describes how this can be observed by adjusting pH.
In one embodiment, the composition comprises a pharmaceutically acceptable carrier which is an aqueous buffered solution.
In one embodiment, the pH of the aqueous buffered solution is at least about 6.5, such as from 6.5 to about 9.0, such as from about 7.0 to about 8.0, such as about 7.4. Buffer systems suitable for maintaining a pH in this range and/or near physiological pH include phosphate buffer systems. Thus, in one embodiment, the aqueous buffered solution is a phosphate buffer system. In one embodiment, the dimeric protein is predominantly in oligomeric form, such as hexameric form, in a phosphate buffer at a pH of about 6.8.
In one embodiment, the pH of the aqueous buffered solution is less than pH 6.5, such as from about 4.0 to 6.4, such as from about 5.0 to about 6.0. Buffer systems suitable for maintaining a pH in this range include citrate, acetate, histidine and/or glycine-based buffer systems. Thus, in one embodiment, the buffer system is an acetate, histidine, glycine, citrate, nicotinate, lactate, and/or succinate based buffer system. Such buffer systems may also be a combination of buffer systems. In one embodiment, the dimeric protein is predominantly in monomeric form, i.e. single dimeric protein, at a pH of less than 6.0, such as about 5.0.
As shown in Example 31, the oligomerization of the dimeric protein according to the invention is a reversible process which may be controlled by pH. This could be useful for application in processing during manufacturing of the dimeric protein, such as the purification steps wherein clotting of e.g. purification columns, translaminar flow filtration, dead-end filtration and/or nanofiltration devices can be avoided by lowering the pH without compromising the efficacy of the final product, such as an antibody. Furthermore, lowering the pH below 6.8, e.g. 5.0 and 5.5, during purification can improve purification yields as the dimeric protein is predominantly in monomeric form and thereby less likely to clot than the hexameric form of the dimeric protein, as demonstrated by Example 32. Furthermore, lowering the pH below 6.8 during purification can enable removal of non-specific aggregates by chromatography, such as using weak cation exchange resins. Thus, once the dimeric protein has been purified at a pH below 6.8, the oligomeric, e.g. hexameric, form may be restored by increasing the pH of the solution to a pH around 6.8.
Mixtures
In some aspects, the invention provides compositions comprising a dimeric protein and a second molecule, wherein the second molecule also comprises a first and a second polypeptide, each comprising at least CH2, CH3 and hinge regions of an immunoglobulin heavy chain. Thus, the dimeric protein is to be understood as the dimeric protein according to any aspect or embodiment of the present invention, such as dimeric protein of a parent dimeric protein, such as a variant dimeric protein of a parent dimeric protein.
Advantageously, the relative amounts of the dimeric protein(s) and the second molecule in the compositions can be adjusted to modulate the average number of units of each different dimeric protein in the oligomers/hexamers formed. This, in turn, provides a means for optimizing effector function and/or target-binding properties, when one or more of the dimeric protein(s) and the second molecules bind(s) a target.
Specific aspects and embodiments of compositions comprising mixtures of a dimeric protein and a second molecule are described below. While applicable to all types of dimeric proteins according to the invention and all types of Fc-containing second molecules, including, e.g., Fc-fusion proteins with ligand-binding regions, antibody molecules are particularly contemplated for both components.
In one aspect, the composition of the invention comprises a first dimeric protein according to any aspect or embodiment of the invention, a second dimeric protein according to any aspect or embodiment of the invention, and a pharmaceutically acceptable carrier.
In one aspect, the second molecule is one in which at least one of the amino acids at the positions corresponding to E345, E430 and S440 in a human IgG1 heavy chain is the one normally present at this position in a native IgG1 heavy chain. For example, the heavy-chain polypeptides of the second molecule may comprise E, E and S at each of the positions corresponding to E345, E430 and S440, respectively. According to this aspect, the second molecule may thus comprise, e.g., the native Fc region sequences of a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE or IgM antibody, or in particular at least IgG1, IgG2, IgG3, or IgG4 Fc sequences without amino acid substitutions in all three of E345, E430 and S440.
In one embodiment, the second molecule is an antibody, in particular a well-known antibody already in clinical or pre-clinical use, such as an antibody which has a suitable safety profile. In a further embodiment, said second antibody may have a suitable safety profile but may not be sufficiently efficacious.
In one embodiment, also the dimeric protein is an antibody, so that the composition comprises a first antibody and a second antibody, wherein only the first antibody is a dimeric protein of the invention. The combination of a first antibody which comprises mutations capable of increasing an effector function and a second antibody which does not comprise such a mutation may, as shown in Example 17 provide an increased effector function. Thus, without being bound by theory, it is believed that e.g. this method may be used to combine a therapeutic antibody, as a second antibody, which has been proven to be safe but not efficient enough on its own for a specific application, with a dimeric protein according to the invention, thereby resulting in a combination which is efficacious.
Examples of suitable second antibodies include but are not limited to any of those selected from the group consisting of: (90Y) clivatuzumab tetraxetan; (90Y) tacatuzumab tetraxetan; (99mTc) fanolesomab; (99mTc) nofetumomab Merpentan; (99mTc) pintumomab; 3F8; 8H9; abagovomab; abatacept; abciximab; Actoxumab; adalimumab; adecatumumab; afelimomab; aflibercept; Afutuzumab; alacizumab pegol; albiglutide; ALD518; alefacept; alemtuzumab; Alirocumab; altumomab; Altumomab pentetate; alvircept sudotox; amatuximab; AMG714/HuMax-IL15; anatumomab mafenatox; Anrukinzumab (=IMA-638); apolizumab; arcitumomab; aselizumab; atacicept; atinumab; Atlizumab (=tocilizumab); atorolimumab; baminercept; Bapineuzumab; basiliximab; bavituximab; bectumomab; belatacept; belimumab; benralizumab; bertilimumab; besilesomab; bevacizumab; Bezlotoxumab; biciromab; bifarcept; bivatuzumab; Bivatuzumab mertansine; blinatumomab; blosozumab; brentuximab vedotin; briakinumab; briobacept; brodalumab; canakinumab; cantuzumab mertansine; cantuzumab ravtansine; caplacizumab; capromab; Capromab pendetide; carlumab; catumaxomab; CC49; cedelizumab; certolizumab pegol; cetuximab; Ch.14.18; citatuzumab bogatox; cixutumumab; Clazakizumab; clenoliximab; Clivatuzumab tetraxetan; conatumumab; conbercept; CR6261; crenezumab; dacetuzumab; daclizumab; dalantercept; dalotuzumab; daratumumab; Demcizumab; denosumab; Detumomab; Dorlimomab aritox; drozitumab; dulaglutide; ecromeximab; eculizumab; edobacomab; edrecolomab; efalizumab; efungumab; elotuzumab; elsilimomab; enavatuzumab; enlimomab; enlimomab pegol; enokizumab; ensituximab; epitumomab; epitumomab cituxetan; epratuzumab; erlizumab; ertumaxomab; etanercept; etaracizumab; etrolizumab; exbivirumab; Fanolesomab; faralimomab; farletuzumab; Fasinumab; FBTA05; felvizumab; Fezakinumab; ficlatuzumab; figitumumab; flanvolumab; fontolizumab; foralumab; foravirumab; fresolimumab; fulranumab; galiximab; ganitumab; gantenerumab; gavilimomab; gemtuzumab; Gemtuzumab ozogamicin; gevokizumab; girentuximab; glembatumumab; Glembatumumab vedotin; golimumab; Gomiliximab; GS6624; anti-CD74 antibodies; anti-cMet antibodies as disclosed in WO 2011/110642; anti-Her2 antibodies as disclosed WO 2011/147986 or WO 2011/147982; anti-IL8 antibodies as disclosed in WO 2004/058797; anti-TAC antibodies as disclosed in WO 2004/045512; anti-tissue factor (TF) antibodies as disclosed in WO 2010/066803 or WO 2011/157741; ibalizumab; ibritumomab tiuxetan; icrucumab; igovomab; Imciromab; inclacumab; indatuximab ravtansine; infliximab; inolimomab; inotuzumab ozogamicin; intetumumab; iodine (124I) girentuximab; ipilimumab; iratumumab; itolizumab; ixekizumab; keliximab; labetuzumab; lebrikizumab; lemalesomab; lenercept; lerdelimumab; lexatumumab; libivirumab; lintuzumab; lorvotuzumab mertansine; lucatumumab; lumiliximab; mapatumumab; maslimomab; matuzumab; mavrilimumab; mepolizumab; metelimumab; milatuzumab; minretumomab; mirococept; mitumomab; mogamulizumab; morolimumab; motavizumab; moxetumomab; pasudotox; muromonab-CD3; nacolomab tafenatox; namilumab; naptumomab estafenatox; narnatumab; natalizumab; nebacumab; necitumumab; nerelimomab; nimotuzumab; Nivolumab; Nofetumomab; merpentan; obinutuzumab; Ocaratuzumab; ocrelizumab; odulimomab; ofatumumab; olaratumab; olokizumab; omalizumab; onartuzumab; onercept; oportuzumab monatox; oregovomab; otelixizumab; oxelumab; ozoralizumab; pagibaximab; palivizumab; panitumumab; panobacumab; pascolizumab; pateclizumab; patritumab; pegsunercept; Pemtumomab; pertuzumab; pexelizumab; Pintumomab; Placulumab; ponezumab; priliximab; pritumumab; PRO 140; quilizumab; racotumomab; radretumab; rafivirumab; ramucirumab; ranibizumab; raxibacumab; regavirumab; reslizumab; RG1507/HuMax-IGF1R; RG1512/HuMax-pSelectin; rilonacept; rilotumumab; rituximab; robatumumab; roledumab; romosozumab; rontalizumab; rovelizumab; ruplizumab; samalizumab; sarilumab; satumomab; Satumomab pendetide; secukinumab; sevirumab; sibrotuzumab; sifalimumab; siltuximab; siplizumab; sirukumab; solanezumab; solitomab; Sonepcizumab; sontuzumab; sotatercept; stamulumab; sulesomab; suvizumab; tabalumab; Tacatuzumab tetraxetan; tadocizumab; talizumab; tanezumab; taplitumomab paptox; tefibazumab; telimomab aritox; tenatumomab; teneliximab; teplizumab; teprotumumab; TGN1412; Ticilimumab (=tremelimumab); tigatuzumab; TNX-650; Tocilizumab (=atlizumab); toralizumab; torapsel; tositumomab; tralokinumab; trastuzumab; trastuzumab emtansine; TRBS07; trebananib; tregalizumab; tremelimumab; tucotuzumab celmoleukin; tuvirumab; ublituximab; urelumab; urtoxazumab; ustekinumab; vapaliximab; vatelizumab; vedolizumab; veltuzumab; vepalimomab; vesencumab; visilizumab; volociximab; Vorsetuzumab mafodotin; votumumab; zalutumumab; zanolimumab; ziralimumab; and zolimomab aritox.
In one aspect, the second molecule is a second dimeric protein according to the invention. Compositions according to this aspect thus comprise a mixture of two or more different dimeric proteins, each according to any aspect or embodiment of the invention, such as described above. Typically, under the right pH and/or target-binding conditions, hexamers comprising two or more different dimeric proteins may then form in the composition, particularly in an aqueous solution or buffer. The first and second dimeric proteins of the present invention will have preference for oligomerization with one another compared to any wildtype or naturally occurring dimeric protein as shown in Example 3.
In one embodiment, the composition comprises a first dimeric protein and a second dimeric protein, optionally further comprising a pharmaceutically acceptable carrier.
In one embodiment the present invention may relate to a composition comprising a first and a second dimeric protein, wherein both the first and the second dimeric proteins comprise a first and a second polypeptide, wherein in one of said first and/or second, such as both, polypeptides of said first and second dimeric protein the amino acids at the positions corresponding to E345 and E430 in a human IgG1 heavy chain, are not E, and the amino acid in at least one position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253, and S254, corresponding to the position in a human IgG1 heavy chain, is Y, W, K or R; not Y; not D or E; not T; not E; not N; not Q; not I; and not S, for each position, respectively. The first and the second dimeric protein may be any dimeric protein according to the present invention.
In one embodiment, one or both of the first and second dimeric proteins comprise heavy-chain polypeptides wherein, for one or both, such as each, polypeptides, the amino acid at the position corresponding to E345 is selected, e.g. separately, from the group consisting of R, Q, N, K, Y, A, C, D, F, G, H, I, L, M, P, S, T, V and W, such as from the group consisting of R, Q, N, K and Y.
In one embodiment, one or both of the first and the second dimeric proteins comprise heavy-chain polypeptides wherein, for one or both, such as each, polypeptides, the amino acid at the position corresponding to E430 is selected, e.g. separately, from the group consisting of G, T, S, F, H, A, C, D, I, K, L, M, N, P, Q, R, V, W and Y, such as from the group consisting of G, T, S, F and H.
In one embodiment, one or both of the first and second dimeric proteins comprise heavy chain polypeptides wherein, for one or both, such as each, polypeptides, the amino acid in at least one of the positions selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253, and S254 corresponds to the position in a human IgG1 heavy chain, is Y, R, K, or W; not Y; not D or E; not T; not E; not N; not Q; not I; and not S, for each position, respectively.
In one embodiment, said first and/or the second, such as both, dimeric proteins comprise heavy chain polypeptides wherein, for one or both, such as each, polypeptide, the amino acids at the positions corresponding to E345, E430 and S440 in a human IgG1 heavy chain, are R, G and Y, respectively.
In one embodiment, in said first and/or second polypeptides of said first and/or second dimeric proteins, the amino acids at the positions corresponding to E345, E430 and S440 in a human IgG1 heavy chain, are K, G and Y, respectively.
In one embodiment, in said first and/or second polypeptides of said first and/or second dimeric proteins, the amino acids at the positions corresponding to E345, E430 and S440 in a human IgG1 heavy chain, are R, S and Y, respectively.
In one embodiment, in said first and/or second polypeptides of said first and/or second dimeric proteins, the amino acids at the positions corresponding to E345, E430 and S440 in a human IgG1 heavy chain, are R, G and W, respectively.
In one embodiment, in said first and/or second polypeptides of said first and/or second dimeric proteins, the amino acids at the positions corresponding to E345, E430 and Y436 in a human IgG1 heavy chain, are R, G and I, respectively.
In another embodiment, one or both of the first and second dimeric proteins comprise heavy chain polypeptides where the amino acids at the positions corresponding to E345, E430 and S440 are R; G; and Y or W, respectively, and where one or at least one of Y436, D/E356, T359, E382, N434, Q438, I253 and S254 is not Y; D or E; T; E; N; Q; I; and S, respectively.
In one embodiment, either said first or the second dimeric protein comprises the indicated amino acids in both said first and second polypeptide, and the other dimeric protein comprises the indicated amino acids in only said first or second polypeptide.
In one embodiment, both said first and second dimeric proteins comprise the indicated amino acids in both said first and second polypeptides.
In some embodiments, the amino acids at certain positions in the heavy chain polypeptides differ between the first and second dimeric proteins to adjust the strength or specificity of the non-covalent association of the two dimeric proteins. This can be achieved, e.g., by using first and/or second dimeric proteins having specific amino acids at the positions corresponding to K439, S440, K447, K448, and/or K449, as described above.
In one embodiment, the polypeptides of the first dimeric protein comprise an amino acid in the position corresponding to K439 which is not K, and the polypeptides of the second dimeric protein comprise an amino acid in the position corresponding to S440 which is not S, with the proviso that the amino acid in S440 is not Y or W. For example, in the first dimeric protein, the amino acid in the position corresponding to K439 can be D or E, and in the second dimeric protein, the amino acid in the position corresponding to S440 can be K, H or R, such as K or R. A similar strategy can be used for combinations of amino acids in the positions corresponding to K447, 448 and 449. Table 2 shows exemplary amino acids for these positions in the first dimeric protein and second dimeric protein to be used together, separated by a “+”-sign. In any one of these aspects and embodiments, one or both of the first and second dimeric proteins can be an antibody (e.g., Ab1 and Ab2, respectively).
TABLE 2
Exemplary positions and amino acids which may further
be present in two dimeric proteins (e.g., Ab1 + Ab2)
Preferred
Amino acid pair (IgG1)
Exemplary substitutions
substitutions
K439 + S440
439DER + 440DEKR
439E + 440K
K447 + K447/448
447DE + 447KRH/448P
447E + 447/448P
K447 + K447/448/449
447DE + 447KRH/
447E +
448KRH/449P
447K/448K/449P
In a further embodiment, in said first and/or second polypeptide of said first dimeric protein the amino acid at the position corresponding to K439 in a human IgG1 heavy chain, is E or D, optionally E, and in said first and/or second polypeptide of said second dimeric protein the amino acid at the position corresponding to S440 in a human IgG1 heavy chain, is K or R, optionally K.
In one embodiment, in said first and/or second polypeptide of said first dimeric protein the amino acid at the positions corresponding to E345, E430, K439, and S440 in a human IgG1 heavy chain, are R, K, Q, N, or Y; G, S, T, F or H; D or E; and Y or W, respectively, and in said first and/or second polypeptide of said second dimeric protein the amino acid at the positions corresponding to E345, E430 and S440 in a human IgG1 heavy chain, are R, K, Q, N, or Y; G, S, T, F or H; and K or R, respectively. In a further embodiment, in said first and/or second polypeptide of the second dimeric protein, the amino acid at the position corresponding to Y436 in a human IgG1 heavy chain, is I.
In one embodiment, in said first and/or second polypeptide of said first dimeric protein the amino acid at the position corresponding to K439 in a human IgG1 heavy chain, is E or D, optionally E, and in said first and/or second polypeptide of said second dimeric protein the amino acid at the position corresponding to S440 in a human IgG1 heavy chain, is K or R, optionally K, and at least one amino acid in a position selected from the group consisting of Y436, D/E356, T359, E382, N434, Q438, I253, and S254 corresponding to the position in a human IgG1 heavy chain, is not Y; D or E; T; E; N; Q; I; and S, respectively.
In one embodiment, in said first and/or second polypeptides of said first dimeric protein the amino acids at the positions corresponding to E345, E430, K439, and S440 in a human IgG1 heavy chain, are R, G, E, and Y, respectively, and in said first and/or second polypeptides of said second dimeric protein the amino acids at the positions corresponding to E345, E430, K439, and S440 in a human IgG1 heavy chain, are R, G, K, and K, respectively.
In one embodiment, in said first and/or second polypeptides of said first dimeric protein the amino acids at the positions corresponding to E345, E430, K439, and S440 in a human IgG1 heavy chain, are R, G, E, and Y, respectively, and in said first and/or second polypeptides of said second dimeric protein the amino acids at the positions corresponding to E345, E430, and S440 in a human IgG1 heavy chain, are R, G, and K, respectively.
In an alternative embodiment, in the first and/or second polypeptides of the first dimeric protein the amino acid at the positions corresponding to E345, E430, and S440 in a human IgG1 human heavy chain, are K, G, and Y, respectively; or alternatively R, G and W, respectively; or alternatively R, G, and K, respectively, or the amino acid in the positions corresponding to E345, E430 and Y436 in a human IgG1 heavy chain, are R, G, and I, respectively. Furthermore, in the first and/or second polypeptide of the second dimeric protein the amino acid at the positions corresponding to E345, E430 and S440 in a human IgG1 heavy chain, are K, G, and K, respectively; or alternatively R, S, and K, respectively; or alternatively R, G, and R, respectively; or alternatively R, S, and R, respectively.
In one embodiment, in said first and/or second polypeptides of the first dimeric protein the amino acids at the positions corresponding to E345, E430, K439, and S440 in a human IgG1 heavy chain, are R, G, E, and Y, respectively, and in said first and/or second polypeptides of said second dimeric protein the amino acids at the positions corresponding to E345, E430, Y436, and S440 in a human IgG1 heavy chain, are R, G, I, and K, respectively.
In a further embodiment, in said first and/or second polypeptides of the first dimeric protein the amino acid at the position corresponding to K447 in a human IgG1 heavy chain, is D or E, and in said first and/or second polypeptides of said second dimeric protein the amino acid at the position corresponding to K447 in a human IgG1 heavy chain, is K, R, or H, and an amino acid at the position corresponding to 448 in a human IgG1 heavy chain, is P.
In one embodiment, in said first and/or second polypeptides of the first dimeric protein the amino acid at the position corresponding to K447 in a human IgG1 heavy chain, is D or E, and in said first and/or second polypeptides of said second dimeric protein the amino acid at the position corresponding to K447 in a human IgG1 heavy chain, is K, R, or H, and amino acid at the position corresponding to 448 in a human IgG1 heavy chain, is K, R, or H, and an amino acid at the position corresponding to 449 in a human IgG1 heavy chain, is P.
In one embodiment, in said first and second polypeptides of the first dimeric protein the amino acids at the positions corresponding to K439 and K447 in a human IgG1 heavy chain, are D or E; and D or E, respectively, and in said first and second polypeptides of said second dimeric protein the amino acids at the positions corresponding to S440, K447, and 448 in a human IgG1 heavy chain, are K or R; K, R, or H; and P, respectively.
In one embodiment, either said first or the second dimeric protein comprises the indicated amino acids in both said first and second polypeptide, and the other dimeric protein comprises the indicated amino acids in only said first or second polypeptide.
In one embodiment, both said first and second dimeric proteins comprise the indicated amino acids in both said first and second polypeptides. In a further embodiment, the first or second dimeric protein may be an antibody, and the other dimeric protein may be a fusion protein or a conjugate as described herein.
In one embodiment, in the first and/or second polypeptide of said first dimeric protein, the amino acid positions corresponding to E345, E430, S440, and K447, in a human IgG1 heavy chain, are R, G, Y, and D/E, respectively, and in the first and/or second polypeptides of said second dimeric protein, the amino acid positions corresponding to E345, E430, S440, K447 and 448, in a human IgG1 heavy chain, are R, G, Y, K/R/H and P, respectively, or vice versa.
In one embodiment, in the first and/or second polypeptide of said first dimeric protein, the amino acid positions corresponding to E345, E430, S440, and K447, in a human IgG1 heavy chain, are R, G, Y, and D/E, respectively, and in the first and/or second polypeptides of said second dimeric protein, the amino acid positions corresponding to E345, E430, S440, K447, 448, and 449, in a human IgG1 heavy chain, are R, G, Y, K/R/H, K/R/H, and P, respectively, or vice versa.
In a particular embodiment, the composition comprising a first and as second dimeric protein, both the first and the second polypeptides of said first and second dimeric proteins comprise the indicated amino acids in the specific positions.
In one embodiment, at least one of said first and second dimeric proteins is an antibody.
In one embodiment, both the first and the second dimeric proteins are antibodies.
In one embodiment, said first and/or second, such as at least one, of said dimeric proteins is a heterodimeric protein, such as a bispecific antibody. It may be any heterodimeric protein described herein.
In one embodiment, said first and second antibodies, bind to the same epitope of the same antigen.
In one embodiment, said first and second antibodies comprise the same variable heavy and light chain region sequences.
In one embodiment, said first and second antibodies bind to different antigens or to different epitopes on the same antigen.
In another embodiment, said first and/or second, such as at least one, of said dimeric proteins is a fusion protein.
The dimeric proteins of the compositions of the preceding aspects or embodiments may contain binding regions binding to a specific target.
In one embodiment, the composition comprises at least one additional dimeric protein according to any aspect or embodiment of the invention, such as three or six, or such as four, five, seven, eight, nine or more dimeric proteins.
In another embodiment, said first and/or second, such as at least one, of said dimeric proteins is an Fc fragment.
In one embodiment, the composition comprises more than two different, such as three, four, five or six, different dimeric proteins according to any aspect or embodiment of the invention.
In one particular embodiment, the composition comprises one or more dimeric proteins and an Fc fragment, wherein said one or more dimeric proteins comprise a first and a second polypeptide, and wherein in the first or second polypeptide the amino acid at the positions corresponding to E345 and E430 in a human IgG1 heavy chain are not E, and the amino acid in at least one position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253, and S254, corresponding to the position in a human IgG1 heavy chain, is Y, W, K or R; not Y; not D or E; not T; not E; not N; not Q; not I; and not S, for each position, respectively; and wherein said Fc fragment comprises a first and second polypeptide wherein in both said first and second polypeptide, the amino acid at the positions corresponding to E345 and E430 in a human IgG1 heavy chain are not E, and the amino acid in at least one position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253, and S254, corresponding to the position in a human IgG1 heavy chain, is Y, W, K or R; not Y; not D or E; not T; not E; not N; not Q; not I; and not S, for each position, respectively. In a further embodiment, the one or more dimeric protein and/or Fc fragment may be a fusion protein or a conjugate.
In one embodiment, composition comprises two dimeric proteins according to any aspect or embodiment of the present invention, wherein said first dimeric protein is linked to a first pro-drug, and said second dimeric protein is linked to a second pro-drug. For example, on of the first and second pro-drug may be capable of activation of the other.
In one embodiment, only one of the first and second dimeric proteins comprises a target-binding region. This can be used, e.g., for pharmaceutical compositions where an “Fc only” dimeric protein is conjugated to a therapeutic or diagnostic compound is mixed with a dimeric protein having binding specificity for a target.
In one embodiment, both the first and the second dimeric protein comprise a target-binding region. If the first and second dimeric proteins are heterodimeric proteins, they may bind to the different epitopes on the same target or to different targets. Any combination with respect to such binding is foreseen. By selecting different epitopes and/or targets for each dimeric protein, hexamer formation can be optimized to primarily occur on cells, bacteria or virions expressing both of the epitopes or targets. This provides for a mechanism to guide an immune response towards specific cell types. Additionally, a mixture of dimeric proteins binding to different epitopes on the same target molecule can provide a similar effect as a polyclonal antibody.
In some embodiments, at least one of the first and second dimeric proteins is an antibody as defined herein.
In one embodiment, both of the first and second dimeric proteins are antibodies, representing a first and second antibody. In one embodiment, the antibodies bind the same epitope of the same antigen. Optionally, the antigen-binding regions of the two antibodies are identical, i.e., comprise the same variable heavy and light chain region sequences.
In another embodiment, the first and second antibodies bind to different antigens or to different epitopes on the same antigen.
In another embodiment, the first and second antibodies bind to different antigens on different cells.
In one embodiment the first and second antibodies may each be selected from the group consisting of but not limited to monospecific, bispecific and multispecific antibodies. Further, in any of the above aspects or embodiments, at least one of the first and second dimeric proteins can be an antibody comprising at least the antigen-binding region of a known antibody in clinical or pre-clinical use, e.g., selected from the “second antibodies” listed above.
Non-Limiting Examples of Compositions Include
a) a first dimeric protein which comprises a binding region;
b) a first and second dimeric protein, wherein said first and second dimeric proteins bind to different epitopes on the same target or to different targets
c) a first dimeric protein of the present invention wherein said first dimeric protein comprises an amino acid in the position corresponding to K439 in a human IgG1 heavy chain which is not K, and the second dimeric protein of the present invention comprises an amino acid in a position corresponding to S440 in a human IgG1 heavy chain which is not S, Y, or W; optionally the amino acid in position K439 is E in the first dimeric protein and the amino acid in position S440 is K in the second dimeric protein.
d) a first dimeric protein of the present invention and a second dimeric protein wherein the amino acid in the second dimeric protein in positions corresponding to E345 and E430 of a human IgG1 heavy chain are not E
e) a first dimeric protein of the present invention and a second dimeric protein, e.g. a second antibody, wherein the amino acids in the second dimeric protein in positions corresponding to E345 or E430 of a human IgG1 heavy chain are not E.
f) A first dimeric protein and a second dimeric protein, wherein either the first or second dimeric protein comprises an amino acid mutation which modulates one or more effector functions and/or pharmacokinetic profile of said first or second dimeric protein.
The first and second dimeric proteins may also include combinations of the aspects described in a) to e). For example the first and second dimeric proteins may in particular comprise both the features described in b) and c).
In one embodiment, the specificity is increased when a combination of the first and second dimeric proteins is bound to its target on a cell or virion expressing the target.
In any of the above aspects or embodiments, the composition may comprise at least one additional dimeric protein according to the invention. For example, the composition may comprise three, four, five, six, seven, eight, nine or more dimeric proteins, each according to an aspect or embodiment of the invention. The relative amounts of each dimeric protein can be adjusted to optimize a desired property of the hexamers formed, e.g., target cell specificity, effector function, hexamer avidity and/or stability. Additionally, a composition comprising dimeric proteins binding to different epitopes on the same target may resemble or function as a composition of polyclonal antibodies.
The dimeric protein and the second molecule comprised in each of the above-described compositions may alternatively be provided as a kit of parts, for simultaneous, separate or sequential use in, e.g., imaging or therapy.
Methods
The present invention also relates to a method of increasing oligomerization in solution and/or an effector function of a parent dimeric protein comprising a first and second polypeptide, each comprising at least CH2 and CH3 of an immunoglobulin heavy chain, the method comprising introducing into the first and/or second polypeptides, amino acid substitutions in at least the positions corresponding to E345, E430, and in a position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254 in a human IgG1 heavy chain.
In one embodiment, the effector function is complement-dependent cytotoxicity (CDC).
In one embodiment, said first and/or second polypeptide may further comprise a region capable of covalent binding between said first and second polypeptide.
In one embodiment, said first and/or second polypeptides may further comprise a hinge region.
In one embodiment, the method comprises introducing to said first and second polypeptide amino acid substitutions in at least the positions corresponding to E345, E430, and in at least one position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254 in a human IgG1 heavy chain.
In one embodiment, the method comprises introducing to said first and second polypeptide amino acid substitutions in at least the positions corresponding to E345, E430, and in at least one position selected from the group consisting of S440, Y436, E356, T359, E382, N434, Q438, I253 and S254 in a human IgG1 heavy chain.
Thus, in one embodiment the present invention also relates to a method of increasing one or both of an effector function or oligomerization in solution of a parent dimeric protein comprising a first and second polypeptide, each comprising at least CH2, CH3, and hinge regions of an immunoglobulin heavy chain, the method comprising introducing into each polypeptide amino acid substitutions in at least the positions corresponding to E345, E430 and S440 in a human IgG1 heavy chain.
In one embodiment, the amino acid substitutions are in at least the positions corresponding to E345, E430 and S440 in a human IgG1 heavy chain.
In one embodiment, the amino acids substitution in the position corresponding to E345 is, for each polypeptide, selected from the group consisting of 345R, 345Q, 345N, 345K, 345Y, 345A, 345C, 345D, 345F, 345G, 345H, 345I, 345L, 345M, 345P, 345S, 345T, 345V and 345W, such as from the group consisting of 345R, 345Q, 345N, 345K and 345Y.
In one embodiment, the amino acid substitution in the position corresponding to E430 is, for each polypeptide, selected from the group consisting of 430G, 430T, 430S, 430F, 430H, 430A, 430C, 430D, 430I, 430K, 430L, 430M, 430N, 430P, 430Q, 430R, 430V, 430W and 430Y, such as from the group consisting of 430G, 430T, 430S, 430F and 430H.
In one embodiment, the amino acid substitution in the position corresponding to S440 is, in each polypeptide, 440Y or 440W.
In one embodiment, the amino acid substitutions in the positions corresponding to E345, E430 and S440 are 345R, 430G and 440Y, respectively.
In one embodiment, the amino acid substitutions in the positions corresponding to E345, E430 and S440 in a human IgG1 heavy chain are 345K, 430G and 440Y, respectively.
In one embodiment, the amino acid substitutions in the positions corresponding to E345, E430 and S440 in a human IgG1 heavy chain are 345R, 430S and 440Y, respectively.
In one embodiment, the amino acid substitutions in the positions corresponding to E345, E430 and S440 in a human IgG1 heavy chain are 345R, 430G and 440W, respectively.
In one embodiment, the amino acid substitutions in the positions corresponding to E345, E430 and Y436 in a human IgG1 heavy chain are 345R, 430G and 436I, respectively.
In one embodiment, the amino acid substitutions in the positions corresponding to E345, E430, Y436, and S440 in a human IgG1 heavy chain are 345R, 430G, 436I, and 440Y respectively.
In any one of the preceding embodiments, the isotype of the heavy chain sequence can selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM and IgE.
In any one of the preceding embodiments, the heavy chain can be of mammalian origin.
In any one of the preceding embodiments, the heavy chain can be of primate or murine origin, such as of human origin.
In any one of the preceding embodiments, each polypeptide may comprise an immunoglobulin heavy-chain variable region associated with an immunoglobulin light chain sequence comprising light chain variable and constant regions to form a first and a second antigen-binding region, optionally binding the same antigen.
In the preceding embodiment, each polypeptide of the dimeric protein may comprise a full-length heavy chain constant region, such as a full-length human IgG1 heavy chain constant region.
In any one of the preceding embodiments, the parent dimeric protein can be an antibody, such as, for example, a full-length IgG1 antibody.
The invention also provides for any dimeric protein according to any aspect or embodiment herein described, prepared by the method of any one of the preceding embodiments.
The invention also provides for a variant dimeric protein, such as a variant antibody prepared by the method of any one of the preceding embodiments. Specifically, introducing mutations in the designated positions in a parent dimeric protein according to a method of the present invention can result in a dimeric protein of the present invention. The dimeric protein may then be regarded as a variant of the parent dimeric protein, e.g. a variant dimeric protein. Thus, the method(s) of the present invention may be performed so as to obtain any dimeric protein as described herein.
The present invention also relates to a method for purification of a dimeric protein according to the present invention comprising purification on a Protein A or Protein G column at a pH below 6.8, such as between 5.0 and 6.5, e.g. between 5.0 and 6.0, e.g. between 5.0 to 5.5, and subsequently raising the pH above 6.8 or above pH 7.0. Buffers for adjusting the pH may be any of those described herein.
Kit-of-Parts
The present invention also relates to a kit-of-parts comprising a first dimeric protein according to any aspect or embodiment described herein, and a second dimeric protein according to any aspect or embodiment described herein, for simultaneous, separate or sequential use in imaging, diagnostic or therapy.
Uses
As described herein the dimeric protein of the present invention forms hexameric structures in solution, thereby resembling IgM molecules. Furthermore, as described above combinations of a first and second dimeric protein of the present invention, or optionally a second molecule which is not a dimeric protein according to the invention, wherein the different components of the combination bind to different epitopes on the same target is foreseen to create compositions which resemble polyclonal antibody compositions. These features and other features of the dimeric protein of the present invention make it particularly suitable for certain applications.
IgM Like Feature
IgM has a major role in immune response to infectious organisms. It is a potent activator of the classical complement pathway.
Antibodies against carbohydrates are often of IgM isotype. Carbohydrates are potential targets for treatment of bacterial, fungal or viral infections, cancer and autoimmune diseases.
IgM antibodies are described to have immune regulatory properties and to be protective in a number of autoimmune diseases, like lupus (SLE) and multiple sclerosis. IgM would also have a protective role in atherosclerosis, myocardial infarction and stroke, cerebral small vessel disease and Alzheimer's disease. (Groenwall et al 2012, Frontiers in Immunology 3, 1-10)
Naturally occurring antibodies to cancer cells are often of IgM isotype.
IgM (and polymeric IgA) has a function in immune exclusion on the luminal side of mucosal surfaces. For passive immunization, protective levels of IgM (and polymeric IgA) can be delivered directly to mucosal surfaces.
IgM based products are being developed for autoimmune, cancer and infection indications.
The dimeric protein of the present invention could mimic the IgM-like features of listed above when in a pH adjusted, such as pH 6.5 to 7.0, solution, and it is therefore foreseen that the dimeric protein of the invention can be used for treatment of any of said indications.
In addition, by adjusting the pH of the solution, the dimeric proteins of the invention can be in monomeric, i.e. as a single dimeric protein, or hexameric form (as described in Example 32). The term “monomeric form” in the context of dimeric protein according to the present invention refers to a single, individual dimeric protein, which is composed of dimeric proteins that do not associate non-covalently with each other. When referring to a “hexameric form” it is to be understood as a complex of six non-covalently associated single dimeric proteins. It is foreseen that standard production and purification methods used for IgG molecules can be used for the dimeric proteins of the invention when they are in monomeric form, such as, but not limited to, the use of protein A resins and protein A variant resins for purification, and the use of protein A and protein A variant based immunoglobulin domain detection assays for example applied in process control, and the use of cation exchange chromatography for concomitant aggregate removal during protein purification, and the use of nanofiltration for viral clearance, thus avoiding problems often encountered when producing or purifying IgM proteins.
Fast Clearance
The hexameric form of dimeric proteins of the present invention is rapidly cleared unless when combined with technologies preventing rapid clearance as described herein. Fab fragment products, also being cleared rapidly, are being developed/used for treatment of poisoning, poison intoxication, and to deplete excess or abundant ligands and/or soluble factors.
Dimeric proteins of the present invention could have similar applications.
Dimeric proteins of the present invention could also be used to deplete soluble/shedded forms of membrane proteins that would form a sink for cell-targeted therapy.
Polyclonal Aspects
Polyclonal antibody products have the potential of synergistic action (better efficacy), and could overcome acquired therapy resistance.
Polyclonal antibody products are developed/being used for treatment of viral or bacterial infections, envenomation, (immune thrombocytopenic purpura), Digoxin toxicity, renal transplant acute rejection and cancer.
The dimeric protein of the present invention, thus, has similar applications and is therefore suitable for use in the treatment of any of said indications.
Thus, in one embodiment the dimeric proteins of the present invention may be used for treatment of any of the following indications: Autoimmune diseases, including systemic lupus erythematodes (SLE), multiple sclerosis, Neuromyelitis optica, Sjögrens syndrome, CREST syndrome, opsoclonus, Inflammatory myopathy, Mixed connective tissue disease, Systemic sclerosis, Primary biliary cirrhosis, Coeliac disease, Miller-Fisher syndrome, Acute motor axonal neuropathy, Multifocal motor neuropathy MMN, Rheumatoid arthritis, Osteoarthritis, Autoimmune hepatitis, Anti-phospholipid syndrome, Wegener's granulomatosis, Microscopic polyangiitis, Churg-Strauss syndrome, Polymyositis, Scleromyositis, Myasthenia gravis, Lambert-Eaton myasthenic syndrome, Hashimoto's thyroiditis, Graves'disease, Paraneoplastic cerebellar syndrome, Stiff person syndrome, Limbic encephalitis, Sydenham's chorea, PANDAS, Encephalitis, limbic encephalitis, Diabetes mellitus type 1, ataxia, Epilepsia partialis continua, Idiopathic thrombocytopenic purpura, Pernicious anemia, Addison's anemia, Autoimmune gonadal failure, Autoimmune hemolytic diseases, such as hematological auto-immune anemia and HIV-associated thrombocytopenia, Pemphigus, Bullous pemphigoid, Dermatitis hepetiformis, Linear IgA dermatosis, Vitiligo, Goodpasture's syndrome, Myocarditis, idiopathic dilated cardiomyopathy, Crohn's disease and ulcerative colitis, cancer, bacterial infections, viral and fungal infections, poisoning and envenomation, or vascular or other diseases.
Examples of cancer, include but are not limited to various cancer types such as: tumors of the central nervous system, head and neck cancer, lung cancer (such as non-small cell lung cancer), breast cancer (such as triple-negative breast cancer), esophageal cancer, stomach cancer, liver and biliary cancer, pancreatic cancer, colorectal cancer, bladder cancer, kidney cancer, prostate cancer, endometrial cancer, ovarian cancer, malignant melanoma, sarcoma (soft tissue eg. bone and muscle), tumors of unknown primary origin (i.e. unknown primaries), leukemia, bone marrow cancer (such as multiple myeloma) acute lymphoblastic leukemia, chronic lymphoblastic leukemia and non-Hodgkin lymphoma, acute myeloid leukemia (AML), skin cancer, glioma, cancer of the brain, uterus, and rectum.
Thus, in one aspect, the present invention relates to a method for preventing or treating a disease, such as cancer, auto-immune diseases, infections, diabetes mellitus, organ transplant rejections, ophthalmological diseases and C1q depletion in the humeral system, comprising administration of a dimeric protein, oligomer, hexamer, composition, kit-of-parts according to any aspect or embodiment of the present invention.
In one embodiment, the cancer is a tumor, such as a brain tumor. The dimeric protein according to the invention may be used to mechanically obstruct blood flow in tumor blood vessels by injection directly into the tumor, such as brain tumors. The dimeric protein according to any aspect or embodiment of the present invention, may be particularly useful to induce mechanical obstruction in tumors due to its capability to form oligomers, such as dimer, trimers, and hexamers. When the dimeric protein, such as an antibody, according to the present invention is used in the treatment of cancer it is particularly useful in overcoming suppression of effector mechanisms due to the low pH of the tumor microenvironment.
In one embodiment, the dimeric protein, oligomer, hexamer, composition or kit-of-parts according to any aspect or embodiment of the present invention, is for use in the treatment of tumors by making use of pH-dependent delivery of toxins or payloads/drugs. In such uses the lower pH at the tumor site can be exploited when the dimeric protein, such as an antibody, of the present invention is fused to a toxin or drug that has optimal function at the lower pH at the tumor site.
In one embodiment, the method comprises the steps of administering to the bloodstream a first dimeric protein according to any aspect or embodiment of the invention linked to a first pro-drug, and a second dimeric protein according to any aspect or embodiment of the invention linked to a second pro-drug.
In one aspect, the present invention relates to a method of inducing an immunomodulatory effector function, such as mediated through CD32b and KIR, wherein the method comprises administration of the dimeric protein, oligomer, hexamer, composition or kit-of-parts according to any aspect or embodiment of the present invention, optionally combined with sialylation of the dimeric protein. In such a method, the dimeric protein of the invention will induce clustering of the target molecules and thereby induce the immunomodulatory effector function. Thus, the dimeric protein according to the invention may be used as an alternative to intravenous immunoglobulin (IVIG).
In one embodiment, the dimeric protein, such as an antibody or Fc-fusion protein, according to any aspect or embodiment of the present invention, may be used to enhance clearance of a target molecule from the bloodstream, such as a ligand, a receptor, a toxin, C1q, IgE, an anti-graft antibody, human anti-human antibodies (HAHA), antidrug antibodies (ADA), human anti murine antibodies (HAMA), human anti chimeric antibodies (HACA), pharmaceutical compounds, and immunomodulatory compounds.
The dimeric protein according to the invention, such as an Fc fragment fused to an antigen, may have improved immunostimulatory effect when in oligomeric form, such as a hexameric molecule, as the oligomer provides antigen complexes that for example stimulate clustering of B-cell receptors directed against said antigen, and facilitate affinity maturation of initial low affinity B-cell receptors by presenting the antigen in a multivalent form. This may be obtained both when the dimeric protein according to the invention is in solution or is presented on the surface of a cell, virion, virus-like particle, embedded in a liposome or in other forms supporting presentation of transmembrane proteins commonly known in the art. Thus, in one embodiment, the dimeric protein, such as an Fc-fragment, according to any aspect or embodiment of the present invention, is for use in vaccination, immunization and immune response stimulation. Thus, in one embodiment the present invention also relates to a method for vaccination, immunization and immune response stimulation comprising administration of the dimeric protein as described herein.
The dimeric protein according to the invention, may be used to create supramolecular structures, that optionally may be assembled in a pH-dependent fashion, both in solution, as well as on a surface, such as, but not limited to, the surface of a cell, virion, virus-like particle, liposome, microchip, solid surface, porous scaffold, or other methods for protein presentation commonly known in the art.
In one embodiment, said first and/or second polypeptide of the dimeric protein may comprise a protein binding domain, such as a Fab domain, specifically binding a Fab domain in a different Fc domain containing polypeptide. The dimeric protein and its target molecule may be used for the formation of supramolecular structures, that may optionally be assembled in a pH controlled fashion.
In one embodiment, the dimeric protein according to any aspect or embodiment of the present invention is for use in protein crystallization. The dimeric protein may be particularly useful due to its capability to form oligomeric structures in a pH controlled fashion.
In one embodiment the present invention relates to a method of using the dimeric protein according any aspect or embodiment herein described for immune complex formation in diagnostical kits, such as an agglutination assay. An example of an agglutination assay is Coombs test.
Examples of bacterial infections, include but are not limited to Staphylococcus aureus infection (S. aureus), e.g. Methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa infection, infections caused by a bacteria selected from the group consisting of S. epidermidis, S. pneumonia, Bacillus anthracis, Chlamydia trachomatis, E. coli, Salmonella, Shigella, Yersinia, S. typhimurium, Neisseria meningitides, and Mycobacterium tuberculosis. Examples of viral and fungal infections, include but are not limited to West Nile virus, Dengue virus, hepatitis C-virus (HCV), human immunodeficiency virus (HIV), RVS, Aspergillus, Candida albicans, Cryptococcus, Histoplasma, human cytomegalovirus (HCMV), herpes simplex virus, human respiratory syncytial virus, human papillomavirus, Epstein-Barr virus, Herpesviruses, poxviruses, and avian influenza virus. Examples of poisoning and envenomation include but are not limited to digoxin, Colchicine, venom from reptiles such as snake venom, venom from insects such as bee, wasp and caterpillar venom, spider venom, Microbial endotoxins and exotoxins, such as botelinum neurotoxins, tetanus toxin, Staphylococcal toxins, alpha toxin, Anthrax toxin, Diphteria toxin, Persussis toxin, Shiga toxin, Shiga-like toxin.
Examples of vascular and other diseases may be e.g. atherosclerosis, myocardial infarction and stroke, cerebral small vessel disease, Alzheimer's disease, and depletion of C1q in high-fat diet induced hepatic insulin resistance and systemic glucose tolerance, and the clearance of anti-graft antibodies before or after organ transplantation.
In one aspect, the present invention relates to the dimeric protein, oligomer, hexamer, composition or kit-of-parts according to any aspect or embodiments described herein, for use in the treatment of a disease, such as a bacterial, viral or parasitic infection, autoimmune disease, cancer, inflammation, and/or reducing the risk for septic shock caused by a bacterial infection.
For the treatment of bacterial infections and/or reducing the risk of septic shock, the dimeric protein of the invention may, for example, comprise a binding region specifically binding to a lipopolysaccharide (LPS), a lipooligosaccharide (LOS), a delta endotoxin, Botulinum toxin, Corynebacterium diphtheriae exotoxin, a bacterial superantigen, a heat-stable enterotoxin, cytolysin, a channel-forming toxin, an enzymatically active toxin or a mycotoxin.
In another aspect, the invention provides for the use of the dimeric protein, hexamer, composition or kit-of-parts according to any one of the preceding embodiments in imaging at least a part of the body of a human or other mammal. In one aspect, the present invention relates to a method for imaging of at least a part of the body of a human or other mammal, comprising administering a dimeric protein, oligomer, hexamer, composition or kit-of-parts according to any aspect or embodiments described herein.
In another aspect, the invention relates to a method for treating a bacterial, viral or parasitic infection, for imaging of at least a part of the body of human or other mammal, or for modulating clearance of a target molecule from the body of a human or other mammal, comprising administering a dimeric protein, oligomer, hexamer, composition or kit-of-parts according to any aspect or embodiment described herein.
In another aspect, the invention relates to a method for preventing or treating a disease, such as cancer, auto-immune diseases, organ transplant rejections, and C1q depletion in the humoral system, comprising administration of a dimeric protein, oligomer, hexamer, composition, kit-of-parts according to any aspect or embodiment described herein.
EXAMPLES
Example 1
Design and Generation of CD38 Antibody 005 Mutants
The human monoclonal antibody HuMab 005 is a fully human IgG1,κ antibody described in WO/2006/099875, that is directed against human CD38. Here, it was used as a model antibody to test the capability of Fc mutations to enhance CDC activity. The tested mutations are listed in Table 3.
DNA constructs for the different mutants were prepared and transiently transfected using the heavy chain of HuMab 005 with IgGlm(f) allotype as a template for mutagenesis reactions. Briefly, mutants were prepared using the Quikchange site-directed mutagenesis kit (Stratagene, US). A forward and a reverse primer encoding the desired mutation were used to replicate full length plasmid DNA template encoding the 005 heavy chain with IgGlm(f) allotype. The resulting DNA mixture was digested using DpnI to remove source plasmid DNA and used to transform E. coli. Mutant plasmid DNA isolated from resulting colonies was checked by DNA sequencing (Agowa, Germany). Plasmid DNA mixtures encoding both heavy and light chain of antibodies were transiently transfected to Freestyle HEK293F cells (Invitrogen, US) using 293fectin (Invitrogen, US) essentially as described by the manufacturer.
To test the functional relevance of oligomeric Fc-Fc interactions in complement activation and CDC, amino acids in the hydrophobic patch at the Fc:Fc interface were mutated to potentially disrupt the Fc-Fc side-on interaction and CDC efficacy of 005. Mutations I253D and H433A were introduced to change the charge at positions that were chosen based on the 1HZH crystal structure and described to be exposed in hydrophobic patches in the CH2-CH3 domain (Burton Mol Immunol 1985 March; 22(3):161-206)).
The 1HZH crystal structure shows that I253 and H433 bind two different pockets on the opposing Fc positions of the partnering antibody. To exclude the possibility that disruption of direct binding sites for C1q were the cause of the observed effects on CDC, mutants K439E and S440K were generated. As shown in FIG. 4, K439 and S440 face each other on opposite sides at the Fc:Fc interface, so K439E and S440K were designed to induce loss of CDC as single mutant by inhibiting the Fc:Fc interaction, but were expected to restore CDC when interacting with each other, due to restored Fc:Fc interactions in the antibody mixture.
TABLE 3
set of mutations that were introduced in the CH2—CH3
domain of 005 (HuMax-CD38).
Mutation
Charge WT aa
Charge mutant aa
I253D
=
−
E345R
−
+
H433A
δ+
=
K439E
+
−
S440K
=
+
(=) no charge
(−) negative charge
(+) positive charge
(δ+) partial positive charge
Example 2
CD38 Binding on Cells by HuMab-005 Mutants
Binding of unpurified antibody samples to CD38-positive Daudi and Raji cells was analyzed by FACS analysis. 105 cells were incubated in 100 μL in polystyrene 96-well round-bottom plates with serial dilutions of antibody preparations (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg/mL) in RPMI1640/0.1% BSA at 4° C. for 30 min. After washing twice in RPMI1640/0.1% BSA, cells were incubated in 50 μL with FITC-conjugated rabbit F(ab′)2 anti-human IgG (cat.no. F0056; DAKO; 1:150) at 4° C. for 30 min. Next, cells were washed twice in PBS/0.1% BSA/0.02% azide, resuspended in 100 μL PBS/0.1% BSA/0.02% azide and analyzed on a FACS Cantoll (BD Biosciences). Binding curves were analyzed using GraphPad Prism V5.01 software. As a negative control, supernatant of mock-transfected cells was used.
Binding of HuMab 005 to Daudi cells was not much affected by the introduction of point mutations in the CH2-CH3 domain. All tested antibodies bound Daudi cells in a dose-dependent manner. Binding was similar to wild type HuMab-005 for all tested mutants, with the exception of 005-E345R, which showed slightly decreased binding. However, without being bound by any theory, the lower binding might be a result of decreased binding by the secondary antibody. The actual binding avidity by 005-E345R might be similar or even increased compared 005-WT, however we could not confirm this because of lack of directly labeled antibodies.
Binding of HuMab-005 to Raji cells was also not much affected by the introduction of point mutations in the CH2-CH3 domain. All tested antibodies bound Raji cells in a dose-dependent manner. Maximal binding was similar to that of wild type 005 for the 005-1253D and H433A mutants and lower for the 005-E435R, K439E, S440K mutants and the combination of 005-K439E+005-S440K. However, without being bound by any theory, the lower binding might be a result of decreased binding by the secondary antibody (shielding of the epitope).
Example 3
CDC Assay on CD38-Positive Cells by Mutants of the CD38 Antibody 005
0.1×106 Daudi or Raji cells were pre-incubated in round-bottom 96-well plates with a concentration series of unpurified antibodies (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg/mL) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of C1q (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
The impact of the E435R mutation on CDC was further analyzed on Wien133 cells with different concentration normal human serum (NHS). 0.1×106 Wien133 cells were pre-incubated for 15 min on a shaker at RT in round-bottom 96-well plates with a concentration series of unpurified antibodies (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg/mL) in a total volume of 50 μL. Next, NHS was added as a source of C1q to reach a final concentration of either 20% or 50% NHS in a total volume of 100 μL. The reaction mixture was incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
FIG. 5 shows that 005-I253D, H443A, K439E and S440K showed complete loss of CDC activity on both Daudi (FIG. 5A) and Raji (FIG. 5B) cells, whereas the 005-E345R mutant showed strongly enhanced CDC activity on both cell lines. Comparable to 7D8 data, a combination of 005-K439E+005-S440K, which both result in loss of CDC as a single mutant, resulted in restored CDC. Surprisingly, 005-E435R even strongly induced CDC on Wien133 cells, for which wild type 005 is not capable to induce killing by CDC (FIG. 5C). CDC killing by 005-E345R on Wien133 cells was observed with both 20% and 50% serum concentrations (FIG. 5C). On Raji cells, both 7D8-E345R and 005-E345R showed enhanced CDC in vitro in 50% serum, with similar efficacy as in 20% serum (FIG. 5D).
As the E345R mutation in the CH2-CH3 region resulted in enhanced CDC activity in both the tested CD20 antibody 7D8 and CD38 antibody 005, the E345R mutation is considered to be a general antibody modification that can be applied to induce or enhance CDC.
Example 4
IgG1 Antibodies Containing the CDC-Enhancing Mutation E345R are Less Sensitive to Inhibition of CDC by Fc Binding Peptide DCAWHLGELVWCT than Wild Type Antibodies
By mutating amino acid positions in the hydrophobic patch at the Fc:Fc interface of IgG, CDC efficacy was found to be either disturbed or enhanced. The involvement of the interactions at the Fc-Fc interface, and thus possibly the formation of an oligomeric (e.g., hexameric ring) structure as observed in the b12 crystal structure, in CDC efficacy was further explored. Therefore, a 13-residue peptide (DCAWHLGELVWCT (SEQ ID NO:7)) was used that targets a consensus binding site in the hydrophobic patch region on the surface of wild type IgG Fc (Delano et al., Science 2000 Feb. 18; 287(5456):1279-83). Indeed, the identification of the consensus binding site on the surface of IgG Fc as an adaptive region that is primed for interaction with a variety of distinct molecules (Delano et al., Science 2000 Feb. 18; 287(5456):1279-83), is consistent with the identification of the core amino acids in the hydrophobic patch that are involved in the Fc-Fc interaction in the IgG1 b12 crystal structure (Saphire et al., Science 2001 Aug. 10; 293(5532):1155-9). Interactions that are present in all of the binding interfaces are mediated by a shared set of six amino acids (Met-252, Ile-253, Ser-254, Asn-434, His-435, and Tyr-436), as well as shared backbone contacts (Delano et al., Science 2000 Feb. 18; 287(5456):1279-83). Accordingly, the Fc binding peptide is expected to affect the Fc-Fc interaction and consequently CDC efficacy.
0.1×106 Daudi cells were pre-incubated in 75 μL with 1.0 μg/mL unpurified antibody in round-bottom 96-well plates for 10 min at room temperature on a shaker. 25 μL of a concentration series (range 0.06-60 μg/mL final concentration) of the Fc binding peptide DCAWHLGELVWCT was added to the opsonized cells and incubated for 10 min on a shaker at RT. Next, 25 μL NHS was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by adding 25 μL ice cold RPMI medium, supplemented with 0.1% BSA. 15 μL propidium iodide was added and cell lysis was determined by FACS analysis.
CDC mediated by wild type 005 (FIG. 6) was found to be inhibited by the Fc-binding peptide DCAWHLGELVWCT in a dose-dependent manner. These competition data suggest again the involvement of the Fc-Fc interactions at the hydrophobic patch of IgG in CDC efficacy. The CDC-enhanced IgG1-005-E345R mutant was less sensitive for competition by the Fc-binding peptide compared to the corresponding wild type antibodies, suggesting that the E345R mutation results in increased stability of the Fc-Fc interaction, and consequently increased CDC.
Example 5
Increased Specificity of Enhanced CDC by Combining E345R with Complementary Inhibiting Mutations K439E and S440K in a Mixture of Two Different Monoclonal Antibodies
As described in Example 3, CD38 antibody 005 mutations K439E and S440K decreased the CDC efficacy as monoclonal antibodies. Mixing 005 antibodies containing these mutations restored CDC. Efficient CDC was thus restricted to cells bound by both mutant antibodies simultaneously. Similar, data have been found for the CD20 antibody 7D8 described in WO 2004/035607 (data not shown).
It can be advantageous to restrict the enhancement of CDC induction to target cells that express two specific antigens simultaneously, exploiting their combined expression to improve selectivity of enhanced CDC induction. It can also be advantageous to restrict the enhancement of CDC induction to target cells that are bound by mixtures of at least two different antibodies simultaneously, said antibodies binding an identical cell surface antigen at two different epitopes simultaneously, or at two cross-competing, similar, or identical epitopes.
Therefore, to restrict enhanced CDC induction to cells bound by both CD20 and CD38 antibodies simultaneously, the CDC enhancing mutation E345R was combined with CDC inhibiting mutations in the antibodies 7D8-E345R/K439E, 7D8-E345R/S440K, 005-E345R/S440K and 005-E345R/K439E. These antibodies were added separately or mixed 1:1 in CDC experiments as follows. 0.1×106 Wien133 cells (other cell types such as Daudi or Raji cells may also be used) were pre-incubated in round-bottom 96-well plates with a concentration series of unpurified antibodies (final concentration 0.056-10,000 ng/mL in 3-fold dilutions for 7D8-E345R/K439E, 7D8-E345R/S440K, 005-E345R/S440K or 005-E345R/K439E) or antibody mixtures (final concentrations 0.01 μg/mL CD20 antibody mixed with 0-333 ng/mL in 3-fold dilutions CD38 antibody; or 3.3 μg/mL CD38 antibody mixed with 0.0056-1,000 ng/mL in 3-fold dilutions CD20 antibody) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
A concentration series of 005-E345R/K439E or 005-E345R/S440K antibody was mixed with a fixed concentration of 0.01 μg/mL 7D8 double mutant antibody (maximal concentration with minimal CDC on Wien133 cells as a single agent as determined from FIG. 7A) to make the complementary combinations 005-E345R/K439E+7D8-E345R/S440K or 005-E345R/S440K+7D8-E345R/K439E. FIG. 7C shows that the 005 double mutant CD38 antibodies induced CDC dose-dependently in the presence of fixed concentration of the complementary 7D8-E345R/K439E or 7D8-E345R/S440K CD20 antibody, respectively. The CDC efficacy by these complementary combinations (FIG. 7C) was comparable to the 005-E345R single mutant (enhancer) antibody as a single agent (FIG. 7B). In contrast, in the presence of irrelevant antibody b12, both 005-E345R/K439E and 005-E345R/S440K showed hardly any CDC in the concentration series tested (comparable to 005-E345R/K439E or 005-E345R/S440K as single agents shown in FIG. 7B).
A concentration series of 7D8-E345R/K439E or 7D8-E345R/S440K antibody was mixed with a fixed concentration of 3.3 μg/mL 005 double mutant antibody (showing a little but limited CDC on Wien133 cells as a single agent as determined from FIG. 7B) to make the complementary combinations 7D8-E345R/K439E+005-E345R/S440K or 7D8-E345R/S440K+005-E345R/K439E. FIG. 7D shows that the 7D8 double mutant CD20 antibodies induced CDC very efficiently in the presence of the complementary 005-E345R/K439E or 005-E345R/S440K CD38 antibody respectively, even at the lowest concentrations tested, resembling not more than a few 7D8 double mutant antibody molecules per cell. To eliminate the contribution of increased Fc-tail density on the cell membrane to the observed enhanced CDC by the mixture of 7D8 and 005 antibodies with complementary K439E and S440K mutations, also antibody combinations with non-complementary mutations were tested. FIG. 7D shows that non-complementary combinations showed much lower CDC efficacy than complementary combinations, as a result of less efficient Fc-Fc interaction than the complementary combinations.
These data suggest that the induction of (enhanced) CDC by therapeutic antibodies can be limited to cells that bind simultaneous a mixture of two complementary antibodies, in this case with different antigen specificities, thereby increasing target cell specificity by requiring co-expression of both antigens.
As can be seen in FIGS. 7A and 7B, 7D8-E345R/K439E, 005-E345R/S440K, 7D8-E345R/S440K and 005-E345R/K439E displayed limited CDC efficiency in comparison to 7D8-E345R alone. It is further seen, that the mixture of 7D8-E345R/K439E and 7D8-E345R/S440K enabled CDC with enhanced efficiency compared to wildtype 7D8 antibody as single agent. Likewise, it was observed that the mixture of 005-E345R/K439E and 005-E345R/S440K enabled CDC with enhanced efficiency compared to wildtype 005 antibody as single agent (data not shown).
Example 6
Use of a Mutant Screening Approach to Identify Mutations Stimulating Fc:Fc Interaction Mediated Antibody Oligomerization Detected by a CDC Assay
As described in Example 3, amino acid mutations were identified that stimulated CDC for an antibody recognizing the target antigens, CD38, on multiple cell lines expressing variable levels of said antigens. Surprisingly, the single point mutation E345R proved sufficient to endow CDC-dependent cell lysis of Wien133 cells to the anti-CD38 antibody 005, which failed to lyse these cells by CDC in wild type IgG1 format.
Other mutations on or at the periphery of the Fc:Fc interface could stimulate oligomerization and CDC in an analogous fashion. Alternatively, mutations could indirectly stimulate oligomerization, for example by allosterically inducing Fc:Fc interactions.
To determine if other amino acid mutations could stimulate Fc-mediated antibody oligomerization, a library of anti-CD38 IgG1-005 mutants was screened using CDC assays, both individually and mixed in a pairwise fashion to select for example amino acid pairs interacting across the Fc:Fc interface. However, the same strategy can be applied to other antibodies, such as another IgG1 or an IgG3 antibody.
A focused library of mutations at the positions indicated in Table 4 was generated. Mutations were introduced into the IgG1-005 Fc region using the Quikchange site-directed mutagenesis kit (Stratagene, US). Briefly, for each desired mutation position, a forward and a reverse primer encoding a degenerate codon at the desired location were used to replicate full length plasmid DNA template of the 005 heavy chain with IgG1m(f) allotype. The resulting DNA mixtures were digested using DpnI to remove source plasmid DNA and used to transform E. coli. Resulting colonies were pooled and cultured and plasmid DNA was isolated from these pools and retransformed into E. coli to obtain clonal colonies. Mutant plasmid DNA isolated from resulting colonies was checked by DNA sequencing (LGC genomics, Berlin, Germany). Expression cassettes were amplified from plasmid DNA by PCR and DNA mixes containing both a mutant heavy and a wildtype light chain of IgG1-005 were transiently transfected to Freestyle HEK293F cells (Invitrogen, US) using 293fectin (Invitrogen, US) essentially as described by the manufacturer. Supernatants of transfected cells containing antibody mutants were collected. Mutant antibody supernatants were screened in CDC assays both individually and in pairwise mixtures as follows.
0.1×106 Daudi or Wien-133 cells (other cells types such as Raji cells may be used) were pre-incubated in round-bottom 96-well plates with 1.0 ug/ml of unpurified antibodies in a total volume of 100 μL for 15 min on a shaker at RT. Next, 30 μL normal human serum was added as a source of complement (30% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μl propidium iodide was added and cell lysis was determined by FACS.
Mutations described in Table 4, Table 5 and Table 6 were selected for their ability to enhance oligomerization as detected by CDC efficiency, either as a single mutant or when mixed with other mutants for example facing the mutation across the Fc:Fc interface. Mutations can optionally be further screened for their ability to not compromise FcRn, Protein-A or Protein-G binding, ADCC, ADCP or other effector functions mediated by the Fc domain. Combining such stimulating point mutations into one Fc domain can stimulate oligomerization and CDC efficiency even further.
Mutations in the CH2-CH3 region incorporated in the CD38 antibody 005 were tested for their ability to inhibit oligomerization as determined by CDC on Daudi cells. Lysis of the mutant antibody was compared to wild type 005, for which lysis was set to 100%. The cutoff for inhibition was set to 66% lysis. Measured in this way, most of the tested mutations inhibited CDC (see Table 4).
Mutations in the CH2-CH3 region incorporated in the CD38 antibody 005 were tested for their ability to enhance oligomerization as determined by CDC on Wien133 cells (Table 5). Wild type CD38 antibody 005 is not able to induce CDC on Wien133 cells. Mutants displaying ≥39% cell lysis were scored as enhancing. Completely unexpectedly, virtually all obtained substitutions of amino acids E345 and E430 stimulated cell lysis by CDC. To verify this result, amino acids E345, E430 and S440 were substituted with each possible mutation by site directed mutagenesis and tested for their ability to enhance oligomerization as determined by CDC of Wien133 cells using a new human serum batch, yielding slightly more efficient lysis (Table 6). Again, all substitutions of E345 and E430 induced efficient CDC of Wien133 cells.
The following preferred mutations caused 39% cell lysis of Wien133 cells: P247G, I253V, S254L, Q311L, Q311W, E345A, E345C, E345D, E345F, E345G, E345H, E345I, E345K, E345L, E345M, E345N, E345P, E345Q, E345R, E345S, E345T, E345V, E345W, E345Y, D/E356G, D/E356R, T359R, E382L, E382V, Q386K, E430A, E430C, E430D, E430F, E430G, E430H, E430I, E430L, E430M, E430N, E430P, E430Q, E430R, E430S, E430T, E430V, E430W, E430Y, Y436I, S440Y and S440W.
TABLE 4
Percentage lysis of Daudi cells in the presence of 1.0 μg/ml IgG1-005 antibody point mutations.
IgG1-005 wildtype lysed 66% of cells under these conditions. For each of the individual
positions which have been substituted by another amino acid are given in the outer left
column. The substituted amino acid for each particular position is given followed by the
measured percentage lysis indicated in paranteses ( ) in the horizontal rows of the individual positions.
Position
P247
A
C
D
F
G
H
I
K
L
M
N
R
S
T
V
W
(42)
(67)
(91)
(93)
(95)
(80)
(89)
(96)
(13)
(83)
(78)
(93)
(93)
(10)
(9)
(82)
1253
A
D
K
M
N
R
S
V
(17)
(12)
(13)
(6)
(5)
(7)
(6)
(94)
S254
E
F
G
H
I
K
L
P
T
W
(14)
(75)
(100)
(46)
(93)
(86)
(99)
(4)
(8)
(7)
H310
K
W
(6)
(87)
Q311
A
C
E
F
G
H
I
K
L
N
P
R
S
T
W
Y
(53)
(72)
(5)
(90)
(68)
(72)
(92)
(93)
(96)
(53)
(97)
(87)
(66)
(54)
(93)
(85)
E345
A
C
F
G
H
I
K
L
M
N
P
R
S
T
V
W
Y
(85)
(91)
(95)
(86)
(83)
(96)
(94)
(98)
(94)
(97)
(74)
(98)
(93)
(82)
(92)
(95)
(95)
D/E356
G
I
L
R
T
V
(88)
(95)
(94)
(97)
(97)
(98)
T359
G
N
P
R
(88)
(93)
(87)
(96)
E382
F
K
L
M
P
V
W
(3)
(3)
(99)
(90)
(3)
(96)
(3)
G385
D
H
Q
R
S
T
(28)
(9)
(24)
(27)
(14)
(10)
Q386
A
C
D
E
F
G
H
I
K
L
N
P
R
S
T
V
W
Y
(56)
(18)
(6)
(9)
(11)
(10)
(26)
(42)
(98)
(15)
(25)
(6)
(10)
(43)
(12)
(53)
(13)
(42)
E430
A
F
G
H
L
P
Q
R
S
V
(97)
(97)
(99)
(98)
(95)
(95)
(90)
(96)
(94)
(98)
N434
D
E
K
R
S
W
(5)
(5)
(5)
(5)
(6)
(98)
Y436
I
K
L
R
S
T
W
(98)
(7)
(10)
(35)
(8)
(7 )
(6)
Q438
E
K
S
T
W
Y
(5)
(6)
(5)
(8)
(10)
(31)
K439
A
D
H
L
P
T
Y
(6)
(5)
(5)
(5)
(8)
(4 )
(7)
S440
A
C
D
E
F
G
I
N
R
T
Y
(61)
(10)
(95)
(24)
(13)
(40)
(8)
(33)
(11)
(28)
(98)
K447
E
*del
(20)
(90)
*where “del” means that there was a deletion of the amino acid residue at the indicated position.
TABLE 5
Percentage lysis of Wien-133 cells in the presence on 1.0 μg/ml IgG1-005 antibody point mutants. IgG1-005 wildtype lysed 3%
of cells under these conditions. For each of the individual positions which have been substituted by another amino acid
are given in the outer left column.The substituted amino acid for each particular position is given followed
by the measured percentage lysis indicated in paranteses ( ) in the horizontal rows of the individualpositions.
Position
P247
A
C
D
F
G
H
I
K
L
M
N
R
S
T
V
W
(5)
(5)
(12)
(16)
(50)
(11)
(10)
(14)
(4)
(13)
(7)
(10)
(7)
(4)
(3)
(9)
1253
A
D
K
M
N
R
S
V
(11)
(9)
(3)
(3)
(3)
(4)
(3)
(51)
S254
E
F
G
H
I
K
L
P
T
W
(14)
(10)
(32)
(2)
(15)
(12)
(65)
(2)
(9)
(9)
H310
K
W
(3)
(13)
Q311
A
C
E
F
G
H
I
K
L
N
P
R
S
T
W
Y
(9)
(4)
(3)
(19)
(4)
(6)
(28)
(16)
(55)
(6)
(12)
(18)
(9)
(3)
(41)
(12)
E345
A
C
F
G
H
I
K
L
M
P
R
S
T
V
W
Y
(57)
(22)
(48)
(47)
(49)
(59)
(42)
(72)
(67)
(51)
(64)
(60)
(53)
(67)
(52)
(70)
D/E356
G
I
L
R
T
V
(39)
(31)
(30)
(64)
(32)
(13)
T359
G
N
P
R
(2)
(3)
(4)
(40)
E382
F
K
L
M
P
V
W
(2)
(2)
(44)
(21)
(3)
(53)
(2)
G385
D
H
N
Q
R
S
T
(5)
(4)
(18)
(4)
(14)
(4)
(4)
Q386
A
C
D
E
F
G
H
I
K
L
N
P
R
S
T
V
W
Y
(3)
(4)
(4)
(4)
(3)
(3)
(3)
(4)
(60)
(3)
(4)
(2)
(4)
(3)
(3)
(3)
(3)
(4)
E430
A
F
G
H
L
P
Q
R
S
V
(54)
(68)
(55)
(57)
(58)
(56)
(31)
(39)
(20)
(53)
N434
D
E
K
R
S
W
(2)
(2)
(2)
(2)
(3)
(18)
Y436
I
K
L
R
S
T
W
(49)
(3)
(4)
(3)
(3)
(2)
(3)
Q438
E
K
S
T
W
Y
(3)
(3)
(2)
(2)
(2)
(2)
K439
A
D
H
L
P
T
Y
(3)
(2)
(2)
(2)
(2)
(2)
(4)
S440
A
C
D
E
F
G
I
N
R
T
Y
(3)
(3)
(6)
(2)
(2)
(3)
(2)
(2)
(2)
(3)
(64)
TABLE 6
Percentage lysis of Wien-133 cells in the presence on 1.0 μg/ml IgG1-005 antibody point mutants. IgG1-005 wildtype lysed
12% of cells under these conditions. Each of the individual positions which have been substituted by another amino
acid are given in the outer left column. The substituted amino acid for each particular position is given followed
by the measured percentage lysis indicated in paranteses ( ) in the horizontal rows of the individual positions.
Position
E345
A
C
D
F
G
H
I
K
L
M
N
P
Q
R
S
T
V
W
Y
(94)
(87)
(76)
(95)
(95)
(94)
(93)
(97)
(94)
(96)
(93)
(97)
(98)
(94)
(93)
(92)
(96)
(93)
(94)
E430
A
C
D
F
G
H
I
K
L
M
N
P
Q
R
S
T
V
W
Y
(95)
(79)
(91)
(96)
(96)
(95)
(96)
(83)
(94)
(75)
(95)
(97)
(86)
(92)
(96)
(97)
(96)
(98)
(97)
S440
A
C
D
E
F
G
H
I
K
L
M
N
P
Q
R
T
V
W
Y
(12)
(8)
(41)
(9)
(7)
(8)
(26)
(7)
(6)
(7)
(8)
(12)
(10)
(21)
(9)
(10)
(7)
(86)
(90)
Example 7
In Vivo Efficacy of IgG1-005-E345R in a Subcutaneous B Cell Lymphoma Xenograft Model
The in vivo anti-tumor efficacy of the IgG1-005-E345R antibody was evaluated in a subcutaneous model with Raji-luc #2D1 cells. These cells show ˜150,000 CD38 molecules per cell (determined by QIFIKIT analysis, data not shown) and high complement defense receptor expression. The protocol for tumor inoculation and measurement is basically the same as described in Example 20. At day 0, 5×106 Raji-luc #2D1 cells in 200 μL PBS were s.c. injected in the right flank of SCID mice. When average tumor volume was 100 mm3 (around day 7), the mice were sorted into groups (n=7) and treated by i.p. injection of a single dose of 500 μg antibody per mouse (25 mg/kg). Treatment groups are shown in Table 7. Tumors were measured until an endpoint tumor volume of 1500 mm3 or until tumors showed ulcerations or serious clinical signs were observed to avoid major discomfort.
FIG. 8A shows mean tumor growth on day 21, when all groups were still complete. Wild type antibody IgG1-005 slightly inhibited tumor growth, although this was not statistically significant. Only IgG1-005-E345R significantly inhibited tumor growth compared to the irrelevant antibody control at day 21 (One-way ANOVA p<0.05).
FIG. 8B shows a Kaplan-Meier plot of the percentage mice with tumor sizes smaller than 500 mm3. Tumor formation was significantly delayed in mice treated with IgG1-005-E345R antibody compared to mice treated with negative control antibody IgG1-b12 (Mantel-Cox analysis p<0.001) or wild type IgG1-005 (p<0.05).
These data show that introduction of the E345R mutation in the CD38 antibody 005 resulted in enhanced in vivo anti-tumor activity.
TABLE 7
Treatment groups and dosing.
Group
Antibody
Dose
1. wild type
IgG1-005-WT
500 μg (=25 mg/kg)
2. CDC-enhancing mutant
IgG1-005-E345R
500 μg (=25 mg/kg)
3. Irrelevant Ab control
IgG1-b12
500 μg (=25 mg/kg)
Example 8
Monovalent Target Binding Further Enhances the CDC Efficacy of E345R Antibodies
A molecular surface of the IgG1 hexameric ring observed in the b12 crystal structure demonstrates that for each IgG in the hexameric ring, one of the two C1q binding sites is facing upwards and the other site is facing downwards of the ring structure, and also one Fab-arm of each antibody is oriented up and one is oriented down, resulting in only one Fab-arm per antibody to take part in antigen binding, suggesting monovalent binding per antibody molecule in the hexameric antibody ring. Monovalency might bring antibodies upon antigen binding in a hexamerization compatible orientation. To test this hypothesis, the CDC efficacy of a bispecific CD38/EGFR antibody with the E345R mutation was tested on CD38-positive, EGFR-negative Wien133 cells, to which this bispecific antibody can only bind monovalently via CD38, and compared to the CDC efficacy of the bivalent binding CD38 antibody, also with the E345R mutation. The human monoclonal antibody HuMax-EGFr (2F8, described in WO 2004/056847) was used as a basis for the EGFR antibodies described in this example.
Bispecific antibodies were generated in vitro according to the DuoBodyn™ platform, i.e. 2-MEA-induced Fab-arm exchange as described in WO 2011/147986. The basis for this method is the use of complementary CH3 domains, which promote the formation of heterodimers under specific assay conditions. To enable the production of bispecific antibodies by this method, IgG1 molecules carrying certain mutations in the CH3 domain were generated: in one of the parental IgG1 antibody the F405L mutation, in the other parental IgG1 antibody the K409R mutation. To generate bispecific antibodies, these two parental antibodies, each antibody at a final concentration of 0.5 mg/mL, were incubated with 25 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 μL TE at 37° C. for 90 min. The reduction reaction is stopped when the reducing agent 2-MEA is removed by using spin columns (Microcon centrifugal filters, 30 k, Millipore) according to the manufacturer's protocol.
For the CDC assay, 0.1×106 Wien133 cells were pre-incubated in round-bottom 96-well plates with a concentration series of antibodies (0.01 to 10.0 μg/mL) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
FIG. 9 shows that, as expected, CD38 antibodies without the E345R mutation (wild type IgG1-005 and IgG-b12-K409R×IgG1-005-F405L) did not induce killing of Wien133 cells. Also the EGFR antibody IgG1-2F8-E345R/F405L, that did not bind the EGFR-negative Wien133 cells (data not shown), did not induce CDC, as expected. The introduction of the K409R mutation did not influence the capacity of the IgG1-005-E345R antibody to induce ˜60% killing on Wien133 cells (described in Example 10). Interestingly, the bispecific CD38/EGFR antibody IgG1-005-E345R/K409R×IgG1-2F8-E345R/F405L, which can only bind monovalently to the CD38-positive, EGFR-negative Wien133 cells, showed increased maximal CDC killing (from ˜60% to ˜100% killing).
These data show that monovalent targeting can further enhance the maximal killing capacity of antibodies containing the CDC enhancing E345R mutation. Furthermore, these data show that the E345R oligomerization enhancing mutation, as measured by enhancing CDC activity, can be applied to other antibody formats, such as DuoBody.
Example 9
The Oligomerization Enhancing E345R Mutation can be Applied to Other Antibody Formats Such as DuoBody™
The effect of the E345R mutation was tested in a bispecific antibody of the DuoBody format. CDC assays were performed with CD20/CD38 bispecific antibodies on CD20-positive, CD38-positive Wien133 and Raji cells.
Bispecific antibodies were generated as described in Example 8. For the CDC assay, 0.1×106 Wien133 or Raji cells were pre-incubated in round-bottom 96-well plates with a concentration series of antibodies (0.01 to 30.0 μg/mL) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
FIG. 10 shows that introduction of the E345R mutation enhanced CDC of the bispecific IgG1-005-F405L×IgG1-7D8-K409R antibody on Wien 133 (FIG. 10A) and Raji (FIG. 10B) cells. These data show that the E345R oligomerization enhancing mutation can be applied to other antibody formats to enhance CDC activity.
Example 10
E345R Rescues CDC by EGFR Antibody 2F8, which can be Further Enhanced by Monovalent Target Binding
As described in Examples 3 and 12, E345R enhanced or rescued CDC for antibodies recognizing different hematological tumor targets (CD20 and CD38). To extend the analysis to a solid tumor antigen, the effect of E345R on the CDC capacity of the EGFR antibody 2F8 was tested on A431 epidermoid carcinoma cells. Furthermore, the effect of monovalent EGFR targeting on E345R-mediated CDC induction was tested using a bispecific EGFRxCD20 antibody (IgG1-2F8-E345R/F405L×IgG1-7D8-E345R/K409R) on EGFR-positive, CD20-negative A431 cells.
Bispecific antibodies were generated as described in Example 8. For the CDC assay, 5×106 A431 cells/mL were labeled with 100 μCi 51Cr for 1 h at 37° C. Cells were washed three times with PBS and resuspended in medium at a concentration of 1×105 cells/mL. 25,000 labeled cells were incubated in round-bottom 96-well plates with a concentration series of unpurified antibodies (0-30 μg/mL in 3-fold dilutions) in a total volume of 100 μL for 15 min at RT. Next, 50 μL normal human serum dilution was added as a source of complement (25% final concentration) and incubated in a 37° C. incubator for 1 h. Cells were spun down (3 min at 300×g) and 25 μL supernatant was added to 100 μL microscint in a white 96 well optiplate (PerkinElmer) for incubation on a shaker (750 rpm) for 15 min. 51Cr release was determined as counts per minute (cpm) on a scintillation counter. Maximum lysis (100%) was determined by the 51Cr level measured in the supernatant of Triton X-100-treated cells. Spontaneous lysis was determined by the 51Cr level measured in the supernatant of cells incubated without antibody. Specific cell lysis was calculated according to the formula: Specific lysis=100×(cpm sample−cpm spont)/(cpm max−cpm spont).
FIG. 11 shows that IgG1-2F8-E345R/F405L is able to lyse A431 cells by CDC, whereas wild type 2F8 is not capable of killing A431 cells. These data show that CDC activity can be rescued in the EGFR antibody 2F8 by introduction of the E345R mutation. This potentially extends the applicability of the CDC enhancing E345R mutation to antibodies targeting solid tumor antigens.
Bispecific EGFRxCD20 antibody IgG-2F8-E345R/F405L×IgG1-7D8-E345R/K409R, showed further enhancement of CDC on the EGFR-positive, CD20-negative A431 cells.
These data further support the hypothesis that monovalency facilitates the formation of Fc-Fc interactions and subsequent CDC induction as postulated for a CD38 binding antibody described in Example 8.
Example 11
E345R Enhances or Rescues CDC by CD38 Antibody 003 and CD20 Antibodies 11B8 and Rituximab
As described in Examples 3 and 12, E345R enhances or induces CDC activity of several antibodies with different target specificities (CD20, CD38 and EGFR), as was tested on multiple cell lines expressing variable levels of said antigens. Therefore, introduction of the E345R mutation was considered to be a general mechanism to enhance or rescues CDC for existing antibodies. To further support this, the effect of the E345R mutation on CDC was tested for more antibodies with variable intrinsic CDC efficacy on Daudi and WIEN133 cells: CD38 antibody 003, described in WO 2006/099875 and CD20 antibodies rituximab (type I) and 11B8 (type II), described in WO 2005/103081. CD20 antibodies can be divided in two subgroups (Beers et al. Seminars in Hematology 47, (2) 2010, 107-114). Type I CD20 antibodies display a remarkable ability to activate complement and elicit CDC by redistributing the CD20 molecules in the plasma membrane into lipid rafts, which cluster the antibody Fc regions and enabling improved C1q binding. Type II CD20 antibodies do not appreciably change CD20 distribution and without concomitant clustering, they are relatively ineffective in CDC.
0.1×106 Daudi or Raji cells were pre-incubated in round-bottom 96-well plates with a concentration series of unpurified antibodies (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0 μg/mL) in a total volume of 70 μL for 15 min on a shaker at RT. Next, 30 μL normal human serum was added as a source of C1q (30% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
FIG. 12 shows that the E345R mutation enhanced CDC for all tested antibodies on both (A) Daudi and (B) Wien133 cells. Interestingly, at the used concentrations all antibodies that did not induce CDC in the wild type format, induced CDC efficiently after introduction of the E345R mutation: CD38 mAb 003 and CD20 type II mAb 11B8 on Daudi cells, and CD38 mAbs 005 and 003 and CD20 type II mAb 11B8 on Wien133 cells. These data suggest that enhancement of antibody oligomerization, more specifically by introduction of an E345R mutation, is a general mechanism to enhance or rescue CDC by existing antibodies.
Example 12
E345R Enhances Internalization of Tissue Factor Antibodies
To test if enhanced oligomerization can induce increased antibody internalization, colocalization studies of wild type and E345R mutated Tissue Factor (TF) antibodies with the lysosomal marker LAMP1 were performed by confocal microscopy.
SK-OV-3 cells were grown on glass coverslips (thickness 1.5 micron, Thermo Fisher Scientific, Braunschweig, Germany) in standard tissue culture medium at 37° C. for 1 day. Cells were pre-incubated for 1 hour with 50 μg/mL leupeptin (Sigma) to block lysosomal activity, after which 10 μg/mL Tissue Factor (TF) antibody (WO 2010/066803) was added. The cells were incubated for an additional 1, 3 or 16 hours at 37° C. Hereafter, cells were washed with PBS and incubated for 30 minutes at room temperature (RT) with 4% formaldehyde (Klinipath). Slides were washed with blocking buffer (PBS supplemented with 0.1% saponin [Roche] and 2% BSA [Roche]) and incubated for 20 minutes with blocking buffer containing 20 mM NH4Cl to quench formaldehyde. Slides were washed again with blocking buffer and incubated for 45 minutes at RT with a cocktail of mouse-anti-human CD107a-APC (BD Pharmingen) to identify lysosomal LAMP1 and goat-anti-human IgG-FITC (Jackson) to identify TF antibodies. Slides were washed again with blocking buffer and mounted overnight on microscope slides using 20 μL mounting medium (6 gram Glycerol [Sigma] and 2.4 gram Mowiol 4-88 [Omnilabo] was dissolved in 6 mL distilled water to which 12 mL 0.2M Tris [Sigma] pH8.5 was added followed by incubation for 10 min at 50-60° C.; mounting medium was aliquoted and stored at −20° C.). Slides were imaged with a Leica SPE-II confocal microscope (Leica Microsystems) equipped with a 63×1.32-0.6 oil immersion objective lens and LAS-AF software.
12-bit grayscale TIFF images were analyzed for colocalization using MetaMorph® software (version Meta Series 6.1, Molecular Devices Inc, Sunnyvale Calif., USA). Images were imported as stacks and background was subtracted. Identical thresholds settings were used (manually set) for all FITC images and all APC images. Colocalization was depicted as the pixel intensity of FITC in the region of interest (ROI), were the ROI is composed of all APC positive regions. To compare different slides stained with different TF antibodies, the images were normalized using the pixel intensity of APC. Mouse-anti-human CD107a-APC was used to stain the lysosomal marker LAMP1 (CD107a). The pixel intensity of LAMP1 should not differ between various TF antibodies imaged.
Normalized values for colocalization of FITC and APC are expressed as arbitrary units according to the formula [(TPI FITC×percentage colocalization)/100]×[1/TPI APC]
Percentage colocalization=TPI FITC that colocalizes with an APC pixel/TPI APC
TPI, total pixel Intensity
FIG. 13 depicts the amount of FITC pixel intensity of wild type and E345R mutated TF antibodies that overlap with APC-labeled lysosomal marker. For each antibody or condition tested, three different images were analyzed from one slide containing ˜1, 3 or >5 cells. Variation was observed between the different images within each slide. Still, it was evident that the E345R mutation for antibodies 011 and 098 resulted in increased lysosomal colocalization after 1 hour incubation, when compared with wild type 011 and 098. These results indicate that mutation E345R induces more rapid internalization and lysosomal colocalization and could therefore potentiate antibody drug conjugates.
Example 13
Enhanced CDC by E345R Mutation in Rituximab in Different B Cell Lines with Similar CD20 Expression but Different Levels of Membrane-Bound Complement Regulatory Proteins
Examples 11 and 14 show that the CDC efficacy of wild type rituximab on Daudi and Wien133 cells was enhanced by introducing the E345R mutation. This enhanced CDC efficacy results from the E345R-mediated stabilization of Fc-Fc interactions. The concomitantly formed hexameric antibody ring structure on the target cell membrane can then promote efficient generation of the membrane attack complex by facilitating the capture and concentration of activated complement components close to the cell membrane. As a result of this efficient complement activation, the inhibiting effects of membrane-bound complement regulatory proteins (mCRP) could be partly overcome. Overexpression of mCRPs, such as CD55, CD46 and CD59, is considered as a barrier for successful immunotherapy with monoclonal anti-tumor antibodies (Jurianz et al., Mol Immunol 1999 36:929-39; Fishelson et al. Mol Immunol 2003 40:109-23, Gorter et al., Immunol Today 1999 20:576-82, Zell et al., Clin Exp Immunol. 2007 December 150(3):576-84). Therefore, the efficacy of rituximab-E345R was compared to that of wild type rituximab on a series of B cell lines with different levels of the mCRPs CD46, CD55 and CD59, but comparable levels of the CD20 target expression.
The B cell lines Daudi, WIL2-S, WSU-NHL, MEC-2 and ARH-77 express comparable amounts of CD20 molecules (˜250.000 specific antibody-binding capacity—sABC) as determined by QIFIKIT analysis (data not shown). To compare the expression levels of complement regulatory proteins between these cell lines, QIFIKIT analysis was performed to determine the levels of CD46 (mouse anti-human CD46, CBL488, clone J4.48 Chemicon), CD55 (mouse anti-human CD55, CBL511, Clone BRIC216, Chemicon), and CD59 (mouse anti-human CD59, MCA1054x, clone MEM-43, Serotec).
For the CDC assay, 0.1×106 of cells were pre-incubated in round-bottom 96-well plates with a saturating antibody concentration series (0.002-40.0 μg/mL in 4-fold dilutions) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS. The maximal CDC-mediated killing was calculated from two independent experiments using the top of best-fit values of a non-linear fit in GraphPad PRISM 5.
FIG. 14A-D shows that introduction of E345R in wild type rituximab resulted in enhanced CDC efficacy as observed by an increased maximal lysis and decreased EC50 for all tested B cell lines.
FIG. 14E shows that the maximal CDC-mediated killing induced by the rituximab-E345R mutant was always higher than by wild type rituximab, independent of the expression levels of the membrane-bound complement regulatory proteins. These data indicate that introduction of E345R enhances the therapeutic potential of monoclonal antibodies as the tumor cells are less effective in evading antibody-mediated complement attack by the E345R containing antibodies.
Example 14
Comparison of CDC Kinetics for Wild Type and E345R Antibodies
Introduction of the Fc:Fc interaction stabilizing E345R mutation has been shown to enhance or rescue CDC as observed by decreased EC50 values and increased maximal lysis for different antibodies on different cell lines described in Example 3 (CD38 antibody 005 on Daudi, Raji and Wien133) and Example 11 (CD38 antibody 003 and CD20 antibodies rituximab and 11B8 on Daudi and Wien133). Next, the kinetics of the CDC reactions were analyzed to further unravel the difference in CDC efficacy between wild type and E345R antibodies.
0.1×106 Raji cells were pre-incubated in round-bottom 96-well plates with antibody at a saturating concentration (10.0 μg/mL) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for different periods of time, varying between 0 and 60 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
FIG. 15A shows that wild type CD20 antibody IgG1-7D8 showed a maximal CDC-mediated killing of 80% of the Raji cells, which was already reached after 5 min under the tested conditions. However, for IgG-7D8-E345R, 80% killing of Raji cells was observed even faster, after 3 min. Maximal lysis by IgG-7D8-E345R (95%) was also reached after 5 minutes.
FIG. 15B shows that also for wild type CD20 antibody rituximab, which is less potent than 7D8 to induce CDC on the used Raji cells, introduction of the E345R mutation resulted in faster killing of the target cells. Wild type rituximab showed a maximal CDC-mediated killing of 32%, which was reached after 20 minutes. Rituximab-E345R reached 32% killing already after approximately 3 minutes and remarkably, maximal lysis by rituximab-E345R (85%) was also reached after 20 minutes.
FIG. 15C+D shows that the used Raji cells, which are resistant for CDC-mediated killing by wild type CD38 antibodies IgG1-003 and IgG1-005, could be killed fast by introducing the E345R mutation. IgG1-003-E345R and IgG1-005-E345R showed maximal CDC (50% and 60%, respectively) already after 5 min.
In summary, E345R antibodies are more potent than their wild type counterparts, which results from a combination of higher efficacy (lower EC50), increased maximal lysis and a faster kinetics of the CDC reaction.
Example 15
Comparison of CDC Kinetics for Bispecific Antibodies with or without the E345R Mutation
In example 9 it is described that the E345R mutation can be applied to the CD38×CD20 bispecific antibody IgG1-005-F405L×IgG1-7D8-K409R that was generated by the DuoBody platform, resulting in an enhanced killing capacity as observed by a decreased EC50 in CDC assays on Raji and Wien133 cells. Next, the kinetics of the CDC reaction was analyzed to further unravel the difference in CDC efficacy between the CD38×CD20 bispecific antibodies with and without E345R.
0.1×106 Raji cells were pre-incubated in round-bottom 96-well plates with antibody at a saturating concentration (10.0 μg/mL) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for different periods of time, varying between 0 and 60 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
FIG. 16 shows that the bispecific antibody IgG1-005-F405L×IgG1-7D8-K409R induced a maximal CDC-mediated killing of 83%, which was reached after 10 minutes. Introduction of E345R resulted in an increased maximal killing by IgG1-005-E345R-F405L×IgG1-7D8-E345R-K409R (98%), which was already reached after 2 minutes. These data indicate that introducing the Fc-Fc stabilizing E345R mutation in the bispecific antibody results in an accelerated CDC-mediated killing of the target cells.
Example 16
Comparison of CDC Kinetics for Monovalent Binding Antibodies with and without E345R
Example 8 shows that monovalent target binding further enhanced the CDC efficacy of E345R antibodies as observed by increased maximal lysis with a CD38×EGFR bispecific antibody on the CD38-positive, EGFR-negative Wien133 cells. Next, the kinetics of the CDC reaction was analyzed to further unravel the difference in CDC-mediated killing capacity between monovalently binding antibodies with and without E345R.
Bispecific CD38×EGFR and CD20×EGFR antibodies, with or without the E345R mutation, were generated in vitro according to the DuoBody platform as described in Example 8. CDC efficacy of the CD38×EGFR bispecific antibodies was tested on the CD38-positive, EGFR-negative Raji cells, to which the bispecific antibodies can only bind monovalently via CD38. 0.1×106 Raji cells were pre-incubated in round-bottom 96-well plates with antibody at a saturating concentration (10.0 μg/mL) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for different periods of time, varying between 0 and 60 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
FIG. 17 shows that bispecific antibody CD38×EGFR (IgG1-005-K409R×IgG1-2F8-F405L) induced a maximal CDC-mediated killing of 55%, which was reached after approximately 10 minutes. Introduction of E345R resulted in an increased maximal killing (96%), which was already reached within 5 minutes.
FIG. 17 shows that bispecific antibody CD20×EGFR (IgG1-7D8-K409R×IgG1-2F8-F405L) induced a maximal CDC-mediated killing of 85%, which was reached after approximately 5 minutes. However, with the CD20×EGFR antibody with introduced E345R, 85% lysis was observed faster, after 2 minutes. Maximal lysis by the E345R CD20×EGFR antibody (97%) was also reached after 5 minutes.
In summary, introduction of the E345R mutation in these monovalent binding antibodies resulted in more potent antibodies, which results from a combination of increased maximal lysis and a faster kinetics of the CDC reaction.
Example 17
CDC by a Combination of Therapeutic and E345R/Q386K Antibodies
As described in Example 6, mutant CD38 antibodies derived from IgG1-005 could induce efficient CDC on Wien133 cells when the E345 position of the wild type antibody was substituted to any amino acid other than Glutamate (E). This suggests that oligomerization, as a prerequisite of CDC, is hindered by the presence of the Glutamate side chain at position 345 of the antibody. Since E345 on one Fc is in close proximity to Q386 on the facing second Fc moiety in the hexameric antibody ring structure, the E345-mediated hindrance of oligomerization in a first antibody could possibly be removed by substitutions at the Q386 position of a second antibody. This would then enable E345 in the first antibody to interact better with the mutated 386 position in the second antibody in case both antibodies are combined. To test this hypothesis, CDC assays were performed on Wien133, in which wild type antibodies (IgG1-003, IgG1-005 or IgG1-11B8) were mixed with IgG1-005-E345R/Q386K or IgG1-005-E345R/Q386K/E430G as an example.
0.1×106 Wien133 cells were pre-incubated in round-bottom 96-well plates with a concentration series of unpurified IgG1-005-E345R/Q386K, IgG1-005-E345R/Q386K/E430G or control antibody (0.0001-20.0 μg/mL in 3.33-fold dilutions) in the presence or absence of 1.0 or 10.0 μg/mL wild type IgG1-003, IgG1-005 or IgG1-11B8 antibody in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
FIG. 18A/B/C shows that CD38 antibody IgG1-005-E345R/Q386K induced CDC-mediated lysis of Wien133 cells in a dose-dependent fashion (dashed line). Combining IgG1-005-E345R/Q386K with 1 or 10 μg/mL wild type CD38 antibody IgG1-003 (FIG. 18A) or wild type CD20 antibody IgG1-11B8 (FIG. 18B) resulted in an increased maximal cell lysis. Combining IgG1-005-E345R/Q386K with wild type IgG1-005 inhibited CDC in a dose-dependent fashion, possibly by competing for the binding site (FIG. 18C).
FIG. 18D/E/F shows similar results for CD38 antibody IgG1-005-E345R/Q386K/E430G.
These data indicate that wild type antibodies IgG1-003 and IgG1-11B8 participated in antibody oligomerization and CDC activation when combined with IgG1-005-E345R/Q386K or IgG1-005-E345R/Q386K/E430G. In such combinations, the hindrance of oligomerization by the E345-position that is present in the wild type antibody could be, at least partly, removed by the Q386K substitution in the mutant antibody. This application is in particular interesting to improve therapies with antibodies that are wild type in the E345 position, such as rituximab, ofatumumab, daratumumab or trastuzumab. Also, such oligomerization-inducing antibodies might promote formation of cell-bound complexes with patient-own antibodies directed against target cells like tumor cells or bacteria.
Example 6 describes multiple amino acids in addition to E345 that enhance CDC upon mutation, for example E430 and S440, of which specific mutations induced efficient CDC on Wien133 cells when incorporated in CD38 antibody IgG1-005. With the exception of 1253 and Y436 mutants, the identified oligomerization-enhancing mutations contact unmutated amino acids on the facing second Fc moiety in the hexameric ring structure. Therefore, the identified oligomerization-enhancing mutations, both alone or combined, can be expected to also promote oligomerization with unmutated antibodies, and further optimization of such mutants could be achieved by a selection strategy similar to that applied in example 6.
Example 18
E345R Induced CDC in IgG2, IgG3 and IgG4 Antibody Isotypes
To test if the introduction of oligomerization-promoting mutations can stimulate the CDC activity of non-IgG1 antibody isotypes, isotypic variants of the CD38 antibody IgG1-005 were generated with constant domains of human IgG2, IgG3 or IgG4 yielding IgG2-005, IgG3-005 and IgG4-005 by methods known in the art. Furthermore, the oligomerization enhancing E345R mutation was introduced in all these antibodies, yielding IgG2-005-E345R, IgG3-005-E345R and IgG4-005-E345R. In a similar way, also IgG2-003 and IgG2-003-E345R were generated from CD38 antibody IgG1-003. CDC efficacy of the different isotypes was compared in an in vitro CDC assay.
0.1×106 Wien133 cells were pre-incubated in round-bottom 96-well plates with 10 μg/mL unpurified antibodies in a total volume of 100 μL for 15 min on a shaker at RT. IgG1-005-E345R was added at 3.0 μg/mL. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
FIG. 19 shows that IgG2-005, IgG2-003, IgG3-005 and IgG4-005 were unable to lyse either (A) Daudi or (B) Wien133 cells efficiently under the tested conditions (the observed ˜20% lysis was considered as background). Introduction of the E345R mutation enabled potent CDC on Daudi cells by all IgG isotypes tested. These results were confirmed using CDC on Wien133 cells, albeit that IgG3-005-E345R displayed limited CDC activity relative to the other isotypic variants. These data indicate that besides IgG1, an oligomerization enhancing mutation such as E345R can also be applied to promote CDC activity of IgG2, IgG3 and IgG4 antibodies.
Example 19
CDC by IgG1-005 and IgG1-005-E345R in an Ex Vivo CDC Assay on Patient-Derived CD38-Positive B Cell Chronic Lymphocytic Leukemia (CLL) Cells
Cryopreserved primary cells from CLL patient samples were obtained from the hematopathology biobank from CDB-IDIBAPS-Hospital Clinic (Dr. Elias Campo, Hematopathology Unit, Department of Pathology, Hospital Clinic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain), or from clinical studies by the National Heart, Lung, and Blood Institute (NHLBI) (Dr. Adrian Wiestner, NHLBI, Hematology Branch of the National Institutes of Health (NIH), Bethesda). Informed consent was obtained from all patients in accordance with the Institutional Ethics Committee of the Hospital Clinic (Barcelona, Spain) or the Institutional Review Board of the NIH and the Declaration of Helsinki. All samples were genetically and immunophenotypically characterized.
The CLL samples were categorized into two groups according to their CD38 expression as determined by FACS: five samples were included in the CD38 high group (between 50% and 98% of the CD38 expression on Daudi cells) and four samples were included in the CD38 low group (between 0.5% and 3% of the CD38 expression on Daudi cells).
Fluorescently labeled CLL cells (labeling with 5 μM Calcein AM) were incubated with a concentration series of antibody (0.01-10 μg/mL in 10-fold dilutions). Next, normal human serum was added to the antibody-opsonized cells (100,000 cells/well) as a source of complement (10% final concentration) and incubated for 45 min at 37° C. Supernatants were recovered and fluorescence was read in a Synergy™ HT fluorometer as a measure for cell lysis. Cell killing was calculated as follows:
Specific lysis=100×(sample−spontaneous lysis)/(max lysis−spontaneous lysis)
where max lysis is determined by a sample of cells treated with 1% Triton, and spontaneous lysis is determined from a sample where cells were incubated in the presence of 10% NHS without antibody.
FIG. 20 shows that IgG1-005-E345R strongly enhanced CDC efficacy compared to wild type IgG1-005 on both CLL primary cells with high CD38 expression and CLL primary cells with low CD38 expression.
Example 20
IgG1-005-E345R/E430G/S440Y Forms Non-Covalent, Hexameric Complexes in Solution
The IgG1-005-E345R/E430G/S440Y triple mutant was prepared using the Quikchange site-directed mutagenesis kit (Stratagene, US). Briefly, forward and reverse primers encoding the desired mutation E345R were used to replicate full length plasmid DNA template encoding the IgG1-005 heavy chain with IgG1m(f) allotype. The resulting DNA mixture was digested using DpnI to remove source plasmid DNA and used to transform E. coli. Mutant plasmid DNA isolated from resulting colonies was checked by DNA sequencing (Agowa, Germany). The E430G mutation was introduced into the IgG1-005-E345R backbone using the same strategy. The S440Y mutation was introduced into the IgG1-005-E345R/E430G backbone using the same strategy. Plasmid DNA mixtures encoding both heavy and light chain of antibodies were transiently transfected to Freestyle HEK293F cells (Invitrogen, US) using 293fectin (Invitrogen, US) essentially as described by the manufacturer. The resulting antibody is a homodimer containing the E345R/E430G/S440Y triple mutation in both heavy chains.
IgG1-005 and IgG1-005-E345R/E430G/S440Y antibodies were purified by protein A affinity chromatography. The cell culture supernatants were filtered over a 0.20 μM dead-end filter, followed by loading on a 5 mL Protein A column (rProtein A FF, GE Healthcare, Uppsala, Sweden) and elution of the IgG with 0.1 M citric acid-NaOH, pH 3. The eluate was immediately neutralized with 2 M Tris-HCl, pH 9 and dialyzed overnight to 12.6 mM sodium phosphate, 140 mM NaCl, pH 7.4 (B. Braun, Oss, The Netherlands). After dialysis, samples were sterile filtered over a 0.20 μM dead-end filter. Purified proteins were analyzed by SDS-PAGE, native PAGE, HP-SEC, multiple angle light scattering (MALS), and dynamic light scattering (DLS).
SDS-PAGE was performed under reducing and non-reducing conditions on 4-12% NuPAGE Bis-Tris gels (Invitrogen, Breda, The Netherlands) using a modified Laemmli method (Laemmli 1970 Nature 227(5259): 680-5), where the samples were run at neutral pH. The SDS-PAGE gels were stained with Coomassie and digitally imaged using the GeneGenius (Synoptics, Cambridge, UK). FIG. 21 shows that IgG1-005-E345R/E430G/S440Y displayed behavior typical of IgG1 antibodies with disulfide bridged heavy and light chains. A single molecular species with apparent MW of approximately 150 kDa was visible under non-reducing conditions, while under reducing conditions a heavy chain with apparent MW of 50 kDa and light chain of 26 kDa were visible. We concluded that, under denaturing conditions, a monomeric molecule is formed displaying behavior highly similar to wild type IgG1 antibodies.
Native PAGE was performed under non-reducing conditions using a Sebia Hydragel 15/30 protein gel (Westburg, Leusden, The Netherlands), acid violet-staining and run on a Hydrasys instrument (Sebia, Vilvoorde, Belgium). FIG. 21 shows that IgG1-005-E345R/E430G/S440Y ran at a height similar to that of the unrelated IgG1-b12 control antibody, albeit slightly more diffuse. The observed diffuse staining could be caused by formation of unstable complexes, but under these PAGE conditions, the IgG1-005-E345R/E430G/S440Y behaved predominantly like a monomeric IgG1 molecule.
HP-SEC fractionation was performed using a Waters Alliance 2975 separation unit (Waters, Etten-Leur, The Netherlands) connected to a TSK HP-SEC column (G3000SWxl; Toso Biosciences, via Omnilabo, Breda, The Netherlands), a Waters 2487 dual λ absorbance detector (Waters), and a Mini Dawn Treos MALS detection unit (Wyatt). 50 μL samples containing 1.25 μg/mL protein were separated at 1 mL/min in 0.1 M Na2SO4/0.1 M sodium phosphate buffered at pH 6.8. Results were processed using Empower software version 2002 and expressed per peak as percentage of total peak area. FIG. 22 shows that >99% of wild type IgG1-005 consisted of intact monomeric IgG, with practically no aggregates formed. FIG. 23 shows that the triple mutant IgG1-005-E345R/E430G/S440Y shows a large fraction oligomer which was estimated at 79%, while 21% of the population eluted in a peak observed at the expected elution time for a monomeric species.
An overlay of the HP-SEC profiles of wild type IgG1-005 and IgG1-005-E345R/E430G/S440Y is shown in FIG. 24 and further illustrates the difference in behavior between the two antibodies. IgG1-005-E345R/E430G/S440Y clearly formed high MW complexes, though these complexes seemed to be sensitive to HP-SEC separation, as indicated by the significant amount of protein eluting between the two peaks. Possibly the shear caused by HP-SEC separation may destabilize the non-covalent complexes formed by assembly of IgG1-005-E345R/E430G/S440Y monomers.
To assess the size of the observed oligomeric complex in the IgG1-005-E345R/E430G/S440Y sample, the average molecular weight of the HP-SEC eluate was determined by multiple angle light scattering (MALS). While the minor monomeric peak eluted with an apparent average MW of 143 kDa (145.4 kDa expected), the multimeric peak eluted with an apparent average MW of 772 kDa, or approximately 5.4 monomeric subunits. The MW of the complex is probably underestimated due to the instability of the complex under these conditions. For example, a co-eluting mixture of 88% hexameric species and 12% monomeric species would result in an observed average complex size of 5.4 monomer units.
To assess the apparent molecular weight in solution, in the absence of the shear possibly induced by interactions with the HP-SEC matrix, dynamic light scattering (DLS) analysis was performed. 45 μL of 0.2 μM filtered IgG1-005 (3.80 mg/mL) or IgG1-005-E345R/E430G/S440Y (2.86 mg/mL) in PBS pH 7.4 was analyzed using a DynaPro-801 instrument (Protein Solutions Inc/Wyatt, Dernbach, Germany) in a 100 μL quartz cuvette, recording twenty consecutive measurements per experiment, in three independent experiments. Calibrated using the MW of BSA as a reference, the apparent MW of IgG1-005 was 141.7 kDa (145.4 expected), while IgG1-005-E345R/E430G/S440Y displayed a MW of approximately 875.6 kDa, or 6.17 monomeric subunits. No indication of oligomerization was observed for the IgG1-005 antibody, while IgG1-005-E345R/E430G/S440Y suggested highly efficient complex formation.
In summary, the biophysical data indicate that mutant IgG1-005-E345R/E430G/S440Y forms disulfide-bridged IgG1-like molecules that are monomeric, i.e. single dimeric protein, under denaturing conditions as observed by SDS-PAGE and form hexameric complexes in solution as observed by DLS. The shear imposed by native PAGE was sufficient to fully dissociate the complexes, while HP-SEC partially destabilized predominantly hexameric complexes, as indicated by the presence of a minor fraction of monomers.
Example 21
Functional Assays with IgG1-005, IgG1-005-E345R/E430G/S440Y and IgG1-005-E345R
C1q Binding ELISA
C1q binding by wild-type IgG1-005, triple mutant IgG1-005-E345R-E430G-S440Y and IgG1-005-E345R was tested in an ELISA in which the purified antibodies were immobilized on the plastic surface, bringing about random antibody multimerization. Pooled human serum was used as a source of C1q.
96-well Microlon ELISA plates (Greiner, Germany) were coated overnight at 4° C. with a dilution series of the antibodies in PBS (range 0.007-25.0 μg/mL in 2.5-fold dilutions). Plates were washed and blocked with 200 μL/well 0.5×PBS supplemented with 0.025% Tween 20 and 0.1% gelatine. With washings in between incubations, plates were sequentially incubated with 3% pooled human serum (Sanquin, product # M0008) for 1 h at 37° C., with 100 μL/well rabbit anti-human C1q (DAKO, product # A0136, 1/4.000) for 1 h at RT, and with 100 μL/well swine anti-rabbit IgG-HRP (DAKO, P0399, 1:10.000) as detecting antibody for 1 h at RT. Development was performed for circa 30 min with 1 mg/mL 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany). The reaction was stopped by the addition of 100 μL 2% oxalic acid. Absorbance was measured at 405 nm in a microplate reader (Biotek, Winooski, Vt.). Log transformed data were analyzed by fitting sigmoidal dose-response curves with variable slope using GraphPad Prism software. From the sigmoidal dose response curves the EC50 values were calculated.
FIG. 25 and Table 8 show that IgG1-005-E345R/E430G/S440Y showed more efficient C1q binding than WT IgG1-005 and IgG1-005-E345R as measured by ELISA (lower EC50 value). Coating efficacy was tested for the three antibodies and was found to be similar (not shown).
TABLE 8
EC50 for C1q binding in ELISA
EC50
Antibody
(μg/mL)
IgG1-005-WT
1.551
IgG1-005-
0.52
E345R/E430G/S440Y
IgG1-005-E345R
0.836
CDC Assay on CD38-Positive Ramos Cells
0.1×106 Ramos cells were pre-incubated in round-bottom 96-well plates with a concentration series of purified antibodies (10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.005, 0.0025, 0.0013, 0.0006 and 0.0003 μg/mL) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
A three-phase model was used to fit the IgG1-005-E345R/E430G/S440Y data and the medium EC50 value was calculated (Table 9). IgG1-005-WT and IgG1-005-E345R could be fitted by fitting sigmoidal dose-response curves with variable slope. From the sigmoidal dose response curve the EC50 value was calculated (Table 9). GraphPad Prism software was used to fit the data (FIG. 26). IgG1-005-E345R/E430G/S440Y showed enhanced CDC activity compared to wild type IgG1-005 and IgG1-005-E345R antibodies on Ramos cells. The three-phase model of IgG1-005-E345R/E430G/S440Y can be explained by the fact that at low concentrations (between 0.0003 and 0.03 μg/mL) the antibodies within a stable hexamer do not all have to bind a target to induce efficient CDC. Effectively, cell surface C1q binding sites are created by IgG1-005-E345R/E430G/S440Y binding already at low antibody concentrations, because clustering of antigens is not needed for antibody hexamerization.
TABLE 9
EC50 for CDC
EC50
Antibody
(μg/mL)
IgG1-005-WT
0.116
IgG1-005-
0.005
E345R/E430G/S440Y
(medium EC50)
IgG1-005-E345R
0.026
ADCC Reporter Assay Using CD38-Positive Raji Cells
ADCC activity of anti-CD38 antibodies opsonized on Raji target cell was measured using an ADCC bioluminescent reporter assay (Promega Madison, Wis., USA) in which biological pathway activation in the effector cells is quantified.
The reporter assay uses as effector cells Jurkat cells stably transfected with the gene for FcγRIIIa receptor, V158 (high affinity) variant, and the firefly luciferase reported gene cloned after an NFAT (nuclear factor of activated T-cells) response element driving luciferase expression. Antibody binding to the FcγRIIIa receptor on the effector cells induces NFAT-mediated gene transcription and thus luciferase expression which is quantified by luminescence readout. Raji cells were incubated with a concentration series of purified antibodies (250, 71.4, 20.4, 5.8, 1.7 and 0.5 ng/mL). See for further description of the materials and methods the technical manual provided by Promega. IgG1-005-E345R/E430G/S440Y induced NFAT pathway activation after FcγRIIIa receptor engagement. FIG. 27 shows that Raji cells opsonized with IgG1-005-E345R/E430G/S440Y induced FcgRIIIa-mediated activation of effector cells as measured in the reported assay. The EC50 value for IgG1-005-E345R/E430G/S440Y was higher than for wild type IgG1-005 and IgG1-005-E345R (Table 10). However, the maximal signal for IgG1-005-E345R/E430G/S440Y was higher than for wild type IgG1-005 and IgG1-005-E345R (Table 10).
TABLE 10
EC50 and maximal signal for ADCC reporter assay
EC50
Maximal signal
Antibody
(ng/mL)
(RLU)
IgG1-005-WT
9.5
334878
IgG1-005-E345R/E430G/S440Y
36
393802
IgG1-005-E345R
4.7
172293
Example 22
Pharmacokinetic (PK) Analysis of IgG1-005-E345R/E430G/S440Y Compared to Wild Type IgG1-005
The mice in this study were housed in a barrier unit of the Central Laboratory Animal Facility (Utrecht, The Netherlands) and kept in filter-top cages with water and food provided ad libitum. All experiments were approved by the Utrecht University animal ethics committee. SCID mice (C.B-17/IcrCrl-scid-BR, Charles-River) were injected intravenously with 500 μg antibody (wild type IgG1-005 or IgG1-005-E345/E430G/S440Y) using 3 mice per group.
50 μL blood samples were collected from the saphenous vein at 10 minutes, 4 hours, 1 day, 2 days, 7 days, 14 days and 21 days after antibody administration. Blood was collected into heparin containing vials and centrifuged for 5 minutes at 10,000 g. Plasma was stored at −20° C. until determination of antibody concentrations.
Specific human IgG concentrations were determined using a total hIgG and CD38 specific sandwich ELISA. For the total hIgG ELISA, mouse mAb anti-human IgG-kappa clone MH16 (# M1268, CLB Sanquin, The Netherlands), coated to 96-well Microlon ELISA plates (Greiner, Germany) at a concentration of 2 μg/mL was used as capturing antibody. After blocking plates with PBS supplemented with 0.2% bovine serum albumin, samples were added, serially diluted ELISA buffer (PBS supplemented with 0.05% Tween 20 and 0.2% bovine serum albumin), and incubated on a plate shaker for 1 h at room temperature (RT). Plates were subsequently incubated with goat anti-human IgG immunoglobulin (#109-035-098, Jackson, West Grace, Pa.) and developed with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany). Absorbance was measured in a microplate reader (Biotek, Winooski, Vt.) at 405 nm. For the specific CD38 ELISA, His-tagged CD38 extracellular domain was coated to 96-well Microlon ELISA plates (Greiner, Germany) at a concentration of 2 μg/mL. After blocking plates with ELISA buffer, samples serially diluted with ELISA buffer were added, and incubated on a plate shaker for 1 h at room temperature (RT). Plates were subsequently incubated with 30 ng/ml mouse anti human IgG1-HRP, (Sanquin M1328, clone MH161-1) and developed with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany). Absorbance was measured in a microplate reader (Biotek, Winooski, Vt.) at 405 nm.
FIG. 28 shows that the plasma human IgG concentrations were considerably lower for mutant IgG1-005-E345R/E430G/S440Y than for IgG1-005 wild type at all tested timepoints. FIG. 29 shows that the clearance rate of IgG1-005-E345R/E430G/S440Y was approximately 50× higher than that of WT IgG1-005.
Example 23
The Oligomeric State of IgG1-005-E345R/E430G/S440Y can be Controlled by Buffer Composition
HP-SEC fractionation of IgG1-005 and IgG1-005-E345R/E430G/S440Y antibodies was performed using a Waters Alliance 2975 separation unit (Waters, Etten-Leur, The Netherlands) connected to a TSK HP-SEC column (G3000SWxl; Toso Biosciences, via Omnilabo, Breda, The Netherlands), a Waters 2487 dual λ absorbance detector (Waters), and a Mini Dawn Treos MALS detection unit (Wyatt). 50 μL samples containing 1.0 μg/mL protein were separated at 1 mL/min under different buffer conditions. Results were processed using Empower software version 2002 and expressed per peak as percentage of total peak area.
FIG. 30 shows the HP-SEC elution profiles recorded in 0.1 M Na2SO4/0.1 M sodium phosphate buffered at pH 6.8. At pH 6.8, >99% of wild type IgG1-005 antibodies eluted as monomeric species. In contrast, the HP-SEC profile of IgG1-005-E345R/E430G/S440Y in this buffer shown in FIG. 31 shows a fraction oligomer of 77%, while 23% of the population eluted as a monomeric species. As described in Example 19, the remaining minor fraction of monomer might be caused by column-induced dissociation, since no trace of IgG1-005-E345R/E430G/S440Y monomer was observed during batch-mode analysis using dynamic light scattering under these conditions.
FIG. 32 shows an overlay of the HP-SEC elution profiles of IgG1-005 recorded in 0.15 M NaCl/0.1 M citrate buffered at pH 6.8 (dashed line) and pH 5.0 (solid line). The HP-SEC profile of IgG1-005 in citrate buffer both at pH 6.8 and pH 5.0 was highly comparable to the behavior in phosphate buffer at pH 6.8, with >99% of the protein eluting as monomeric species.
FIG. 33 shows an overlay of the HP-SEC elution profiles of IgG1-005-E345R/E430G/S440Y recorded in 0.15 M NaCl/0.1 M citrate buffered at pH 6.8 (dashed line) and pH 5.0 (solid line). Consistent with the behavior in phosphate at pH 6.8, in citrate pH 6.8 the antibody displayed 84% oligomerization. In stark contrast, lowering the pH to 5.0 dramatically reversed the oligomerization of IgG1-005-E345R/E430G/S440Y. The fraction multimer dropped to less than 1%, with >99% of the protein eluting as a monomeric species. The disassembly of oligomers was specific to low pH conditions and not caused by using citrate as a buffer component, as shown by the efficient oligomerization in citrate buffered at pH 6.8.
In summary, lowering the pH from 6.8 to 5.0 suffices to completely disassemble solution-phase antibody hexamers, an effect that might be explained by the charge modification of histidine amino acids present at the Fc:Fc interface crucial for hexameric antibody assembly. In addition, this behavior was specific to antibody variants containing mutations that induced Fc mediated self assembly, like IgG1-005-E345R/E430G/S440Y, while wild type antibodies remained monomeric at both pH levels.
Example 24
Introduction of the Fc-Fc Stable Hexamer Mutations E345R/E430G/S440Y Will Result in Increased Bactericidal Activity of IgG Antibodies Against Bacteria that Express Fc-Binding Surface Proteins
The complement cascade system is an important host defense mechanism against pathogens and can be divided in three different activation routes to recognize pathogens: i) the antibody-mediated classical pathway, which is activated upon C1q binding to the pathogen-bound antibody, ii) the lectin and iii) the alternative pathway, in which the complement system directly recognizes and is triggered by the pathogen in the absence of antibody. The three pathways converge at the step of C3 cleavage and C3b deposition. Microorganisms have developed multiple mechanisms of complement evasion, one of which is mediated by Protein A [Joiner Ann. Rev. Microbiol. (1988) 42:201-30; Foster Nat Rev Microbiol (2005) December; 3(12):948-58]. Protein A was first identified in the cell wall of Staphylococcus aureus and is well known for its binding to the Fc region of IgG (Deisenhofer et al., Biochem (1981) 20, 2361-70; Uhlen et al., J. Biol. Chem (1984) 259, 1695-1702). So far, the antiphagocytotic effect of Protein A and its role in the pathogenesis of S. aureus was explained by the interaction between Protein A and IgG, which results in an incorrect antibody orientation to be recognized by the neutrophil Fc receptor (Foster Nat Rev Microbiol (2005) December; 3(12):948-58). In example 4, it was shown that CDC mediated by B cell-specific IgG1 antibodies was inhibited by the competing Fc-binding peptide DCAWHLGELVWCT. The peptide targets the consensus binding site on IgG Fc that coincides with the binding site for Protein A, Protein G and rheumatoid factor (Delano et al., Science 2000 Feb. 18; 287(5456):1279-83). Based on these data, it was speculated that the Protein A-mediated bacterial complement evasion mechanism could work by competing for Fc binding, resulting in destabilization of the Fc-Fc interaction of a microbe-specific antibody, and consequently inhibition of antibody-mediated complement activation. Moreover, in example 4, it was also shown that B cell-specific IgG1 antibodies containing the CDC-enhancing E345R mutation were less sensitive to inhibition of CDC by the competing Fc-binding peptide DCAWHLGELVWCT than the parent wild type antibodies. By extrapolating these results to Fc binding proteins expressed on microbes, increased stabilization of the IgG1 Fc-Fc interactions by the E345R mutation would make microbe-specific antibodies less prone to complement inhibition by an escape strategy of the pathogen via Fc binding competition by microbial surface proteins, such as Protein A. Consequently, introduction of the E345R mutation in IgG antibodies directed against a bacterium would result in increased C3b deposition on bacteria and increased bactericidal activity compared to the parent wild type antibodies. It is expected, that a stabilized hexamer (IgG-E345R/E430G/S440Y) that is already oligomerized before binding to a microbe target would be even more resilient to e.g. Protein A binding than IgG antibodies containing the single E345R mutation. Consequently, introduction of the E345R/E430G/S440Y mutations in IgG antibodies directed against microbes would result in increased C3b deposition on microbes and increased microbial activity compared to the parent wild type antibodies.
To test if IgG1-005-E345R/E430/S440Y oligomerization can inhibit binding to protein A, purified protein preparations of IgG1-005 and IgG1-005-E345R/E430/S440Y were analyzed by two orthogonal methods:
1) IgG concentration determination by measuring absorbance at 280 nm wavelength using a Nanodrop ND-1000 spectrophotometer (Isogen Life Science, Maarssen, The Netherlands).
2. IgG concentration determination using an Octet QK instrument (Fortebio, Menlo Park, USA), in Octet Sample Diluent and using ready-to-use protein A sensortips (Fortebio, Menlo Park, USA) in direct comparison with an IgG standard (Siemens) reference curve.
By determining the ratio between the concentration determined by A280 over the concentration determined using Octet-Protein A, it was observed that IgG1-005-E345R/E430/S440Y is indeed less prone to bind Protein A than IgG1-005 (table 11).
TABLE 11
Antibody concentrations by absorbance at 280 nm and Octet-Protein A
A280
Octet-Protein A
A280/Protein A ×
Antibody
(μg/ml)
(μg/ml)
100 (%)
IgG1-005-E345R-E430G-
2905
2052.5
0.71
S440Y
IgG1-005
3818
3619.6
0.95
As an in vitro measure for complement-mediated bacterial killing, both phagocytosis by neutrophils and the generation of C3a in the plasma, which coincides with C3b deposition on the bacteria, can be determined. Indeed, it has been described that C3b deposition on S. aureus results in enhanced phagocytosis and correlates with bacterial killing (Rooijakkers et. al., Nature Immunology 2005: 6, 920-927).
S. aureus will be labelled with FITC by incubating an exponentially growing bacterial culture with 100 μg/mL FITC for 1 h at 37° C. in 0.1 M carbonate buffer (pH 9.6). Human polymorph nuclear cells (PMN) will be isolated using a Ficoll gradient. FITC-labelled bacteria will be opsonized with a concentration series of specific antibodies with or without the mutation E345R/E430G/S440Y. Phagocytosis will be performed in vitro by incubating 1×108 opsonized FITC-labelled bacteria with human PMN in the presence of 25% IgG-depleted serum as complement source for 25 min at 37° C. in a total volume of 200 μL under vigorous shaking. The cells will be fixed and erythrocytes lyzed by incubation with BD FACS lysing solution for 15 min at room temperature. After washing, phagocytosis will be measured by FACS. The neutrophil population will be selected through forward and side scatter gating and phagocytosis will be expressed as the mean fluorescence in the neutrophil population. Alternatively, C3a generation will be measured in the samples by ELISA as a measure for complement activation and C3b deposition.
It is expected that the S. aureus-specific antibodies containing the E345R/E430G/S440Y mutation will induce more complement activation and phagocytosis by neutrophils than the parent wild type antibodies. An example of an antibody that could be used in such experiments is the chimeric monoclonal IgG1 pagibaximab (BSYX-A110; Biosynexus), targeting Lipoteichoic acid (LTA) that is embedded in the cell wall of staphylococci (Baker, Nat Biotechnol. 2006 December; 24(12):1491-3; Weisman et al., Int Immunopharmacol. 2009 May; 9(5):639-44).
Example 25
The Formation of Non-Covalent Hexameric IgG Complexes by Introducing the E345R/E430G/S440Y Triple Mutation Occurs Irrespective of the Fab Domain
The formation in solution of non-covalently bound, hexameric antibody complexes by introducing the E345R/E430G/S440Y (RGY) triple mutation was demonstrated for CD38 antibody 005 in Example 20. In this experiment formation of non-covalent hexameric IgG complexes of other human IgG1 antibodies that differ in their Fab domains: CD20 antibodies 7D8 and rituximab, EGFR antibody 2F8 and C. albicans mannan antibody M1g1. The generation and purification of the triple mutant antibodies and HP-SEC analyses were performed essentially as described in Example 20.
FIG. 34 shows that all triple mutant antibodies showed a large fraction oligomer: IgG1-7D8-RGY (67.2%) (FIG. 34A), IgG1-ritux-RGY (83.6%) (FIG. 34B), IgG1-2F8-RGY (74.5%) (FIG. 34C), and IgG1-M1-RGY (74.5%) (FIG. 34D). These data indicate that the concept of E345R/E430G/S440Y for inducing self-assembly of antibodies into hexamers in solution can be generally applied to IgG1 sequences, irrespective of Fab domain primary structure.
Example 26
CDC by CD20 Antibodies Containing the E345R/E430G/S440Y Triple Mutation in an Ex Vivo CDC Assay on Patient-Derived CD20-Positive CLL Cells
Frozen CLL PB CD19+/CD5+ B cells (Relapsed/Refractory) isolated from CLL peripheral blood mononuclear cells were purchased from Allcells, Emeryville, Calif. CDC was performed as described in Example 21, with the exception that 20,000 cells per 96-well plate were used. CD20 expression was determined as 39,000 Specific Antibody-Binding Capacity (sABC) by QIFIKIT, Dako, Glostrup, Denmark. FIG. 35 shows that CD20 antibodies containing the E345R/E430G/S440Y triple mutation showed functional CDC activity on CD20-positive primary CLL cells. Introduction of the E345R/E430G/S440Y mutation resulted in more efficient CDC-mediated killing (lower EC50) of primary CLL cells by 7D8 (FIG. 35A) and enabled potent CDC by rituximab, which did not show any killing activity in the WT format (FIG. 35B).
Example 27
Introduction of E345R/E430G/S440Y Triple Mutation for Induction of Hexamerization and Increased CDC can be Applied to Different Antibody Isotypes
Isotypic variants of the CD38 antibody IgG1-005 were generated with constant domains of human IgG2, IgG3 or IgG4 yielding IgG2-005, IgG3-005 and IgG4-005 by methods known in the art. Furthermore, the triple mutation E345R/E430G/S440Y was introduced in all these antibodies, yielding IgG2-005-RGY, IgG3-005-RGY and IgG4-005-RGY.
HP-SEC analysis of the different isotypes was performed as described in Example 20.
FIG. 36 shows that the tested isoforms containing the E345R/E430G/S440Y triple mutation formed hexameric complexes in solution: IgG1-005-RGY (79.2% multimeric) (FIG. 36A), IgG2-005-RGY (46.1% multimeric) (FIG. 36B), IgG3-005-RGY (37.8% multimeric) (FIG. 36C), and IgG4-005-RGY (84.4% multimeric) (FIG. 36D).
CDC efficacy of the different isotypes was compared by testing unpurified antibody concentration series (0.0003-10 μg/mL in 2-fold dilutions) in an in vitro CDC assay as described in Example 18. FIG. 37 shows that introduction of the RGY triple mutation enabled potent CDC on Daudi cells (FIG. 37A) by all IgG isotypes tested. These results were confirmed using CDC on Wien133 cells (FIG. 37B), albeit that IgG3-005-RGY displayed limited CDC activity relative to other isotypic variants. These data for the RGY triple mutants are similar as shown for E345R mutants in Example 18, FIG. 19.
Example 28
Combinations of Mutations for Inducing the Formation of Non-Covalent Oligomeric Complexes in Solution
Example 20 describes that antibody IgG1-005 containing the three mutations E345R, E430G, and S440Y (RGY) forms oligomeric complexes in solution. Antibodies containing variants of this triple mutation with an amino acid substitution at either one of these three positions were tested for their capacity to form oligomeric complexes in solution. As an example of the class of possible E345R substitutions, consisting of E345 to A/C/D/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, E345K was tested in combination with E430G/S440Y. As an example of the class of possible E430G substitutions, consisting of E430 to A/C/D/F/G/H/I/K/L/M/N/P/Q/R/S/T/V/W/Y, E430S was tested in combination with E345R/S440Y. The class of possible S440Y substitutions consists of proteins with an amino acid in at least one position selected from the group consisting of S440, Y436, D/E356, T359, E382, N434, Q438, I253 and S254, that is Y or W; not Y; not D or E; not T; not E; not N; not Q; not I; and not S, for each position, respectively. As an example of this class of S440Y substitutions, Y436I and S440W were tested in combination with E345R/E430G. Mutation combinations E345K/E430G/S440Y (denoted RGY), E345R/E430S/S440Y (denoted RSY), E345R/E430G/S440W (denoted RGW), or E345R/E430G/Y436I (denoted RGI) were introduced in the CD38 antibody IgG1-005 by methods known in the art, yielding IgG1-005-KGY, IgG1-005-RSY, IgG1-005-RGW, and IgG1-005-RGI, respectively.
HP-SEC analysis was performed as described in Example 20. FIG. 38 shows that similar to IgG1-005-RGY (Example 20, FIG. 23), IgG1-005-KGY (FIG. 38A), IgG1-005-RSY (FIG. 38B), and IgG1-005-RGW (FIG. 38C) formed oligomeric complexes in solution with varying efficiency. For mutants IgG1-005-KGY and IgG1-005-RGW, the observed A280 signal migrating in between the oligomer and monomer peaks suggested that the HP-SEC method may contribute to destabilization of oligomeric complexes, as described in Example 20.
CDC efficacy of the antibodies was compared by testing unpurified antibody concentration series (0.0003-10 μg/mL in 3-fold dilutions) in an in vitro CDC assay as described in Example 18. FIG. 39A shows that all tested triple mutation combinations endowed IgG-005 with the capacity to kill Wien133 cells in an in vitro CDC assay, where wild type IgG-005 does not show any killing. FIG. 39B shows that also Ramos cells were killed more efficiently by the tested triple mutant antibodies as compared to wild type IgG1-005.
These data show that oligomerization in solution and/or induction of CDC can be induced by IgG1-005-KGY, IgG1-005-RSY, IgG1-005-RGW and IgG1-005-RGI, suggesting that mutations selected from any possible naturally occurring amino acid E345R substitutions, any possible naturally occurring amino acid E430G substitutions, or the amino acids tryptophan or tyrosine may be possible amino acid substitutions for S440, can substitute for E345R, E430G, and S440Y, respectively. Furthermore, the HP-SEC data suggest that such substitutions can modulate the interaction strength between the Fc-containing polypeptide subunits of the oligomeric complex.
Example 29
Antibodies Containing E345R/E430G/S440Y Triple Mutations can be Assembled into Hetero-Oligomeric Rings
Example 5, FIG. 7 demonstrates that antibodies containing one of the two complementary mutations K439E or S440K, illustrated in FIG. 4, are inhibited in their CDC activity, whereas they could form complexes capable of CDC activation when mixed. Example 20 describes the construction of antibody IgG1-005-E345R/E430G/S440Y (here referred to as IgG1-005-RGY), which formed oligomeric, most likely hexameric, complexes in solution, that also showed enhanced CDC activity compared to wild type IgG1-005 (Example 21, FIG. 26). Example 28 describes that also IgG1-005-KGY, IgG1-005-RSY, IgG1-005-RGW and IgG-005-RGI showed enhanced CDC compared to IgG1-005. To test if solution-phase oligomerization could be restricted to mixtures of non-self-interacting antibodies, antibody variants of IgG1-005-RGY were generated that each contained one of the two complementary mutations K439E or S440K that prohibited self-oligomerization.
Mutation K439E was introduced into IgG1-005-E345R/E430G/S440Y by methods known in the art, yielding IgG1-005-E345R/E430G/K439E/S440Y (IgG1-005-RGEY). Mutation S440K was introduced into IgG1-005-E345R/E430G by methods known in the art, yielding IgG1-005-E345R/E430G/S440K (IgG1-005-RGK). Mutations Y436I and S440K were introduced into IgG1-005-E345R/E430G by methods known in the art, yielding IgG1-005-E345R/E430G/Y436I/S440K (IgG1-005-RGIK). The rationale to include Y436I in IgG1-005-RGIK was to compensate for the absence of the oligomerization enhancing mutation S440Y.
HP-SEC analysis of the different antibody variants and equimolar antibody mixtures was performed as described in Example 20, but using PBS (12.6 mM sodium phosphate, 140 mM NaCl, pH 7.4; B. Braun, Oss, The Netherlands) as the mobile phase. FIG. 40 shows that the introduction of K439E in IgG1-005-RGY prohibited self-oligomerization of IgG1-005-RGEY (2.7% multimers). Likewise, substituting S440Y in IgG1-005-RGY with mutation S440K prohibited self-oligomerization of IgG1-005-RGK (2.2% multimers). Remarkably, a mixture of the solution-phase monomeric (i.e. single dimeric antibodies) IgG1-005-RGEY plus IgG1-005-RGK formed oligomeric species with equivalent HP-SEC mobility as IgG1-005-RGY (65% multimers), albeit with lower efficiency than IgG1-005-RGY (84% multimers).
FIG. 41 shows that the introduction of Y436I plus S440K into IgG1-005-E345R/E430G prohibited self-oligomerization of IgG1-005-RGIK (1.8% multimers). Again, a mixture of the solution-phase monomeric IgG1-005-RGEY plus IgG1-005-RGIK formed oligomeric species with equivalent HP-SEC mobility as IgG1-005-RGY, but now with high efficiency (93% multimers).
FIG. 42 shows a direct comparison of mixture IgG1-005-RGEY plus IgG1-005-RGK with mixture IgG1-005-RGEY plus IgG1-005-RGIK, demonstrating that IgG1-005-RGIK (65% multimers) could induce heteromeric oligomerization with IgG1-005-RGEY more efficiently than IgG1-005-RGK (93% multimers). The presence of the extra oligomerization and CDC-enhancing mutation Y436I in IgG1-005-RGIK apparently stabilized the formation of complexes with IgG1-005-E345R/E430G/K439E/S440Y.
In summary, IgG1-005 antibodies containing mutations E345R/E430G and the additional self-oligomerization inhibiting mutations K439E/S440Y, or S440K, or Y436I/S440K, could be reassembled into multimeric complexes, by mixing antibody molecules with complementary mutations (K439E in one, S440K in the other antibody).
Example 30
Fc Fragments can be Recruited to the Cell Surface by Cell Binding Antibodies, if Both Components Contain the E345R/E430G/S440Y Triple Mutations
The three mutations E345R, E430G and S440Y were introduced in a IgG1m(f) Fc fragment by methods known in the art, creating Fc-RGY. Protein was expressed and purified as described in Example 20. HP-SEC analysis of the Fc-RGY sample was performed as described in Example 20. FIG. 43 shows that under the used HP-SEC conditions, Fc-RGY showed approximately 28% monomers and 72% oligomers distributed over multiple states, as measured by fraction of peak area to total area.
Next it was tested whether Fc-RGY fragments could be recruited in oligomeric complexes with IgG1-RGY antibodies in solution. Therefore, 6 μg/mL Alexa-647-labeled Fc-RGY (Fc-RGY-A647),) was mixed 1:1 with a concentration series (0.001-3 μg/mL in 3-fold dilutions) of an EGFR-specific or CD20-specific antibody. Immediately after mixing, the samples were added to 0.1×106 EGFR-positive A431 or CD20-positive Daudi cells and incubated for 45 minutes at 4° C. After washing the cells twice with RPMI1640/0.1% BSA (3 minutes, 1200 rpm), the cells were resuspended in PBS/0.1% BSA/0.02% azide and analyzed on a FACS Canto II (BD Biosciences).
FIG. 44A shows that mixing Fc-RGY-A647 with EGFR-specific IgG1-2F8-RGY antibody resulted in a dose-dependent fluorescent signal on EGFR-positive A431 cells. In contrast, the CD20-specific IgG1-7D8-RGY was not able to recruit Fc-RGY-A647 to the CD20-negative A431 cells. None of the other tested control combinations of Fc-RGY-A647 mixed with either IgG1-2F8 or IgG1-2F8-E345R resulted in a fluorescent signal. Also, neither of the control combinations of CD20-specific IgG1-RTX-A647 mixed with IgG1-2F8 nor IgG1-2F8-RGY induced a fluorescent signal on A431 cells.
These data indicate that the labeled Fc-RGY fragment was specifically recruited to the A431 cells by incorporation into oligomeric complexes with IgG1-2F8-RGY antibodies that bind EGFR on A431 cells.
Similarly, FIG. 44B shows that the CD20-specific antibodies IgG1-7D8-RGY and IgG1-RTX-RGY were able to recruit Fc-RGY-A647 fragments to CD20-positive Daudi cells. In contrast, the EGFR-specific IgG1-2F8-RGY was not able to recruit Fc-RGY-A647 to the EGFR-negative Daudi cells. Negative control samples of either Fc-RGY-A647 alone, or Fc-RGY-A647 mixed with IgG1-7D8 or IgG1-RTX, did not yield a fluorescent signal on Daudi cells.
In summary, these data show that Fc-RGY molecules can form complexes in solution, and can be recruited to cells by RGY-containing antibodies that specifically bind the cells.
Example 31
Reversable Oligomerization of Antibody Molecules with Fc-Fc Interaction Enhancing Mutations can be Controlled by pH
Example 23 showed that antibody IgG1-005-E345R/E430G/S440Y, here abbreviated to IgG1-005-RGY, was capable of hexamerization at pH 6.8, while lowering the pH to 5.0 dissolved the hexameric complex in individual monomeric subunits. To characterize this property in detail, 50 mM citric acid and 100 mM Na2HPO4 were mixed in different ratios to generate mobile phase buffers at pH 5.0, 5.5, 6.0, 6.5 and 7.0. IgG1-005-RGY samples were exchanged into these buffers and separated by HP-SEC using the matching mobile phase. FIG. 45A shows that lowering the pH resulted in disassembly of multimeric complexes into monomeric subunits; that a pH of approximately 5.0 was needed to eliminate multimers from the mixture; and that at pH 6.0, approximately half of the complexes had disassembled.
The ability to control the antibody oligomeric state by lowering and raising the pH in a reversible fashion could be useful for applications in upstream or downstream processing during manufacturing. To test if pH-mediated disassembly was reversible, a sample with antibody hexamers was brought to pH 5.0 and split into two samples, one of which was brought back to pH 7.0. FIG. 45B shows that the sample that was exposed to pH 5.0 and subsequently brought back to pH 7.0 (pH 7.0 rev), formed antibody complexes with an efficiency highly similar to the reference sample kept at pH7.0.
Example 32
IgG1-RGY Protein Purification and Downstream Processing Efficiency can be Controlled by Choice of Buffer pH Condition
Protein A purification is a cornerstone of antibody downstream processing and implemented in a large number of antibody manufacturing processes. Because the protein A binding site partially overlaps with the Fc:Fc interaction interface mediating hexamerization of IgG1-005-E345R/E430G/S440Y, here abbreviated to IgG1-005-RGY, loading of protein A columns was attempted at pH 7.4, permissive of hexamerization and at pH 5.0, which blocks hexamerization as demonstrated in Example 23.
In example 20, the cloning of antibody IgG1-005-E345R/E430G/S440Y, here abbreviated to IgG1-005-RGY, was described. IgG1-005-RGY was expressed in EXPI293F cells essentially as described by the manufacturer (Invitrogen), after which the supernatant was collected by centrifugation at 300 g for 10 min. Supernatant was concentrated 4-fold using a MiniKros M155-260-01P Hollow Fiber Tangential Flow Filtration device with a 50 kDa cutoff membrane (SpectrumLabs, Rancho Dominguez Calif., USA), yielding supernatant with a protein concentration of 1.1 g/L. The supernatant was split in two parts, one of which was kept at the original pH of 7.5, while the other batch was pH adjusted to pH 5.0 by dropwise addition of 1.0 M citric acid-NaOH pH 3.0. Both batches were filtered over a 0.20 μM dead-end filter.
The supernatant batch kept at pH 7.5 was loaded at a low flow rate of 109 cm/h to mimic downstream processing conditions at manufacturing scale, on a 1.0 mL Protein A column (HiTrap MabSelectSuRe, GE Healthcare, Uppsala, Sweden), which was consecutively washed with PBS (12.6 mM sodium phosphate, 140 mM NaCl, pH 7.4; B. Braun, Oss, The Netherlands), after which bound IgG protein was eluted using 0.1 M citric acid-NaOH, pH 3.0. The eluate was immediately neutralized with 2 M Tris-HCl, pH 9.0 and dialyzed overnight to PBS. After dialysis, the sample was sterile filtered over a 0.20 μM dead-end filter.
The batch brought to pH 5.0 was loaded at a flow rate of 109 cm/h on the same 1.0 mL Protein A column (HiTrap MabSelectSuRe), which was consecutively washed with 20 mM citric acid/citrate pH 5.0, after which bound IgG protein was eluted using 0.1 M citric acid-NaOH, pH 3.0. The eluate was immediately neutralized with 2 M Tris-HCl, pH 9.0 and dialyzed overnight to PBS. After dialysis, the sample was sterile filtered over a 0.20 μM dead-end filter.
Flow-throughs of both protein purifications were collected and purified using a 5.0 mL MabSelect SuRe column yielding approximately 50 mg of protein, demonstrating that the 1.0 mL MabSelectSuRe column had been saturated effectively.
The yields of IgG1-005-RGY were determined by measuring A280 of the dialyzed elution samples using a Nanodrop device (ThermoScientific, Wilmington Del., USA). Protein A purification at 1.0 mL scale at pH 7.0 yielded 21.45 mg of IgG1-005-RGY, while purification at pH 5.0 yielded 29.14 mg of IgG1-005-RGY. In conclusion, the protein yield was increased approximately 36% by performing the binding of antibody to protein A under conditions keeping IgG1-005-RGY monomeric.
Example 33
Programmed Cell Death (PDC) by Stable Hexameric IgG2-005
To test if different isotypic variants of IgG antibodies containing the triple mutation E345R/E430G/S440Y could induce programmed cell death (PCD), antibody IgG2-005-E345R/E430G/S440Y (IgG2-005-RGY) was generated by methods known in the art. 1.0×105 Ramos cells expressing CD38 were cultured for 24 hours in 96-well, U-bottom plates (Nalgene Nunc) in the presence of a dilution series (10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.005, and 0.0025 μg/mL) of wild type IgG2-005, IgG2-005-RGY, hexameric IgM-005 or human control antibodies IgG1-2F8 and IgG1-2F8-RGY, recognizing EGFR, which is not expressed on Ramos cells. PCD was quantified after these 24 hours by staining with annexin V-FITC (Annexin binding assay; BD Biosciences, San Diego, Calif., USA) according to the manufacturer's instructions. The amount of annexin V-FITC-positive cells was determined using a FACS (BD).
FIG. 46 shows that IgG2-005-RGY demonstrated enhanced programmed cell death capacity compared to wild type IgG2-005 and control antibodies IgG1-2F8 and IgG1-2F8-RGY. Hexameric IgM did not induce PCD under the conditions tested.
Example 34
IgG-005-RGY Against CD38 Out Performs Hexameric IgM-005 in a CDC Assay on B Cells
To compare the CDC efficacy of IgG1-005-RGY to that of IgM, the VH domain of IgG1-005 was cloned into an IgM backbone by methods know in the art, and expressed in the absence of J-chain to produce IgM hexamers against CD38. The construction of IgG1-005-E345R/E430G/S440Y (here referred to IgG1-005-RGY) was described in Example 20.
HP-SEC analysis of the different antibodies was performed as described in Example 20, but using PBS (12.6 mM sodium phosphate, 140 mM NaCl, pH 7.4; B. Braun, Oss, The Netherlands) as the mobile phase. FIG. 47 shows that IgM-005 expressed in the absence of J-chain yielded a molecule with slightly higher mobility in HP-SEC than IgG1-005-RGY, as could be expected due to the higher molecular weight of hexameric IgM compared to hexameric IgG1-005-RGY.
CDC efficacy of IgG1-005-RGY was compared to wild type IgG1-005 and hexameric IgM-005 by testing antibody concentration series (0.0003-10 μg/mL in 2-fold dilutions) in an in vitro CDC assay as described in Example 18. FIG. 48 shows that IgG1-005-RGY showed more potent CDC activity on Daudi and Wien133 cells than hexameric IgM-005. Wild-type IgG1-005 showed lower CDC efficacy than IgG1-005-RGY and IgM-005 on Daudi cells, and no killing activity on Wien133 cells. IgG1-b12 was used as a non-cell binding negative control antibody. Monomeric (i.e. single dimeric protein) IgG1-005, hexameric IgG1-005-RGY and hexameric IgM-005 concentrations are indicated as C1q binding equivalents to enable comparison of non-covalent IgG and covalent IgM complexes with different molecular weight.
In summary, IgG1-005-RGY could induce complement-mediated lysis of target cells more efficiently than IgM-005 at antibody concentrations binding equivalent amounts of C1q.
Example 35
Introduction of the Triple Mutations E345R/E430G/S440Y into Anti-EGFR Antibody IgG1-2F8 Enhances Efficacy of CDC-Mediated Lysis of EGFR-Positive Solid Tumor Cell Lines
To test if introduction of the triple mutations E345R/E430G/S440Y into a solid tumor target antibody could lead to activation of complement-mediated lysis, IgG1-2F8-E345R/E430G/S440Y (here referred to as IgG1-2F8-RGY) was generated by methods known in the art.
CDC efficacy by IgG1-2F8-RGY was tested on EGFR-positive A431 and Difi tumor cell lines and was compared to wild type IgG1-2F8 and the control antibodies IgG1-005 and IgG1-005-RGY. The control antibodies recognize CD38, which is expressed on neither A431 nor Difi cells.
After the solid tumor cells were detached by using trypsin-EDTA in phosphate-buffered saline (PBS), the cells were washed and passed through a 40 μm nylon cell strainer (DB Falcon™) and resuspended in PBS at a concentration of 1.0×106 cells/mL. Cells were stained for 30 minutes at 37° C. using SYBR Green (SYBR Green 57563 in DMSO, Invitrogen, 25000× diluted). After centrifugation (1200 rpm, 5 minutes at RT), cells were resuspended in RPMI1640/0.1% BSA at a concentration of 3.0×105 cells/mL. Antibody serial dilutions (0.0003-10 μg/mL) were prepared in RPMI/0.1% BSA supplemented with TOPRO-3 (TOPRO-3 iodide T3605, diluted 1600×). Cells were seeded at 30,000 cells per well into flat bottom 96 wells plates (black 96-Well ABI™ 4315480 FMAT Plates); after addition of the antibody serial dilutions, plates were incubated for 15′ on a shaker (300 rpm, RT). Normal Human Serum (NHS, Sanquin) was added at 20% final concentration. Plates were incubated for 45 minutes at 37° C. The amounts of dead cells (TOPRO-3 positive) and total cells (SYBR Green positive) were determined using a Celigo® imaging cytometer (Brooks Life Science Systems). Results were analyzed using GraphPad Prism 5.04.
FIG. 49 shows that the efficacy to induce complement-mediated lysis of EGFR-positive solid tumor cells was considerably higher for IgG1-2F8-RGY than wild type IgG1-2F8.
Example 36
IgG1-005-RGY Shows Target-Independent Complement Activation in Contrast to Wild Type IgG1-005
In example 20, the cloning of antibody IgG1-005-E345R/E430G/S440Y, here abbreviated to IgG1-005-RGY, was described. To test if IgG1-005-RGY could activate complement in solution in the absence of target cells, the formation of C4d, a marker for classical complement pathway activation, was analyzed. Complement activation was determined by measuring C4d concentrations after incubating 100 μg/mL antibody in 90% normal human serum for 1 hour at 37° C. in low protein binding 96 wells polypropylene microplates (U-shaped and sterile; Greiner 650261). C4d concentrations were measured in an ELISA (MicroVue C4d EIA kit, Quidel Corporation) according to the manufacturer's instructions. A heat aggregated IgG (HAG) sample was used as positive control for complement activation in solution. FIG. 50 shows that HAG induced efficient C4d production, while wild type IgG-005 did not show complement activation under these conditions. In contrast, IgG1-005-RGY induced elevated C4d levels, indicative of complement activation in solution.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. Any and all combination of embodiments disclosed in dependent claims is also contemplated to be within the scope of the invention.
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14413178
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genmab b.v.
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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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Apr 1st, 2022 06:04PM
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Apr 1st, 2022 06:04PM
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Genmab A/S
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Health Care
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Pharmaceuticals & Biotechnology
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cph:gen
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Genmab A/S
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Apr 4th, 2017 12:00AM
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Jul 9th, 2013 12:00AM
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https://www.uspto.gov?id=US09611327-20170404
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Anti-DR5 family antibodies, bispecific or multivalent anti-DR5 family antibodies and methods of use thereof
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Anti-DR5 family member antibodies and bispecific antibodies comprising one or more anti-DR5 family member antibodies are disclosed. These antibodies can be used to trigger cell death on DR5 positive cells.
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9611327
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1. A composition comprising at least one or two polypeptides binding specifically a DR5 receptor, wherein the at least one or two polypeptides comprise two immunoglobulin binding domains comprising:
a first binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of amino acid sequence FAS, a CDR3 of sequence SEQ ID NO: 17, and
a second binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of amino acid sequence RTS, a CDR3 of sequence SEQ ID NO: 21,
wherein
the at least one polypeptide comprises both immunoglobulin binding domains; or
the at least two polypeptides comprise a first polypeptide comprising the first binding domain and a second polypeptide comprising the second binding domain;
and a pharmaceutically carrier, diluent or excipient.
2. The composition of claim 1, comprising:
one or more of amino acid sequence pairs SEQ ID NO: 2 and 4 and SEQ ID NO: 6 and 8,
the pair of amino acid sequences SEQ ID NO: 2 and 4,
the pair of amino acid sequences SEQ ID NO: 6 and 8, or
both amino acid sequence pairs SEQ ID NO: 2 and 4 and SEQ ID NO: 6 and 8; or
one or more of amino acid sequence pairs SEQ ID NO: 35 and 37 and SEQ ID NO: 39 and 41,
the pair of amino acid sequences SEQ ID NO: 35 and 37,
the pair of amino acid sequences SEQ ID NO: 39 and 41, or
both amino acid sequence pairs SEQ ID NO: 35 and 37 and SEQ ID NO: 39 and 41.
3. The composition of claim 1 comprising a bispecific antibody comprising the amino acid sequence pair SEQ ID NO: 2 and 4 and the amino acid sequence pair SEQ ID NO: 6 and 8.
4. The composition of claim 1 comprising a bispecific antibody comprising the amino acid sequence pair SEQ ID NO: 35 and 37 and the amino acid sequence pair SEQ ID NO: 39 and 41.
5. The composition of claim 1, wherein the polypeptide or polypeptides is/are selected from the group of an Fv, an Fab, an F(ab′)2, an scFv, and an antibody.
6. The composition of claim 5, wherein the antibody is a monoclonal antibody.
7. The composition of claim 1, wherein the first or second binding domain comprises the amino acid sequence pairs SEQ ID NO:2 and 4, or SEQ ID NO: 35 and 37.
8. The composition of claim 1, wherein the first or second binding domain comprises the amino acid sequence pairs SEQ ID NO: 6 and 8, or SEQ ID NO: 39 and 41.
9. The composition of claim 1, wherein one binding domain comprises the pair of amino acid sequences SEQ ID NO: 2 and 4, and the other binding domain comprises the pair of amino acid sequences SEQ ID NO: 6 and 8.
10. The composition of claim 1, wherein one binding domain comprises the pair of amino acid sequences SEQ ID NO: 35 and 37, and the other binding domain comprises the pair of amino acid sequences SEQ ID NO: 39 and 41.
11. A polypeptide binding specifically a DR5 receptor, comprising one or two binding domains comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of amino acid sequence FAS, a CDR3 of sequence SEQ ID NO: 17.
12. A polypeptide binding specifically a DR5 receptor, comprising one or two binding domains comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of amino acid sequence RTS, a CDR3 of sequence SEQ ID NO: 21.
13. A bispecific or multivalent polypeptide binding specifically a DR5 receptor, which comprises:
a first binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14 CDR1, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of amino acid sequence FAS, a CDR3 of sequence SEQ ID NO: 17, and
a second binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of amino acid sequence RTS, a CDR3 of sequence SEQ ID NO: 21.
14. A composition comprising two polypeptides, or antibodies or fragments thereof, both having the capability to bind to DR5 receptor,
wherein a first polypeptide or antibody or fragment thereof, comprises a first antigen-biding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of amino acid sequence FAS, a CDR3 of sequence SEQ ID NO: 17, and
wherein a second polypeptide or antibody or fragment thereof, comprises a second antigen-binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of amino acid sequence RTS, a CDR3 of sequence SEQ ID NO: 21.
15. A bispecific antibody, or fragment thereof, having the capability to bind to DR5 receptor, said antibody comprising a first antigen-binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of amino acid sequence FAS, a CDR3 of sequence SEQ ID NO: 17, and a second antigen-binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of amino acid sequence RTS, a CDR3 of sequence SEQ ID NO: 21.
16. A method for treating a mammal having a disease or disorder in which DR5 is expressed, wherein the disease or disorder is selected from the group consisting of cancer, an autoimmune disease, an inflammatory condition, a viral infection, and a viral disease, wherein the method comprises the administration to the mammal of an effective amount of a composition according to claim 1.
17. The method of claim 16, wherein the disease or disorder is a solid cancer.
18. The method of claim 16, wherein the composition comprises at least two polypeptides, and wherein the at least two polypeptides are administered simultaneously, separately, or sequentially to the mammal.
19. The method of claim 16, wherein the mammal is a human.
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RELATED APPLICATIONS
This application is a 371 filing of International Application No. PCT/EP2013/064466, filed Jul. 9, 2013, which claims priority to U.S. Provisional Patent Application No. 61/669,866, filed Jul. 10, 2012 and European Patent Application No. 12305821.6, filed Jul. 9, 2012, the contents of each of which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
The present invention relates to the fields of immunology, oncology, and more specifically, to monospecific, bispecific or multivalent antibody molecules that can be used to advantage in the treatment of various cancers, autoimmune diseases, and infectious diseases that express DR5 antigen. The present invention is related to novel polypeptides binding specifically to the DR5 receptor also called TRAIL receptor 2. The invention relates in particular to a polypeptide having two different binding domains or a combination of polypeptides having these different binding domains, which bind to different epitopes of the DR5 receptor, whereby apoptosis is induced. The invention also relates to pharmaceutical compositions containing these polypeptides and the treatment of cancer, autoimmune diseases and viral infections using these polypeptides and compositions.
BACKGROUND OF THE INVENTION
Apoptosis, or programmed cell death, is a physiologic process essential to the normal development and homeostasis of multicellular organisms. Derangements of apoptosis contribute to the pathogenesis of several human diseases including cancer, neurodegenerative disorders, and acquired immune deficiency syndrome.
The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily of cytokines, is a type 2 membrane protein that is expressed in the majority of normal tissues and can undergo protease cleavage, resulting in a soluble form able to bind to TRAIL receptors, (Wiley S R. et al., Immunity. 1995; 3:673-682; Daniel P T et al., J Immunol. 1994; 152:5624).
Ligands of this family generally recognize and bind to a limited subset of cognate receptors on the cell surface, leading to signal transduction cascades downstream of the receptor, allowing the activation of a large panel of signalling pathways including NF-kB or caspase activation. TRAIL induces apoptosis of certain transformed cells, including a number of different types of cancer cells as well as virally infected cells, while not inducing apoptosis of a number of normal cell types and is thus of particular interest in the development of cancer therapies, (Walczak et al., Nature Medecine. 1999; 5/157-163, Ashkenazi A. et al., J Clin Invest. 1999; 104:155).
There are four known cell surface receptors for TRAIL. TRAIL Receptor 1 (TRAIL-R1, DR4) and Trail Receptor 2 (TRAIL-R2, DR5, Apo-2, TRICK2, Killer, TR6, Tango-63) have a cytoplasmic death domain and are able to trigger apoptosis in tumor cells via downstream caspase activation. The other two receptors, TRAIL Receptor 3 (TRAIL-R3, DcR1, TR5, TRIDD, LIT) and TRAIL Receptor 4 (TRAIL-R4, DcR2, TRUNDD) lack a cytoplasmic death domain and do not mediate apoptosis. In addition, osteoprotegerin (OPG), a soluble (secreted) member of the TNF receptor family of proteins, also binds TRAIL.
The intracytoplasmic domains of DR4 and DR5 each include a so-called death domain. After activation of the receptors DR4 and DR5, the fas-associated death domain adapter molecule is recruited to the receptor, leading to an autoproteolytic cleavage and activation of initiator caspase-8. DR4 and DR5 have been reported to transduce an apoptotic signal to TRAIL sensitive cancer cells, upon binding of TRAIL. Active caspase-8 in turn triggers the proteolytic activation of downstream caspases including caspase-3. Downstream caspases ultimately degrade a broad range of cellular proteins, and apoptosis is finalized.
Expression of either DR4 or DR5 is frequently detected in human cancers, including colon, gastric, pancreatic, ovarian, breast, and non-small-cell lung cancer with low or no expression in normal tissues.
In the development or progression of many diseases it is often the case that cells are not deleted. In many autoimmune diseases and inflammatory conditions, the surviving activated cells attack normal tissues or cells. Further, progression of tumorigenesis and the proliferative pannus formation of rheumatoid arthritis are characterized by the unchecked proliferation of cells. Thus insufficient apoptosis leads to the development of disease, and the uses of apoptosis-inducing ligand or agonistic MAb to enhance apoptosis are considered as a potential therapeutic strategy for eliminating those unwanted cells
TRAIL induces apoptosis in a wide range of haematopoietic and solid tumor cells, while sparing most normal cells. TRAIL has strong apoptosis-inducing activity against cancer cells in vitro and potent antitumor activity against tumor xenografts of various cancers in vivo.
TRAIL and its derivatives, including agonistic antibodies targeting TRAIL receptors are attractive compounds for cancer therapy due to their ability to induce tumor regression without significant side effects.
There are many instances in the patent literature of efforts to use polypeptides derived from the TRAIL ligand as a therapy against cancerous cells (US20090131317; U.S. Pat. No. 6,469,144; U.S. Pat. No. 6,740,739; US20070026000; U.S. Pat. No. 6,444,640; US20050244857; US20050233958; U.S. Pat. No. 7,736,637).
TRAIL polypeptides have been used to induce the TRAIL apoptotic pathway, but they have the drawback of a short half-life.
Currently, a great deal of attention has focused on the development of novel immunotherapy strategies for the treatment of cancer. One such strategy is antibody-based cancer therapy.
The most prominent determinant of the above targeting properties is the size of the antibody-based molecule relative the degree of specificity, the retention in tumors and their clearance. Another important feature of antibody-based molecules is valence, as significantly greater tumor retention has been associated with multivalent binding to target, (Adams et al., Cancer Res. 1993; 51:6363-6371; Wolf et al., Cancer Res. 1993; 53:2560-2565).
As mentioned earlier, agonistic antibodies against DR4 or DR5 have been produced and represent a new generation of cancer therapy. Works have been conducted also on the use of agonistic antibodies directed against the TRAIL receptors in order to induce the TRAIL apoptotic pathway.
Agonistic monoclonal antibodies that specifically bind to DR4 or DR5 are supposed to be able to directly induce apoptosis of targeted tumor cells, (Buchsbaum D J et al., Future Oncol. 2006; 2:493; Rowinsky E K et al., J Clin Oncol. 2005; 23:9394).
Other patents relate to the use of agonistic antibodies directed against DR4 or DR5, or DR4 and DR5, or to the combined use of antibodies against DR5 and another chemotherapeutic agent: US20040147725; US 20090022707; US20080248037; US20020155109; U.S. Pat. No. 6,461,823; U.S. Pat. No. 6,872,568; U.S. Pat. No. 7,064,189; U.S. Pat. No. 6,521,228; U.S. Pat. No. 7,704,502.
Combined treatment with agonistic antibodies directed against different TRAIL receptors, for example DR4 and DR5, have been developed as well. Agonistic bispecific antibodies that bind DR4 or DR5 (or hybridomas producing such agonistic MAbs) may be employed as starting materials in various procedures (WO 2002/0155109).
These include anti-DR5 MAb lexatumumab, (Plummer R. et al., Clin Cancer Res. 2007; 13:6187), the anti-DR5 MAb apomab, (Adams C. et al., Cell Death Differ. 2008; 15:751), the anti-DR5 MAb LBy135, (Li J. et al., AACR Meeting Abstracts. 2007. Abstract 4874), the anti-DR5 MAb WD-1, (Wang J. et al., Cell Mol Immunol. 2008; 5:55) and the anti-DR5 MAb AMG655, (Wall J. et al., AACR Meeting Abstracts. 2008. Abstract 1326, Kaplan-Lefko P. et al., AACR Meeting Abstracts. 2008. Abstract 399). A consistent finding from all these studies is the considerable variability in the sensitivity of various tumor cell lines to anti-DR5-mediated cytotoxicity.
Anti-DR4 or anti-DR5 agonistic antibodies, including mapatumumab or lexatumumab respectively are also well tolerated in patients (Herbst R. S. et al., J Clin Oncol. 2006; 24(18S)/3013; Hotte S. J. et al., Clin Cancer Res. 2008; 14/3450-3455; Wakelee H. A et al., Ann Oncol. 2010; 21/376-381; Fox N. L. et al., Expert Opin Biol Ther. 2010; 10/1-18).
Lexatumumab (also known as ETR2-ST01) is an agonistic human monoclonal antibody against DR5 used in the treatment of cancer. HGS-ETR2 antibodies were generated by HGS through collaboration with Cambridge Antibody Technology.
Tigatuzumab (CS-1008) is a humanized IgG1 monoclonal antibody composed of the CDR regions of mTRA-8. The murine anti-DR5 monoclonal antibody, TRA-8 (mTRA-8), was selected from a series of anti-DR5 monoclonal antibodies based on its specificity, ability to trigger apoptosis in vitro without the use of crosslinking reagents, and lack of toxicity to human hepatocytes, (Buchsbaum D J et al., Clin Cancer Res. 2003; 9:3731; Ichikawa K. et al., Nat Med. 2001; 7:954).
Tigatuzumab mediates a very similar pattern of in vitro cytotoxicity and in vivo antitumor efficacy as mTRA-8. It was shown to have potent in vitro cytotoxicity to a variety of human tumor cell lines and in vivo antitumor efficacy in murine xenograft models of human cancers. Its in vitro cytotoxicity and in vivo antitumor efficacy can be substantially enhanced in combination with a variety of chemotherapeutic agents and/or radiation, (Buchsbaum D J et al., Clin Cancer Res. 2003; 9:3731; DeRosier L C et al., Clin Cancer Res. 2007; 13:5535s).
Anti-DR4 and anti-DR5 antibodies have been tested in associations, together or with other chemotherapeutic agents or therapies. A combined treatment of colorectal tumors with two agonistic antibodies HGS-ETR1 (anti-DR4) and HGS-ETR2 (anti-DR5) and radiotherapy let to enhanced effects in vitro and dose-dependent growth delay in vivo (Marini P et al., Oncogene. 2006; 25 (37):5145-54). Fully human agonistic antibodies to DR4 and DR5 demonstrated in primary and cultured lymphoma cells induction of apoptosis and enhancement of doxorubicin- and bortezomib-induced cell death (Georgakis G V et al., Oncogene. 2006; 25(37):5145-54).
It has been found that the expression of DR5 and susceptibility to TRAIL-induced apoptosis of breast cancer cells is enhanced by the radiation, suggesting that combined with radiation, the efficiency of TRAIL would be increased in cancer therapy (Chinnaiyan A. M et al., PNAS. 2000; 97/1754-1759).
The combination of antibody and chemotherapy usually enhances the degree of apoptosis and can partially reverse resistance in some cell lines (Buchsbaum D J et al., J Clin Cancer Res. 2003; 9:3731; DeRosier L C et al., Clin Cancer Res. 2007; 13:5535s; Oliver P G et al., Clin Cancer Res. 2008; 14:2180; Derosier L C et al., Mol Cancer Ther. 2007; 6:3198; Long J W. et al., J Surg Res. 2007; 137:167).
SUMMARY OF THE INVENTION
The present inventors have now found that unexpectedly, it is possible to induce the DR5 apoptotic pathway by using two antibodies directed against at least two different epitopes of the DR5 receptor. The binding to both epitopes on the same receptor has an agonistic action on the receptor and induces apoptosis in an efficient way. Combination of antibodies DR5-01 and DR5-05 as disclosed herein revealed a stronger agonistic action than the ligand itself.
An unexpected and synergistic action has been observed by using two antibodies directed each against a different epitope on the DR5 receptor, with respect to one antibody against one single epitope. Without wishing to be bound to the theory, it is postulated that the binding to the two epitopes of DR5 allows for a synergistic agonist function, leading to an unexpectedly elevated apoptosis induction. It has been found that the unexpected and synergistic action may be beneficial for therapeutic treatment or for integration to a therapeutic protocol. It has thus been found that the combination of the antibodies may lead to a synergic increase of inhibition of cancer cells proliferation in particular in glioma. It has also been found that the combination of the antibodies and a chemotherapeutic drug may lead to a synergic increase of inhibition of cancer cells proliferation in the case of cancers that are difficult to treat, such as glioma, lung and breast cancers that more or less resist to chemotherapeutic drugs. It has also been found that the combination of antibodies and drug may allow getting a therapeutic effect, such as inhibition of cell proliferation, which is stably obtained over a wide range of drug and/or antibodies dosages.
It is thus now possible to provide for pharmaceutical compositions comprising two polypeptides or antibodies acting as agonist by binding to the two different epitopes on DR5, or bispecific antibodies acting as agonist by binding to the two different epitopes on DR5 and pharmaceutical compositions containing the same.
An “agonist” or an “agonistic polypeptide or antibody” for a natural receptor is a compound which binds the receptor to form a receptor-agonist complex and which activates said receptor, initiating a pathway signaling and further biological process. In the context of the present invention, an agonist function is obtained owing the simultaneous or sequential interaction between the polypeptides or antibodies of the invention and two different epitopes of the DR5 receptor, initiating the DR5 apoptosis pathway.
An object of the invention is thus a composition comprising two polypeptides, or antibodies or fragment thereof, both having the capability to bind to DR5, a first polypeptide or antibody comprising a first antigen-binding site that binds to a first epitope of said DR5, and a second polypeptide or antibody comprising a second different antigen-binding site that binds to a second epitope of said DR5. Each of said first and second antigen-binding sites binds to a different epitope on the same DR5 molecule. The two polypeptides, or antibodies or fragments thereof are for a simultaneous, separate or sequential administration to a mammal, including human.
The composition or pharmaceutical composition may further contain a pharmaceutically acceptable carrier, diluent, or excipient. The polypeptides or antibodies are synergistically agonistic in combination, which means that they have the capability upon binding to both epitopes of a DR5 molecule to induce the DR5 apoptotic pathway.
An object of the invention is also a bispecific or biparatopic antibody, or fragment thereof, having the capability to bind to DR5, said antibody comprising a first antigen-binding site that binds to a first epitope of said DR5, and a second different antigen-binding site that binds to a second epitope of said DR5. Each of said first and second antigen-binding sites binds to a different epitope on the same DR5 molecule. The polypeptides or antibodies are synergistically agonistic in combination, which means that they have the capability upon binding of both to their specific epitopes of a DR5 molecule to induce the DR5 apoptotic pathway.
The invention encompasses the binding of one bispecific antibody to the two different epitopes of the same DR5 molecule, or of two bispecific antibodies to the two epitopes of the same DR5 molecule, one antibody to a first epitope, the second to the second epitope of the same DR5 molecule.
The bispecific antibody may be formulated in a pharmaceutical composition further containing a pharmaceutically acceptable carrier, diluent, or excipient.
Without wishing to be bound to theory, it is deemed that, regarding the mechanism of action, antibody combination or bispecific antibodies according to the invention may promote DR5 clustering. These components may promote DR5 amassing of higher concentration compared with a monospecific antibody.
Antibody combination or bispecific antibodies may promote also a conformation change inducing a higher incidence to trigger apoptosis signalling or to reverse the resistance of cancer cell to the apoptosis. These components may promote DR5 amassing of higher concentration compared with a monospecific antibody.
Another object of the invention encompasses the binding at least of two, three, four, five or more monovalent binding polypeptides, or antibodies or fragment thereof, both having the capability to bind to DR5, a first polypeptide or antibody comprising a first antigen-binding site that binds to a first epitope of said DR5, and a second polypeptide or antibody comprising a second different antigen-binding site that binds to a second epitope of said DR5.
Another object of the invention is thus a composition comprising at least one chemotherapeutic drug and two polypeptides, or antibodies or fragment thereof, both having the capability to bind to DR5, a first polypeptide or antibody comprising a first antigen-binding site that binds to a first epitope of said DR5, and a second polypeptide or antibody comprising a second different antigen-binding site that binds to a second epitope of said DR5. Each of said first and second antigen-binding sites binds to a different epitope on the same DR5 molecule. The drug and the two polypeptides, or antibodies or fragments thereof are for a simultaneous, separate or sequential administration to a mammal, including human. In this object, the two polypeptides may be replaced by a bispecific or biparatopic antibody, or fragment thereof, as disclosed herein.
The polypeptides, especially antibodies, according to the invention may be further defined by the CDRs of the VH and VL regions of the murine antibodies DR5-01 and DR5-05 or by their complete VH and VL regions.
An object of the invention is to a composition comprising at least one or two polypeptides binding specifically a DR5 receptor, wherein the at least one or two polypeptides comprise two immunoglobulin binding domains comprising:
a first binding domain comprising a pair of VH and VL chains wherein
the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 13, a CDR2 comprising or consisting of sequence SEQ ID NO: 14 CDR1, a CDR3 comprising or consisting of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 16, a CDR2 comprising or consisting of sequence FAS, a CDR3 comprising or consisting of sequence SEQ ID NO: 17; or
wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO:22, a CDR2 comprising or consisting of sequence SEQ ID NO: 23, a CDR3 comprising or consisting of sequence SEQ ID NO: 24; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 25, a CDR2 comprising or consisting of sequence SEQ ID NO: 26, a CDR3 comprising or consisting of sequence SEQ ID NO: 17, or
wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 32, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 24; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 16, a CDR2 comprising or consisting of sequence FAS, a CDR3 comprising or consisting of sequence SEQ ID NO: 17,
and
a second binding domain comprising a pair of VH and VL chains wherein
the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 18, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 20, a CDR2 comprising or consisting of sequence RTS, a CDR3 comprising or consisting of sequence SEQ ID NO: 21, or
wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 27, a CDR2 comprising or consisting of sequence SEQ ID NO: 28, a CDR3 comprising or consisting of sequence SEQ ID NO: 29; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 30, a CDR2 comprising or consisting of sequence SEQ ID NO: 31, a CDR3 comprising or consisting of sequence SEQ ID NO: 21, or
wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 33, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 29; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 20, a CDR2 comprising or consisting of sequence RTS, a CDR3 comprising or consisting of sequence SEQ ID NO: 21,
wherein
the at least one polypeptide comprises both immunoglobulin binding domains, or
the at least two polypeptides comprise a first polypeptide comprising the first binding domain and a second polypeptide comprising the second binding domain for a simultaneous, separate or sequential administration to a mammal, including man,
and a pharmaceutically carrier, diluent or excipient. In an embodiment, the composition comprises further a chemotherapeutic drug for a simultaneous, separate or sequential administration to a mammal, including man.
Other objects of the invention are the individual polypeptides or antibodies and their various combinations in accordance with the invention, kits comprising at least two polypeptides or antibodies, and kits comprising at least one polypeptide or antibody and at least one drug, wherein antibodies or polypeptides and drugs are separated or not.
The polypeptides or antibodies of the invention may comprise one or several, preferably two, binding sites or domains or paratopes. An object of the present invention is a polypeptide binding specifically a DR5 receptor, comprising one or more, preferably one or two, immunoglobulin binding domain(s) comprising:
a binding domain comprising a pair of VH and VL chains wherein:
the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 13, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 16, a CDR2 comprising or consisting of sequence FAS, a CDR3 comprising or consisting of sequence SEQ ID NO: 17 (DR5-01 type CDRs), or
the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO:22, a CDR2 comprising or consisting of sequence SEQ ID NO: 23, a CDR3 comprising or consisting of sequence SEQ ID NO: 24; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 25, a CDR2 comprising or consisting of sequence SEQ ID NO: 26, a CDR3 comprising or consisting of sequence SEQ ID NO: 17, (DR5-01 type CDRs), or
the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 32, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 24; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 16, a CDR2 comprising or consisting of sequence FAS, a CDR3 comprising or consisting of sequence SEQ ID NO: 17 (DR5-01 type CDRs);
and/or
a binding domain comprising a pair of VH and VL chains wherein:
the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 18, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 20, a CDR2 comprising or consisting of sequence RTS, a CDR3 comprising or consisting of sequence SEQ ID NO: 21 (DR5-05 type CDRs), or
the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 27, a CDR2 comprising or consisting of sequence SEQ ID NO: 28, a CDR3 comprising or consisting of sequence SEQ ID NO: 29; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 30, a CDR2 comprising or consisting of sequence SEQ ID NO: 31, a CDR3 comprising or consisting of sequence SEQ ID NO: 21 (DR5-05 type CDRs), or
the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 33, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 29; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 20, a CDR2 comprising or consisting of sequence RTS, a CDR3 comprising or consisting of sequence SEQ ID NO: 21 (DR5-05 type CDRs). The binding domain is best defined by the VH and VL chains comprising the CDRs defined based on the same method, either IMGT®, Kabat® or common numbering system, see CDR table infra.
The VH and VL chains together define a single binding site. Each one of these binding domain binds specifically to a different epitope on the DR5 receptor. The polypeptides are synergistically agonistic, which means that they have the capability upon binding to both epitopes of a DR5 molecule to induce the DR5 apoptotic pathway.
By “immunoglobulin binding domain” or “binding domain” it is meant the paratope of an immunoglobulin made of the two variable light (VL) and variable heavy (VH) chains. The paratope is able to specifically bind to the targeted epitope.
In accordance with the invention, the VL and VH chains have a conventional structure of a light chain or a heavy chain of an immunoglobulin, with the framework regions FR. The structure may be defined as the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In a preferred embodiment, the polypeptide of the invention comprises one or more, preferably one or two, immunoglobulin binding domain(s) comprising the VH+VL region of mDR5-01 and/or the VH+VL region of mDR5-05. In an embodiment, the polypeptide comprise one or two binding domain(s) comprising the VH+VL region of mDR5-01. In an embodiment, the polypeptide comprise one or two binding domain(s) comprising the VH+VL region of mDR5-05. In an embodiment, the polypeptide comprise two binding domain(s) comprising the VH+VL region of mDR5-05, on the one hand, and the VH+VL region of mDR5-01, on the other hand. In a preferred embodiment, the polypeptide of the invention comprises one or more, preferably one or two, immunoglobulin binding domain(s) comprising the VH+VL region of HzDR5-01 and/or the VH+VL region of HzDR5-05. In an embodiment, the polypeptide comprise one or two binding domain(s) comprising the VH+VL region of HzDR5-01. In an embodiment, the polypeptide comprise one or two binding domain(s) comprising the VH+VL region of HzDR5-05. In an embodiment, the polypeptide comprise two binding domain(s) comprising the VH+VL region of HzDR5-05, on the one hand, and the VH+VL region of HzDR5-01, on the other hand.
The anti-DR5 polypeptide thus comprises one or two binding domains. In an embodiment, the binding domains are specific of the same epitope on the DR5 receptor. These binding domains comprise the same set of 3 CDRs on the VH and VL as disclosed and provided therein and may be identical or slightly different in the framework regions, as soon as this does not affect the specificity to bind the targeted epitope.
The anti-DR5 polypeptide may be in particular an antibody, preferably a monoclonal antibody, or a suitable antibody fragment, such as a Fv, a Fab, a F(ab′)2, a single-chain variable fragment (scFv).
The invention also encompasses the combined use of polypeptides or antibodies or of bispecific polypeptides or antibodies or fragments and the like, making use of the synergic activity linked to binding to the two epitopes revealed by the present invention. This use may be further combined with the administration of a chemotherapeutic drug, as disclosed herein.
Another object of the invention encompasses the binding at least of two, three, four, five or more monovalent binding polypeptides, or antibodies or fragment thereof, both having the capability to bind to DR5, a first polypeptide or antibody comprising a first antigen-binding site that binds to a first epitope of said DR5, and a second polypeptide or antibody comprising a second different antigen-binding site that binds to a second epitope of said DR5.
Thus, another object of the invention is a composition comprising two polypeptides, or antibodies or fragment thereof, both having the capability to bind to DR5, a first polypeptide or antibody comprising a first antigen-binding site that binds to a first epitope of said DR5, this first epitope being the one to which specifically binds a binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14 CDR1, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17, and a second polypeptide or antibody comprising a second different antigen-binding site that binds to a second epitope of said DR5, this epitope being the one to which specifically binds a binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21, for a simultaneous, separate or sequential administration to a mammal, including man. As an alternative, one may replace herein above the definition of the CDRs by those according to Kabat® or Common numbering System as per Tables 1 and 2.
Another object of the invention is a bispecific antibody, or fragment thereof, having the capability to bind to DR5, said antibody comprising a first antigen-binding site that binds to a first epitope of said DR5, this first epitope being the one to which specifically binds a binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14 CDR1, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17, and a second different antigen-binding site that binds to a second epitope of said DR5, this epitope being the one to which specifically binds a binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21. As an alternative, one may replace hereinabove the definition of the CDRs by those according to Kabat® or Common numbering System as per Tables 1 and 2.
Another object of the invention is the method of treatments, comprising the administration of an effective or sufficient amount of at least two polypeptides or antibodies as disclosed herein, or of at least one bispecific or biparatopic polypeptide or antibody as disclosed herein, or of at least two polypeptides or antibodies and at least one drug, as disclosed herein, or of at least one bispecific or biparatopic polypeptide or antibody and at least one drug, as disclosed herein. By treatment is meant in particular treatment of various cancers, autoimmune diseases, infectious diseases that express DR5 antigen.
DEFINITIONS
The terms “apoptosis” and “apoptotic activity” are used in a broad sense and refer to the orderly or controlled form of cell death in mammals that is typically accompanied by one or more characteristic cell changes, including condensation of cytoplasm, loss of plasma membrane microvilli, segmentation of the nucleus, degradation of chromosomal DNA or loss of mitochondrial function. This activity can be determined and measured, for instance, by cell viability assays, FACS analysis or DNA electrophoresis, and more specifically by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies).
As used herein, the term “synergy” or “synergism” or “synergistically” refers to the interaction of two or more agents so that their combined effect is greater than the sum of their individual effects.
The term “agonist” and “agonistic” when used herein refer to or describe a molecule which is capable of, directly or indirectly, substantially inducing, promoting or enhancing DR5 biological activity or activation. Optionally, an “agonist DR5 antibody” is an antibody which has activity at least comparable to the ligand for DR5, known as Apo-2 ligand (TRAIL), or is capable of activating DR5 receptor which results in an activation of one more intracellular signaling pathway which may include activation of caspase 3, caspase 8, caspase 10 or FADD.
The terms “antagonist” and “antagonistic” when used herein refer to or describe a molecule which is capable of, directly or indirectly, substantially counteracting, reducing or inhibiting DR5 biological activity of DR5 activation. Optionally, an antagonist is a molecule which neutralizes the biological activity resulting from DR5 activation or formation of a complex between DR5 and its ligand, such as Apo-2 ligand.
The term “antibody” is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multivalent antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
“Native antibodies” and “native immunoglobulins” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH).
As used herein, an “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
With respect to antibodies of the invention, the term “immunologically specific” or “specifically binds” refers to antibodies that bind to one or more epitopes of a protein of interest (e.g., DR5/TRAIL R2), but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
The “epitope DR5-01” and the “epitope DR5-05” are the regions in the extracellular domain of DR5 to which the DR5-01 and the DR5-05 antibodies bind respectively.
The term “bispecific antibody” as used herein refers to an antibody comprising two antigen-binding sites, a first binding site having affinity for a first antigen or epitope and a second binding site having binding affinity for a second antigen or epitope distinct from the first.
“Bispecific antibodies” or “biparatopic antibodies” are single, divalent antibodies which have two different specific antigen binding sites. According to this invention, these antibodies have two different binding sites, each one directed against a specific and different epitope on the DR5 molecule. This definition also encompasses the fragments of a bispecific or biparatopic antibody that comprise both binding site and wherein each of these binding sites has the capability of binding to the corresponding epitope on DR5. Such a fragment may be for example a F(ab′)2 antibody fragment.
The term “bivalent, bispecific antibody” as used herein refers to an antibody as described above in which each of the two pairs of heavy chain and light chain (HC/LC) are specifically binding to a different epitope, i.e. the first heavy and light chains are specifically binding together to a first epitope, and, the second heavy and light chains are specifically binding together to a second epitope; such bivalent, bispecific antibodies are capable of specifically binding to two different epitopes, at the same time or not.
According to the invention, the ratio of a desired bivalent, bispecific antibody compared to undesired side products can be improved by the replacement of certain domains in only one pair of heavy chain and light chain (HC/LC). While the first of the two HC/LC pairs originates from an antibody specifically binding to a first epitope and is left essentially unchanged, the second of the two HC/LC pairs originates from an antibody specifically binding to a second epitope, and is altered by the following replacement:
Light chain: replacement of the variable light chain domain VL by the variable heavy chain domain VH of said antibody specifically binding to a second epitope, and the constant light chain domain CL by the constant heavy chain domain CH of said antibody specifically binding to a second epitope and
Heavy chain: replacement of the variable heavy chain domain VH by the variable light chain domain VL of said antibody specifically binding to a second epitope, and the constant heavy chain domain CH by the constant light chain domain CL of said antibody specifically binding to a second epitope.
Engineered proteins, such as bi- or multivalent antibodies capable of binding two or more antigens or epitopes are known in the art. Such multivalent binding proteins can be generated using cell fusion, chemical conjugation, or recombinant DNA techniques.
In one approach bispecific antibodies that are very similar to natural antibodies have been produced using the quadroma technology, (Milstein C. et al., Nature. 1983; 305:537-40) based on the somatic fusion of two different hybridoma cell lines expressing murine monoclonal antibodies with the desired specificities of the bispecific antibody. Because of the random pairing of two different antibody heavy and light chains within the resulting hybrid-hybridoma (or quadroma) cell line, up to ten different antibodies species are generated of which only one is the desired, functional bispecific antibody. Due to the presence of mispaired byproducts, and significantly reduced production yields, means sophisticated purification procedures are required, (Morrison S. L., Nature Biotech. 2007; 25:1233-1234). In general the same problem of mispaired byproducts remains if recombinant expression techniques are used.
An approach to circumvent the problem of mispaired byproducts, which is known as “knobs-into-holes”, aims at forcing the pairing of two different antibody heavy chains by introducing mutations into the CH3 domains to modify the contact interface. On one chain bulky amino acids are replaced by amino acids with short side chains to create a “hole”. Conversely, amino acids with large side chains are introduced into the other CH3 domain, to create a “knob”. By coexpressing these two heavy chains (and two identical light chains, which have to be appropriate for both heavy chains), high yields of heterodimer formation (“knob-hole”) versus homodimer formation (“hole-hole” or “knob-knob”) may be observed, (Ridgway, J B et al., Protein Eng. 1996; 9:617-621; and WO 96/027011).
“Antibody fragments” comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. A suitable “antibody fragment” is a fragment of antibody that has the capability to bind to the DR5 epitope and initiate the apoptosis pathway.
Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies, (Zapata et al., Protein Eng. 1995; 8(10):1057-1062); single-chain antibody molecules; and multivalent antibodies formed from antibody fragments.
An “intact” antibody is one which comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each comprising a single antigen-binding site and a CL and a CH1 region, and a residual Fc fragment. Pepsin treatment yields an “F(ab′)2” fragment that has two antigen-binding sites and is still capable of cross-linking antigen.
“Fv” is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions (CDRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions or CDRs confer antigen-binding specificity to the antibody.
The “Fab” fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain and has one antigen-binding site only.
“Fab” fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known (Hermanson et al., Bioconjugate Techniques, Academic Press, 1996, U.S. Pat. No. 4,342,566).
“Single-chain Fv” or “scFv” antibody fragments comprise the VH and the VL domains of an antibody wherein these domains are present in a single polypeptide chain. Preferably, the scFv comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers as well as to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid. The term also includes variants on the traditional peptide linkage joining the amino acids making up the polypeptide. Preferred “peptides”, “polypeptides”, and “proteins” are chains of amino acids whose carbons are linked through peptide bonds.
The terminal amino acid at one end of the chain (amino terminal) therefore has a free amino group, while the terminal amino acid at the other end of the chain (carboxy terminal) has a free carboxyl group. As used herein, the term “amino terminus” (abbreviated N-terminus) refers to the free α-amino group on an amino acid at the amino terminal of a peptide or to the α-amino group (amino group when participating in a peptide bond) of an amino acid at any other location within the peptide. Similarly, the term “carboxy terminus” refers to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other location within the peptide. Peptides also include essentially any polyamino acid including, but not limited to peptide mimetics such as amino acids joined by ether as opposed to an amine bond.
The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated, in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies, (Kabat et al., NIH Publ. 1991; No. 91-3242, Vol. 1, 647-669). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effectors functions, such as participation of the antibody in antibody-dependent cellular toxicity.
The monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
Other preferred forms of “chimeric antibodies” encompassed by the present invention are those in which the constant region has been modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to C1q binding and/or Fc receptor (FcR) binding.
Such chimeric antibodies are also referred to as “class-switched antibodies”. Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art. See, e.g., Morrison, S. L et al., Proc. Natl. Acad. Sci. USA 1984; 81:6851-6855; U.S. Pat. No. 5,202,238 and U.S. Pat. No. 5,204,244. WO 2006/093794 relates to heterodimeric protein binding compositions. WO 99/37791 describes multipurpose antibody derivatives. Morrison et al., the J. Immunolog. 1998; 160:2802-2808 refers to the influence of variable region domain exchange on the functional properties of IgG.
“Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
In a preferred embodiment, a murine CDR is grafted into the framework region of a human antibody to prepare the “humanized antibody”. See, e.g., Riechmann, L. et al., Nature. 1988; 332: 323-327; and Neuberger, M S et al., Nature. 1985; 314: 268-270. Particularly preferred CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric antibodies. Other forms of “humanized antibodies” encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to C1q binding and/or Fc receptor (FcR) binding.
Furthermore, humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature. 1986; 321:522-525; Reichmann et al., Nature. 1988; 332:323-329; and Presta et al., Curr. Op. Struct. Biol. 1992; 2:593-596.
Immune effector functions which have been shown to contribute to antibody-mediated cytotoxicity include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).
Cytotoxicity may also be mediated via antiproliferative effects. The mechanism of antibody modulation of tumor cell proliferation is poorly understood. However, advances in understanding the interactions of antibodies with Fcg receptors (FcgR) on immune effector cells have allowed the engineering of antibodies with significantly improved effector function.
The mechanism of action of MAbs is complex and appears to vary for different MAbs. There are multiple mechanisms by which MAbs cause target cell death. These include apoptosis, CDC, ADCC and inhibition of signal transduction.
Effector functions such as CDC and ADCC are effector functions that may be important for the clinical efficacy of MAbs. All of these effector functions are mediated by the antibody Fc region and let authors to attempt amino acid modifications with more or less success. Glycosylation, especially fucosylation of the Fc region have a dramatic influence on the efficacy of an antibody. This let the authors to modify the conditions of production of the antibodies in the CHO cells in order to change the glycosylation profile in an attempt here again to improve some effector functions, with more or less success one again.
Previous research has shown that a polymorphism of the FcgRIIIa gene encodes for either a phenylalanine (F) or a valine (V) at amino acid 158. Expression of the valine isoform correlates with increased affinity and binding to MAbs (Rowland A J, et al. 1993. Cancer Immunol Immunother. 37(3):195-202; Sapra P, Allen T M. 2002. Cancer Res 62: 7190-4; Mølhøj M, et al. 2007. Mol Immunol. 44(8):1935-43). Some clinical studies have supported this finding, with greater clinical response to rituximab in patients with non-Hodgkin's lymphoma who display the V/V polymorphism (Bargou R, et al. 2008. Science. 321:974-7; Bruenke J, 2005. Br J Haematol. 130(2):218-28; Cartron G, Blood. 2002 Feb. 1; 99(3):754-8; Hekman A, et al. 1991. Cancer Immunol Immunother 32:364-72).
WO1999051642 describes a variant human IgG Fc region comprising an amino acid substitution at positions 270 or 329, or at two or more of positions 270, 322, 329, and 331. These modifications aim at increasing the CDC and ADCC effector functions
“Treatment” or “therapy” refer to both therapeutic treatment and prophylactic or preventative measures.
“Mammal” for purposes of treatment or therapy refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.
The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, renal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
The term “nucleic acid” or “oligonucleotide” or grammatical equivalents herein refer to at least two nucleotides covalent linked together. A nucleic acid of the present invention is preferably single-stranded or double stranded and will generally contain phosphodiester bonds.
Amino acid sequence “variants” (or mutants) of the antibody are prepared by introducing appropriate nucleotide changes into the antibody DNA, or by nucleotide synthesis. Such modifications can be performed, however, only in a very limited range, e.g. as described above. For example, the modifications do not alter the above mentioned antibody characteristics such as the IgG isotype and antigen binding, but may improve the yield of the recombinant production, protein stability or facilitate the purification.
DETAILED DESCRIPTION OF THE INVENTION
The CDR sequences may be defined in accordance with IMGT®, Kabat® or the Common numbering system which retain the common sequence between IMGT® and Kabat®.
The CDRs for the anti-DR5 antibodies mDR5-01 a chimeric antibody with murine VH and VL and human Fc) and HzDR5-01 (a humanized antibody with murine CDRs and human FR with or without back mutation and Fc optimized or not) of the invention comprises the following CDRs:
TABLE 1
Sequence
SEQ
SEQ
SEQ
(Common
ID
Sequence
ID
Sequence
ID
numbering
NO:
IMGT ®
NO:
Kabat ®
NO:
system)
VH mDR5-01 - VH HzDR5-01
CDR1
13
GFNIKDTF
22
DTFIH
32
KDTF
CDR2
14
IDPANGNT
23
RIDPANGNTKYDPKFQG
14
IDPANGNT
CDR3
15
VRGLYTYYFDY
24
GLYTYYFDY
24
GLYTYYFDY
VL mDR5-01 - VH HzDR5-01
CDR1
16
QSISNN
25
RASQSISNNLH
16
QSISNN
CDR2
FAS
26
FASQSIS
FAS
CDR3
17
QQGNSWPYT
17
QQGNSWPYT
17
QQGNSWPYT
The CDRs for the anti-DR5 antibodies mDR5-05 and HzDR5-05 of the invention comprises the following CDRs:
TABLE 2
Sequence
SEQ
SEQ
SEQ
(Common
ID
Sequence
ID
Sequence
ID
numbering
NO:
IMGT ®
NO:
Kabat ®
NO:
system)
VH mDR5-05 - VH HzDR5-05
CDR1
18
GFNIKDTH
27
DTHIH
33
KDTH
CDR2
14
IDPANGNT
28
RIDPANGNTEYDPKFQG
14
IDPANGNT
CDR3
19
ARWGTNVYFAY
29
WGTNVYFAY
29
WGTNVYFAY
VL mDR5-05 - VH HzDR5-05
CDR1
20
SSVSY
30
SASSSVSYMY
20
SSVSY
CDR2
RTS
31
RTSNLAS
RTS
CDR3
21
QQYHSYPPT
21
QQYHSYPPT
21
QQYHSYPPT
By definition, these CDRs include variant CDRs, by deletion, substitution or addition of one or more amino acid(s), which variant keeps the specificity of the original CDR. The common numbering system provides for a CDR definition having the shortest amino acid sequences or the minimal CDR definition.
mDR5-01, mDR5-05, HzDR5-01 and HzDR5-05 have the VH and VL amino acid sequences and nucleic acid sequences are depicted on the following tables:
TABLE 3
Amino acid
Amino acid
sequence VH
sequence VL
mDR5-01
SEQ ID NO: 4
SEQ ID NO: 2
mDR5-05
SEQ ID NO: 8
SEQ ID NO: 6
HzDR5-01
SEQ ID NO: 35
SEQ ID NO: 37
HzDR5-05
SEQ ID NO: 39
SEQ ID NO: 41
TABLE 4
Nucleic acid
Nucleic acid
sequence VH
sequence VL
mDR5-01
SEQ ID NO: 3
SEQ ID NO: 1
mDR5-05
SEQ ID NO: 7
SEQ ID NO: 5
HzDR5-01
SEQ ID NO: 34
SEQ ID NO: 36
HzDR5-05
SEQ ID NO: 38
SEQ ID NO: 40
DR5-01 and DR5-05 have the CH and CL amino acid sequences and nucleic acid sequences are depicted on the following tables:
TABLE 5
CH
CL
Amino acid sequence
SEQ ID NO: 10
SEQ ID NO: 12
Nucleic acid sequence
SEQ ID NO: 9
SEQ ID NO: 11
In an embodiment, the polypeptide comprises one or two binding domains comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17. This polypeptide binds specifically to a first epitope on the DR5 receptor. In an embodiment, the polypeptide comprises two such binding domains.
In an embodiment, the polypeptide comprises one or two binding domains comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 22, a CDR2 of sequence SEQ ID NO: 23, a CDR3 of sequence SEQ ID NO: 24; and the VL chain contains a CDR1 of sequence SEQ ID NO: 25, a CDR2 of sequence SEQ ID NO: 26, a CDR3 of sequence SEQ ID NO: 17. This polypeptide binds specifically to a first epitope on the DR5 receptor. In an embodiment, the polypeptide comprises two such binding domains.
In an embodiment, the polypeptide comprises one or two binding domains comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 32, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 24; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17. This polypeptide binds specifically to a first epitope on the DR5 receptor. In an embodiment, the polypeptide comprises two such binding domains.
In another embodiment, the polypeptide comprises a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21. This polypeptide binds specifically to a second and different epitope on the DR5 receptor. In an embodiment, the polypeptide comprises two such binding domains.
In another embodiment, the polypeptide comprises a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 27, a CDR2 of sequence SEQ ID NO: 28, a CDR3 of sequence SEQ ID NO: 29; and the VL chain contains a CDR1 of sequence SEQ ID NO: 30, a CDR2 of sequence SEQ ID NO: 31, a CDR3 of sequence SEQ ID NO: 21. This polypeptide binds specifically to a second and different epitope on the DR5 receptor. In an embodiment, the polypeptide comprises two such binding domains.
In another embodiment, the polypeptide comprises a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 33, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 29; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21. This polypeptide binds specifically to a second and different epitope on the DR5 receptor. In an embodiment, the polypeptide comprises two such binding domains.
In another embodiment, the anti-DR5 polypeptide comprises two binding domains and these two binding domains are each specific of a different epitope on the DR5 receptor. These binding domains comprise a specific set of 3 CDRs on the VH and VL as disclosed and provided therein and may be identical or slightly different in the framework regions.
The anti-DR5 polypeptide may be in particular a F(ab′)2, Fab, Fv, a divalent single-chain variable fragment (scFv), an antibody, preferably a monoclonal antibody, fragment, nanobody, multimeric scFv.
In this embodiment, the anti-DR5 polypeptide, preferably antibody, is bispecific or biparatopic and comprises:
a first binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 13, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 16, a CDR2 comprising or consisting of sequence FAS, a CDR3 comprising or consisting of sequence SEQ ID NO: 17, and
a second binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 18, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 20, a CDR2 comprising or consisting of sequence RTS, a CDR3 comprising or consisting of sequence SEQ ID NO: 21.
In an embodiment, the anti-DR5 polypeptide, preferably antibody, is bispecific or biparatopic and comprises:
a first binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO:22, a CDR2 comprising or consisting of sequence SEQ ID NO: 23, a CDR3 comprising or consisting of sequence SEQ ID NO: 24; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 25, a CDR2 comprising or consisting of sequence SEQ ID NO: 26, a CDR3 comprising or consisting of sequence SEQ ID NO: 17, and
a second binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 27, a CDR2 comprising or consisting of sequence SEQ ID NO: 28, a CDR3 comprising or consisting of sequence SEQ ID NO: 29; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 30, a CDR2 comprising or consisting of sequence SEQ ID NO: 31, a CDR3 comprising or consisting of sequence SEQ ID NO: 21.
In an embodiment, the anti-DR5 polypeptide, preferably antibody, is bispecific or biparatopic and comprises:
a first binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 32, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 24; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 16, a CDR2 comprising or consisting of sequence FAS, a CDR3 comprising or consisting of sequence SEQ ID NO: 17, and
a second binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 33, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 29; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 20, a CDR2 comprising or consisting of sequence RTS, a CDR3 comprising or consisting of sequence SEQ ID NO: 21.
This bispecific or biparatopic anti-DR5 polypeptide or antibody comprises the two different domains of the invention and may bind specifically to either one of the two different epitopes or to both different epitopes at the same time.
In some embodiments, the anti-DR5 polypeptides preferably antibody of the invention comprises:
one or more of amino acid sequence pairs SEQ ID NO: 2 and 4 (VL and VH from DR5-01) and SEQ ID NO: 6 and 8 (VH and VL from DR5-05),
the pair of amino acid sequences SEQ ID NO: 2 and 4, (VL and VH from DR5-01)
the pair of amino acid sequences SEQ ID NO: 6 and 8, (VL and VH from DR5-05) or
both amino acid sequence pairs SEQ ID NO: 2 and 4 (VL and VH from DR5-01) and SEQ ID NO: 6 and 8 (VL and VH from DR5-05);
one or more of amino acid sequence pairs SEQ ID NO: 35 and 37 (VH and VL from HzDR5-01) and SEQ ID NO: 39 and 41 (VH and VL from HzDR5-05),
the pair of amino acid sequences SEQ ID NO: 35 and 37, (VH and VL from HzDR5-01)
the pair of amino acid sequences SEQ ID NO: 39 and 41, (VH and VL from HzDR5-05) or
both amino acid sequence pairs SEQ ID NO: 35 and 37 (VH and VL from HzDR5-01) and SEQ ID NO: 39 and 41 (VL and VH from HzDR5-05).
In some embodiments, the anti-DR5 polypeptide, preferably antibody of the invention comprises:
two of each amino acid sequences SEQ ID NO: 4, 10, 2 and 12 (e.g. the whole or intact DR5-01 antibody)
amino acid sequences SEQ ID NO: 4, 10, 2 and 12 (single chain Fv based on DR5-01);
two of each amino acid sequences SEQ ID NO: 8, 10, 6 and 12 (e.g. the whole or intact DR5-05 antibody)
amino acid sequences SEQ ID NO: 8, 10, 6 and 12 (single chain Fv based on DR5-05);
amino acid sequences SEQ ID NO: 4, 8, 2, 6, 10 and 12 (bispecific antibody), especially the bispecific antibody comprises SEQ ID NO: 4, 8, 2, 6 (one of each) and 10, 12 (two of each);
amino acid sequences SEQ ID NO: 2 and 12 (light chain);
amino acid sequences SEQ ID NO: 6 and 12 (light chain);
amino acid sequences SEQ ID NO: 4 and 10 (heavy chain);
amino acid sequences SEQ ID NO: 8 and 10 (heavy chain).
two of each amino acid sequences SEQ ID NO: 35, 10, 37 and 12 (e.g. the whole or intact HzDR5-01 antibody)
amino acid sequences SEQ ID NO: 35, 10, 37 and 12 (single chain Fv based on HzDR5-01);
two of each amino acid sequences SEQ ID NO: 39, 10, 41 and 12 (e.g. the whole or intact HzDR5-05 antibody)
amino acid sequences SEQ ID NO: 39, 10, 41 and 12 (single chain Fv based on HzDR5-05);
amino acid sequences SEQ ID NO: 35, 39, 37, 41, 10 and 12 (bispecific antibody), especially the bispecific antibody comprises SEQ ID NO: 35, 39, 37, 41 (one of each) and 10, 12 (two of each);
amino acid sequences SEQ ID NO: 37 and 12 (light chain);
amino acid sequences SEQ ID NO: 41 and 12 (light chain);
amino acid sequences SEQ ID NO: 35 and 10 (heavy chain);
amino acid sequences SEQ ID NO: 39 and 10 (heavy chain).
The anti-DR5 polypeptides, preferably antibodies, of the invention may be fully murine, say they comprise amino acid sequences that match with the amino acid sequence of the maternal or original murine antibody. The polypeptides of the invention may also be chimeric or humanized, say they can comprise human-derived amino acid sequences. Specifically, the polypeptide may comprise framework regions and/or constant regions of a human-derived antibody.
Another object of the invention is a composition or pharmaceutical composition comprising one, two or more polypeptides according to the invention, as disclosed above and provided herein, and a pharmaceutically acceptable carrier, diluent or excipient. Embodiments of these compositions are defined by using the CDRs definitions according to IMGT®. However, the invention encompasses and relates also to the equivalent or alternative compositions wherein the IMGT® numbering is replaced either by the Kabat® numbering or the Common numbering system, using the sequences indicated supra. Therefore, in the following embodiments of a composition, other embodiments are part of the invention in which one replaces the CDRs defined with IMGT® numbering, by the Kabat® numbering, in accordance with the table supra. Also, in the following embodiments of a composition, other embodiments are part of the invention in which one replaces the CDRs defined with IMGT® numbering, by the Common numbering system, in accordance with the table supra.
In a first embodiment, the composition comprises a polypeptide, preferably antibody, having one or two binding domain(s) comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17. In a second embodiment, the composition comprises a polypeptide, preferably antibody, having one or two binding domain(s) comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21. In a third embodiment, the composition comprises these two polypeptides or antibodies in mixture. As an alternative, one may replace hereinabove the definition of the CDRs by those according to Kabat® or Common numbering System as per Tables 1 and 2.
In another embodiment, the composition comprises a anti-DR5 bispecific polypeptide, preferably antibody, comprising a first binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17, and a second binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21. As an alternative, one may replace hereinabove the definition of the CDRs by those according to Kabat® or Common numbering System as per Tables 1 and 2.
In an embodiment, the composition comprises an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 2 and 4, an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 6 and 8, and a pharmaceutically carrier, diluents or excipient. In an embodiment, the composition comprises an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 35 and 37, an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 39 and 41, and a pharmaceutically carrier, diluents or excipient.
The present invention also relates to these compositions comprising at least two polypeptides, preferably antibodies, for a simultaneous, separate or sequential administration to a mammal, including man.
A particular object is a composition comprising a bispecific anti-DR5 antibody comprising an amino acid sequence pair SEQ ID NO: 2 and 4 and an amino acid sequence pair SEQ ID NO: 6 and 8, or comprising an amino acid sequence pair SEQ ID NO: 35 and 37 and an amino acid sequence pair SEQ ID NO: 39 and 41, and a pharmaceutically acceptable carrier.
An object of the invention is especially a composition comprising at least one or two polypeptides binding specifically a DR5 receptor, wherein the at least one or two polypeptides comprise two immunoglobulin binding domains comprising:
a first binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14 CDR1, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17, and
a second binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21,
wherein
the at least one polypeptide comprises both immunoglobulin binding domains, or
the at least two polypeptides comprise a first polypeptide comprising the first binding domain and a second polypeptide comprising the second binding domain for a simultaneous, separate or sequential administration to a mammal, including man,
and a pharmaceutically carrier, diluent or excipient. As an alternative, one may replace hereinabove the definition of the CDRs by those according to Kabat® or Common numbering System as per Tables 1 and 2.
In some embodiments, the composition of the invention comprises an anti-DR5 polypeptide, preferably antibody comprising:
two of each amino acid sequences SEQ ID NO: 4, 10, 2 and 12 (e.g. the whole or intact DR5-01 antibody)
amino acid sequences SEQ ID NO: 4, 10, 2 and 12 (single chain Fv based on DR5-01);
two of each amino acid sequences SEQ ID NO: 8, 10, 6 and 12 (e.g. the whole or intact DR5-05 antibody)
amino acid sequences SEQ ID NO: 8, 10, 6 and 12 (single chain Fv based on DR5-05);
amino acid sequences SEQ ID NO: 4, 8, 2, 6, 10 and 12 (bispecific antibody), especially the bispecific antibody comprises SEQ ID NO: 4, 8, 2, 6 (one of each) and 10, 12 (two of each);
amino acid sequences SEQ ID NO: 2 and 12 (light chain);
amino acid sequences SEQ ID NO: 6 and 12 (light chain);
amino acid sequences SEQ ID NO: 4 and 10 (heavy chain);
amino acid sequences SEQ ID NO: 8 and 10 (heavy chain);
two of each amino acid sequences SEQ ID NO: 35, 10, 37 and 12 (e.g. the whole or intact HzDR5-01 antibody)
amino acid sequences SEQ ID NO: 35, 10, 37 and 12 (single chain Fv based on HzDR5-01);
two of each amino acid sequences SEQ ID NO: 39, 10, 41 and 12 (e.g. the whole or intact HzDR5-05 antibody)
amino acid sequences SEQ ID NO: 39, 10, 41 and 12 (single chain Fv based on HzDR5-05);
amino acid sequences SEQ ID NO: 35, 39, 37, 41, 10 and 12 (bispecific antibody), especially the bispecific antibody comprises SEQ ID NO: 35, 39, 37, 41 (one of each) and 10, 12 (two of each);
amino acid sequences SEQ ID NO: 37 and 12 (light chain);
amino acid sequences SEQ ID NO: 41 and 12 (light chain);
amino acid sequences SEQ ID NO: 35 and 10 (heavy chain);
amino acid sequences SEQ ID NO: 39 and 10 (heavy chain).
These compositions may comprise at least one additional polypeptide or antibody directed against another target and/or at least one chemotherapeutic drug (such as small molecule), for a simultaneous, separate or sequential administration with polypeptide(s) or antibody(ies) of the invention, to a mammal, including man. As additional active principle, one may cite doxorubicine, gemcitabine, camptothecin, paclitaxel. The composition may comprise two polypeptides, or antibodies or fragments thereof, both having the capability to bind to DR5, modified to comprise a variant human optimized IgG Fc region, preferably IgG1 Fc region, wherein this variant region comprises an amino acid substitution to modulate PDCC, ADCC and/or CDC. In particular, two polypeptides, or antibodies or fragments thereof, have the capability to bind to DR5, and conjugate to cellular cytotoxic components (ADC)z.
The compositions or pharmaceutical compositions according to the invention are intended for use as a medicament, especially to induce apoptosis of a tumor cell. The compositions or pharmaceutical compositions according to the invention are intended for use as a medicament, especially to treat cancer, preferably a solid cancer.
The isolated nucleic acid sequences disclosed and provided herein are also object of the invention.
Thus the invention also relates to an isolated nucleotide sequence comprising the following nucleotide sequences SEQ ID NO: 1, 3, 5, or 7 or combinations of nucleotide sequences linked together; SEQ ID NO: 9 and 7, or 9 and 3, SEQ ID NO: 11 and 1, or 11 and 5. The invention also relates to an isolated nucleotide sequence comprising the following nucleotide sequences SEQ ID NO: 34, 36, 38 or 40 or combinations of nucleotide sequences linked together; SEQ ID NO: 9 and 34, or 9 and 38, SEQ ID NO: 11 and 36, or 11 and 40.
The present invention also relates to a method of prevention and/or treatment of a disease wherein inducing apoptosis of some cell is beneficial to the mammal, in particular the human in terms of prevention or treatment (therapeutic or prophylactic). Those diseases are in particular cancer, especially one of those listed in the Definitions supra, autoimmune diseases, inflammatory conditions, viral infections and viral diseases. This method comprises the administration to a mammal, including human, of an effective amount of a composition as disclosed and provided herein. The method comprises the administration of the two polypeptides, preferably antibodies directed against the two different epitopes according to the invention, or of the bispecific polypeptide, preferably antibody directed against the two different epitopes according to the invention. Embodiments of these compositions are defined by using the CDRs definitions according to IMGT®. However, the invention encompasses and relates also to the equivalent or alternative methods wherein the IMGT® numbering is replaced either by the Kabat® numbering or the Common numbering system, using the sequences indicated supra. Therefore, in the following embodiments of a method, other embodiments are part of the invention in which one replaces the CDRs defined with IMGT® numbering, by the Kabat® numbering, in accordance with the table supra. Also, in the following embodiments of a method, other embodiments are part of the invention in which one replaces the CDRs defined with IMGT® numbering, by the Common numbering system, in accordance with the table supra.
In a first embodiment, the method comprises the administration of a composition which comprises a polypeptide, preferably antibody, having one or two binding domain(s) comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17. In a second embodiment, the method comprises the administration of a composition which comprises a polypeptide, preferably antibody, having one or two binding domain(s) comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21. In a third embodiment, the method comprises the administration of a composition which comprises these two polypeptides or antibodies in mixture, or of two compositions, one containing the first mentioned polypeptide or antibody, and the second comprising the second mentioned polypeptide or antibody. As an alternative, one may replace hereinabove the definition of the CDRs by those according to Kabat® or Common numbering System as per Tables 1 and 2.
In another embodiment, the method comprises the administration of a composition which comprises a bispecific polypeptide, preferably antibody, comprising a first binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14 CDR1, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17, and a second binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21. As an alternative, one may replace hereinabove the definition of the CDRs by those according to Kabat® or Common numbering System as per Tables 1 and 2.
In an embodiment, the method provides for the administration of a composition which comprises an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 2 and 4 and an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 6 and 8, and a pharmaceutically carrier, diluent or excipient. In another embodiment, the method provides for the administration of two compositions, one which comprises an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 2 and 4 and another which comprises an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 6 and 8. In an embodiment, the method provides for the administration of a composition which comprises an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 35 and 37 and an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 39 and 41, and a pharmaceutically carrier, diluent or excipient. In another embodiment, the method provides for the administration of two compositions, one which comprises an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 35 and 37 and another which comprises an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 39 and 41.
In another embodiment, the method provides for the administration of a composition comprising a bispecific anti-DR5 antibody comprising an amino acid sequence pair SEQ ID NO: 2 and 4 and an amino acid sequence pair SEQ ID NO: 6 and 8, and a pharmaceutically acceptable carrier. In another embodiment, the method provides for the administration of a composition comprising a bispecific anti-DR5 antibody comprising an amino acid sequence pair SEQ ID NO: 35 and 37 and an amino acid sequence pair SEQ ID NO: 39 and 41, and a pharmaceutically acceptable carrier.
In another embodiment, the method provides for the administration of a composition comprising the DR5-01 and the DR5-05 antibodies as disclosed and provided herein, or similar antibodies produced through genetic engineering as described herein, based on nucleotide sequences SEQ ID NO: 9, 3, 11 and 1, or SEQ ID NO: 9, 34, 11 and 36 for DR5-01, and SEQ ID NO: 9, 7, 11 and 5, or SEQ ID NO: 9, 38, 11 and 40 for DR5-05; use can be made of a composition comprising these antibodies defined by their amino acid sequences and comprising SEQ ID NO: 4, 10, 2 and 12 for DR5-01 and SEQ ID NO: 8, 10, 6 and 12 for DR5-05, or SEQ ID NO: 35, 10, 37 and 12 for HzDR5-01 and SEQ ID NO: 39, 10, 41 and 12 for HzDR5-05.
The pharmaceutical compositions, uses and methods of treatment are thus intended for the prevention and/or treatment of cancer. A list of cancers that may beneficiate from the invention is given supra in the Definitions.
The pharmaceutical compositions, uses and methods of treatment are thus also intended for the prevention and/or treatment of autoimmune diseases and inflammatory conditions. The following diseases are in particular concerned.
The pharmaceutical compositions, uses and methods of treatment are thus also intended for the prevention and/or treatment of viral infection or viral diseases. Viral infections and diseases include, but are not limited to, infections with cytomegalovirus, influenza, Newcastle, disease virus, vesicular stomatitis virus, herpes simplex virus, hepatitis, adenovirus-2, bovine viral diarrhoea virus, human immunodeficiency virus (HIV), and Epstein-Barr virus.
In a particular embodiment, the polypeptides, antibodies or bispecific antibodies of this invention can also be used to specifically label cancer cells, solid tumors, and the like, and more generally, to specifically target/deliver any conjugated or otherwise coupled effector (e.g. radioisotope, label, cytotoxin, drug, liposome, antibody, nucleic acid, dendrimer, etc. . . . ) to cancer cells including, but not limited to, isolated cancer cells, metastatic cells, solid tumor cells, and the like.
Therefore, another object of the invention is a complex of a polypeptide according to the invention and a molecule, which is an effector molecule, which function may beneficiate from the targeting of the DR5 receptor by the polypeptide. Such an effector molecule may be a radioisotope, a label, a cytotoxin, a drug, a liposome, an antibody, a nucleic acid, a dendrimer. The invention also concerns a pharmaceutical composition containing this complex and a pharmaceutically acceptable vehicle, diluent or excipient.
The invention also concern the use of such composition, and a method as well, to prevent or treat a cancer, such as one of those cited supra in the Definitions.
A polypeptide or the polypeptides of this invention may be used to identify other polypeptides or antibodies that bind to one of the epitopes against which the DR5-01 and the DR5-05 are directed. Thus, in certain embodiments, a polypeptide or antibody of this invention, directed against one epitope, can be used or paired with another antibody with binding specificity for the other epitope DR5.
A polypeptide or the polypeptides of this invention may be used to identify other polypeptides or antibodies that bind to another epitope on DR5, which upon binding of polypeptides or antibodies on these various epitopes on DR5, induce apoptosis. DR5-01 and/or the DR5-05 are directed. Thus, in certain embodiments, a polypeptide or antibody of this invention, directed against one epitope (DR5-01 or DR5-05), or polypeptides or antibodies of this invention, directed against both epitopes (DR5-01 and DR5-05) can be used with another antibody with binding specificity for another epitope on DR5.
One or more polypeptides, antibodies, bispecific antibodies, and/or functionalized bispecific antibodies, and/or chimeric moieties of this invention, or pharmaceutical compositions containing the same, can be administered by injection, that is, intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally or intraperitoneally. Also in certain embodiments, the compounds can be administered by inhalation, for example, intranasally. Other pharmaceutical delivery systems can also be employed, for example, liposomes.
Targeting DR5 with the polypeptides or antibodies of the present invention in combination with existing chemotherapeutic treatments will be more effective in killing the tumor cells than chemotherapy alone. A wide variety of drugs have been employed in chemotherapy of cancer. Examples include, but are not limited to, cisplatin, taxol, etoposide, mitoxantrone, actinomycin D, campthotecin, methotrexate, gemcitabine, mitomycin, dacarbazine, 5-fluorouracil, doxorubicine and daunomycin.
In one approach, antibody combination or bispecific antibody anti-DR5 MAb is added to a standard chemotherapy regimen, in treating a cancer patient. For those combinations in which the antibody and additional anti-cancer agent(s) exert a synergistic effect against cancer cells, the dosage of the additional agent(s) may be reduced, compared to the standard dosage of the second agent when administered alone. The antibody may be co-administered with an amount of an anti-cancer drug that is effective in enhancing sensitivity of cancer cells to the antibody combination or bispecific antibody.
In one method of the invention, targeting DR5 with antibody combination or bispecific antibody, is administered to the patient prior to administration of a second anti-cancer agent. One alternative method comprises administering the second anti-cancer agent prior to administering the antibody combination or the bispecific antibody and second agent on an alternative schedule. In another embodiment, the antibody combination or bispecific antibody and second agent are administered simultaneously.
The method of the invention may provide for the inclusion in a therapeutic regimen involving the use of at least one other treatment method, such as irradiation, chemotherapy with small molecule or antibody. The method of the invention may directly include the administration of a sufficient amount of at least one additional polypeptide or antibody directed against another target and/or at least one chemotherapeutic drug (such as small molecule), for a simultaneous, separate or sequential administration with polypeptide(s) or antibody(ies) of the invention, to a mammal, including man. As additional active principle, one may cite doxorubicine, gemcitabine, camptothecin, paclitaxel or the other drugs mentioned above. In an embodiment, lung cancer and breast cancer is treated using such combination. This combination more generally is useful for cancers (in particular aggressive cancers) which do not respond well to treatment with the drug alone or the antibodies/antibody of the invention alone, and for which the combination leads to a synergistic effect.
In one method of the invention, targeting DR5 with antibody combination or bispecific antibody or multivalent antibody fragment, may be employed in treating viral infections and associated conditions arising from viral infections. Viral infections, include, but are not limited to, infections with cytomegalovirus, influenza, Newcastle, disease virus, vesicular stomatitus virus, herpes simplex virus, hepatitis, adenovirus-2, bovine viral diarrhea virus, human immunodeficiency virus (HIV), and Epstein-Barr virus.
Mammalian cells are the preferred hosts for production of therapeutic glycoproteins, due to their capability to glycosylate proteins in the most compatible form for human applications. Bacteria very rarely glycosylate proteins, and like other type of common hosts, such as yeasts, filamentous fungi, insect and plant cells yield glycosylation patterns associated with rapid clearance from the blood stream.
Among mammalian cells, Chinese hamster ovary (CHO) cells are the most commonly used. In addition to giving suitable glycosylation patterns, these cells allow consistent generation of genetically stable, highly productive clonal cell lines. They can be cultured to high densities in simple bioreactors using serum-free media, and permit the development of safe and reproducible bioprocesses. Other commonly used animal cells include baby hamster kidney (BHK) cells, NSO- and SP2/0-mouse myeloma cells.
In an embodiment, the polypeptides and antibodies according to the invention are produced or expressed in mammal cells, preferably wild-type mammal cells, preferably of rodent origin, especially CHO cells.
Modifications and changes may be made in the structure of a polypeptide of the present invention and still obtain a molecule having like characteristics. For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a polypeptide with like properties.
In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics.
It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, for example, enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid may be substituted by another amino acid having a similar hydropathic index and still obtain a biologically functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within +2 is preferred, those which are within +1 are particularly preferred, and those within +0.5 are even more particularly preferred.
Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biologically functionally equivalent peptide or polypeptide thereby created is intended for use in immunological embodiments. U.S. Pat. No. 4,554,101, incorporated herein by reference or to which the person skilled in the art: may refer, states that the greatest local average hydrophilicity of a polypeptide, as governed by the hydrophilicity of its adjacent amino acids, correlate with its immunogenicity and antigenicity, i.e. with a biological property of the polypeptide.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 +1); glutamate (+3.0 +1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (−0.5 +1); threonine (−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent, polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within +2 is preferred, those which are within +1 are particularly preferred, and those within +0.5 are even more particularly preferred.
As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
TABLE 5
Amino Acid
Index
isoleucine L
(+4.5)
valine V
(+4.2)
leucine L
(+3.8)
phenylalanine
(+2.8)
cysteine C
(+2.5)
methionine M
(+1.9)
alanine A
(+1.8)
glycine G
(−0.4)
threonine T
(−0.7)
serine S
(−0.8)
tryptophan W
(−0.9)
tyrosine Y
(−1.3)
proline P
(−1.6)
histidine H
(−3.2)
glutamate E
(−3.5)
glutamine Q
(−3.5)
aspartate D
(−3.5)
asparagine N
(−3.5)
lysine K
(−3.9)
arginine R
(−4.5)
Amino acid substitution may be chosen or selected differently. Possible substitutions have been documented in WO99/51642, WO2007024249 and WO2007106707.
By definition, the CDRs of the invention include variant CDRs, by deletion, substitution or addition of one or more amino acid(s), which variant keeps the specificity of the original CDR. The common numbering system provides for a CDR definition having the shortest amino acid sequences or the minimal CDR definition.
The antibody may be a monoclonal antibody, a chimeric antibody, a humanized antibody, a full human antibody, a bispecific antibody, an antibody drug conjugate or an antibody fragment. A “humanized antibody” or “chimeric humanized antibody” shall mean an antibody derived from a non human antibody, typically a murine antibody, that retains or substantially retains the antigen-binding properties of the parental antibody, but which is less immunogenic in humans.
Methods for producing the polypeptides and antibodies are known from the person skilled in the art. The mammal cells, preferably rodent cells such as CHO cells, preferably wild-type cells are transfected with one or several expression vectors. Preferably, the cells are co-transfected with an expression vector for light chain and with an expression vector for heavy chain.
Cell transfection is also known from the person skilled in the art. As transfection that may be performed, one may mention without limitation standard transfection procedures, well-known from the man skilled in the art, such as calcium phosphate precipitation, DEAE-Dextran mediated transfection, electroporation, magnetofection, nucleofection (AMAXA Gmbh, GE), liposome-mediated transfection (using Dreamfect®, Lipofectin® or Lipofectamine® technology for example) or microinjection.
Expression vectors are known. As vectors that may be used, one may mention without limitation: pcDNA3.3, pOptiVEC, pFUSE, pMCMVHE, pMONO, pSPORT1, pcDV1, pcDNA3, pcDNA1, pRc/CMV, pSEC. One may use a single expression vector or several expression vectors expressing different parts of the polypeptide or antibody.
An expression vector for the CH1, hinge region, CH2 and CH3 comprises SEQ ID NO: 9 or comprises a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 10.
An expression vector contains a nucleic acid sequence encoding a variable region VH of the invention. In an embodiment, the vector comprises SEQ ID NO: 3 or comprises a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 4. In another embodiment, it comprises SEQ ID NO: 7 or comprises a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 8. In an embodiment, the vector comprises SEQ ID NO: 34 or comprises a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 35. In another embodiment, it comprises SEQ ID NO: 38 or comprises a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 39.
A set of expression vectors encoding a heavy chain, comprise an expression vector which comprises SEQ ID NO: 9 (or comprises a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 10), and either SEQ ID NO: 3 (or a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 4) or SEQ ID NO: 7 (or a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 8). A set of expression vectors encoding a heavy chain, comprise an expression vector which comprises SEQ ID NO: 9 (or comprises a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 10), and either SEQ ID NO: 34 (or a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 35) or SEQ ID NO: 38 (or a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 39).
A single expression vector for the heavy chain contains a nucleic acid sequence which encodes VH, CH1, hinge region, CH2, CH3. In an embodiment, the vector comprises SEQ ID NO: 3 and 9 or comprises a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 4 and 10. In another embodiment, it comprises SEQ ID NO: 7 and 9 or comprises a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 8 and 10. In an embodiment, the vector comprises SEQ ID NO: 34 and 9 or comprises a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 35 and 10. In another embodiment, it comprises SEQ ID NO: 38 and 9 or comprises a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 39 and 10.
An expression vector for the light constant chain comprises SEQ ID NO: 11 or comprises a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 12.
An expression vector contains a nucleic acid sequence encoding a variable region VL of the invention. In an embodiment, the vector comprises SEQ ID NO: 1 or a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 2. In another embodiment, it comprises SEQ ID NO: 5 or a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 6. In an embodiment, the vector comprises SEQ ID NO: 36 or a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 37. In another embodiment, it comprises SEQ ID NO: 40 or a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 41.
An expression vector contains a nucleic acid sequence encoding a light chain of the invention. In an embodiment, the vector comprises SEQ ID NO: 1 and 11 or a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 2 and 12. In another embodiment, it comprises SEQ ID NO: 5 and 11 or a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 6 and 12. In an embodiment, the vector comprises SEQ ID NO: 36 and 11 or a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 37 and 12. In another embodiment, it comprises SEQ ID NO: 40 and 11 or a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 41 and 12.
A set of expression vectors for producing a complete antibody comprise several vectors, for example two or three.
A single expression vector may also be used, which comprise either SEQ ID NO: 3, 9, 1 and 11 (or a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 4, 10, 2 and 12), or SEQ ID NO: 7, 9, 5 and 11 (or a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 8, 10, 6 and 12). A single expression vector may also be used, which comprise either SEQ ID NO: 34, 9, 36 and 11 (or a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 35, 10, 37 and 12), or SEQ ID NO: 38, 9, 40 and 11 (or a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 39, 10, 41 and 12).
The expression vector comprises a nucleic acid sequence or nucleic acid sequences which code(s) for the variable region that is wished. Various embodiments of variable regions which can be expressed by the vector are presented below. Embodiments of these vectors are defined by using the CDRs definitions according to IMGT®. However, the invention encompasses and relates also to the equivalent or alternative vectors wherein the IMGT® numbering is replaced either by the Kabat® numbering or the Common numbering system, using the sequences indicated supra. Therefore, in the following embodiments of a vector, other embodiments are part of the invention in which one replaces the CDRs defined with IMGT® numbering, by the Kabat® numbering, in accordance with the table supra. Also, in the following embodiments of a vector, other embodiments are part of the invention in which one replaces the CDRs defined with IMGT® numbering, by the Common numbering system, in accordance with the table supra.
An expression vector codes for a VH comprising a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14 CDR1, a CDR3 of sequence SEQ ID NO: 15.
An expression vector codes for a VL comprising a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17.
A set of expression vectors comprise an expression vector which codes for a VH comprising a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14 CDR1, a CDR3 of sequence SEQ ID NO: 15, and an expression vector which codes for a VL comprising a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17.
An expression vector comprises a nucleic acid sequence coding for a VH comprising a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14 CDR1, a CDR3 of sequence SEQ ID NO: 15, and a nucleic acid sequence coding for a VL comprising a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17.
An expression vector codes for a VH comprising a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19.
An expression vector codes for a VL comprising a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21.
A set of expression vectors comprise an expression vector which codes for a VH comprising a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19, and an expression vector which codes for a VL comprising a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21.
An expression vector comprises a nucleic acid sequence coding for a VH comprising a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19, and a nucleic acid sequence coding for a VL comprising a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21.
The invention thus comprises the use of one single vector or a set of vectors to produce the polypeptides or antibodies of the invention. These vectors are also objects of the invention, alone or as a set of vectors.
Another object of the invention is a host cell containing a vector or a set of vectors of the invention. The host cell may be a mammal cell, preferably a rodent cell, more preferably CHO cell. Still more preferably, the host cell may be a wild-type mammal cell, preferably a wild-type rodent cell, most preferably a wild-type CHO cell.
The person skilled in the art fully owns the methods to generate the antibodies according to the invention using such a vector or vectors and cells such as CHO cells.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will now be described in further detail by way of examples referring to the figure. Note that in the block diagrams, the blocks appear from the left to the right in the same order than indicated in the legend in the diagrams where the legend is put in a box.
FIG. 1 shows the FACS analysis of anti-DR5 antibody panel in human glioma cell lines (H4, HS683, A172, T98G, U87MG).
FIG. 2 shows the FACS analysis of anti-DR5 expression in some cancer cell lines such as human kidney adenocarcinoma (A704, ACHN, Caki1), human colon carcinoma (SW948, HCT 116), human urinary bladder carcinoma (5637) and human breast adenocarcinoma (MCF7).
FIG. 3 is a graph showing the results of an ELISA assay evaluating binding of MAbs (1 μg/mL) to Fas (50 ng/mL), FasL (100 ng/mL), TRAIL (100 ng/mL) and to DR4, DR5, DcR1 or DcR2 (50 ng/mL), (mean+/−SD, n=2).
FIG. 4 is a bar diagram showing percent (%) of the inhibition of biotinylated anti-DR5 MAb binding (1 μg/mL, FACS analysis) in the presence of other unconjugated antibody anti-DR5 (5 μg/mL) using the T98G cells (1·106 cells/mL), (mean+/−SD, n=2).
FIG. 5 is a bar diagram showing percent (%) of the inhibition of TRAIL binding (100 ng/mL, FACS analysis) in the presence of antibody (MAb anti-TRAIL, MAb anti-DR5 MAb) tested at different concentrations using H4 cells (5·105 cells/mL), (mean+/−SD, n=2).
FIG. 6 is a bar diagram showing percent (%) of the cell proliferation inhibition (ATP bioluminescent bioassay, 72 hours) of anti-DR5 antibody alone or combined tested at 1 μg/mL as compared to TRAIL (10 ng/mL) using H4 cells (5·104 cells/mL), (mean+/−SD, n=2).
FIG. 7 is a bar diagram showing percent (%) of the cell proliferation inhibition (BrDU bioassay, 72 hours) of selective anti-DR5 agonistic antibody combination (mDR5-01+mDR5-05) versus neutral antibody combination (mDR5-05+mDR5-04) tested at different concentrations using H4 cells (5·104 cells/mL), (mean+/−SD, n=2).
FIG. 8 is a bar diagram showing percent (%) of apoptosis (propidium iodide staining, 72 hours) of selective anti-DR5 agonistic antibody combination (mDR5-01+mDR5-05) versus neutral antibody combination (mDR5-05+mDR5-04) tested at 1 μg/mL and also compared to TRAIL (10 ng/mL) using H4 cells (1·105 cells/mL), (mean+/−SD, n=2).
FIG. 9 is a bar diagram showing percent (%) of cleaved caspase 3 (FACS analysis, 48 hours) of selective anti-DR5 agonistic antibody combination (mDR5-01+mDR5-05) versus neutral antibody combination (mDR5-05+mDR5-04) and also compared to TRAIL using H4 cells (1·105 cells/mL), (representative experiment, n=2).
FIG. 10 is a western blot showing the cleaved PARP induced or not with the presence of selective anti-DR5 agonistic antibody combination (mDR5-01+mDR5-05) versus neutral antibody combination (mDR5-05+mDR5-04) using H4 cells (2·105 cells/mL, 5 hours).
FIG. 11 is a bar diagram showing percent (%) of the cell proliferation inhibition (ATP bioluminescent bioassay, 72 hours) with the selective anti-DR5 agonistic antibody combination (10 μg/mL mDR5-05+0.1 μg/mL mDR5-01), in the presence or not of anti-DR5 MAb (mDR5-01, mDR5-02, mDR5-04 or mDR5-05, 1 μg/mL) using H4 cells (5·104 cells/mL), (mean+/−SD, n=2).
FIG. 12 is a bar diagram showing percent (%) of the cell proliferation inhibition (ATP bioluminescent bioassay, 72 hours) of selective antibody anti-DR5 agonistic antibody combination (10 μg/mL mDR5-05+0.1 μg/mL mDR5-01) and then diluted at ½ compared to TRAIL (20 ng/mL) and then diluted at ½ using H4, HS683, A172, T98G or U87MG glioma cells (5·104 cells/mL), (mean+/−SD, n=2).
FIG. 13 is a bar diagram showing percent (%) of the proliferation inhibition (ATP bioluminescent bioassay, 72 hours) of chimeric antibody (chDR5-01 or chDR5-05 MAb) tested alone at 5 μg/mL then diluted at ½ versus antibody combination (5 μg/mL chDR5-05+0.05 μg/mL chDR5-01) then diluted at ½ using glioma H4 cells (5·104 cells/mL), (mean+/−SD, n=2).
FIG. 14 is a bar diagram showing percent (%) of the cell proliferation inhibition (ATP bioluminescent bioassay, 72 hours) of anti-DR5 antibody alone or combined tested at 10 μg/mL (ratio 1/10) as compared to TRAIL (50 ng/mL) using ex-vivo human glioma cells (5·104 cells/mL), (mean+/−SD, n=3 from three independent ex vivo GBM cells).
FIGS. 15-18 are bar diagrams showing percent (%) of the cell proliferation inhibition (ATP bioluminescent bioassay, 72 hours) in the presence of mouse anti-DR5 antibody combined tested at 10 μg/mL diluted at 1/10, in the presence of drug alone (1 μg/mL diluted at 1/10) or in association mouse anti-DR5 antibody combined and drug using HS683, A172, 42MGBA or T98G glioma cells, (5·104 cells/mL), (mean+/−SD, n=2), (Campothecin (CPT)).
FIGS. 19-22 are bar diagrams showing percent (%) of the cell proliferation inhibition (ATP bioluminescent bioassay, 72 hours) in the presence of mouse anti-DR5 antibody combined tested at 10 μg/mL diluted at 1/10, in the presence of drug alone (1 μg/mL diluted at 1/10) or in association mouse anti-DR5 antibody combined and drug using human breast cell lines (MCF7, MDAMB231) or human lung adenocarcinoma cell lines (NCIH1703, A549), (5·104 cells/mL), (mean+/−SD, n=2).
FIG. 23 is a bar diagram showing percent (%) of the cell proliferation inhibition (ATP bioluminescent bioassay, 72 hours) of mouse anti-DR5 antibody alone or combined as compared to humanized anti-DR5 antibody alone or combined tested at 1 μg/mL (ratio 1/1 in combined) then diluted at 1/2 using H4 glioma cells cells (5·104 cells/mL), (mean+/−SD, n=2).
FIG. 24 is a survival curve of nude mice orthotopic engrafted with SC2 human glioma treated with or without mouse anti-DR5 antibody combined. MAb treatment was administrated by intraperitoneal injection (IP) at 5 mg/kg per mouse until mice euthanasia due to loss of weight and was applied during 36 days maximum. Survival times obtained with control group were compared to survival times obtained with treated groups (mDR5-01+mDR5-05 versus mDR5-04+mDR5-05) using Kaplan Meier method and Wilcoxon statistical test (JMP software).
FIG. 25 Amino acid and nucleic acid sequence for VH HzDR5-01 with description of the FR1, CDR1, FR2, CDR2, FR3, CDR3 defining according IMGT®.
FIG. 26 Amino acid and nucleic acid sequence for VL HzDR5-01 with description of the FR1, CDR1, FR2, CDR2, FR3, CDR3 defining according IMGT®.
FIG. 27 Amino acid and nucleic acid sequence for VH HzDR5-05 with description of the FR1, CDR1, FR2, CDR2, FR3, CDR3 defining according IMGT®.
FIG. 28 Amino acid and nucleic acid sequence for VL HzDR5-05 with description of the FR1, CDR1, FR2, CDR2, FR3, CDR3 defining according IMGT®.
EXAMPLES
The following examples are offered to illustrate, but not to limit the claimed invention.
Example 1
Preparation of Murine MAb Anti-DR5
This example illustrates the preparation of hybridoma cell lines secreting anti-DR5 antibodies.
Antibodies.
The anti-DR5 antibodies, murine monoclonal antibodies specific for DR5 were produced using standard hybridoma techniques (Zola et al., Aust J. Exp Biol Med Sci. 1981; 59:303-6). Briefly, mice were given i.p. injections of recombinant DR5 (10 μg), (R&D Systems, Lille, France) on weeks 0, 2 and 4. This was followed by an i.v. injection of recombinant DR5 (10 μg) and the splenocytes were fused with mouse myeloma line X63-Ag8.653. Hybridoma supernatants were screened for DR5 binding by ELISA and by flow cytomery on DR5 positive cell lines. A murine MAb panel anti-DR5 noted mDR5-01, mDR5-02, mDR5-04 and mDR5-05 were obtained.
Example 2
Cell Culture
Various tumor-derived cell lines are among the target cells that may be contacted with TRAIL, anti-DR5 MAb alone, MAb combination, in such assay procedures.
Cell lines. The established human neuroglioma cells H4, HS683 or A172 (available from ATCC) and the established human lung adenocarcinoma cells A549 were grown in Dulbecco's Modified Eagle's Medium (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycine (Sigma, St Quentin Fallavier, France). The established human glioblastoma astrocytoma cells U87MG or T98G, the human kidney adenocarcinoma cells A704, the human kidney adenocarcinoma cells ACHN and the human breast adenocarcinoma cells MCF7 (available from ATCC) were grown in Eagle's Minimum Essential Medium (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycine (Sigma, St Quentin Fallavier, France). The established human colon adenocarcinoma cells SW948 and the human breast adenocarcinoma cells MDAMB231 (available from ATCC) were grown in Leibovitz's L-15 (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycine (Sigma, St Quentin Fallavier, France). The established human kidney carcinoma cells Caki-1 and the human colorectal carcinoma cells HCT-116 (available from ATCC) were grown in McCoy's 5A Medium Modified (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycine (Sigma, St Quentin Fallavier, France). The established human urinary bladder carcinoma cells 5637 and the established human lung adenocarcinoma cells NCIH1703 (available from ATCC) were grown in RPMI-1640 Medium (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycine (Sigma, St Quentin Fallavier, France). The established human glioma cells 42MGBA (available from DSMZ) were grown in 80% mixture of RPMI-1640 Medium and Eagle's Minimum Essential Medium at 1:1 (Sigma, St Quentin Fallavier, France) supplemented with 20% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycine (Sigma, St Quentin Fallavier, France).
Example 3
Antibody Binding Assays (FCM, ELISA)
This example describes methods to determine the MAb specificity anti-DR5 by ELISA with coated antigens, to investigate on DR5 cellular expression at the cell surface. and to determine epitopes following MAb competition analyzed by flow cytometry.
Flow cytometry experiments for DR5 cellular expression. Briefly, 2×105 cells per 96 wells are incubated with a dilution of unconjugated anti-DR5 MAb at 10 μg/mL then diluted at 1/10. Unbound antibodies were washed away with PBS (Invitrogen, Villebon sur Yvette, France) supplemented by 1% Bovine Serum Albumin (Sigma, St Quentin Fallavier, France). Subsequently, cells are centrifuged (5 min at 400 g) and bound antibody is detected with Fluorescein Isothiocyanate (FITC) conjugated goat (Fab′)2 polyclonal anti mouse (MP Biomedical, Illkirch, France) at 4° C. for 30 min. Detection reagent is washed away and cells are centrifuged (5 min at 400 g) and resuspended in 300 μL PBS. Bound detection antibody is quantified on a FACSCAN (BD Biosciences, Rungis, France), (FL1 channel, 2000 events per acquisition). During the experiment, the respective isotype controls are included to exclude any unspecific binding events.
Results of experiments are shown in FIG. 1 (at 10 μg/mL), FIG. 2 and Table 6 (at 5 μg/mL) shows as for example the cell staining with MAb concentration or at 5 μg/mL. Various cancer cell lines express different subsets of TRAIL receptors. Expression patterns varied from cell line to cell lines. In the present study DR5 was expressed on all cell lines tested. Whatever the MAb tested anti-DR5 (mDR5-01, mDR5-02, mDR5-04 or mDR5-05), similar cellular pattern was observed.
Table 6 shows the FACS analysis of DR5 expression using 5 μg/mL of anti-DR5 antibody in other solid tumour cell lines (1×106 cells/mL) i.e. human breast adenocarcinoma cell lines (MCF7, MDAMB231) and on human lung adenocarcinoma cell lines (NCIH1703, A549).
TABLE 6
Breast cancer cell line
Lung cancer cell line
MCF7
MDAMB231
NCIH1703
A549
Mab
%
MFI
%
MFI
%
MFI
%
MFI
mIgG1
1
176
0
119
0
100
0
190
CTRL
mDR5-
51
225
89
238
64
152
82
323
01
mDR5-
33
192
82
224
74
166
71
274
05
Analysis of MAb specificity by using coated antigens ELISA. The specific binding properties of antibodies were evaluated in an ELISA with coated Fas (50 ng/mL) (R&D Systems, Lille, France), FasL (100 ng/mL) (Tebu-bio, Le Perray en Yvelines, France), TRAIL (100 ng/mL) (R&D Systems, Lille, France), DR4 (50 ng/mL) (R&D Systems, Lille, France), DR5 (50 ng/mL) (R&D Systems, Lille, France), DcR1 (50 ng/mL) (R&D Systems, Lille, France) or DcR2 (50 ng/mL) (R&D Systems, Lille, France) antigens. The anti-DR5 MAb panel was tested at 1 μg/mL and revealed by using a goat polyclonal anti mouse IgG1 Horse Radish Peroxydase (HRP) conjugated (AbD Serotec, Colmar, France).
Results of experiments are shown in FIG. 3. The mDR5-01, mDR5-02, mDR5-04 and mDR5-05 antibodies (1 μg/mL) only reacted with DR5 coated antigens (50 ng/mL). No reactivity was observed with other apoptotic related antigens (FAS, FASL, TRAIL, DR4, DcR1, DcR2), (mean+/−SD on 2 independent experiments).
Flow cytometry experiments for MAb competition binding. Briefly, 2×105 cells T98G per 96 wells are incubated with a dilution of biotinylated murine antibody anti-DR5 (10 μg/mL then diluted at 1/10) as a reference and with or without unconjugated antibody at 5 μg/mL and incubated at 4° C. for 30 min. Only data obtained with 1 μg/mL of biotinylated antibody is shown. Unbound antibody is washed away with PBS (Invitrogen, Villebon sur Yvette, France) supplemented by 1% Bovine Serum Albumin (Sigma, St Quentin Fallavier, France). Subsequently, cells are centrifuged (5 min at 400 g) and bound antibody is detected with Phycoerythrin conjugated Streptavidin (Interchim, Montluçon, France) at 4° C. for 30 min. Detection reagent is washed away and cells are centrifuged (5 min at 400 g) and resuspended in 300 μL PBS. Bound detection antibody is quantified on a FACSCAN (BD Biosciences, Rungis, France), (FL2 channel, 2000 events per acquisition). During the experiment, the respective isotype controls are included to exclude any unspecific binding events.
Results of experiments are shown in FIG. 4. For example the unconjugated mDR5-02 and mDR5-05 antibodies (5 μg/mL) are not in competition with the biotinylated mDR5-01 antibody (1 μg/mL). By contrast, unconjugated mDR5-01 and mDR5-04 are in competition with the biotinylated mDR5-01 antibody. Therefore, the epitopes DR5-01 and DR5-04 are common or adjacent, whereas the epitopes DR5-02 and DR5-05 are two separate epitopes. Moreover the epitope DR5-01 is also distinct of the epitope DR5-05, (mean+/−SD on two independent experiments).
Example 3
In Vitro Biologic MAb Activity
This example illustrates methods of evaluating the anti-DR5 MAb impact on TRAIL cellular binding on their ability to trigger cellular cytotoxic effect on cancer cells. These components may be assayed for anti-tumour activity, using any of a number of suitable assays, including but not limited to assays for the ability to slow tumour growth or to kill cancer cells in vitro. Various tumour-derived cell lines are among the target cells that may be contacted with MAb combination, in such assay procedures.
To identify or select anti-DR5 antibody combination which induce apoptosis, loss of membrane integrity as indicated by, e.g. PI is assessed relative to control (untreated cells) and compared to recombinant TRAIL (FIG. 8). The ability to slow tumour growth is assessed by ATP or BrDU quantification (FIG. 6, FIG. 7). The apoptotic response is assessed by quantification of cleaved caspase 3 (FIG. 9) or cleaved Poly-(ADP-Ribose)-Polymerase (PARP), (FIG. 10).
Biochemical reagents. Biochemical reagents used for the apoptosis studies were: propidium iodide (PI), (Sigma, St Quentin Fallavier, France), Caspase 3 antibody (Ozyme, Saint Quentin Yvelines, France), Cell proliferation ELISA-BrdU (Roche Diagnostics, Meylan, France), Cell Titer GLo-ATP (Promega, Charbonnières-les-bains, France) and the polyclonal anti Poly-(ADP-Ribose)-Polymerase (PARP) (Roche Diagnostics, Meylan, France).
Flow cytometry experiments of MAb impact on TRAIL binding. H4 cell lines were seeded at a density of 1×105 per 96-wells. Cells were incubated for 30 min at 4° C. with or without MAb anti-TRAIL or anti-DR5 (mDR5-01, mDR5-02, mDR5-3 mDR5-4) tested at 1 μg/mL then diluted at 1/10. Unbound antibodies were washed away with PBS (Invitrogen, Villebon sur Yvette, France) supplemented by 1% Bovine Serum Albumin (Sigma, St Quentin Fallavier, France). Subsequently, cells are incubated with the recombinant TRAIL (100 ng/mL), (R&D Systems, Lille, France) for 30 min at 4° C. Unbound antibodies were washed away with PBS (Invitrogen, Villebon sur Yvette, France) supplemented by 1% Bovine Serum Albumin (Sigma, St Quentin Fallavier, France). The bound recombinant TRAIL is detected with biotinylated conjugated anti TRAIL MAb B-S23 (iDD biotech, Dardilly, France). After washings, Phycoerthrin conjugated Streptavidin (Interchim, Montluçon, France) was added at 4° C. for 30 min. Detection reagent is washed away and cells are centrifuged (5 min at 400 g) and resuspended in 300 μL PBS. Bound detection antibody is quantified on a FACSCAN (BD Biosciences, Rungis, France), (FL2 channel, 2 000 events per acquisition). During the experiment, the respective isotype controls are included to exclude any unspecific binding events.
Human H4 expressing DR5 at the cell surface was used to determine the agonist or antagonist activity of the four anti-DR5 antibodies denoted mDR5-01, mDR5-02, mDR5-04 and mDR5-05. Results of experiments are shown in FIG. 5. The recombinant TRAIL binding at the cell surface was inhibited with the antagonist anti TRAIL MAb B-T24 (iDD biotech, Dardilly, France). Among the anti-DR5 MAb panel tested, the MAbs mDR5-01, mDR5-04 and mDR-5-05 inhibited the recombinant TRAIL binding, without any mDR5-02 MAb impact.
Cell viability analysis following ATP level determination. The CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Charbonnières les Bains, France) was used to determine the number of viable cells in culture based on quantification of the ATP present, an indicator of metabolically active cells. Detection is based on using the luciferase reaction to measure the amount of ATP from viable cells. Within minutes after a loss of membrane integrity, cells lose the ability to synthesize ATP, and endogenous ATPases destroy any remaining ATP; thus the levels of ATP fall precipitously. Cell cultures (5×104 cells/mL) are incubated for 72 hours alone or with anti-DR5 MAb alone (1 μg/mL) or with two combined MAb at 1 μg/mL for each MAb (FIG. 6). The TRAIL ligand concentration was used at 10 ng/mL. The CellTiter-Glo® reagent was added directly to cells in culture at a ratio of 50 μL of reagent to 200 μL of culture medium. The assay plates are incubated at room temperature for 10 min and the bioluminescent signal is recorded using a standard multiwell fluorometer Mithras LB940, (Berthold, Thoiry, France).
Results of experiments to determine the agonist activity of the four anti-DR5 antibodies are shown in FIG. 6. None of the anti-DR5 MAb alone tested was capable of inducing cellular cytotoxicity in H4 cells. By contrast, only the anti-DR5 MAb combination mDR5-01 and mDR5-05 triggered apoptosis in H4 cells. The ability of this restricted anti-DR5 MAb combination (1/10) was not related to the level of MAb staining (FIG. 1). Interestingly the MAbs mDR5-01 and mDR5-05 recognize two different epitopes (FIG. 4). However MAb combination of mDR5-05 with other mDR5 MAb such as mDR5-02 recognizing also distinct epitope failed to trigger H4 apoptosis (FIG. 6).
Cell viability analysis following BrDU incorporation determination. The H4 target cells (5×104 cells/mL) were cultured with the MAb combination mDR5-05 and mDR5-01 or with the MAb combination mDR5-05 and mDR5-04 at different range of MAb concentration. Cell growth is determining using the Cell proliferation ELISA-BrdU (Roche Diagnostics, Meylan, France), according to the manufacturer's instructions. This method is based on the incorporation of the pyrimidine analogue BrdU instead of thymidine into the DNA of proliferating cells. After its incorporation into DNA, BrdU is detected with a MAb anti-BrdU. At the end of revelation, the bioluminescent signal is recorded using a standard multiwell fluorometer Mithras LB940, (Berthold, Thoiry, France).
Results of experiments are shown in FIG. 7. Specific MAb combination mDR5-01 and mDR5-05 synergistically induced apoptosis in H4 cell line as evidence by BrDU quantification, (mean+/−SD on 2 independent experiments). No significant impact was observed with the MAb combination mDR5-05 and mDR5-04.
Propidium iodide uptake by flow cytometry for measuring MAb induced apoptosis. H4 cell lines were seeded at a density of 2×104 per 96-wells. Cells were incubated for a 3 day time period with or without MAb anti-DR5. Each anti-DR5 MAb was tested alone or following MAb combination at 1 μg/mL (FIG. 8). The TRAIL ligand concentration was used at 10 ng/mL. Cells were then centrifuged at 2000 rpm for 5 min at 4° C., the pellet resuspended in 70% ethanol (Sigma, St Quentin Fallavier, France) for permeabilization. After a new centrifugation, cells were incubated with 100 μL of PI (100 μg/mL) and 100 μL of Rnase (100 μg/mL), (Sigma, St Quentin Fallavier, France) per well for 15 min. Cells are centrifuged (5 min at 2000 rpm) and resuspended in 300 μL PBS. Bound detection antibody is quantified on a FACSCAN (BD Biosciences, Rungis, France), (FL2 channel, 3000 events per acquisition).
Results of experiments are shown in FIG. 8. Whereas no PCD was obtained with MAb tested alone, specific MAb combination mDR5-01 and mDR5-05 synergistically induced apoptosis in H4 cell line as evidence by PI uptake, (mean+/−SD on 2 independent experiments). No MAb cross linking was required. No PCD was obtained with MAb combination mDR5-05 and mDR5-04.
Cleaved caspase-3 quantification by flow cytometry for measuring MAb induced apoptosis. H4 cell lines were seeded at a density of 2×104 per 96-wells. Cells were incubated for a 48 hours with or without MAb anti-DR5. Each anti-DR5 MAb was tested alone or in the presence of MAb combination at 1 μg/mL for mDR5-05 with 0.01 μg/mL for mDR5-01 or mDR5-04 (FIG. 9). The TRAIL ligand concentration was used at 10 ng/mL. Cells were then centrifuged at 2000 rpm for 5 min at 4° C., the pellet resuspended in 90% methanol (Sigma, St Quentin Fallavier, France) for permeabilization. Cells were then centrifuged at 2000 rpm for 5 min at 4° C. and incubated at 4° C. for 30 min with the MAb anti-active caspase-3 antibodies alexa fluor 488 conjugated (Ozyme, Saint Quentin Yvelines, France). Cells are centrifuged (5 min at 2000 rpm) and resuspended in 300 μL PBS. Bound detection antibody is quantified on a FACSCAN (BD Biosciences, Rungis, France), (FL2 channel, 3000 events per acquisition).
When apoptosis is activated, caspases cleave multiple protein substrates, which leads to the loss of cellular structure and function, and ultimately results in cell death. In particular, caspases-8, -9, and -3 have been implicated in apoptosis: caspase-9 in the mitochondrial pathway, caspase-8 in the Fas/CD95 pathway, and caspase-3 more downstream, activated by multiple pathways. Specific MAb combination mDR5-01 and mDR5-05 synergistically induced apoptosis in H4 cell line as evidence by cleaved caspase 3 quantification (FIG. 9), (mean+/−SD on 2 independent experiments). As compared to TRAIL (called also Apo2L), only the MAb combination mDR5-01 and mDR5-05 triggered cell apoptosis compared to the MAb combination mDR5-04 and mDR5-05.
PARP Western blotting. H4 cell lines were seeded at a density of 1·106 per flask T25 cm2. Cells were incubated for a 5 hours with or without MAb anti-DR5. Cell extracts were resuspended in Tris-HCl 50 mM, KCl 150 mM at pH7 and submitted to sonication and incubated for 15 min at 65° C. Samples (10 μg) were subjected to reducing SDS-PAGE and transferred to PVDF membrane using standard methods. After blocking in milk 5%, the blots were incubated in the anti-Poly-(ADP-Ribose)-Polymerase (PARP) (Roche Diagnostics, Meylan, France) at 1/2000. After washing, the membranes were incubated in PAb sheep anti-rabbit IgG horseradish peroxidase conjugated antibody at 1/10000, (AbD Serotec, Colmar, France). The blots were developed with ECL Advance Western blotting using enhanced luminol-based chemiluminescent substrate for detection of horseradish peroxidase (GE Healthcare, St Cyr au Mont d'Or, France).
Many target-specific substrates for caspase have been identified, including the DNA repair enzyme, poly (ADP-ribose) polymerase (PARP). Western blot detection of PARP cleavage has been used extensively as an indicator of apoptosis. PARP is cleaved between Asp213 and Gly 214 in the human sequence, producing two fragments of apparent molecular weights of 24 and 89 kDa. From H4 cells treated with the MAb combination mDR5-01 and mDR5-05, the fragments of cleaved PARP were detected, whereas no similar effect was observed from the untreated cells or treated with the MAb combination mDR5-05 an mDR5-04, (FIG. 10).
As shown in FIG. 11, the MAb mDR5-02 (1 μg/mL) blocked apoptosis triggered with the MAb combination mDR5-01 and mDR5-05 tested at the ratio 1/100 (10 μg/mL+0.1 μg/mL). No significant impact was observed with the other anti-DR5 MAbs (mDR5-01, mDR5-04 or mDR5-05). Cell viability was evaluated based on quantification of the ATP present, an indicator of metabolically active cells.
The susceptibility of five of the glioma cell lines, H4, HS683, A172, T98G and U87MG to TRAIL or anti-DR5 MAb combination (mDR5-01+mDR5-05 versus mDR5-05+mDR5-04) tested at the ratio 1/100 (10 μg/mL+0.1 μg/mL) were evaluated based on quantification of the ATP present, an indicator of metabolically active cells (FIG. 12). Whereas four cell lines (HS683, A172, T98G and U87MG) are resistant or very low sensitive to TRAIL-induced apoptosis, the use of the MAb anti-DR5 combination mDR5-01 and mDR5-05 bypass this regulatory mechanism.
The susceptibility of ex vivo glioma cells from patients to mouse anti-DR5 MAb combination (mDR5-01+mDR5-05) tested at 10 μg/mL (ratio 1/10) were evaluated based on quantification of the ATP present, an indicator of metabolically active cells (FIG. 14).
The susceptibility of four glioma cell lines, (HS683, A172, 42MGBA, T98G) to mouse anti-DR5 MAb combination (mDR5-01+mDR5-05) tested at 10 μg/mL then diluted at 1/10 (ratio 1/1) were evaluated alone or in association with Camptothecin (FIG. 15-18). These cell lines exhibited different levels apoptosis induced with mouse anti-DR5 MAb combination or in the presence of Camptothecin. The use of MAb anti-DR5 combination in association with Camptothecin bypassed this regulatory mechanism and enhanced the level of apoptosis.
The susceptibility of other solid tumor cell lines expressing DR5 such as on human breast adenocarcinoma cell lines (MCF7, MDAMB231) and on human lung adenocarcinoma cell lines (NCIH1703, A549) to mouse anti-DR5 MAb combination (mDR5-01+mDR5-05) tested at 10 μg/mL then diluted at 1/10 (ratio 1/1) were evaluated alone or in association with Paclitaxel, Gemcitabine or Doxorobucine (FIG. 19-22). These cell lines exhibited different levels apoptosis induced with mouse anti-DR5 MAb combination or in the presence of the different drugs tested. The use of MAb anti-DR5 combination in association with these drugs bypassed this regulatory mechanism and enhanced the level of apoptosis.
Example 4
Preparation of Chimeric Monoclonal Antibodies Directed Against DR5
DNA encoding the monoclonal antibodies is 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 murine antibodies). The hybridoma cells serve as a preferred source of such DNA.
Conversion of murine MAb to native chimeric MAb: cDNA corresponding to the variable region of the hybridoma was obtained using two approaches. The first approach consists of using PCR with a degenerate N-terminal amino acid related primer set to generate the N-Terminal sequencing product. The second approach consists of using PCR with a degenerate primer set generated by IMGT® primer database and specific primers previously described (Essono et al., J Immunol Methods. 2003; 203: 279:251-66, Wang et al., Mol Immunol. 1991; 28:1387-97). The sequence of N-terminal variable region was determined by Edman degradation. Total RNA extraction was carried out using the Tri Reagent kit according to the protocol described by the supplier Sigma. The amplified VL and VH fragments were cloned into the TOPO-TA cloning vector (Invitrogen) for sequence analyses by the dideoxytermination method (Sanger et al., Nature. 1977; 265:687-95). Then antibody variant constructs were amplified by PCR and cloned into the expression vector.
Positions are numbered according to IMGT® and to Kabat® index (Identical V region amino acid sequences and segments of sequences in antibodies of different specificities). Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites were analyzed (Kabat et al., NIH Publ. 1991; No. 91-3242, Vol. 1, 647-669).
As shown in FIG. 13, the chimeric MAb combination chDR5-01 and chDR5-05 triggered H4 cell apoptosis tested at the ratio 1/100 (5 μg/mL+0.05 μg/mL). No significant MAb impact was observed with the chimeric MAb tested alone. Cell viability was evaluated based on quantification of the ATP present, an indicator of metabolically active cells.
The nucleic acid sequence or amino acid sequence regarding on the chimeric MAbs DR5-01 and DR5-05 are shown in the Sequence Listing:
nucleotide sequence of the variable murine light chain of DR5-01 antibody anti-DR5 (SEQ ID NO:1) and its derived amino acid sequence (SEQ ID NO:2).
nucleotide sequence of the variable murine heavy chain of DR5-01 antibody anti-DR5 (SEQ ID NO:3) and its derived amino acid sequence (SEQ ID NO:4).
nucleotide sequence of the variable murine light chain of DR5-05 antibody anti-DR5 (SEQ ID NO:5) and its derived amino acid sequence (SEQ ID NO:6).
nucleotide sequence of the variable murine heavy chain of DR5-05 antibody anti-DR5 (SEQ ID NO:7) and its derived amino acid sequence (SEQ ID NO:8).
nucleotide sequence of the constant human heavy chain of DR5-01 or DR5-05 antibody anti-DR5 (SEQ ID NO:9) and its derived amino acid sequence (SEQ ID NO:10).
nucleotide sequence of the constant human light chain of DR5-01 or DR5-05 antibody anti-DR5 (SEQ ID NO:11) and its derived amino acid sequence (SEQ ID NO:12).
Example 5
MAb Production and Protein A Purification
Mammalian cells are the preferred hosts for production of therapeutic glycoproteins, due to their capability to glycosylate proteins in the most compatible form for human applications (Jenkins et al., Nat Biotech. 1996; 14:975-81). Mammalian host cells that could be used include, human Hela, 283, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1 African green monkey cells, quail QC1-3 cells, mouse L cells and Chinese hamster ovary cells. Bacteria very rarely glycosylates proteins, and like other type of common hosts, such as yeasts, filamentous fungi, insect and plant cells yield glycosylation patterns associated with rapid clearance from the blood stream.
The Chinese hamster ovary (CHO) cells allow consistent generation of genetically stable, highly productive clonal cell lines. They can be cultured to high densities in simple bioreactors using serum-free media, and permit the development of safe and reproducible bioprocesses. Other commonly used animal cells include baby hamster kidney (BHK) cells, NSO- and SP2/0-mouse myeloma cells. Production from transgenic animals has also been tested (Jenkins et al., Nat Biotech. 1996; 14:975-81).
A typical mammalian expression vector contains the promoter element (early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses e.g. RSV, HTLV1, HIV1 and the early promoter of the cytomegalovirus (mCMV, hCMV), which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript (BGH polyA, Herpes thimidine kinase gene of Herpes simplex virus polyA (TKpa), Late SV40 polyA and 3′ UTR_Beta_Globin_polyA). Additional elements include enhancers (Eμ, hIE1), Kozak sequences, signal peptide and intervening sequences flanked by donor and acceptor sites for RNA splicing. Suitable expression vectors for use in practise in practising the present invention include, for examples, vectors such as pcDNA3.1, pcDNA3.3, pOptiVEC, pRSV, pEμMCMV, pMCMVHE-UTR-BG, pHCMVHE-UTR-BG, pMCMV-UTR-BG, pHCMV-UTR-BG, pMCMVHE-5V40, pHCMVHE-5V40, pMCMV-5V40, pHCMV-5V40, pMCMVHE-TK, pHCMVHE-TK, pMCMV-TK, pHCMV-TK, pMCMVHE-BGH, pHCMVHE-BGH, pMCMV-BGH, pHCMV-UTR-BGH).
The empty CHO Easy C cells (purchased by the CCT collection) were co-transfected with MAb expression vector for light and heavy chains following transient or stable transfection procedure established in our laboratory. Secretion of H and L chains were enabled by the respective human IgH leader sequence. The coding regions for light and heavy chains of MAb anti-DR5 are introduced into the MAb expression vector in the multiple cloning site. The transformants are analyzed for correct orientation and reading frame, the expression vector may be transfected into CHO cell line.
Protein A chromatography from murine ascitic fluid. The murine ascitic fluid is adjusted at pH 8.3 with the equilibration buffer 0.1 M Tris and 1.5 M Sulfate Ammonium and then loaded onto the rProtein A Sepharose Fast Flow column (GE Healthcare, Saint Cyr au Mont d'or, France). The non binding proteins are flowed through and removed by several washings with equilibration buffer. The MAb anti-DR5 is eluted off the Protein A column using the elution buffer 0.1 M Citrate Sodium at pH 3.5. Column eluent is monitored by A280. The anti-DR5 MAb peak is pooled.
Protein A chromatography from harvested CHO cell culture fluid. The harvested cell culture fluid produced from CHO cells is loaded onto the Hi Trap rProtein A column (GE Healthcare, Saint Cyr au Mont d'Or, France) that is equilibrated with Phosphate buffered saline, pH 7.2. The non binding proteins are flowed through and removed by several washings with PBS buffer followed. The MAb anti-DR5 is eluted off the Protein A column using a step of elution of 0.1 M Citric acid at pH 3.0. Column eluent is monitored by A280. The anti-DR5 MAb peak is pooled.
Example 6
Preparation of Humanized Monoclonal Antibodies Directed Against DR5
Antibody CDR and FR regions have been determined according to various numbering approaches such as IMGT (ImMunoGeneTics Information System® http://imgt.cines.fr), Kabat or Common Numbering System. However, IMGT determined CDRs for a given antibody are not necessarily identical to the CDRs defined by the other numbering systems. The variable domain CDRs and framework regions have been identified by the inventor thanks to IMGT numbering systems.
Conversion of chimeric MAb to Humanized MAb: Humanized DR5 antibody H and L chain was generated using CDR-grafting by the PCR method. In order to generate a humanized antibody in which the CDRs of a mouse monoclonal antibody is grafted onto a human antibody, there is preferably a high homology between the variable region of a mouse monoclonal antibody and the variable region of a human antibody. Thus, the H chain and L chain V regions of a mouse anti-human DR5 monoclonal antibody are compared to the V region of all known human antibodies using the software IMGT/DomainGapAlign. When a mouse antibody is humanized by a conventional technology, the amino acid sequence of some of the V region FRs of a mouse antibody supporting the CDR may be grafted onto the FR of a human V region, as desired.
For both of the humanized H chain and L chain V regions, it is possible to select the Land H chain V regions and J region, IGKV3-D-15*01, IGHV1-3*01, IGKJ2*01 and IGHJ4*01 respectively, having a high homology with the H and L chain V region and J region of the mDR5 antibody and IGKV1-16*01, IGHV1-3*01, IGKJ4*01 and IGHJ4*01, having a high homology with the H and L chain V region and J region of the mDR5-05 antibody.
After sequence of the Humanized variable region of HzDR5-01 and HzDR5-05 is determined. The variables regions of H and L of HzDR5-01 and Hz-DR5-05 were amplified by PCR and cloned into the expression vector p3U containing the human IgG1 constant region.
In the case of human CDR-grafted antibodies, the binding activity is decreased by grafting of the amino acid sequence of CDR in the mouse antibody alone. In order to avoid this reduction, among the amino acid residues in FR different between a human antibody and a mouse antibody, amino acid residues considered to have influences on the binding activity are grafted together with the amino acid sequence of CDR. Accordingly, an attempt was also made in this example to identify the amino acid residues in FR considered to have influences on the binding activity.
The susceptibility of the glioma cell line H4 to mouse or humanized anti-DR5 MAb combination (mDR5-01+mDR5-05) tested at 1 μg/mL then diluted at 1/2 (ratio 1/1) were evaluated based on quantification of the ATP present, an indicator of metabolically active cells (FIG. 23). The humanized MAb combination (hzDR5-01 and hzDR5-05) triggered cell apoptosis at a higher level compared to the mouse MAb combination (mDR5-01 and mDR5-05).
Example 7
In Vivo Biologic MAb Activity
Orthotopic human glioma xenograft mouse model was obtained by intracerebral injection in nude mouse of 100 000 isolated cell coming from heterotypic human glioma xenograft mouse model Sc2. MAb treatment was administrated by intraperitoneal injection (IP) at 5 mg/kg per mouse until mice euthanasia due to loss of weight and was applied during 36 days maximum. Survival times obtained with control group were compared to survival times obtained with treated groups (mDR5-01+mDR5-05 versus mDR5-04+mDR5-05) using Kaplan Meier method and Wilcoxon statistical test (JMP software), (FIG. 24). This study demonstrated anti-tumor activity of mouse anti-DR5 MAb combination (mDR5-01+mDR5-05) on intracerebral glioma.
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14413194
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genmab b.v.
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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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Apr 1st, 2022 06:04PM
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Apr 1st, 2022 06:04PM
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Genmab A/S
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Health Care
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Pharmaceuticals & Biotechnology
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cph:gen
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Genmab A/S
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May 12th, 2020 12:00AM
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Feb 17th, 2017 12:00AM
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https://www.uspto.gov?id=US10647774-20200512
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Anti-DR5 family antibodies, bispecific or multivalent anti-DR5 family antibodies and methods of use thereof
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Anti-DR5 family member antibodies and bispecific antibodies comprising one or more anti-DR5 family member antibodies are disclosed. These antibodies can be used to trigger cell death on DR5 positive cells.
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10647774
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1. An isolated nucleic acid encoding a variable light (VL) region or a variable heavy (VH) region of an antibody capable of binding a DR5 receptor, wherein the isolated nucleic acid encoding the VL region comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 5, 36, and 40, and the isolated nucleic acid encoding the VH region comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, 7, 34, and 38.
2. An expression vector comprising the isolated nucleic acid encoding the VL region, the isolated nucleic acid encoding the VH region, or the isolated nucleic acid encoding the VL region and the isolated nucleic acid encoding the VH region according to claim 1.
3. A host cell comprising the vector according to claim 2.
4. A method of producing an antibody capable of binding a DR5 receptor, the method comprising
culturing the host cell of claim 3 in a culture medium under conditions sufficient to produce the antibody, wherein the host cell comprises the isolated nucleic acid encoding the VL region selected from the group consisting of SEQ ID NO: 1 and 36, and the isolated nucleic acid encoding the VH region selected from the group consisting of SEQ ID NO: 3 and 34, and
isolating the antibody from the culture medium, thereby producing the antibody capable of binding a DR5 receptor.
5. The method of claim 4, wherein the isolated nucleic acid encoding the VL region comprises the nucleotide sequence set forth in SEQ ID NO: 1, and the isolated nucleic acid encoding the VH region comprises the nucleotide sequence set forth in SEQ ID NO: 3.
6. The method of claim 4, wherein the isolated nucleic acid encoding the VL region comprises the nucleotide sequence set forth in SEQ ID NO: 36, and the isolated nucleic acid encoding the VH region comprises the nucleotide sequence set forth in SEQ ID NO: 34.
7. A method of producing an antibody capable of binding a DR5 receptor, the method comprising
culturing the host cell of claim 3 in a culture medium under conditions sufficient to produce the antibody, wherein the host cell comprises the isolated nucleic acid encoding the VL region selected from the group consisting of SEQ ID NO: 5 and 40, and the isolated nucleic acid encoding the VH region selected from the group consisting of SEQ ID NO: 7 and 38, and
isolating the antibody from the culture medium, thereby producing the antibody capable of binding a DR5 receptor.
8. The method of claim 7, wherein the isolated nucleic acid encoding the VL region comprises the nucleotide sequence set forth in SEQ ID NO: 5, and the isolated nucleic acid encoding the VH region comprises the nucleotide sequence set forth in SEQ ID NO: 7.
9. The method of claim 7, wherein the isolated nucleic acid encoding the VL region comprises the nucleotide sequence set forth in SEQ ID NO: 40, and the isolated nucleic acid encoding the VH region comprises the nucleotide sequence set forth in SEQ ID NO: 38.
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9
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 14/413,194, filed Jan. 6, 2015, which is a 371 National Stage filing of International Patent Application No. PCT/EP2013/064466, filed Jul. 9, 2013, which claims priority to U.S. Provisional Patent Application Ser. No. 61/669,866, filed Jul. 10, 2012, and European Patent Application No. 12305821.6, filed Jul. 9, 2012, the contents of which are hereby incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to the fields of immunology, oncology, and more specifically, to monospecific, bispecific or multivalent antibody molecules that can be used to advantage in the treatment of various cancers, autoimmune diseases, and infectious diseases that express DR5 antigen. The present invention is related to novel polypeptides binding specifically to the DR5 receptor also called TRAIL receptor 2. The invention relates in particular to a polypeptide having two different binding domains or a combination of polypeptides having these different binding domains, which bind to different epitopes of the DR5 receptor, whereby apoptosis is induced. The invention also relates to pharmaceutical compositions containing these polypeptides and the treatment of cancer, autoimmune diseases and viral infections using these polypeptides and compositions.
BACKGROUND OF THE INVENTION
Apoptosis, or programmed cell death, is a physiologic process essential to the normal development and homeostasis of multicellular organisms. Derangements of apoptosis contribute to the pathogenesis of several human diseases including cancer, neurodegenerative disorders, and acquired immune deficiency syndrome.
The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily of cytokines, is a type 2 membrane protein that is expressed in the majority of normal tissues and can undergo protease cleavage, resulting in a soluble form able to bind to TRAIL receptors, (Wiley S R. et al., Immunity. 1995; 3:673-682; Daniel P T et al., J Immunol. 1994; 152:5624).
Ligands of this family generally recognize and bind to a limited subset of cognate receptors on the cell surface, leading to signal transduction cascades downstream of the receptor, allowing the activation of a large panel of signalling pathways including NF-kB or caspase activation. TRAIL induces apoptosis of certain transformed cells, including a number of different types of cancer cells as well as virally infected cells, while not inducing apoptosis of a number of normal cell types and is thus of particular interest in the development of cancer therapies, (Walczak et al., Nature Medecine. 1999; 5/157-163, Ashkenazi A. et al., J Clin Invest. 1999; 104:155).
There are four known cell surface receptors for TRAIL. TRAIL Receptor 1 (TRAIL-R1, DR4) and Trail Receptor 2 (TRAIL-R2, DR5, Apo-2, TRICK2, Killer, TR6, Tango-63) have a cytoplasmic death domain and are able to trigger apoptosis in tumor cells via downstream caspase activation. The other two receptors, TRAIL Receptor 3 (TRAIL-R3, DcR1, TR5, TRIDD, LIT) and TRAIL Receptor 4 (TRAIL-R4, DcR2, TRUNDD) lack a cytoplasmic death domain and do not mediate apoptosis. In addition, osteoprotegerin (OPG), a soluble (secreted) member of the TNF receptor family of proteins, also binds TRAIL.
The intracytoplasmic domains of DR4 and DR5 each include a so-called death domain. After activation of the receptors DR4 and DR5, the fas-associated death domain adapter molecule is recruited to the receptor, leading to an autoproteolytic cleavage and activation of initiator caspase-8. DR4 and DR5 have been reported to transduce an apoptotic signal to TRAIL sensitive cancer cells, upon binding of TRAIL. Active caspase-8 in turn triggers the proteolytic activation of downstream caspases including caspase-3. Downstream caspases ultimately degrade a broad range of cellular proteins, and apoptosis is finalized.
Expression of either DR4 or DR5 is frequently detected in human cancers, including colon, gastric, pancreatic, ovarian, breast, and non-small-cell lung cancer with low or no expression in normal tissues.
In the development or progression of many diseases it is often the case that cells are not deleted. In many autoimmune diseases and inflammatory conditions, the surviving activated cells attack normal tissues or cells. Further, progression of tumorigenesis and the proliferative pannus formation of rheumatoid arthritis are characterized by the unchecked proliferation of cells. Thus insufficient apoptosis leads to the development of disease, and the uses of apoptosis-inducing ligand or agonistic MAb to enhance apoptosis are considered as a potential therapeutic strategy for eliminating those unwanted cells
TRAIL induces apoptosis in a wide range of haematopoietic and solid tumor cells, while sparing most normal cells. TRAIL has strong apoptosis-inducing activity against cancer cells in vitro and potent antitumor activity against tumor xenografts of various cancers in vivo.
TRAIL and its derivatives, including agonistic antibodies targeting TRAIL receptors are attractive compounds for cancer therapy due to their ability to induce tumor regression without significant side effects.
There are many instances in the patent literature of efforts to use polypeptides derived from the TRAIL ligand as a therapy against cancerous cells (US20090131317; U.S. Pat. Nos. 6,469,144; 6,740,739; US20070026000; U.S. Pat. No. 6,444,640; US20050244857; US20050233958; U.S. Pat. No. 7,736,637).
TRAIL polypeptides have been used to induce the TRAIL apoptotic pathway, but they have the drawback of a short half-life.
Currently, a great deal of attention has focused on the development of novel immunotherapy strategies for the treatment of cancer. One such strategy is antibody-based cancer therapy.
The most prominent determinant of the above targeting properties is the size of the antibody-based molecule relative the degree of specificity, the retention in tumors and their clearance. Another important feature of antibody-based molecules is valence, as significantly greater tumor retention has been associated with multivalent binding to target, (Adams et al., Cancer Res. 1993; 51:6363-6371; Wolf et al., Cancer Res. 1993; 53:2560-2565).
As mentioned earlier, agonistic antibodies against DR4 or DR5 have been produced and represent a new generation of cancer therapy. Works have been conducted also on the use of agonistic antibodies directed against the TRAIL receptors in order to induce the TRAIL apoptotic pathway.
Agonistic monoclonal antibodies that specifically bind to DR4 or DR5 are supposed to be able to directly induce apoptosis of targeted tumor cells, (Buchsbaum D J et al., Future Oncol. 2006; 2:493; Rowinsky E K et al., J Clin OncoL 2005; 23:9394).
Other patents relate to the use of agonistic antibodies directed against DR4 or DR5, or DR4 and DR5, or to the combined use of antibodies against DR5 and another chemotherapeutic agent: US20040147725; US 20090022707; US20080248037; US20020155109; U.S. Pat. Nos. 6,461,823; 6,872,568; 7,064,189; 6,521,228; 704,502.
Combined treatment with agonistic antibodies directed against different TRAIL receptors, for example DR4 and DR5, have been developed as well. Agonistic bispecific antibodies that bind DR4 or DR5 (or hybridomas producing such agonistic MAbs) may be employed as starting materials in various procedures (WO 2002/0155109).
These include anti-DR5 MAb lexatumumab, (Plummer R. et al., Clin Cancer Res. 2007; 13:6187), the anti-DR5 MAb apomab, (Adams C. et al., Cell Death Differ. 2008; 15:751), the anti-DR5 MAb LBy135, (Li J. et al., AACR Meeting Abstracts. 2007. Abstract 4874), the anti-DR5 MAb WD-1, (Wang J. et al., Cell Mol Immunol. 2008; 5:55) and the anti-DR5 MAb AMG655, (Wall J. et al., AACR Meeting Abstracts. 2008. Abstract 1326, Kaplan-Lefko P. et al., AACR Meeting Abstracts. 2008. Abstract 399). A consistent finding from all these studies is the considerable variability in the sensitivity of various tumor cell lines to anti-DR5-mediated cytotoxicity.
Anti-DR4 or anti-DR5 agonistic antibodies, including mapatumumab or lexatumumab respectively are also well tolerated in patients (Herbst R. S. et al., J Clin Oncol. 2006; 24(18S)/3013; Hotte S. J. et al., Clin Cancer Res. 2008; 14/3450-3455; Wakelee H. A et al., Ann Oncol. 2010; 21/376-381; Fox N. L. et al., Expert Opin Biol Ther. 2010; 10/1-18).
Lexatumumab (also known as ETR2-ST01) is an agonistic human monoclonal antibody against DR5 used in the treatment of cancer. HGS-ETR2 antibodies were generated by HGS through collaboration with Cambridge Antibody Technology.
Tigatuzumab (CS-1008) is a humanized IgG1 monoclonal antibody composed of the CDR regions of mTRA-8. The murine anti-DR5 monoclonal antibody, TRA-8 (mTRA-8), was selected from a series of anti-DR5 monoclonal antibodies based on its specificity, ability to trigger apoptosis in vitro without the use of crosslinking reagents, and lack of toxicity to human hepatocytes, (Buchsbaum D J et al., Clin Cancer Res. 2003; 9:3731; Ichikawa K. et al., Nat Med. 2001; 7:954).
Tigatuzumab mediates a very similar pattern of in vitro cytotoxicity and in vivo antitumor efficacy as mTRA-8. It was shown to have potent in vitro cytotoxicity to a variety of human tumor cell lines and in vivo antitumor efficacy in murine xenograft models of human cancers. Its in vitro cytotoxicity and in vivo antitumor efficacy can be substantially enhanced in combination with a variety of chemotherapeutic agents and/or radiation, (Buchsbaum D J et al., Clin Cancer Res. 2003; 9:3731; DeRosier L C et al., Clin Cancer Res. 2007; 13:5535s).
Anti-DR4 and anti-DR5 antibodies have been tested in associations, together or with other chemotherapeutic agents or therapies. A combined treatment of colorectal tumors with two agonistic antibodies HGS-ETR1 (anti-DR4) and HGS-ETR2 (anti-DR5) and radiotherapy let to enhanced effects in vitro and dose-dependent growth delay in vivo (Marini P et al., Oncogene. 2006; 25 (37):5145-54). Fully human agonistic antibodies to DR4 and DR5 demonstrated in primary and cultured lymphoma cells induction of apoptosis and enhancement of doxorubicin- and bortezomib-induced cell death (Georgakis G V et al., Oncogene. 2006; 25(37):5145-54).
It has been found that the expression of DR5 and susceptibility to TRAIL-induced apoptosis of breast cancer cells is enhanced by the radiation, suggesting that combined with radiation, the efficiency of TRAIL would be increased in cancer therapy (Chinnaiyan A. M et al., PNAS. 2000; 97/1754-1759).
The combination of antibody and chemotherapy usually enhances the degree of apoptosis and can partially reverse resistance in some cell lines (Buchsbaum D J et al., J Clin Cancer Res. 2003; 9:3731; DeRosier L C et al., Clin Cancer Res. 2007; 13:5535s; Oliver P G et al., Clin Cancer Res. 2008; 14:2180; Derosier L C et al., Mol Cancer Ther. 2007; 6:3198; Long J W. et al., J Surg Res. 2007; 137:167).
SUMMARY OF THE INVENTION
The present inventors have now found that unexpectedly, it is possible to induce the DR5 apoptotic pathway by using two antibodies directed against at least two different epitopes of the DR5 receptor. The binding to both epitopes on the same receptor has an agonistic action on the receptor and induces apoptosis in an efficient way. Combination of antibodies DR5-01 and DR5-05 as disclosed herein revealed a stronger agonistic action than the ligand itself.
An unexpected and synergistic action has been observed by using two antibodies directed each against a different epitope on the DR5 receptor, with respect to one antibody against one single epitope. Without wishing to be bound to the theory, it is postulated that the binding to the two epitopes of DR5 allows for a synergistic agonist function, leading to an unexpectedly elevated apoptosis induction. It has been found that the unexpected and synergistic action may be beneficial for therapeutic treatment or for integration to a therapeutic protocol. It has thus been found that the combination of the antibodies may lead to a synergic increase of inhibition of cancer cells proliferation in particular in glioma. It has also been found that the combination of the antibodies and a chemotherapeutic drug may lead to a synergic increase of inhibition of cancer cells proliferation in the case of cancers that are difficult to treat, such as glioma, lung and breast cancers that more or less resist to chemotherapeutic drugs. It has also been found that the combination of antibodies and drug may allow getting a therapeutic effect, such as inhibition of cell proliferation, which is stably obtained over a wide range of drug and/or antibodies dosages.
It is thus now possible to provide for pharmaceutical compositions comprising two polypeptides or antibodies acting as agonist by binding to the two different epitopes on DR5, or bispecific antibodies acting as agonist by binding to the two different epitopes on DR5 and pharmaceutical compositions containing the same.
An “agonist” or an “agonistic polypeptide or antibody” for a natural receptor is a compound which binds the receptor to form a receptor-agonist complex and which activates said receptor, initiating a pathway signaling and further biological process. In the context of the present invention, an agonist function is obtained owing the simultaneous or sequential interaction between the polypeptides or antibodies of the invention and two different epitopes of the DR5 receptor, initiating the DR5 apoptosis pathway.
An object of the invention is thus a composition comprising two polypeptides, or antibodies or fragment thereof, both having the capability to bind to DR5, a first polypeptide or antibody comprising a first antigen-binding site that binds to a first epitope of said DR5, and a second polypeptide or antibody comprising a second different antigen-binding site that binds to a second epitope of said DR5. Each of said first and second antigen-binding sites binds to a different epitope on the same DR5 molecule. The two polypeptides, or antibodies or fragments thereof are for a simultaneous, separate or sequential administration to a mammal, including human.
The composition or pharmaceutical composition may further contain a pharmaceutically acceptable carrier, diluent, or excipient. The polypeptides or antibodies are synergistically agonistic in combination, which means that they have the capability upon binding to both epitopes of a DR5 molecule to induce the DR5 apoptotic pathway.
An object of the invention is also a bispecific or biparatopic antibody, or fragment thereof, having the capability to bind to DR5, said antibody comprising a first antigen-binding site that binds to a first epitope of said DR5, and a second different antigen-binding site that binds to a second epitope of said DR5. Each of said first and second antigen-binding sites binds to a different epitope on the same DR5 molecule. The polypeptides or antibodies are synergistically agonistic in combination, which means that they have the capability upon binding of both to their specific epitopes of a DR5 molecule to induce the DR5 apoptotic pathway.
The invention encompasses the binding of one bispecific antibody to the two different epitopes of the same DR5 molecule, or of two bispecific antibodies to the two epitopes of the same DR5 molecule, one antibody to a first epitope, the second to the second epitope of the same DR5 molecule.
The bispecific antibody may be formulated in a pharmaceutical composition further containing a pharmaceutically acceptable carrier, diluent, or excipient.
Without wishing to be bound to theory, it is deemed that, regarding the mechanism of action, antibody combination or bispecific antibodies according to the invention may promote DR5 clustering. These components may promote DR5 amassing of higher concentration compared with a monospecific antibody.
Antibody combination or bispecific antibodies may promote also a conformation change inducing a higher incidence to trigger apoptosis signalling or to reverse the resistance of cancer cell to the apoptosis. These components may promote DR5 amassing of higher concentration compared with a monospecific antibody.
Another object of the invention encompasses the binding at least of two, three, four, five or more monovalent binding polypeptides, or antibodies or fragment thereof, both having the capability to bind to DR5, a first polypeptide or antibody comprising a first antigen-binding site that binds to a first epitope of said DR5, and a second polypeptide or antibody comprising a second different antigen-binding site that binds to a second epitope of said DR5.
Another object of the invention is thus a composition comprising at least one chemotherapeutic drug and two polypeptides, or antibodies or fragment thereof, both having the capability to bind to DR5, a first polypeptide or antibody comprising a first antigen-binding site that binds to a first epitope of said DR5, and a second polypeptide or antibody comprising a second different antigen-binding site that binds to a second epitope of said DR5. Each of said first and second antigen-binding sites binds to a different epitope on the same DR5 molecule. The drug and the two polypeptides, or antibodies or fragments thereof are for a simultaneous, separate or sequential administration to a mammal, including human. In this object, the two polypeptides may be replaced by a bispecific or biparatopic antibody, or fragment thereof, as disclosed herein.
The polypeptides, especially antibodies, according to the invention may be further defined by the CDRs of the VH and VL regions of the murine antibodies DR5-01 and DR5-05 or by their complete VH and VL regions.
An object of the invention is to a composition comprising at least one or two polypeptides binding specifically a DR5 receptor, wherein the at least one or two polypeptides comprise two immunoglobulin binding domains comprising:
a first binding domain comprising a pair of VH and VL chains wherein
the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 13, a CDR2 comprising or consisting of sequence SEQ ID NO: 14 CDR1, a CDR3 comprising or consisting of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 16, a CDR2 comprising or consisting of sequence FAS, a CDR3 comprising or consisting of sequence SEQ ID NO: 17; or
wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO:22, a CDR2 comprising or consisting of sequence SEQ ID NO: 23, a CDR3 comprising or consisting of sequence SEQ ID NO: 24; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 25, a CDR2 comprising or consisting of sequence SEQ ID NO: 26, a CDR3 comprising or consisting of sequence SEQ ID NO: 17, or
wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 32, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 24; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 16, a CDR2 comprising or consisting of sequence FAS, a CDR3 comprising or consisting of sequence SEQ ID NO: 17,
and
a second binding domain comprising a pair of VH and VL chains wherein
the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 18, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 20, a CDR2 comprising or consisting of sequence RTS, a CDR3 comprising or consisting of sequence SEQ ID NO: 21, or
wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 27, a CDR2 comprising or consisting of sequence SEQ ID NO: 28, a CDR3 comprising or consisting of sequence SEQ ID NO: 29; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 30, a CDR2 comprising or consisting of sequence SEQ ID NO: 31, a CDR3 comprising or consisting of sequence SEQ ID NO: 21, or
wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 33, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 29; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 20, a CDR2 comprising or consisting of sequence RTS, a CDR3 comprising or consisting of sequence SEQ ID NO: 21,
wherein
the at least one polypeptide comprises both immunoglobulin binding domains, or
the at least two polypeptides comprise a first polypeptide comprising the first binding domain and a second polypeptide comprising the second binding domain for a simultaneous, separate or sequential administration to a mammal, including man,
and a pharmaceutically carrier, diluent or excipient. In an embodiment, the composition comprises further a chemotherapeutic drug for a simultaneous, separate or sequential administration to a mammal, including man.
Other objects of the invention are the individual polypeptides or antibodies and their various combinations in accordance with the invention, kits comprising at least two polypeptides or antibodies, and kits comprising at least one polypeptide or antibody and at least one drug, wherein antibodies or polypeptides and drugs are separated or not.
The polypeptides or antibodies of the invention may comprise one or several, preferably two, binding sites or domains or paratopes. An object of the present invention is a polypeptide binding specifically a DR5 receptor, comprising one or more, preferably one or two, immunoglobulin binding domain(s) comprising:
a binding domain comprising a pair of VH and VL chains wherein:
the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 13, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 16, a CDR2 comprising or consisting of sequence FAS, a CDR3 comprising or consisting of sequence SEQ ID NO: 17 (DR5-01 type CDRs), or
the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO:22, a CDR2 comprising or consisting of sequence SEQ ID NO: 23, a CDR3 comprising or consisting of sequence SEQ ID NO: 24; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 25, a CDR2 comprising or consisting of sequence SEQ ID NO: 26, a CDR3 comprising or consisting of sequence SEQ ID NO: 17, (DR5-01 type CDRs), or
the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 32, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 24; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 16, a CDR2 comprising or consisting of sequence FAS, a CDR3 comprising or consisting of sequence SEQ ID NO: 17 (DR5-01 type CDRs);
and/or
a binding domain comprising a pair of VH and VL chains wherein:
the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 18, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 20, a CDR2 comprising or consisting of sequence RTS, a CDR3 comprising or consisting of sequence SEQ ID NO: 21 (DR5-05 type CDRs), or
the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 27, a CDR2 comprising or consisting of sequence SEQ ID NO: 28, a CDR3 comprising or consisting of sequence SEQ ID NO: 29; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 30, a CDR2 comprising or consisting of sequence SEQ ID NO: 31, a CDR3 comprising or consisting of sequence SEQ ID NO: 21 (DR5-05 type CDRs), or
the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 33, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 29; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 20, a CDR2 comprising or consisting of sequence RTS, a CDR3 comprising or consisting of sequence SEQ ID NO: 21 (DR5-05 type CDRs).
The binding domain is best defined by the VH and VL chains comprising the CDRs defined based on the same method, either IMGT®, Kabat® or common numbering system, see CDR table infra.
The VH and VL chains together define a single binding site. Each one of these binding domain binds specifically to a different epitope on the DR5 receptor. The polypeptides are synergistically agonistic, which means that they have the capability upon binding to both epitopes of a DR5 molecule to induce the DR5 apoptotic pathway.
By “immunoglobulin binding domain” or “binding domain” it is meant the paratope of an immunoglobulin made of the two variable light (VL) and variable heavy (VH) chains. The paratope is able to specifically bind to the targeted epitope.
In accordance with the invention, the VL and VH chains have a conventional structure of a light chain or a heavy chain of an immunoglobulin, with the framework regions FR. The structure may be defined as the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In a preferred embodiment, the polypeptide of the invention comprises one or more, preferably one or two, immunoglobulin binding domain(s) comprising the VH+VL region of mDR5-01 and/or the VH+VL region of mDR5-05. In an embodiment, the polypeptide comprise one or two binding domain(s) comprising the VH+VL region of mDR5-01. In an embodiment, the polypeptide comprise one or two binding domain(s) comprising the VH+VL region of mDR5-05. In an embodiment, the polypeptide comprise two binding domain(s) comprising the VH+VL region of mDR5-05, on the one hand, and the VH+VL region of mDR5-01, on the other hand. In a preferred embodiment, the polypeptide of the invention comprises one or more, preferably one or two, immunoglobulin binding domain(s) comprising the VH+VL region of HzDR5-01 and/or the VH+VL region of HzDR5-05. In an embodiment, the polypeptide comprise one or two binding domain(s) comprising the VH+VL region of HzDR5-01. In an embodiment, the polypeptide comprise one or two binding domain(s) comprising the VH+VL region of HzDR5-05. In an embodiment, the polypeptide comprise two binding domain(s) comprising the VH+VL region of HzDR5-05, on the one hand, and the VH+VL region of HzDR5-01, on the other hand.
The anti-DR5 polypeptide thus comprises one or two binding domains. In an embodiment, the binding domains are specific of the same epitope on the DR5 receptor. These binding domains comprise the same set of 3 CDRs on the VH and VL as disclosed and provided therein and may be identical or slightly different in the framework regions, as soon as this does not affect the specificity to bind the targeted epitope.
The anti-DR5 polypeptide may be in particular an antibody, preferably a monoclonal antibody, or a suitable antibody fragment, such as a Fv, a Fab, a F(ab)2, a single-chain variable fragment (scFv).
The invention also encompasses the combined use of polypeptides or antibodies or of bispecific polypeptides or antibodies or fragments and the like, making use of the synergic activity linked to binding to the two epitopes revealed by the present invention. This use may be further combined with the administration of a chemotherapeutic drug, as disclosed herein.
Another object of the invention encompasses the binding at least of two, three, four, five or more monovalent binding polypeptides, or antibodies or fragment thereof, both having the capability to bind to DR5, a first polypeptide or antibody comprising a first antigen-binding site that binds to a first epitope of said DR5, and a second polypeptide or antibody comprising a second different antigen-binding site that binds to a second epitope of said DR5.
Thus, another object of the invention is a composition comprising two polypeptides, or antibodies or fragment thereof, both having the capability to bind to DR5, a first polypeptide or antibody comprising a first antigen-binding site that binds to a first epitope of said DR5, this first epitope being the one to which specifically binds a binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14 CDR1, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17, and a second polypeptide or antibody comprising a second different antigen-binding site that binds to a second epitope of said DR5, this epitope being the one to which specifically binds a binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21, for a simultaneous, separate or sequential administration to a mammal, including man. As an alternative, one may replace herein above the definition of the CDRs by those according to Kabat® or Common numbering System as per Tables 1 and 2.
Another object of the invention is a bispecific antibody, or fragment thereof, having the capability to bind to DR5, said antibody comprising a first antigen-binding site that binds to a first epitope of said DR5, this first epitope being the one to which specifically binds a binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14 CDR1, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17, and a second different antigen-binding site that binds to a second epitope of said DR5, this epitope being the one to which specifically binds a binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21. As an alternative, one may replace hereinabove the definition of the CDRs by those according to Kabat® or Common numbering System as per Tables 1 and 2.
Another object of the invention is the method of treatments, comprising the administration of an effective or sufficient amount of at least two polypeptides or antibodies as disclosed herein, or of at least one bispecific or biparatopic polypeptide or antibody as disclosed herein, or of at least two polypeptides or antibodies and at least one drug, as disclosed herein, or of at least one bispecific or biparatopic polypeptide or antibody and at least one drug, as disclosed herein. By treatment is meant in particular treatment of various cancers, autoimmune diseases, infectious diseases that express DR5 antigen.
Definitions
The terms “apoptosis” and “apoptotic activity” are used in a broad sense and refer to the orderly or controlled form of cell death in mammals that is typically accompanied by one or more characteristic cell changes, including condensation of cytoplasm, loss of plasma membrane microvilli, segmentation of the nucleus, degradation of chromosomal DNA or loss of mitochondrial function. This activity can be determined and measured, for instance, by cell viability assays, FACS analysis or DNA electrophoresis, and more specifically by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies).
As used herein, the term “synergy” or “synergism” or “synergistically” refers to the interaction of two or more agents so that their combined effect is greater than the sum of their individual effects.
The term “agonist” and “agonistic” when used herein refer to or describe a molecule which is capable of, directly or indirectly, substantially inducing, promoting or enhancing DR5 biological activity or activation. Optionally, an “agonist DR5 antibody” is an antibody which has activity at least comparable to the ligand for DR5, known as Apo-2 ligand (TRAIL), or is capable of activating DR5 receptor which results in an activation of one more intracellular signaling pathway which may include activation of caspase 3, caspase 8, caspase 10 or FADD.
The terms “antagonist” and “antagonistic” when used herein refer to or describe a molecule which is capable of, directly or indirectly, substantially counteracting, reducing or inhibiting DR5 biological activity of DR5 activation. Optionally, an antagonist is a molecule which neutralizes the biological activity resulting from DR5 activation or formation of a complex between DR5 and its ligand, such as Apo-2 ligand.
The term “antibody” is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multivalent antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
“Native antibodies” and “native immunoglobulins” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH).
As used herein, an “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
With respect to antibodies of the invention, the term “immunologically specific” or “specifically binds” refers to antibodies that bind to one or more epitopes of a protein of interest (e.g., DR5/TRAIL R2), but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
The “epitope DR5-01” and the “epitope DR5-05” are the regions in the extracellular domain of DR5 to which the DR5-01 and the DR5-05 antibodies bind respectively.
The term “bispecific antibody” as used herein refers to an antibody comprising two antigen-binding sites, a first binding site having affinity for a first antigen or epitope and a second binding site having binding affinity for a second antigen or epitope distinct from the first.
“Bispecific antibodies” or “biparatopic antibodies” are single, divalent antibodies which have two different specific antigen binding sites. According to this invention, these antibodies have two different binding sites, each one directed against a specific and different epitope on the DR5 molecule. This definition also encompasses the fragments of a bispecific or biparatopic antibody that comprise both binding site and wherein each of these binding sites has the capability of binding to the corresponding epitope on DR5. Such a fragment may be for example a F(ab′)2 antibody fragment.
The term “bivalent, bispecific antibody” as used herein refers to an antibody as described above in which each of the two pairs of heavy chain and light chain (HC/LC) are specifically binding to a different epitope, i.e. the first heavy and light chains are specifically binding together to a first epitope, and, the second heavy and light chains are specifically binding together to a second epitope; such bivalent, bispecific antibodies are capable of specifically binding to two different epitopes, at the same time or not.
According to the invention, the ratio of a desired bivalent, bispecific antibody compared to undesired side products can be improved by the replacement of certain domains in only one pair of heavy chain and light chain (HC/LC). While the first of the two HC/LC pairs originates from an antibody specifically binding to a first epitope and is left essentially unchanged, the second of the two HC/LC pairs originates from an antibody specifically binding to a second epitope, and is altered by the following replacement:
Light chain: replacement of the variable light chain domain VL by the variable heavy chain domain VH of said antibody specifically binding to a second epitope, and the constant light chain domain CL by the constant heavy chain domain CH of said antibody specifically binding to a second epitope and
Heavy chain: replacement of the variable heavy chain domain VH by the variable light chain domain VL of said antibody specifically binding to a second epitope, and the constant heavy chain domain CH by the constant light chain domain CL of said antibody specifically binding to a second epitope.
Engineered proteins, such as bi- or multivalent antibodies capable of binding two or more antigens or epitopes are known in the art. Such multivalent binding proteins can be generated using cell fusion, chemical conjugation, or recombinant DNA techniques.
In one approach bispecific antibodies that are very similar to natural antibodies have been produced using the quadroma technology, (Milstein C. et al., Nature. 1983; 305:537-40) based on the somatic fusion of two different hybridoma cell lines expressing murine monoclonal antibodies with the desired specificities of the bispecific antibody. Because of the random pairing of two different antibody heavy and light chains within the resulting hybrid-hybridoma (or quadroma) cell line, up to ten different antibodies species are generated of which only one is the desired, functional bispecific antibody. Due to the presence of mispaired byproducts, and significantly reduced production yields, means sophisticated purification procedures are required, (Morrison S. L., Nature Biotech. 2007; 25:1233-1234). In general the same problem of mispaired byproducts remains if recombinant expression techniques are used.
An approach to circumvent the problem of mispaired byproducts, which is known as “knobs-into-holes”, aims at forcing the pairing of two different antibody heavy chains by introducing mutations into the CH3 domains to modify the contact interface. On one chain bulky amino acids are replaced by amino acids with short side chains to create a “hole”. Conversely, amino acids with large side chains are introduced into the other CH3 domain, to create a “knob”. By coexpressing these two heavy chains (and two identical light chains, which have to be appropriate for both heavy chains), high yields of heterodimer formation (“knob-hole”) versus homodimer formation (“hole-hole” or “knob-knob”) may be observed, (Ridgway, J B et al., Protein Eng. 1996; 9:617-621; and WO 96/027011).
“Antibody fragments” comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. A suitable “antibody fragment” is a fragment of antibody that has the capability to bind to the DR5 epitope and initiate the apoptosis pathway.
Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies, (Zapata et al., Protein Eng. 1995; 8(10):1057-1062); single-chain antibody molecules; and multivalent antibodies formed from antibody fragments.
An “intact” antibody is one which comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each comprising a single antigen-binding site and a CL and a CH1 region, and a residual Fc fragment. Pepsin treatment yields an “F(ab′)2” fragment that has two antigen-binding sites and is still capable of cross-linking antigen.
“Fv” is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions (CDRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions or CDRs confer antigen-binding specificity to the antibody.
The “Fab” fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain and has one antigen-binding site only.
“Fab” fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known (Hermanson et al., Bioconjugate Techniques, Academic Press, 1996, U.S. Pat. No. 4,342,566).
“Single-chain Fv” or “scFv” antibody fragments comprise the VH and the VL domains of an antibody wherein these domains are present in a single polypeptide chain. Preferably, the scFv comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers as well as to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid. The term also includes variants on the traditional peptide linkage joining the amino acids making up the polypeptide. Preferred “peptides”, “polypeptides”, and “proteins” are chains of amino acids whose carbons are linked through peptide bonds.
The terminal amino acid at one end of the chain (amino terminal) therefore has a free amino group, while the terminal amino acid at the other end of the chain (carboxy terminal) has a free carboxyl group. As used herein, the term “amino terminus” (abbreviated N-terminus) refers to the free α-amino group on an amino acid at the amino terminal of a peptide or to the α-amino group (amino group when participating in a peptide bond) of an amino acid at any other location within the peptide. Similarly, the term “carboxy terminus” refers to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other location within the peptide. Peptides also include essentially any polyamino acid including, but not limited to peptide mimetics such as amino acids joined by ether as opposed to an amine bond.
The term “variable” refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated, in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies, (Kabat et al., NIH Publ. 1991; No. 91-3242, Vol. 1, 647-669). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effectors functions, such as participation of the antibody in antibody-dependent cellular toxicity.
The monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
Other preferred forms of “chimeric antibodies” encompassed by the present invention are those in which the constant region has been modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to C1q binding and/or Fc receptor (FcR) binding.
Such chimeric antibodies are also referred to as “class-switched antibodies”. Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art. See, e.g., Morrison, S. L et al., Proc. Natl. Acad. Sci. USA 1984; 81:6851-6855; U.S. Pat. Nos. 5,202,238 and 5,204,244. WO 2006/093794 relates to heterodimeric protein binding compositions. WO 99/37791 describes multipurpose antibody derivatives. Morrison et al., the J. Immunolog. 1998; 160:2802-2808 refers to the influence of variable region domain exchange on the functional properties of IgG.
“Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
In a preferred embodiment, a murine CDR is grafted into the framework region of a human antibody to prepare the “humanized antibody”. See, e.g., Riechmann, L. et al., Nature. 1988; 332: 323-327; and Neuberger, M S et al., Nature. 1985; 314: 268-270. Particularly preferred CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric antibodies. Other forms of “humanized antibodies” encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to C1q binding and/or Fc receptor (FcR) binding.
Furthermore, humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature. 1986; 321:522-525; Reichmann et al., Nature. 1988; 332:323-329; and Presta et al., Curr. Op. Struct. Biol. 1992; 2:593-596.
Immune effector functions which have been shown to contribute to antibody-mediated cytotoxicity include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).
Cytotoxicity may also be mediated via antiproliferative effects. The mechanism of antibody modulation of tumor cell proliferation is poorly understood. However, advances in understanding the interactions of antibodies with Fcg receptors (FcgR) on immune effector cells have allowed the engineering of antibodies with significantly improved effector function.
The mechanism of action of MAbs is complex and appears to vary for different MAbs. There are multiple mechanisms by which MAbs cause target cell death. These include apoptosis, CDC, ADCC and inhibition of signal transduction.
Effector functions such as CDC and ADCC are effector functions that may be important for the clinical efficacy of MAbs. All of these effector functions are mediated by the antibody Fc region and let authors to attempt amino acid modifications with more or less success. Glycosylation, especially fucosylation of the Fc region have a dramatic influence on the efficacy of an antibody. This let the authors to modify the conditions of production of the antibodies in the CHO cells in order to change the glycosylation profile in an attempt here again to improve some effector functions, with more or less success one again.
Previous research has shown that a polymorphism of the FcgRIIIa gene encodes for either a phenylalanine (F) or a valine (V) at amino acid 158. Expression of the valine isoform correlates with increased affinity and binding to MAbs (Rowland A J, et al. 1993. Cancer Immunol Immunother. 37(3):195-202; Sapra P, Allen T M. 2002. Cancer Res 62: 7190-4; Mølhøj M, et al. 2007. Mol Immunol. 44(8):1935-43). Some clinical studies have supported this finding, with greater clinical response to rituximab in patients with non-Hodgkin's lymphoma who display the V/V polymorphism (Bargou R, et al. 2008. Science. 321:974-7; Bruenke J, 2005. Br J Haematol. 130(2):218-28; Cartron G, Blood. 2002 Feb. 1; 99(3):754-8; Hekman A, et al. 1991. Cancer Immunol Immunother 32:364-72).
WO1999051642 describes a variant human IgG Fc region comprising an amino acid substitution at positions 270 or 329, or at two or more of positions 270, 322, 329, and 331. These modifications aim at increasing the CDC and ADCC effector functions
“Treatment” or “therapy” refer to both therapeutic treatment and prophylactic or preventative measures.
“Mammal” for purposes of treatment or therapy refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.
The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, renal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
The term “nucleic acid” or “oligonucleotide” or grammatical equivalents herein refer to at least two nucleotides covalent linked together. A nucleic acid of the present invention is preferably single-stranded or double stranded and will generally contain phosphodiester bonds.
Amino acid sequence “variants” (or mutants) of the antibody are prepared by introducing appropriate nucleotide changes into the antibody DNA, or by nucleotide synthesis. Such modifications can be performed, however, only in a very limited range, e.g. as described above. For example, the modifications do not alter the above mentioned antibody characteristics such as the IgG isotype and antigen binding, but may improve the yield of the recombinant production, protein stability or facilitate the purification.
DETAILED DESCRIPTION OF THE INVENTION
The CDR sequences may be defined in accordance with IMGT®, Kabat® or the Common numbering system which retain the common sequence between IMGT® and Kabat®.
The CDRs for the anti-DR5 antibodies mDR5-01 a chimeric antibody with murine VH and VL and human Fc) and HzDR5-01 (a humanized antibody with murine CDRs and human FR with or without back mutation and Fc optimized or not) of the invention comprises the following CDRs:
TABLE 1
Sequence
SEQ
SEQ
SEQ
(Common
ID
Sequence
ID
Sequence
ID
numbering
NO:
IMGT®
NO:
Kabat®
NO:
system)
VH mDR5-01-VH HzDR5-01
CDR1
13
GFNIKDTF
22
DTFIH
32
KDTF
CDR2
14
IDPANGNT
23
RIDPANGNT
14
IDPANGNT
KYDPKFQG
CDR3
15
VRGLYTYYFDY
24
GLYTYYFDY
24
GLYTYYFDY
VL mDR5-01-VH HzDR5-01
CDR1
16
QSISNN
25
RASQSISNN
16
QSISNN
LH
CDR2
FAS
26
FASQSIS
FAS
CDR3
17
QQGNSWPYT
17
QQGNSWPYT
17
QQGNSWPYT
The CDRs for the anti-DR5 antibodies mDR5-05 and HzDR5-05 of the invention comprises the following CDRs:
TABLE 2
Sequence
SEQ
SEQ
SEQ
(Common
ID
Sequence
ID
Sequence
ID
numbering
NO:
IMGT®
NO:
Kabat®
NO:
system)
VH mDR5-05-VH HzDR5-05
CDR1
18
GFNIKDTH
27
DTHIH
33
KDTH
CDR2
14
IDPANGNT
28
RIDPANGNT
14
IDPANGNT
EYDPKFQG
CDR3
19
ARWGTNVYFAY
29
WGTNVYFAY
29
WGTNVYFAY
VL mDR5-05-VH HzDR5-05
CDR1
20
SSVSY
30
SASSSVSYMY
20
SSVSY
CDR2
RTS
31
RTSNLAS
RTS
CDR3
21
QQYHSYPPT
21
QQYHSYPPT
21
QQYHSYPPT
By definition, these CDRs include variant CDRs, by deletion, substitution or addition of one or more amino acid(s), which variant keeps the specificity of the original CDR. The common numbering system provides for a CDR definition having the shortest amino acid sequences or the minimal CDR definition.
mDR5-01, mDR5-05, HzDR5-01 and HzDR5-05 have the VH and VL amino acid sequences and nucleic acid sequences are depicted on the following tables:
TABLE 3
Amino acid sequence VH
Amino acid sequence VL
mDR5-01
SEQ ID NO: 4
SEQ ID NO: 2
mDR5-05
SEQ ID NO: 8
SEQ ID NO: 6
HzDR5-01
SEQ ID NO: 35
SEQ ID NO: 37
HzDR5-05
SEQ ID NO: 39
SEQ ID NO: 41
TABLE 4
Nucleic acid sequence VH
Nucleic acid sequence VL
mDR5-01
SEQ ID NO: 3
SEQ ID NO: 1
mDR5-05
SEQ ID NO: 7
SEQ ID NO: 5
HzDR5-01
SEQ ID NO: 34
SEQ ID NO: 36
HzDR5-05
SEQ ID NO: 38
SEQ ID NO: 40
DR5-01 and DR5-05 have the CH and CL amino acid sequences and nucleic acid sequences are depicted on the following tables:
TABLE 5
CH
CL
Amino acid sequence
SEQ ID NO: 10
SEQ ID NO: 12
Nucleic acid sequence
SEQ ID NO: 9
SEQ ID NO: 11
In an embodiment, the polypeptide comprises one or two binding domains comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17. This polypeptide binds specifically to a first epitope on the DR5 receptor. In an embodiment, the polypeptide comprises two such binding domains.
In an embodiment, the polypeptide comprises one or two binding domains comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 22, a CDR2 of sequence SEQ ID NO: 23, a CDR3 of sequence SEQ ID NO: 24; and the VL chain contains a CDR1 of sequence SEQ ID NO: 25, a CDR2 of sequence SEQ ID NO: 26, a CDR3 of sequence SEQ ID NO: 17. This polypeptide binds specifically to a first epitope on the DR5 receptor. In an embodiment, the polypeptide comprises two such binding domains.
In an embodiment, the polypeptide comprises one or two binding domains comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 32, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 24; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17. This polypeptide binds specifically to a first epitope on the DR5 receptor. In an embodiment, the polypeptide comprises two such binding domains.
In another embodiment, the polypeptide comprises a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21. This polypeptide binds specifically to a second and different epitope on the DR5 receptor. In an embodiment, the polypeptide comprises two such binding domains.
In another embodiment, the polypeptide comprises a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 27, a CDR2 of sequence SEQ ID NO: 28, a CDR3 of sequence SEQ ID NO: 29; and the VL chain contains a CDR1 of sequence SEQ ID NO: 30, a CDR2 of sequence SEQ ID NO: 31, a CDR3 of sequence SEQ ID NO: 21. This polypeptide binds specifically to a second and different epitope on the DR5 receptor. In an embodiment, the polypeptide comprises two such binding domains.
In another embodiment, the polypeptide comprises a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 33, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 29; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21. This polypeptide binds specifically to a second and different epitope on the DR5 receptor. In an embodiment, the polypeptide comprises two such binding domains.
In another embodiment, the anti-DR5 polypeptide comprises two binding domains and these two binding domains are each specific of a different epitope on the DR5 receptor. These binding domains comprise a specific set of 3 CDRs on the VH and VL as disclosed and provided therein and may be identical or slightly different in the framework regions.
The anti-DR5 polypeptide may be in particular a F(ab′)2, Fab, Fv, a divalent single-chain variable fragment (scFv), an antibody, preferably a monoclonal antibody, fragment, nanobody, multimeric scFv.
In this embodiment, the anti-DR5 polypeptide, preferably antibody, is bispecific or biparatopic and comprises:
a first binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 13, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 16, a CDR2 comprising or consisting of sequence FAS, a CDR3 comprising or consisting of sequence SEQ ID NO: 17, and
a second binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 18, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 20, a CDR2 comprising or consisting of sequence RTS, a CDR3 comprising or consisting of sequence SEQ ID NO: 21.
In an embodiment, the anti-DR5 polypeptide, preferably antibody, is bispecific or biparatopic and comprises:
a first binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO:22, a CDR2 comprising or consisting of sequence SEQ ID NO: 23, a CDR3 comprising or consisting of sequence SEQ ID NO: 24; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 25, a CDR2 comprising or consisting of sequence SEQ ID NO: 26, a CDR3 comprising or consisting of sequence SEQ ID NO: 17, and
a second binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 27, a CDR2 comprising or consisting of sequence SEQ ID NO: 28, a CDR3 comprising or consisting of sequence SEQ ID NO: 29; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 30, a CDR2 comprising or consisting of sequence SEQ ID NO: 31, a CDR3 comprising or consisting of sequence SEQ ID NO: 21.
In an embodiment, the anti-DR5 polypeptide, preferably antibody, is bispecific or biparatopic and comprises:
a first binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 32, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 24; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 16, a CDR2 comprising or consisting of sequence FAS, a CDR3 comprising or consisting of sequence SEQ ID NO: 17, and
a second binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 33, a CDR2 comprising or consisting of sequence SEQ ID NO: 14, a CDR3 comprising or consisting of sequence SEQ ID NO: 29; and the VL chain contains a CDR1 comprising or consisting of sequence SEQ ID NO: 20, a CDR2 comprising or consisting of sequence RTS, a CDR3 comprising or consisting of sequence SEQ ID NO: 21.
This bispecific or biparatopic anti-DR5 polypeptide or antibody comprises the two different domains of the invention and may bind specifically to either one of the two different epitopes or to both different epitopes at the same time.
In some embodiments, the anti-DR5 polypeptides preferably antibody of the invention comprises:
one or more of amino acid sequence pairs SEQ ID NO: 2 and 4 (VL and VH from DR5-01) and SEQ ID NO: 6 and 8 (VH and VL from DR5-05),
the pair of amino acid sequences SEQ ID NO: 2 and 4, (VL and VH from DR5-01)
the pair of amino acid sequences SEQ ID NO: 6 and 8, (VL and VH from DR5-05) or
both amino acid sequence pairs SEQ ID NO: 2 and 4 (VL and VH from DR5-01) and SEQ ID NO: 6 and 8 (VL and VH from DR5-05);
one or more of amino acid sequence pairs SEQ ID NO: 35 and 37 (VH and VL from HzDR5-01) and SEQ ID NO: 39 and 41 (VH and VL from HzDR5-05),
the pair of amino acid sequences SEQ ID NO: 35 and 37, (VH and VL from HzDR5-01)
the pair of amino acid sequences SEQ ID NO: 39 and 41, (VH and VL from HzDR5-05) or
both amino acid sequence pairs SEQ ID NO: 35 and 37 (VH and VL from HzDR5-01) and SEQ ID NO: 39 and 41 (VL and VH from HzDR5-05).
In some embodiments, the anti-DR5 polypeptide, preferably antibody of the invention comprises:
two of each amino acid sequences SEQ ID NO: 4, 10, 2 and 12 (e.g. the whole or intact DR5-01 antibody)
amino acid sequences SEQ ID NO: 4, 10, 2 and 12 (single chain Fv based on DR5-01),
two of each amino acid sequences SEQ ID NO: 8, 10, 6 and 12 (e.g. the whole or intact DR5-05 antibody)
amino acid sequences SEQ ID NO: 8, 10, 6 and 12 (single chain Fv based on DR5-05);
amino acid sequences SEQ ID NO: 4, 8, 2, 6, 10 and 12 (bispecific antibody), especially the bispecific antibody comprises SEQ ID NO: 4, 8, 2, 6 (one of each) and 10, 12 (two of each);
amino acid sequences SEQ ID NO: 2 and 12 (light chain);
amino acid sequences SEQ ID NO: 6 and 12 (light chain);
amino acid sequences SEQ ID NO: 4 and 10 (heavy chain);
amino acid sequences SEQ ID NO: 8 and 10 (heavy chain).
two of each amino acid sequences SEQ ID NO: 35, 10, 37 and 12 (e.g. the whole or intact HzDR5-01 antibody)
amino acid sequences SEQ ID NO: 35, 10, 37 and 12 (single chain Fv based on HzDR5-01);
two of each amino acid sequences SEQ ID NO: 39, 10, 41 and 12 (e.g. the whole or intact HzDR5-05 antibody)
amino acid sequences SEQ ID NO: 39, 10, 41 and 12 (single chain Fv based on HzDR5-05);
amino acid sequences SEQ ID NO: 35, 39, 37, 41, 10 and 12 (bispecific antibody), especially the bispecific antibody comprises SEQ ID NO: 35, 39, 37, 41 (one of each) and 10, 12 (two of each);
amino acid sequences SEQ ID NO: 37 and 12 (light chain);
amino acid sequences SEQ ID NO: 41 and 12 (light chain);
amino acid sequences SEQ ID NO: 35 and 10 (heavy chain);
amino acid sequences SEQ ID NO: 39 and 10 (heavy chain).
The anti-DR5 polypeptides, preferably antibodies, of the invention may be fully murine, say they comprise amino acid sequences that match with the amino acid sequence of the maternal or original murine antibody. The polypeptides of the invention may also be chimeric or humanized, say they can comprise human-derived amino acid sequences. Specifically, the polypeptide may comprise framework regions and/or constant regions of a human-derived antibody.
Another object of the invention is a composition or pharmaceutical composition comprising one, two or more polypeptides according to the invention, as disclosed above and provided herein, and a pharmaceutically acceptable carrier, diluent or excipient. Embodiments of these compositions are defined by using the CDRs definitions according to IMGT®. However, the invention encompasses and relates also to the equivalent or alternative compositions wherein the IMGT® numbering is replaced either by the Kabat® numbering or the Common numbering system, using the sequences indicated supra. Therefore, in the following embodiments of a composition, other embodiments are part of the invention in which one replaces the CDRs defined with IMGT® numbering, by the Kabat® numbering, in accordance with the table supra. Also, in the following embodiments of a composition, other embodiments are part of the invention in which one replaces the CDRs defined with IMGT® numbering, by the Common numbering system, in accordance with the table supra.
In a first embodiment, the composition comprises a polypeptide, preferably antibody, having one or two binding domain(s) comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17. In a second embodiment, the composition comprises a polypeptide, preferably antibody, having one or two binding domain(s) comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21. In a third embodiment, the composition comprises these two polypeptides or antibodies in mixture. As an alternative, one may replace hereinabove the definition of the CDRs by those according to Kabat® or Common numbering System as per Tables 1 and 2.
In another embodiment, the composition comprises a anti-DR5 bispecific polypeptide, preferably antibody, comprising a first binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17, and a second binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21. As an alternative, one may replace hereinabove the definition of the CDRs by those according to Kabat® or Common numbering System as per Tables 1 and 2.
In an embodiment, the composition comprises an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 2 and 4, an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 6 and 8, and a pharmaceutically carrier, diluents or excipient. In an embodiment, the composition comprises an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 35 and 37, an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 39 and 41, and a pharmaceutically carrier, diluents or excipient.
The present invention also relates to these compositions comprising at least two polypeptides, preferably antibodies, for a simultaneous, separate or sequential administration to a mammal, including man.
A particular object is a composition comprising a bispecific anti-DR5 antibody comprising an amino acid sequence pair SEQ ID NO: 2 and 4 and an amino acid sequence pair SEQ ID NO: 6 and 8, or comprising an amino acid sequence pair SEQ ID NO: 35 and 37 and an amino acid sequence pair SEQ ID NO: 39 and 41, and a pharmaceutically acceptable carrier.
An object of the invention is especially a composition comprising at least one or two polypeptides binding specifically a DR5 receptor, wherein the at least one or two polypeptides comprise two immunoglobulin binding domains comprising:
a first binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14 CDR1, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17, and
a second binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21,
wherein
the at least one polypeptide comprises both immunoglobulin binding domains, or
the at least two polypeptides comprise a first polypeptide comprising the first binding domain and a second polypeptide comprising the second binding domain for a simultaneous, separate or sequential administration to a mammal, including man,
and a pharmaceutically carrier, diluent or excipient. As an alternative, one may replace hereinabove the definition of the CDRs by those according to Kabat® or Common numbering System as per Tables 1 and 2.
In some embodiments, the composition of the invention comprises an anti-DR5 polypeptide, preferably antibody comprising:
two of each amino acid sequences SEQ ID NO: 4, 10, 2 and 12 (e.g. the whole or intact DR5-01 antibody)
amino acid sequences SEQ ID NO: 4, 10, 2 and 12 (single chain Fv based on DR5-01),
two of each amino acid sequences SEQ ID NO: 8, 10, 6 and 12 (e.g. the whole or intact DR5-05 antibody)
amino acid sequences SEQ ID NO: 8, 10, 6 and 12 (single chain Fv based on DR5-05);
amino acid sequences SEQ ID NO: 4, 8, 2, 6, 10 and 12 (bispecific antibody), especially the bispecific antibody comprises SEQ ID NO: 4, 8, 2, 6 (one of each) and 10, 12 (two of each);
amino acid sequences SEQ ID NO: 2 and 12 (light chain);
amino acid sequences SEQ ID NO: 6 and 12 (light chain);
amino acid sequences SEQ ID NO: 4 and 10 (heavy chain);
amino acid sequences SEQ ID NO: 8 and 10 (heavy chain);
two of each amino acid sequences SEQ ID NO: 35, 10, 37 and 12 (e.g. the whole or intact HzDR5-01 antibody)
amino acid sequences SEQ ID NO: 35, 10, 37 and 12 (single chain Fv based on HzDR5-01);
two of each amino acid sequences SEQ ID NO: 39, 10, 41 and 12 (e.g. the whole or intact HzDR5-05 antibody)
amino acid sequences SEQ ID NO: 39, 10, 41 and 12 (single chain Fv based on HzDR5-05);
amino acid sequences SEQ ID NO: 35, 39, 37, 41, 10 and 12 (bispecific antibody), especially the bispecific antibody comprises SEQ ID NO: 35, 39, 37, 41 (one of each) and 10, 12 (two of each);
amino acid sequences SEQ ID NO: 37 and 12 (light chain);
amino acid sequences SEQ ID NO: 41 and 12 (light chain);
amino acid sequences SEQ ID NO: 35 and 10 (heavy chain);
amino acid sequences SEQ ID NO: 39 and 10 (heavy chain).
These compositions may comprise at least one additional polypeptide or antibody directed against another target and/or at least one chemotherapeutic drug (such as small molecule), for a simultaneous, separate or sequential administration with polypeptide(s) or antibody(ies) of the invention, to a mammal, including man. As additional active principle, one may cite doxorubicine, gemcitabine, camptothecin, paclitaxel. The composition may comprise two polypeptides, or antibodies or fragments thereof, both having the capability to bind to DR5, modified to comprise a variant human optimized IgG Fc region, preferably IgG1 Fc region, wherein this variant region comprises an amino acid substitution to modulate PDCC, ADCC and/or CDC. In particular, two polypeptides, or antibodies or fragments thereof, have the capability to bind to DR5, and conjugate to cellular cytotoxic components (ADC)z.
The compositions or pharmaceutical compositions according to the invention are intended for use as a medicament, especially to induce apoptosis of a tumor cell. The compositions or pharmaceutical compositions according to the invention are intended for use as a medicament, especially to treat cancer, preferably a solid cancer.
The isolated nucleic acid sequences disclosed and provided herein are also object of the invention.
Thus the invention also relates to an isolated nucleotide sequence comprising the following nucleotide sequences SEQ ID NO: 1, 3, 5, or 7 or combinations of nucleotide sequences linked together; SEQ ID NO: 9 and 7, or 9 and 3, SEQ ID NO: 11 and 1, or 11 and 5. The invention also relates to an isolated nucleotide sequence comprising the following nucleotide sequences SEQ ID NO: 34, 36, 38 or 40 or combinations of nucleotide sequences linked together; SEQ ID NO: 9 and 34, or 9 and 38, SEQ ID NO: 11 and 36, or 11 and 40.
The present invention also relates to a method of prevention and/or treatment of a disease wherein inducing apoptosis of some cell is beneficial to the mammal, in particular the human in terms of prevention or treatment (therapeutic or prophylactic). Those diseases are in particular cancer, especially one of those listed in the Definitions supra, autoimmune diseases, inflammatory conditions, viral infections and viral diseases. This method comprises the administration to a mammal, including human, of an effective amount of a composition as disclosed and provided herein. The method comprises the administration of the two polypeptides, preferably antibodies directed against the two different epitopes according to the invention, or of the bispecific polypeptide, preferably antibody directed against the two different epitopes according to the invention. Embodiments of these compositions are defined by using the CDRs definitions according to IMGT®. However, the invention encompasses and relates also to the equivalent or alternative methods wherein the IMGT® numbering is replaced either by the Kabat® numbering or the Common numbering system, using the sequences indicated supra. Therefore, in the following embodiments of a method, other embodiments are part of the invention in which one replaces the CDRs defined with IMGT® numbering, by the Kabat® numbering, in accordance with the table supra. Also, in the following embodiments of a method, other embodiments are part of the invention in which one replaces the CDRs defined with IMGT® numbering, by the Common numbering system, in accordance with the table supra.
In a first embodiment, the method comprises the administration of a composition which comprises a polypeptide, preferably antibody, having one or two binding domain(s) comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17. In a second embodiment, the method comprises the administration of a composition which comprises a polypeptide, preferably antibody, having one or two binding domain(s) comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21. In a third embodiment, the method comprises the administration of a composition which comprises these two polypeptides or antibodies in mixture, or of two compositions, one containing the first mentioned polypeptide or antibody, and the second comprising the second mentioned polypeptide or antibody. As an alternative, one may replace hereinabove the definition of the CDRs by those according to Kabat® or Common numbering System as per Tables 1 and 2.
In another embodiment, the method comprises the administration of a composition which comprises a bispecific polypeptide, preferably antibody, comprising a first binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14 CDR1, a CDR3 of sequence SEQ ID NO: 15; and the VL chain contains a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17, and a second binding domain comprising a pair of VH and VL chains wherein the VH chain contains a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19; and the VL chain contains a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21. As an alternative, one may replace hereinabove the definition of the CDRs by those according to Kabat® or Common numbering System as per Tables 1 and 2.
In an embodiment, the method provides for the administration of a composition which comprises an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 2 and 4 and an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 6 and 8, and a pharmaceutically carrier, diluent or excipient. In another embodiment, the method provides for the administration of two compositions, one which comprises an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 2 and 4 and another which comprises an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 6 and 8. In an embodiment, the method provides for the administration of a composition which comprises an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 35 and 37 and an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 39 and 41, and a pharmaceutically carrier, diluent or excipient. In another embodiment, the method provides for the administration of two compositions, one which comprises an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 35 and 37 and another which comprises an anti-DR5 polypeptide, preferably antibody, comprising the amino acid sequence pair SEQ ID NO: 39 and
In another embodiment, the method provides for the administration of a composition comprising a bispecific anti-DR5 antibody comprising an amino acid sequence pair SEQ ID NO: 2 and 4 and an amino acid sequence pair SEQ ID NO: 6 and 8, and a pharmaceutically acceptable carrier. In another embodiment, the method provides for the administration of a composition comprising a bispecific anti-DR5 antibody comprising an amino acid sequence pair SEQ ID NO: 35 and 37 and an amino acid sequence pair SEQ ID NO: 39 and 41, and a pharmaceutically acceptable carrier.
In another embodiment, the method provides for the administration of a composition comprising the DR5-01 and the DR5-05 antibodies as disclosed and provided herein, or similar antibodies produced through genetic engineering as described herein, based on nucleotide sequences SEQ ID NO: 9, 3, 11 and 1, or SEQ ID NO: 9, 34, 11 and 36 for DR5-01, and SEQ ID NO: 9, 7, 11 and 5, or SEQ ID NO: 9, 38, 11 and 40 for DR5-05; use can be made of a composition comprising these antibodies defined by their amino acid sequences and comprising SEQ ID NO: 4, 10, 2 and 12 for DR5-01 and SEQ ID NO: 8, 10, 6 and 12 for DR5-05, or SEQ ID NO: 35, 10, 37 and 12 for HzDR5-01 and SEQ ID NO: 39, 10, 41 and 12 for HzDR5-05.
The pharmaceutical compositions, uses and methods of treatment are thus intended for the prevention and/or treatment of cancer. A list of cancers that may beneficiate from the invention is given supra in the Definitions.
The pharmaceutical compositions, uses and methods of treatment are thus also intended for the prevention and/or treatment of autoimmune diseases and inflammatory conditions. The following diseases are in particular concerned.
The pharmaceutical compositions, uses and methods of treatment are thus also intended for the prevention and/or treatment of viral infection or viral diseases. Viral infections and diseases include, but are not limited to, infections with cytomegalovirus, influenza, Newcastle, disease virus, vesicular stomatitis virus, herpes simplex virus, hepatitis, adenovirus-2, bovine viral diarrhoea virus, human immunodeficiency virus (HIV), and Epstein-Barr virus.
In a particular embodiment, the polypeptides, antibodies or bispecific antibodies of this invention can also be used to specifically label cancer cells, solid tumors, and the like, and more generally, to specifically target/deliver any conjugated or otherwise coupled effector (e.g. radioisotope, label, cytotoxin, drug, liposome, antibody, nucleic acid, dendrimer, etc. . . . ) to cancer cells including, but not limited to, isolated cancer cells, metastatic cells, solid tumor cells, and the like.
Therefore, another object of the invention is a complex of a polypeptide according to the invention and a molecule, which is an effector molecule, which function may beneficiate from the targeting of the DR5 receptor by the polypeptide. Such an effector molecule may be a radioisotope, a label, a cytotoxin, a drug, a liposome, an antibody, a nucleic acid, a dendrimer. The invention also concerns a pharmaceutical composition containing this complex and a pharmaceutically acceptable vehicle, diluent or excipient.
The invention also concern the use of such composition, and a method as well, to prevent or treat a cancer, such as one of those cited supra in the Definitions.
A polypeptide or the polypeptides of this invention may be used to identify other polypeptides or antibodies that bind to one of the epitopes against which the DR5-01 and the DR5-05 are directed. Thus, in certain embodiments, a polypeptide or antibody of this invention, directed against one epitope, can be used or paired with another antibody with binding specificity for the other epitope DR5.
A polypeptide or the polypeptides of this invention may be used to identify other polypeptides or antibodies that bind to another epitope on DR5, which upon binding of polypeptides or antibodies on these various epitopes on DR5, induce apoptosis. DR5-01 and/or the DR5-05 are directed. Thus, in certain embodiments, a polypeptide or antibody of this invention, directed against one epitope (DR5-01 or DR5-05), or polypeptides or antibodies of this invention, directed against both epitopes (DR5-01 and DR5-05) can be used with another antibody with binding specificity for another epitope on DR5.
One or more polypeptides, antibodies, bispecific antibodies, and/or functionalized bispecific antibodies, and/or chimeric moieties of this invention, or pharmaceutical compositions containing the same, can be administered by injection, that is, intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally or intraperitoneally. Also in certain embodiments, the compounds can be administered by inhalation, for example, intranasally. Other pharmaceutical delivery systems can also be employed, for example, liposomes.
Targeting DR5 with the polypeptides or antibodies of the present invention in combination with existing chemotherapeutic treatments will be more effective in killing the tumor cells than chemotherapy alone. A wide variety of drugs have been employed in chemotherapy of cancer. Examples include, but are not limited to, cisplatin, taxol, etoposide, mitoxantrone, actinomycin D, campthotecin, methotrexate, gemcitabine, mitomycin, dacarbazine, 5-fluorouracil, doxorubicine and daunomycin.
In one approach, antibody combination or bispecific antibody anti-DR5 MAb is added to a standard chemotherapy regimen, in treating a cancer patient. For those combinations in which the antibody and additional anti-cancer agent(s) exert a synergistic effect against cancer cells, the dosage of the additional agent(s) may be reduced, compared to the standard dosage of the second agent when administered alone. The antibody may be co-administered with an amount of an anti-cancer drug that is effective in enhancing sensitivity of cancer cells to the antibody combination or bispecific antibody.
In one method of the invention, targeting DR5 with antibody combination or bispecific antibody, is administered to the patient prior to administration of a second anti-cancer agent. One alternative method comprises administering the second anti-cancer agent prior to administering the antibody combination or the bispecific antibody and second agent on an alternative schedule. In another embodiment, the antibody combination or bispecific antibody and second agent are administered simultaneously.
The method of the invention may provide for the inclusion in a therapeutic regimen involving the use of at least one other treatment method, such as irradiation, chemotherapy with small molecule or antibody. The method of the invention may directly include the administration of a sufficient amount of at least one additional polypeptide or antibody directed against another target and/or at least one chemotherapeutic drug (such as small molecule), for a simultaneous, separate or sequential administration with polypeptide(s) or antibody(ies) of the invention, to a mammal, including man. As additional active principle, one may cite doxorubicine, gemcitabine, camptothecin, paclitaxel or the other drugs mentioned above. In an embodiment, lung cancer and breast cancer is treated using such combination. This combination more generally is useful for cancers (in particular aggressive cancers) which do not respond well to treatment with the drug alone or the antibodies/antibody of the invention alone, and for which the combination leads to a synergistic effect.
In one method of the invention, targeting DR5 with antibody combination or bispecific antibody or multivalent antibody fragment, may be employed in treating viral infections and associated conditions arising from viral infections. Viral infections, include, but are not limited to, infections with cytomegalovirus, influenza, Newcastle, disease virus, vesicular stomatitus virus, herpes simplex virus, hepatitis, adenovirus-2, bovine viral diarrhea virus, human immunodeficiency virus (HIV), and Epstein-Barr virus.
Mammalian cells are the preferred hosts for production of therapeutic glycoproteins, due to their capability to glycosylate proteins in the most compatible form for human applications. Bacteria very rarely glycosylate proteins, and like other type of common hosts, such as yeasts, filamentous fungi, insect and plant cells yield glycosylation patterns associated with rapid clearance from the blood stream.
Among mammalian cells, Chinese hamster ovary (CHO) cells are the most commonly used. In addition to giving suitable glycosylation patterns, these cells allow consistent generation of genetically stable, highly productive clonal cell lines. They can be cultured to high densities in simple bioreactors using serum-free media, and permit the development of safe and reproducible bioprocesses. Other commonly used animal cells include baby hamster kidney (BHK) cells, NSO- and SP2/0-mouse myeloma cells.
In an embodiment, the polypeptides and antibodies according to the invention are produced or expressed in mammal cells, preferably wild-type mammal cells, preferably of rodent origin, especially CHO cells.
Modifications and changes may be made in the structure of a polypeptide of the present invention and still obtain a molecule having like characteristics. For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a polypeptide with like properties.
In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics.
It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, for example, enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid may be substituted by another amino acid having a similar hydropathic index and still obtain a biologically functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within +2 is preferred, those which are within +1 are particularly preferred, and those within +0.5 are even more particularly preferred.
Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biologically functionally equivalent peptide or polypeptide thereby created is intended for use in immunological embodiments. U.S. Pat. No. 4,554,101, incorporated herein by reference or to which the person skilled in the art: may refer, states that the greatest local average hydrophilicity of a polypeptide, as governed by the hydrophilicity of its adjacent amino acids, correlate with its immunogenicity and antigenicity, i.e. with a biological property of the polypeptide.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (−0.5+1); threonine (−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent, polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within +2 is preferred, those which are within +1 are particularly preferred, and those within +0.5 are even more particularly preferred.
As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
TABLE 5
Amino Acid
Index
isoleucine L
(+4.5)
valine V
(+4.2)
leucine L
(+3.8)
phenylalanine
(+2.8)
cysteine C
(+2.5)
methionine M
(+1.9)
alanine A
(+1.8)
glycine G
(−0.4)
threonine T
(−0.7)
serine S
(−0.8)
tryptophan W
(−0.9)
tyrosine Y
(−1.3)
proline P
(−1.6)
histidine H
(−3.2)
glutamate E
(−3.5)
glutamine Q
(−3.5)
aspartate D
(−3.5)
asparagine N
(−3.5)
lysine K
(−3.9)
arginine R
(−4.5)
Amino acid substitution may be chosen or selected differently. Possible substitutions have been documented in WO99/51642, WO2007024249 and WO2007106707.
By definition, the CDRs of the invention include variant CDRs, by deletion, substitution or addition of one or more amino acid(s), which variant keeps the specificity of the original CDR. The common numbering system provides for a CDR definition having the shortest amino acid sequences or the minimal CDR definition.
The antibody may be a monoclonal antibody, a chimeric antibody, a humanized antibody, a full human antibody, a bispecific antibody, an antibody drug conjugate or an antibody fragment. A “humanized antibody” or “chimeric humanized antibody” shall mean an antibody derived from a non human antibody, typically a murine antibody, that retains or substantially retains the antigen-binding properties of the parental antibody, but which is less immunogenic in humans.
Methods for producing the polypeptides and antibodies are known from the person skilled in the art. The mammal cells, preferably rodent cells such as CHO cells, preferably wild-type cells are transfected with one or several expression vectors. Preferably, the cells are co-transfected with an expression vector for light chain and with an expression vector for heavy chain.
Cell transfection is also known from the person skilled in the art. As transfection that may be performed, one may mention without limitation standard transfection procedures, well-known from the man skilled in the art, such as calcium phosphate precipitation, DEAE-Dextran mediated transfection, electroporation, magnetofection, nucleofection (AMAXA Gmbh, GE), liposome-mediated transfection (using Dreamfect®, Lipofectin® or Lipofectamine® technology for example) or microinjection.
Expression vectors are known. As vectors that may be used, one may mention without limitation: pcDNA3.3, pOptiVEC, pFUSE, pMCMVHE, pMONO, pSPORT1, pcDV1, pcDNA3, pcDNA1, pRc/CMV, pSEC. One may use a single expression vector or several expression vectors expressing different parts of the polypeptide or antibody.
An expression vector for the CH1, hinge region, CH2 and CH3 comprises SEQ ID NO: 9 or comprises a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 10.
An expression vector contains a nucleic acid sequence encoding a variable region VH of the invention. In an embodiment, the vector comprises SEQ ID NO: 3 or comprises a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 4. In another embodiment, it comprises SEQ ID NO: 7 or comprises a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 8. In an embodiment, the vector comprises SEQ ID NO: 34 or comprises a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 35. In another embodiment, it comprises SEQ ID NO: 38 or comprises a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 39.
A set of expression vectors encoding a heavy chain, comprise an expression vector which comprises SEQ ID NO: 9 (or comprises a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 10), and either SEQ ID NO: 3 (or a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 4) or SEQ ID NO: 7 (or a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 8). A set of expression vectors encoding a heavy chain, comprise an expression vector which comprises SEQ ID NO: 9 (or comprises a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 10), and either SEQ ID NO: 34 (or a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 35) or SEQ ID NO: 38 (or a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 39).
A single expression vector for the heavy chain contains a nucleic acid sequence which encodes VH, CH1, hinge region, CH2, CH3. In an embodiment, the vector comprises SEQ ID NO: 3 and 9 or comprises a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 4 and 10. In another embodiment, it comprises SEQ ID NO: 7 and 9 or comprises a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 8 and 10. In an embodiment, the vector comprises SEQ ID NO: 34 and 9 or comprises a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 35 and 10. In another embodiment, it comprises SEQ ID NO: 38 and 9 or comprises a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 39 and 10.
An expression vector for the light constant chain comprises SEQ ID NO: 11 or comprises a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 12.
An expression vector contains a nucleic acid sequence encoding a variable region VL of the invention. In an embodiment, the vector comprises SEQ ID NO: 1 or a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 2. In another embodiment, it comprises SEQ ID NO: 5 or a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 6. In an embodiment, the vector comprises SEQ ID NO: 36 or a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 37. In another embodiment, it comprises SEQ ID NO: 40 or a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 41.
An expression vector contains a nucleic acid sequence encoding a light chain of the invention. In an embodiment, the vector comprises SEQ ID NO: 1 and 11 or a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 2 and 12. In another embodiment, it comprises SEQ ID NO: 5 and 11 or a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 6 and 12. In an embodiment, the vector comprises SEQ ID NO: 36 and 11 or a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 37 and 12. In another embodiment, it comprises SEQ ID NO: 40 and 11 or a nucleic acid sequence encoding amino acid sequences SEQ ID NO: 41 and 12.
A set of expression vectors for producing a complete antibody comprise several vectors, for example two or three.
A single expression vector may also be used, which comprise either SEQ ID NO: 3, 9, 1 and 11 (or a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 4, 10, 2 and 12), or SEQ ID NO: 7, 9, 5 and 11 (or a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 8, 10, 6 and 12). A single expression vector may also be used, which comprise either SEQ ID NO: 34, 9, 36 and 11 (or a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 35, 10, 37 and 12), or SEQ ID NO: 38, 9, 40 and 11 (or a nucleic acid sequence encoding amino acid sequence SEQ ID NO: 39, 10, 41 and 12).
The expression vector comprises a nucleic acid sequence or nucleic acid sequences which code(s) for the variable region that is wished. Various embodiments of variable regions which can be expressed by the vector are presented below. Embodiments of these vectors are defined by using the CDRs definitions according to IMGT®. However, the invention encompasses and relates also to the equivalent or alternative vectors wherein the IMGT® numbering is replaced either by the Kabat® numbering or the Common numbering system, using the sequences indicated supra. Therefore, in the following embodiments of a vector, other embodiments are part of the invention in which one replaces the CDRs defined with IMGT® numbering, by the Kabat® numbering, in accordance with the table supra. Also, in the following embodiments of a vector, other embodiments are part of the invention in which one replaces the CDRs defined with IMGT® numbering, by the Common numbering system, in accordance with the table supra.
An expression vector codes for a VH comprising a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14 CDR1, a CDR3 of sequence SEQ ID NO: 15.
An expression vector codes for a VL comprising a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17.
A set of expression vectors comprise an expression vector which codes for a VH comprising a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14 CDR1, a CDR3 of sequence SEQ ID NO: 15, and an expression vector which codes for a VL comprising a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17.
An expression vector comprises a nucleic acid sequence coding for a VH comprising a CDR1 of sequence SEQ ID NO: 13, a CDR2 of sequence SEQ ID NO: 14 CDR1, a CDR3 of sequence SEQ ID NO: 15, and a nucleic acid sequence coding for a VL comprising a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence FAS, a CDR3 of sequence SEQ ID NO: 17.
An expression vector codes for a VH comprising a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19.
An expression vector codes for a VL comprising a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21.
A set of expression vectors comprise an expression vector which codes for a VH comprising a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19, and an expression vector which codes for a VL comprising a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21.
An expression vector comprises a nucleic acid sequence coding for a VH comprising a CDR1 of sequence SEQ ID NO: 18, a CDR2 of sequence SEQ ID NO: 14, a CDR3 of sequence SEQ ID NO: 19, and a nucleic acid sequence coding for a VL comprising a CDR1 of sequence SEQ ID NO: 20, a CDR2 of sequence RTS, a CDR3 of sequence SEQ ID NO: 21.
The invention thus comprises the use of one single vector or a set of vectors to produce the polypeptides or antibodies of the invention. These vectors are also objects of the invention, alone or as a set of vectors.
Another object of the invention is a host cell containing a vector or a set of vectors of the invention. The host cell may be a mammal cell, preferably a rodent cell, more preferably CHO cell. Still more preferably, the host cell may be a wild-type mammal cell, preferably a wild-type rodent cell, most preferably a wild-type CHO cell.
The person skilled in the art fully owns the methods to generate the antibodies according to the invention using such a vector or vectors and cells such as CHO cells.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will now be described in further detail by way of examples referring to the figure. Note that in the block diagrams, the blocks appear from the left to the right in the same order than indicated in the legend in the diagrams where the legend is put in a box.
FIG. 1 shows the FACS analysis of anti-DR5 antibody panel in human glioma cell lines (H4, HS683, A172, T98G, U87MG).
FIG. 2 shows the FACS analysis of anti-DR5 expression in some cancer cell lines such as human kidney adenocarcinoma (A704, ACHN, Caki1), human colon carcinoma (SW948, HCT 116), human urinary bladder carcinoma (5637) and human breast adenocarcinoma (MCF7).
FIG. 3 is a graph showing the results of an ELISA assay evaluating binding of MAbs (1 μg/mL) to Fas (50 ng/mL), FasL (100 ng/mL), TRAIL (100 ng/mL) and to DR4, DR5, DcR1 or DcR2 (50 ng/mL), (mean+/−SD, n=2).
FIG. 4 is a bar diagram showing percent (%) of the inhibition of biotinylaled anti-DR5 MAb binding (1 μg/mL, FACS analysis) in the presence of other unconjugated antibody anti-DR5 (5 μg/mL) using the T98G cells (1.106 cells/mL), (mean+/−SD, n=2).
FIG. 5 is a bar diagram showing percent (%) of the inhibition of TRAIL binding (100 ng/mL, FACS analysis) in the presence of antibody (MAb anti-TRAIL, MAb anti-DR5 MAb) tested at different concentrations using H4 cells (5.105 cells/mL), (mean+/−SD, n=2).
FIG. 6 is a bar diagram showing percent (%) of the cell proliferation inhibition (ATP bioluminescent bioassay, 72 hours) of anti-DR5 antibody alone or combined tested at 1 μg/mL as compared to TRAIL (10 ng/mL) using H4 cells (5.104 cells/mL), (mean+/−SD, n=2).
FIG. 7 is a bar diagram showing percent (%) of the cell proliferation inhibition (BrDU bioassay, 72 hours) of selective anti-DR5 agonistic antibody combination (mDR5-01+mDR5-05) versus neutral antibody combination (mDR5-05+mDR5-04) tested at different concentrations using H4 cells (5.104 cells/mL), (mean+/−SD, n=2).
FIG. 8 is a bar diagram showing percent (%) of apoptosis (propidium iodide staining, 72 hours) of selective anti-DR5 agonistic antibody combination (mDR5-01+mDR5-05) versus neutral antibody combination (mDR5-05+mDR5-04) tested at 1 μg/mL and also compared to TRAIL (10 ng/mL) using H4 cells (1.105 cells/mL), (mean+/−SD, n=2).
FIG. 9 is a bar diagram showing percent (%) of cleaved caspase 3 (FACS analysis, 48 hours) of selective anti-DR5 agonistic antibody combination (mDR5-01+mDR5-05) versus neutral antibody combination (mDR5-05+mDR5-04) and also compared to TRAIL using H4 cells (1.105 cells/mL), (representative experiment, n=2).
FIG. 10 is a western blot showing the cleaved PARP induced or not with the presence of selective anti-DR5 agonistic antibody combination (mDR5-01+mDR5-05) versus neutral antibody combination (mDR5-05+mDR5-04) using H4 cells (2.105 cells/mL, 5 hours).
FIG. 11 is a bar diagram showing percent (%) of the cell proliferation inhibition (ATP bioluminescent bioassay, 72 hours) with the selective anti-DR5 agonistic antibody combination (10 μg/mL mDR5-05+0.1 μg/mL mDR5-01), in the presence or not of anti-DR5 MAb (mDR5-01, mDR5-02, mDR5-04 or mDR5-05, 1 μg/mL) using H4 cells (5.104 cells/mL), (mean+/−SD, n=2).
FIG. 12 is a bar diagram showing percent (%) of the cell proliferation inhibition (ATP bioluminescent bioassay, 72 hours) of selective antibody anti-DR5 agonistic antibody combination (10 μg/mL mDR5-05+0.1 μg/mL mDR5-01) and then diluted at 1/2 compared to TRAIL (20 ng/mL) and then diluted at 1/2 using H4, HS683, A172, T98G or U87MG glioma cells (5.104 cells/mL), (mean+/−SD, n=2).
FIG. 13 is a bar diagram showing percent (%) of the proliferation inhibition (ATP bioluminescent bioassay, 72 hours) of chimeric antibody (chDR5-01 or chDR5-05 MAb) tested alone at 5 μg/mL then diluted at 1/2 versus antibody combination (5 μg/mL chDR5-05+0.05 μg/mL chDR5-01) then diluted at 1/2 using glioma H4 cells (5.104 cells/mL), (mean+/−SD, n=2).
FIG. 14 is a bar diagram showing percent (%) of the cell proliferation inhibition (ATP bioluminescent bioassay, 72 hours) of anti-DR5 antibody alone or combined tested at 10 μg/mL (ratio 1/10) as compared to TRAIL (50 ng/mL) using ex-vivo human glioma cells (5.104 cells/mL), (mean+/−SD, n=3 from three independent ex vivo GBM cells).
FIGS. 15-18 are bar diagrams showing percent (%) of the cell proliferation inhibition (ATP bioluminescent bioassay, 72 hours) in the presence of mouse anti-DR5 antibody combined tested at 10 μg/mL diluted at 1/10, in the presence of drug alone (1 μg/mL diluted at 1/10) or in association mouse anti-DR5 antibody combined and drug using HS683, A172, 42MGBA or T98G glioma cells, (5.104 cells/mL), (mean+/−SD, n=2), (Campothecin (CMT)).
FIGS. 19-22 are bar diagrams showing percent (%) of the cell proliferation inhibition (ATP bioluminescent bioassay, 72 hours) in the presence of mouse anti-DR5 antibody combined tested at 10 μg/mL diluted at 1/10, in the presence of drug alone (1 μg/mL diluted at 1/10) or in association mouse anti-DR5 antibody combined and drug using human breast cell lines (MCF7, MDAMB231) or human lung adenocarcinoma cell lines (NCIH1703, A549), (5.104 cells/mL), (mean+/−SD, n=2).
FIG. 23 is a bar diagram showing percent (%) of the cell proliferation inhibition (ATP bioluminescent bioassay, 72 hours) of mouse anti-DR5 antibody alone or combined as compared to humanized anti-DR5 antibody alone or combined tested at 1 μg/mL (ratio 1/1 in combined) then diluted at 1/2 using H4 glioma cells (5.104 cells/mL), (mean+/−SD, n=2).
FIG. 24 is a survival curve of nude mice orthotopic engrafted with SC2 human glioma treated with or without mouse anti-DR5 antibody combined. MAb treatment was administrated by intraperitoneal injection (IP) at 5 mg/kg per mouse until mice euthanasia due to loss of weight and was applied during 36 days maximum. Survival times obtained with control group were compared to survival times obtained with treated groups (mDR5-01+mDR5-05 versus mDR5-04+mDR5-05) using Kaplan Meier method and Wilcoxon statistical test (JMP software).
FIG. 25 Amino acid and nucleic acid sequence for VH HzDR5-01 with description of the FR1, CDR1, FR2, CDR2, FR3, CDR3 defining according IMGT®.
FIG. 26 Amino acid and nucleic acid sequence for VL HzDR5-01 with description of the FR1, CDR1, FR2, CDR2, FR3, CDR3 defining according IMGT®.
FIG. 27 Amino acid and nucleic acid sequence for VH HzDR5-05 with description of the FR1, CDR1, FR2, CDR2, FR3, CDR3 defining according IMGT®.
FIG. 28 Amino acid and nucleic acid sequence for VL HzDR5-05 with description of the FR1, CDR1, FR2, CDR2, FR3, CDR3 defining according IMGT®.
EXAMPLES
The following examples are offered to illustrate, but not to limit the claimed invention.
Example 1: Preparation of Murine MAb Anti-DR5
This example illustrates the preparation of hybridoma cell lines secreting anti-DR5 antibodies.
Antibodies. The anti-DR5 antibodies, murine monoclonal antibodies specific for DR5 were produced using standard hybridoma techniques (Zola et al., Aust J. Exp Biol Med Sci. 1981; 59:303-6). Briefly, mice were given i.p. injections of recombinant DR5 (10 μg), (R&D Systems, Lille, France) on weeks 0, 2 and 4. This was followed by an i.v. injection of recombinant DR5 (10 μg) and the splenocytes were fused with mouse myeloma line X63-Ag8.653. Hybridoma supernatants were screened for DR5 binding by ELISA and by flow cytomery on DR5 positive cell lines. A murine MAb panel anti-DR5 noted mDR5-01, mDR5-02, mDR5-04 and mDR5-05 were obtained.
Example 2: Cell Culture
Various tumor-derived cell lines are among the target cells that may be contacted with TRAIL, anti-DR5 MAb alone, MAb combination, in such assay procedures.
Cell lines. The established human neuroglioma cells H4, HS683 or A172 (available from ATCC) and the established human lung adenocarcinoma cells A549 were grown in Dulbecco's Modified Eagle's Medium (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycine (Sigma, St Quentin Fallavier, France). The established human glioblastoma astrocytoma cells U87MG or T98G, the human kidney adenocarcinoma cells A704, the human kidney adenocarcinoma cells ACHN and the human breast adenocarcinoma cells MCF7 (available from ATCC) were grown in Eagle's Minimum Essential Medium (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycine (Sigma, St Quentin Fallavier, France). The established human colon adenocarcinoma cells SW948 and the human breast adenocarcinoma cells MDAMB231 (available from ATCC) were grown in Leibovitz's L-15 (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycine (Sigma, St Quentin Fallavier, France). The established human kidney carcinoma cells Caki-1 and the human colorectal carcinoma cells HCT-116 (available from ATCC) were grown in McCoy's 5A Medium Modified (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycine (Sigma, St Quentin Fallavier, France). The established human urinary bladder carcinoma cells 5637 and the established human lung adenocarcinoma cells NCIH1703 (available from ATCC) were grown in RPMI-1640 Medium (Sigma, St Quentin Fallavier, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycine (Sigma, St Quentin Fallavier, France). The established human glioma cells 42MGBA (available from DSMZ) were grown in 80% mixture of RPMI-1640 Medium and Eagle's Minimum Essential Medium at 1:1 (Sigma, St Quentin Fallavier, France) supplemented with 20% heat-inactivated fetal bovine serum (FBS) (Sigma, St Quentin Fallavier, France), 4 nM L-glutamine (Sigma, St Quentin Fallavier, France) and 100 U/mL, 100 μg/mL penicillin-streptomycine (Sigma, St Quentin Fallavier, France).
Example 3: Antibody Binding Assays (FCM, ELISA)
This example describes methods to determine the MAb specificity anti-DR5 by ELISA with coated antigens, to investigate on DR5 cellular expression at the cell surface. and to determine epitopes following MAb competition analyzed by flow cytometry.
Flow cytometry experiments for DR5 cellular expression. Briefly, 2×105 cells per 96 wells are incubated with a dilution of unconjugated anti-DR5 MAb at 10 μg/mL then diluted at 1/10. Unbound antibodies were washed away with PBS (Invitrogen, Villebon sur Yvette, France) supplemented by 1% Bovine Serum Albumin (Sigma, St Quentin Fallavier, France). Subsequently, cells are centrifuged (5 min at 400 g) and bound antibody is detected with Fluorescein Isothiocyanate (FITC) conjugated goat (Fab)2 polyclonal anti mouse (MP Biomedical, Illkirch, France) at 4° C. for 30 min. Detection reagent is washed away and cells are centrifuged (5 min at 400 g) and resuspended in 300 μL PBS. Bound detection antibody is quantified on a FACSCAN (BD Biosciences, Rungis, France), (FL1 channel, 2000 events per acquisition). During the experiment, the respective isotype controls are included to exclude any unspecific binding events.
Results of experiments are shown in FIG. 1 (at 10 μg/mL), FIG. 2 and Table 6 (at 5 μg/mL) shows as for example the cell staining with MAb concentration or at 5 μg/mL. Various cancer cell lines express different subsets of TRAIL receptors. Expression patterns varied from cell line to cell lines. In the present study DR5 was expressed on all cell lines tested. Whatever the MAb tested anti-DR5 (mDR5-01, mDR5-02, mDR5-04 or mDR5-05), similar cellular pattern was observed.
Table 6 shows the FACS analysis of DR5 expression using 5 μg/mL of anti-DR5 antibody in other solid tumour cell lines (1×106 cells/mL) i.e. human breast adenocarcinoma cell lines (MCF7, MDAMB231) and on human lung adenocarcinoma cell lines (NCIH1703, A549).
TABLE 6
Breast cancer cell line
Lung cancer cell line
MCF7
MDAMB231
NCIH1703
A549
Mab
%
MFI
%
MFI
%
MFI
%
MFI
mIgG1
1
176
0
119
0
100
0
190
CTRL
mDR5-
51
225
89
238
64
152
82
323
01
mDR5-
33
192
82
224
74
166
71
274
05
Analysis of MAb specificity by using coated antigens ELISA. The specific binding properties of antibodies were evaluated in an ELISA with coated Fas (50 ng/mL) (R&D Systems, Lille, France), FasL (100 ng/mL) (Tebu-bio, Le Perray en Yvelines, France), TRAIL (100 ng/mL) (R&D Systems, Lille, France), DR4 (50 ng/mL) (R&D Systems, Lille, France), DR5 (50 ng/mL) (R&D Systems, Lille, France), DcR1 (50 ng/mL) (R&D Systems, Lille, France) or DcR2 (50 ng/mL) (R&D Systems, Lille, France) antigens. The anti-DR5 MAb panel was tested at 1 μg/mL and revealed by using a goat polyclonal anti mouse IgG1 Horse Radish Peroxydase (HRP) conjugated (AbD Serotec, Colmar, France).
Results of experiments are shown in FIG. 3. The mDR5-01, mDR5-02, mDR5-04 and mDR5-05 antibodies (1 μg/mL) only reacted with DR5 coated antigens (50 ng/mL). No reactivity was observed with other apoptotic related antigens (FAS, FASL, TRAIL, DR4, DcR1, DcR2), (mean+/−SD on 2 independent experiments).
Flow cytometry experiments for MAb competition binding. Briefly, 2×105 cells T98G per 96 wells are incubated with a dilution of biotinylated murine antibody anti-DR5 (10 μg/mL then diluted at 1/10) as a reference and with or without unconjugated antibody at 5 μg/mL and incubated at 4° C. for 30 min. Only data obtained with 1 μg/mL of biotinylated antibody is shown. Unbound antibody is washed away with PBS (Invitrogen, Villebon sur Yvette, France) supplemented by 1% Bovine Serum Albumin (Sigma, St Quentin Fallavier, France). Subsequently, cells are centrifuged (5 min at 400 g) and bound antibody is detected with Phycoerythrin conjugated Streptavidin (Interchim, Montlugon, France) at 4° C. for 30 min. Detection reagent is washed away and cells are centrifuged (5 min at 400 g) and resuspended in 300 μL PBS. Bound detection antibody is quantified on a FACSCAN (BD Biosciences, Rungis, France), (FL2 channel, 2000 events per acquisition). During the experiment, the respective isotype controls are included to exclude any unspecific binding events.
Results of experiments are shown in FIG. 4. For example the unconjugated mDR5-02 and mDR5-05 antibodies (5 μg/mL) are not in competition with the biotinylaled mDR5-01 antibody (1 μg/mL). By contrast, unconjugated mDR5-01 and mDR5-04 are in competition with the biotinylaled mDR5-01 antibody. Therefore, the epitopes DR5-01 and DR5-04 are common or adjacent, whereas the epitopes DR5-02 and DR5-05 are two separate epitopes. Moreover the epitope DR5-01 is also distinct of the epitope DR5-05, (mean+/−SD on two independent experiments).
Example 3: In Vitro Biologic MAb Activity
This example illustrates methods of evaluating the anti-DR5 MAb impact on TRAIL cellular binding on their ability to trigger cellular cytotoxic effect on cancer cells. These components may be assayed for anti-tumour activity, using any of a number of suitable assays, including but not limited to assays for the ability to slow tumour growth or to kill cancer cells in vitro. Various tumour-derived cell lines are among the target cells that may be contacted with MAb combination, in such assay procedures.
To identify or select anti-DR5 antibody combination which induce apoptosis, loss of membrane integrity as indicated by, e.g. PI is assessed relative to control (untreated cells) and compared to recombinant TRAIL (FIG. 8). The ability to slow tumour growth is assessed by ATP or BrDU quantification (FIG. 6, FIG. 7). The apoptotic response is assessed by quantification of cleaved caspase 3 (FIG. 9) or cleaved Poly-(ADP-Ribose)-Polymerase (PARP), (FIG. 10).
Biochemical reagents. Biochemical reagents used for the apoptosis studies were: propidium iodide (PI), (Sigma, St Quentin Fallavier, France), Caspase 3 antibody (Ozyme, Saint Quentin Yvelines, France), Cell proliferation ELISA-BrdU (Roche Diagnostics, Meylan, France), Cell Titer GLo-ATP (Promega, Charbonnières-les-bains, France) and the polyclonal anti Poly-(ADP-Ribose)-Polymerase (PARP) (Roche Diagnostics, Meylan, France).
Flow cytometry experiments of MAb impact on TRAIL binding. H4 cell lines were seeded at a density of 1×105 per 96-wells. Cells were incubated for 30 min at 4° C. with or without MAb anti-TRAIL or anti-DR5 (mDR5-01, mDR5-02, mDR5-3 mDR5-4) tested at 1 μg/mL then diluted at 1/10. Unbound antibodies were washed away with PBS (Invitrogen, Villebon sur Yvette, France) supplemented by 1% Bovine Serum Albumin (Sigma, St Quentin Fallavier, France). Subsequently, cells are incubated with the recombinant TRAIL (100 ng/mL), (R&D Systems, Lille, France) for 30 min at 4° C. Unbound antibodies were washed away with PBS (Invitrogen, Villebon sur Yvette, France) supplemented by 1% Bovine Serum Albumin (Sigma, St Quentin Fallavier, France). The bound recombinant TRAIL is detected with biotinylated conjugated anti TRAIL MAb B-S23 (iDD biotech, Dardilly, France). After washings, Phycoerthrin conjugated Streptavidin (Interchim, Montlugon, France) was added at 4° C. for 30 min. Detection reagent is washed away and cells are centrifuged (5 min at 400 g) and resuspended in 300 μL PBS. Bound detection antibody is quantified on a FACSCAN (BD Biosciences, Rungis, France), (FL2 channel, 2000 events per acquisition). During the experiment, the respective isotype controls are included to exclude any unspecific binding events.
Human H4 expressing DR5 at the cell surface was used to determine the agonist or antagonist activity of the four anti-DR5 antibodies denoted mDR5-01, mDR5-02, mDR5-04 and mDR5-05. Results of experiments are shown in FIG. 5. The recombinant TRAIL binding at the cell surface was inhibited with the antagonist anti TRAIL MAb B-T24 (iDD biotech, Dardilly, France). Among the anti-DR5 MAb panel tested, the MAbs mDR5-01, mDR5-04 and mDR-5-05 inhibited the recombinant TRAIL binding, without any mDR5-02 MAb impact.
Cell viability analysis following ATP level determination. The CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Charbonnières les Bains, France) was used to determine the number of viable cells in culture based on quantification of the ATP present, an indicator of metabolically active cells. Detection is based on using the luciferase reaction to measure the amount of ATP from viable cells. Within minutes after a loss of membrane integrity, cells lose the ability to synthesize ATP, and endogenous ATPases destroy any remaining ATP; thus the levels of ATP fall precipitously. Cell cultures (5×104 cells/mL) are incubated for 72 hours alone or with anti-DR5 MAb alone (1 μg/mL) or with two combined MAb at 1 μg/mL for each MAb (FIG. 6). The TRAIL ligand concentration was used at 10 ng/mL. The CellTiter-Glo® reagent was added directly to cells in culture at a ratio of 504 of reagent to 200 μL of culture medium. The assay plates are incubated at room temperature for 10 min and the bioluminescent signal is recorded using a standard multiwell fluorometer Mithras LB940, (Berthold, Thoiry, France).
Results of experiments to determine the agonist activity of the four anti-DR5 antibodies are shown in FIG. 6. None of the anti-DR5 MAb alone tested was capable of inducing cellular cytotoxicity in H4 cells. By contrast, only the anti-DR5 MAb combination mDR5-01 and mDR5-05 triggered apoptosis in H4 cells. The ability of this restricted anti-DR5 MAb combination (1/10) was not related to the level of MAb staining (FIG. 1). Interestingly the MAbs mDR5-01 and mDR5-05 recognize two different epitopes (FIG. 4). However MAb combination of mDR5-05 with other mDR5 MAb such as mDR5-02 recognizing also distinct epitope failed to trigger H4 apoptosis (FIG. 6).
Cell viability analysis following BrDU incorporation determination. The H4 target cells (5×104 cells/mL) were cultured with the MAb combination mDR5-05 and mDR5-01 or with the MAb combination mDR5-05 and mDR5-04 at different range of MAb concentration. Cell growth is determining using the Cell proliferation ELISA-BrdU (Roche Diagnostics, Meylan, France), according to the manufacturer's instructions. This method is based on the incorporation of the pyrimidine analogue BrdU instead of thymidine into the DNA of proliferating cells. After its incorporation into DNA, BrdU is detected with a MAb anti-BrdU. At the end of revelation, the bioluminescent signal is recorded using a standard multiwell fluorometer Mithras LB940, (Berthold, Thoiry, France).
Results of experiments are shown in FIG. 7. Specific MAb combination mDR5-01 and mDR5-05 synergistically induced apoptosis in H4 cell line as evidence by BrDU quantification, (mean+/−SD on 2 independent experiments). No significant impact was observed with the MAb combination mDR5-05 and mDR5-04.
Propidium iodide uptake by flow cytometry for measuring MAb induced apoptosis. H4 cell lines were seeded at a density of 2×104 per 96-wells. Cells were incubated for a 3 day time period with or without MAb anti-DR5. Each anti-DR5 MAb was tested alone or following MAb combination at 1 μg/mL (FIG. 8). The TRAIL ligand concentration was used at 10 ng/mL. Cells were then centrifuged at 2000 rpm for 5 min at 4° C., the pellet resuspended in 70% ethanol (Sigma, St Quentin Fallavier, France) for permeabilization. After a new centrifugation, cells were incubated with 1004 of PI (100 μg/mL) and 1004 of Rnase (100 μg/mL), (Sigma, St Quentin Fallavier, France) per well for 15 min. Cells are centrifuged (5 min at 2000 rpm) and resuspended in 300 μL PBS. Bound detection antibody is quantified on a FACSCAN (BD Biosciences, Rungis, France), (FL2 channel, 3000 events per acquisition).
Results of experiments are shown in FIG. 8. Whereas no PCD was obtained with MAb tested alone, specific MAb combination mDR5-01 and mDR5-05 synergistically induced apoptosis in H4 cell line as evidence by PI uptake, (mean+/−SD on 2 independent experiments). No MAb cross linking was required. No PCD was obtained with MAb combination mDR5-05 and mDR5-04.
Cleaved caspase-3 quantification by flow cytometry for measuring MAb induced apoptosis. H4 cell lines were seeded at a density of 2×104 per 96-wells. Cells were incubated for a 48 hours with or without MAb anti-DR5. Each anti-DR5 MAb was tested alone or in the presence of MAb combination at 1/mL for mDR5-05 with 0.01 μg/mL for mDR5-01 or mDR5-04 (FIG. 9). The TRAIL ligand concentration was used at 10 ng/mL. Cells were then centrifuged at 2000 rpm for 5 min at 4° C., the pellet resuspended in 90% methanol (Sigma, St Quentin Fallavier, France) for permeabilization. Cells were then centrifuged at 2000 rpm for 5 min at 4° C. and incubated at 4° C. for 30 min with the MAb anti-active caspase-3 antibodies alexa fluor 488 conjugated (Ozyme, Saint Quentin Yvelines, France). Cells are centrifuged (5 min at 2000 rpm) and resuspended in 300 μL PBS. Bound detection antibody is quantified on a FACSCAN (BD Biosciences, Rungis, France), (FL2 channel, 3000 events per acquisition).
When apoptosis is activated, caspases cleave multiple protein substrates, which leads to the loss of cellular structure and function, and ultimately results in cell death. In particular, caspases-8, -9, and -3 have been implicated in apoptosis: caspase-9 in the mitochondrial pathway, caspase-8 in the Fas/CD95 pathway, and caspase-3 more downstream, activated by multiple pathways. Specific MAb combination mDR5-01 and mDR5-05 synergistically induced apoptosis in H4 cell line as evidence by cleaved caspase 3 quantification (FIG. 9), (mean+/−SD on 2 independent experiments). As compared to TRAIL (called also Apo2L), only the MAb combination mDR5-01 and mDR5-05 triggered cell apoptosis compared to the MAb combination mDR5-04 and mDR5-05.
PARP Western blotting. H4 cell lines were seeded at a density of 1.106 per flask T25 cm2. Cells were incubated for a 5 hours with or without MAb anti-DR5. Cell extracts were resuspended in Tris-HCl 50 mM, KCl 150 mM at pH7 and submitted to sonication and incubated for 15 min at 65° C. Samples (10 μg) were subjected to reducing SDS-PAGE and transferred to PVDF membrane using standard methods. After blocking in milk 5%, the blots were incubated in the anti-Poly-(ADP-Ribose)-Polymerase (PARP) (Roche Diagnostics, Meylan, France) at 1/2000. After washing, the membranes were incubated in PAb sheep anti-rabbit IgG horseradish peroxidase conjugated antibody at 1/10000, (AbD Serotec, Colmar, France). The blots were developed with ECL Advance Western blotting using enhanced luminol-based chemiluminescent substrate for detection of horseradish peroxidase (GE Healthcare, St Cyr au Mont d'Or, France).
Many target-specific substrates for caspase have been identified, including the DNA repair enzyme, poly (ADP-ribose) polymerase (PARP). Western blot detection of PARP cleavage has been used extensively as an indicator of apoptosis. PARP is cleaved between Asp213 and Gly 214 in the human sequence, producing two fragments of apparent molecular weights of 24 and 89 kDa. From H4 cells treated with the MAb combination mDR5-01 and mDR5-05, the fragments of cleaved PARP were detected, whereas no similar effect was observed from the untreated cells or treated with the MAb combination mDR5-05 an mDR5-04, (FIG. 10).
As shown in FIG. 11, the MAb mDR5-02 (1 μg/mL) blocked apoptosis triggered with the MAb combination mDR5-01 and mDR5-05 tested at the ratio 1/100 (10 μg/mL+0.1 μg/mL). No significant impact was observed with the other anti-DR5 MAbs (mDR5-01, mDR5-04 or mDR5-05). Cell viability was evaluated based on quantification of the ATP present, an indicator of metabolically active cells.
The susceptibility of five of the glioma cell lines, H4, HS683, A172, T98G and U87MG to TRAIL or anti-DR5 MAb combination (mDR5-01+mDR5-05 versus mDR5-05+mDR5-04) tested at the ratio 1/100 (10 μg/mL+0.1 μg/mL) were evaluated based on quantification of the ATP present, an indicator of metabolically active cells (FIG. 12). Whereas four cell lines (HS683, A172, T98G and U87MG) are resistant or very low sensitive to TRAIL-induced apoptosis, the use of the MAb anti-DR5 combination mDR5-01 and mDR5-05 bypass this regulatory mechanism.
The susceptibility of ex vivo glioma cells from patients to mouse anti-DR5 MAb combination (mDR5-01+mDR5-05) tested at 10 μg/mL (ratio 1/10) were evaluated based on quantification of the ATP present, an indicator of metabolically active cells (FIG. 14).
The susceptibility of four glioma cell lines, (HS683, A172, 42MGBA, T98G) to mouse anti-DR5 MAb combination (mDR5-01+mDR5-05) tested at 10 μg/mL then diluted at 1/10 (ratio 1/1) were evaluated alone or in association with Camptothecin (FIG. 15-18). These cell lines exhibited different levels apoptosis induced with mouse anti-DR5 MAb combination or in the presence of Camptothecin. The use of MAb anti-DR5 combination in association with Camptothecin bypassed this regulatory mechanism and enhanced the level of apoptosis.
The susceptibility of other solid tumor cell lines expressing DR5 such as on human breast adenocarcinoma cell lines (MCF7, MDAMB231) and on human lung adenocarcinoma cell lines (NCIH1703, A549) to mouse anti-DR5 MAb combination (mDR5-01+mDR5-05) tested at 10 μg/mL then diluted at 1/10 (ratio 1/1) were evaluated alone or in association with Paclitaxel, Gemcitabine or Doxorobucine (FIG. 19-22). These cell lines exhibited different levels apoptosis induced with mouse anti-DR5 MAb combination or in the presence of the different drugs tested. The use of MAb anti-DR5 combination in association with these drugs bypassed this regulatory mechanism and enhanced the level of apoptosis.
Example 4: Preparation of Chimeric Monoclonal Antibodies Directed Against DR5
DNA encoding the monoclonal antibodies is 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 murine antibodies). The hybridoma cells serve as a preferred source of such DNA.
Conversion of murine MAb to native chimeric MAb: cDNA corresponding to the variable region of the hybridoma was obtained using two approaches. The first approach consists of using PCR with a degenerate N-terminal amino acid related primer set to generate the N-Terminal sequencing product. The second approach consists of using PCR with a degenerate primer set generated by IMGT® primer database and specific primers previously described (Essono et al., J Immunol Methods. 2003; 203: 279:251-66, Wang et al., Mol Immunol. 1991; 28:1387-97). The sequence of N-terminal variable region was determined by Edman degradation. Total RNA extraction was carried out using the Tri Reagent kit according to the protocol described by the supplier Sigma. The amplified VL and VH fragments were cloned into the TOPO-TA cloning vector (Invitrogen) for sequence analyses by the dideoxytermination method (Sanger et al., Nature. 1977; 265:687-95). Then antibody variant constructs were amplified by PCR and cloned into the expression vector.
Positions are numbered according to IMGT® and to Kabat® index (Identical V region amino acid sequences and segments of sequences in antibodies of different specificities). Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites were analyzed (Kabat et al., NIH Publ. 1991; No. 91-3242, Vol. 1, 647-669).
As shown in FIG. 13, the chimeric MAb combination chDR5-01 and chDR5-05 triggered H4 cell apoptosis tested at the ratio 1/100 (5 μg/mL+0.05 μg/mL). No significant MAb impact was observed with the chimeric MAb tested alone. Cell viability was evaluated based on quantification of the ATP present, an indicator of metabolically active cells.
The nucleic acid sequence or amino acid sequence regarding on the chimeric MAbs DR5-01 and DR5-05 are shown in the Sequence Listing:
nucleotide sequence of the variable murine light chain of DR5-01 antibody anti-DR5 (SEQ ID N0:1) and its derived amino acid sequence (SEQ ID NO:2).
nucleotide sequence of the variable murine heavy chain of DR5-01 antibody anti-DR5 (SEQ ID NO:3) and its derived amino acid sequence (SEQ ID NO:4).
nucleotide sequence of the variable murine light chain of DR5-05 antibody anti-DR5 (SEQ ID NO:5) and its derived amino acid sequence (SEQ ID NO:6).
nucleotide sequence of the variable murine heavy chain of DR5-05 antibody anti-DR5 (SEQ ID NO:7) and its derived amino acid sequence (SEQ ID NO:8).
nucleotide sequence of the constant human heavy chain of DR5-01 or DR5-05 antibody anti-DR5 (SEQ ID NO:9) and its derived amino acid sequence (SEQ ID NO:10).
nucleotide sequence of the constant human light chain of DR5-01 or DR5-05 antibody anti-DR5 (SEQ ID NO:11) and its derived amino acid sequence (SEQ ID NO:12).
Example 5: MAb Production and Protein A Purification
Mammalian cells are the preferred hosts for production of therapeutic glycoproteins, due to their capability to glycosylate proteins in the most compatible form for human applications (Jenkins et al., Nat Biotech. 1996; 14:975-81). Mammalian host cells that could be used include, human Hela, 283, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1 African green monkey cells, quail QC1-3 cells, mouse L cells and Chinese hamster ovary cells. Bacteria very rarely glycosylates proteins, and like other type of common hosts, such as yeasts, filamentous fungi, insect and plant cells yield glycosylation patterns associated with rapid clearance from the blood stream.
The Chinese hamster ovary (CHO) cells allow consistent generation of genetically stable, highly productive clonal cell lines. They can be cultured to high densities in simple bioreactors using serum-free media, and permit the development of safe and reproducible bioprocesses. Other commonly used animal cells include baby hamster kidney (BHK) cells, NSO- and SP2/0-mouse myeloma cells. Production from transgenic animals has also been tested (Jenkins et al., Nat Biotech. 1996; 14:975-81).
A typical mammalian expression vector contains the promoter element (early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses e.g. RSV, HTLV1, HIV1 and the early promoter of the cytomegalovirus (mCMV, hCMV), which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript (BGH polyA, Herpes thimidine kinase gene of Herpes simplex virus polyA (TKpa), Late SV40 polyA and 3′ UTR_Beta_Globin_polyA). Additional elements include enhancers (Eμ, hIE1), Kozak sequences, signal peptide and intervening sequences flanked by donor and acceptor sites for RNA splicing. Suitable expression vectors for use in practise in practising the present invention include, for examples, vectors such as pcDNA3.1, pcDNA3.3, pOptiVEC, pRSV, pEμMCMV, pMCMVHE-UTR-BG, pHCMVHE-UTR-BG, pMCMV-UTR-BG, pHCMV-UTR-BG, pMCMVHE-SV40, pHCMVHE-SV40, pMCMV-SV40, pHCMV-SV40, pMCMVHE-TK, pHCMVHE-TK, pMCMV-TK, pHCMV-TK, pMCMVHE-BGH, pHCMVHE-BGH, pMCMV-BGH, pHCMV-UTR-BGH).
The empty CHO Easy C cells (purchased by the CCT collection) were co-transfected with MAb expression vector for light and heavy chains following transient or stable transfection procedure established in our laboratory. Secretion of H and L chains were enabled by the respective human IgH leader sequence. The coding regions for light and heavy chains of MAb anti-DR5 are introduced into the MAb expression vector in the multiple cloning site. The transformants are analyzed for correct orientation and reading frame, the expression vector may be transfected into CHO cell line.
Protein A chromatography from murine ascitic fluid. The murine ascitic fluid is adjusted at pH 8.3 with the equilibration buffer 0.1 M Tris and 1.5 M Sulfate Ammonium and then loaded onto the rProtein A Sepharose Fast Flow column (GE Healthcare, Saint Cyr au Mont d'or, France). The non binding proteins are flowed through and removed by several washings with equilibration buffer. The MAb anti-DR5 is eluted off the Protein A column using the elution buffer 0.1 M Citrate Sodium at pH 3.5. Column eluent is monitored by A280. The anti-DR5 MAb peak is pooled.
Protein A chromatography from harvested CHO cell culture fluid. The harvested cell culture fluid produced from CHO cells is loaded onto the Hi Trap rProtein A column (GE Healthcare, Saint Cyr au Mont d'Or, France) that is equilibrated with Phosphate buffered saline, pH 7.2. The non binding proteins are flowed through and removed by several washings with PBS buffer followed. The MAb anti-DR5 is eluted off the Protein A column using a step of elution of 0.1 M Citric acid at pH 3.0. Column eluent is monitored by A280. The anti-DR5 MAb peak is pooled.
Example 6: Preparation of Humanized Monoclonal Antibodies Directed Against DR5
Antibody CDR and FR regions have been determined according to various numbering approaches such as IMGT (ImMunoGeneTics Information System® http://imgt.cines.fr), Kabat or Common Numbering System. However, IMGT determined CDRs for a given antibody are not necessarily identical to the CDRs defined by the other numbering systems. The variable domain CDRs and framework regions have been identified by the inventor thanks to IMGT numbering systems.
Conversion of chimeric MAb to Humanized MAb: Humanized DR5 antibody H and L chain was generated using CDR-grafting by the PCR method. In order to generate a humanized antibody in which the CDRs of a mouse monoclonal antibody is grafted onto a human antibody, there is preferably a high homology between the variable region of a mouse monoclonal antibody and the variable region of a human antibody. Thus, the H chain and L chain V regions of a mouse anti-human DR5 monoclonal antibody are compared to the V region of all known human antibodies using the software IMGT/DomainGapAlign. When a mouse antibody is humanized by a conventional technology, the amino acid sequence of some of the V region FRs of a mouse antibody supporting the CDR may be grafted onto the FR of a human V region, as desired.
For both of the humanized H chain and L chain V regions, it is possible to select the L and H chain V regions and J region, IGKV3-D-15*01, IGHV1-3*01, IGKJ2*01 and IGHJ4*01 respectively, having a high homology with the H and L chain V region and J region of the mDR5 antibody and IGKV1-16*01, IGHV1-3*01, IGKJ4*01 and IGHJ4*01, having a high homology with the H and L chain V region and J region of the mDR5-05 antibody.
After sequence of the Humanized variable region of HzDR5-01 and HzDR5-05 is determined. The variables regions of H and L of HzDR5-01 and Hz-DR5-05 were amplified by PCR and cloned into the expression vector p3U containing the human IgG1 constant region.
In the case of human CDR-grafted antibodies, the binding activity is decreased by grafting of the amino acid sequence of CDR in the mouse antibody alone. In order to avoid this reduction, among the amino acid residues in FR different between a human antibody and a mouse antibody, amino acid residues considered to have influences on the binding activity are grafted together with the amino acid sequence of CDR. Accordingly, an attempt was also made in this example to identify the amino acid residues in FR considered to have influences on the binding activity.
The susceptibility of the glioma cell line H4 to mouse or humanized anti-DR5 MAb combination (mDR5-01+mDR5-05) tested at 1 μg/mL then diluted at 1/2 (ratio 1/1) were evaluated based on quantification of the ATP present, an indicator of metabolically active cells (FIG. 23). The humanized MAb combination (hzDR5-01 and hzDR5-05) triggered cell apoptosis at a higher level compared to the mouse MAb combination (mDR5-01 and mDR5-05).
Example 7: In Vivo Biologic MAb Activity
Orthotopic human glioma xenograft mouse model was obtained by intracerebral injection in nude mouse of 100000 isolated cell coming from heterotypic human glioma xenograft mouse model Sc2. MAb treatment was administrated by intraperitoneal injection (IP) at 5 mg/kg per mouse until mice euthanasia due to loss of weight and was applied during 36 days maximum. Survival times obtained with control group were compared to survival times obtained with treated groups (mDR5-01+mDR5-05 versus mDR5-04+mDR5-05) using Kaplan Meier method and Wilcoxon statistical test (JMP software), (FIG. 24). This study demonstrated anti-tumor activity of mouse anti-DR5 MAb combination (mDR5-01+mDR5-05) on intracerebral glioma.
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15436583
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genmab b.v.
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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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Apr 1st, 2022 06:04PM
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Apr 1st, 2022 06:04PM
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Genmab A/S
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Health Care
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Pharmaceuticals & Biotechnology
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cph:gen
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Genmab A/S
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Jan 5th, 2021 12:00AM
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Jun 25th, 2019 12:00AM
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https://www.uspto.gov?id=US10882913-20210105
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Anti-DR5 antibodies and methods of use thereof
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The present invention relates to monospecific or bispecific antibody molecules that specifically bind the human DR5 antigen. The invention relates in particular to DR5-specific antibody molecules of the IgG1 isotype having a mutation in the Fc region that enhances clustering of IgG molecules after cell-surface antigen binding leading to the induction of DR5 signaling, apoptosis and cell death. The invention further relates to a combination of antibody molecules binding different epitopes on DR5. The invention also relates to pharmaceutical compositions containing these molecules and the treatment of cancer using these compositions.
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10882913
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1. An antibody which binds to human DR5 and comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH and VL regions comprise VHCDR1, VHCDR2, and VHCDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 1, 8, and 3, respectively, and VLCDR1, VLCDR2 and VLCDR3 domains comprising the amino acid sequences set forth in SEQ ID NO: 5, the sequence FAS, and SEQ ID NO: 6, respectively, and
a Fc region of a human immunoglobulin IgG, wherein in the Fc region, the amino acid at the position corresponding to E430 in human IgG1 is mutated to G, wherein the numbering is according to the EU Index.
2. The antibody of claim 1, wherein the antibody is an IgG1, IgG2, IgG3, IgG4, IgE, IgD or IgM isotype.
3. The antibody of claim 1, wherein the antibody is humanized or chimeric.
4. A composition comprising at least one antibody according to claim 1 and a carrier.
5. A nucleic acid construct comprising a nucleic acid encoding the heavy and light chain variable regions of the antibody according to claim 1.
6. An expression vector comprising one or more nucleic acid constructs according to claim 5.
7. A host cell comprising the expression vector of claim 6.
8. A kit comprising the antibody of claim 1, and instructions for use.
9. The composition of claim 4, which comprises
(a) a first antibody that binds to human DR5 and comprises VH and VL regions, wherein the VH region comprises VHCDR1, VHCDR2, and VHCDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 1, 8, and 3, respectively, and the VL region comprises VLCDR1, VLCDR2, and VLCDR3 domains comprising the amino acid sequences set forth in SEQ ID NO: 5, the sequence FAS, and SEQ ID NO: 6, respectively, and
(b) a second antibody that binds to human DR5 and comprises VH and VL regions, wherein the VH region comprises VHCDR1, VHCDR2, and VHCDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 10, 2, and 11, respectively, and the VL region comprises VLCDR1, VLCDR2, and VLCDR3 domains comprising the amino acid sequences set forth in SEQ ID NO: 13, the sequence RTS, and SEQ ID NO: 14, respectively,
wherein both the first antibody and second antibody comprise a Fc region of a human immunoglobulin IgG, and wherein in the Fc region of both the first antibody and second antibody, the amino acid at the position corresponding to E430 in human IgG1 is mutated to G.
10. The antibody of claim 1, wherein the antibody comprises a VH region and a VL region comprising the amino acid sequences set forth in SEQ ID NOs: 9 and 7, respectively.
11. The antibody of claim 1, wherein the antibody comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 38 and 39, respectively.
12. A composition comprising the antibody of claim 11 and a carrier.
13. The composition of claim 12, further comprising an antibody which comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 42 and 43, respectively.
14. The composition of claim 9, wherein the second antibody comprises a VH region and a VL region comprising the amino acid sequences set forth in SEQ ID NOs: 12 and 15, respectively.
15. The composition of claim 9, wherein the second antibody comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 42 and 43, respectively.
16. The composition of claim 9, wherein the first antibody comprises a VH region and a VL region comprising the amino acid sequences set forth in SEQ ID NOs: 9 and 7, respectively.
17. The composition of claim 16, wherein the second antibody comprises a VH region and a VL region comprising the amino acid sequences set forth in SEQ ID NOs: 12 and 15, respectively.
18. The composition of claim 16, wherein the second antibody comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 42 and 43, respectively.
19. The composition of claim 9, wherein the first antibody comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 38 and 39, respectively.
20. The composition of claim 19, wherein the second antibody comprises a VH region and a VL region comprising the amino acid sequences set forth in SEQ ID NOs: 12 and 15, respectively.
21. A composition comprising:
(a) a first antibody that binds to human DR5 and comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region comprises VHCDR1, VHCDR2, and VHCDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 1, 8, and 3, respectively, and the VL region comprises VLCDR1, VLCDR2, and VLCDR3 domains comprising the amino acid sequences set forth in SEQ ID NO: 5, the sequence FAS, and SEQ ID NO: 6, respectively, and
(b) a second antibody that binds to human DR5 and comprises VH and VL regions, wherein the VH region comprises VHCDR1, VHCDR2, and VHCDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 10, 2, and 11, respectively, and the VL region comprises VLCDR1, VLCDR2, and VLCDR3 domains comprising the amino acid sequences set forth in SEQ ID NO: 13, the sequence RTS, and SEQ ID NO: 14, respectively,
wherein both the first antibody and second antibody comprise a Fc region of a human immunoglobulin IgG, and wherein in the Fc region of both the first antibody and second antibody, the amino acid at the position corresponding to E430 in human IgG1 is mutated to G.
22. A composition comprising:
(a) a first antibody that binds to human DR5 and comprises a heavy chain variable (VH) region and a light chain variable (VL) region comprising the amino acid sequences set forth in SEQ ID NOs: 9 and 7, respectively, and
(b) a second antibody that binds to human DR5 and comprises a VH region and a VL region comprising the amino acid sequences set forth in SEQ ID NOs: 12 and 15, respectively,
wherein both the first antibody and second antibody comprise a Fc region of a human immunoglobulin IgG, and wherein in the Fc region of both the first antibody and second antibody, the amino acid at the position corresponding to E430 in human IgG1 is mutated to G.
23. A composition comprising:
(a) a first antibody that binds to human DR5 and comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 38 and 39, respectively, and
(b) a second antibody that binds to human DR5 and comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 42 and 43, respectively.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a Continuation of application Ser. No. 15/780,268, filed May 31, 2018, which is a 35 U.S.C. 371 national stage filing of International Application No. PCT/EP2016/079518, filed Dec. 1, 2016, which claims the benefit of Danish Patent Application Nos. PA 2015 00771, filed Dec. 1, 2015, PA 2015 00787, filed Dec. 7, 2015, PA 2015 00788, filed Dec. 7, 2015, PA 2016 00701, filed Nov. 10, 2016, and PA 2016 00702, filed Nov. 10, 2016. The contents of the aforementioned applications are hereby incorporated by reference.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 25, 2019, is named GMI_169AUSCN_SEQ.txt and is 144,175 bytes in size.
FIELD OF THE INVENTION
The present invention relates to monospecific or bispecific antibody molecules that specifically bind the human DR5 antigen. The invention relates in particular to DR5-specific antibody molecules of the IgG1 isotype having a mutation in the Fc region that enhances clustering of IgG molecules after cell surface antigen binding. The invention further relates to a combination of antibody molecules binding different epitopes on human DR5. The invention also relates to pharmaceutical compositions containing these molecules and the treatment of cancer and other diseases using these compositions.
BACKGROUND OF THE INVENTION
DR5, also known as death receptor 5, Tumor necrosis factor receptor superfamily member 10B, TNFRSF10B, TNF-related apoptosis-inducing ligand receptor 2, TRAIL receptor 2, TRAIL-R2 and CD262, is a cell surface receptor of the TNF receptor superfamily that binds tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and mediates apoptosis. DR5 is a single-pass type I membrane protein with three extracellular cysteine-rich domains (CRDs), a transmembrane domain (TM) and a cytoplasmic domain containing a death domain (DD). In the absence of ligand, DR5 exists in the cell membrane either as monomer or as pre-assembled complexes of two or three receptors through interactions of the first cysteine-rich domain, also known as pre-ligand assembly domain (PLAD) (Wassenaar et al., Proteins. 2008 Feb. 1; 70(2):333-43; Valley et al., J Biol Chem. 2012 Jun. 15; 287(25):21265-78; Sessler et al., Pharmacol Ther. 2013 November; 140(2):186-99). A Crystal structure of TRAIL in complex with the DR5 ectodomain showed that TRAIL binds to CRD2 and CRD3 in the extracellular domain of DR5 in a complex containing a trimeric receptor and a trimeric ligand (Hymowitz et al., Mol Cell. 1999 October; 4(4):563-71). The DR5 trimers can further cluster into higher-order receptor aggregates in lipid macrodomains, so-called lipid rafts (Sessler et al., Pharmacol Ther. 2013 November; 140(2):186-99). In the ligand-bound conformation, the cytoplasmic death domain-containing adaptor protein FADD associate with the intracellular DD surface of the oligomerized DR5 molecules and engage initiator caspases caspase-8 and caspase-10 to form the death-inducing signaling complex (DISC).
Based on the sensitivity of cancer cells to TRAIL-mediated apoptosis, numerous agents were developed to activate this pathway to induce apoptosis selectively in cancer cells. Human recombinant TRAIL (hrTRAIL), is being developed as dulanermin, and a series of conventional (monospecific, bivalent) anti-DR5 antibodies have been developed and tested in the clinic (reviewed in Ashkenazi et al., Nat Rev Drug Discov. 2008 December; 7(12):1001-12; Trivedi et al., Front Oncol. 2015 Apr. 2; 5:69): DR5 antibodies include lexatumumab (HGS-ETR2), HGS-TR2J, conatumumab (AMG655), tigatuzumab (CS-1008), drozitumab (Apomab) and LBY-135. Clinical studies with these compounds demonstrated that DR5 antibodies were generally well tolerated but failed to show convincing and significant clinical benefit. Efforts to enhance the efficacy of DR5 targeting antibodies mainly focus on (i) improving the sensitivity of cancer cells to DR5 agonists through combination treatment, (ii) developing biomarkers for better patient stratification, and (iii) the development of DR5-targeting agents that activate DR5 signaling and apoptosis-induction more effectively (reviewed in Lim et al., Expert Opin Ther Targets. 2015 May 25: 1-15; Twomey et al., Drug Resist Updat. 2015 March; 19:13-21; Reddy et al., PLoS One. 2015 Sep. 17; 10(9)). Different therapeutic formats for increasing DR5 activation have been described and include oligomerization of synthetic DR5 binding peptides, linear fusions of DR5-specific scaffolds, nanoparticle-based delivery systems of rhTRAIL or conatumumab and multivalent DR5 antibody-based formats (reviewed in Holland et al., Cytokine Growth Factor Rev. 2014 April; 25(2):185-93). APG880 and derivatives exist of two single chain TRAIL receptor binding (scTRAIL-RBD) molecules (TRAIL mimics) fused to the Fc part of a human IgG. Each scTRAIL-RBD has three receptor binding sites resulting in a hexavalent binding mode in the fusion protein (WO 2010/003766 A2). A prototype scTRAIL-RBD (APG350) has been described to induce FcγR-independent antitumor efficacy in vivo (Gieffers et al., Mol Cancer Ther, 2013. 12(12): p. 2735-47). A tetravalent anti-DR5 antibody fragment-derived construct, assembled by fusion of an anti-DR5 scFv fragment, human serum albumin residues and the tetramerization domain of human p53, has been shown to induce apoptosis more potently than the monovalent construct (Liu et al., Biomed Pharmacother. 2015 March; 70:41-5). Nanobody molecules are single domain antibody fragments (VHH) derived from camelid heavy chain-only antibodies, which, similarly to scFvs, can be linked to form multivalent molecules. Preclinical in vitro studies showed that TAS266, a tetravalent anti-DR5 Nanobody® molecule, was more potent than TRAIL or crosslinked DR5 antibody LBY-135, which was attributed to more rapid caspase activation kinetics (Huet et al., MAbs. 2014; 6(6):1560-70). TAS266 was also more potent in vivo than the parental murine mAb of LBY-135. MultYbody™ molecules (MultYmab technology) are based on the fusion of a homomultimerizing peptide to the Fc of one heavy chains in an IgG heterodimer (knob into hole), making MultYbody molecules intrinsically multivalent in solution. An anti-DR5 MultYbody was shown to induce potent killing in vitro. Dual-affinity re-targeting (DART) molecules are covalently-linked Fv-based diabodies. DR5 targeting tetravalent Fc DARTs comprising either tetravalency for a single (mono-epitopic DARTs) or two DR5 epitopes (bi-epitopic DARTs) were shown to be more potent than TRAIL and a conatumumab variant in inducing in cytotoxicity in vitro and in vivo (Li et al., AACR Annual Meeting Apr. 20, 2015, Poster abstract #2464). Alternatively, FcγR-independent avidity-driven DR5 hyperclustering can be mediated by a bispecific DR5×FAP antibody (RG7386) through simultaneous binding to DR5 on the cancer cell and to fibroblast activation protein (FAP) that is expressed on fibroblasts in the tumor microenvironment (Friess et al., AACR Annual Meeting Apr. 19, 2015, Presentation abstract #952; Wartha et al., Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr. 5-9; San Diego, Calif. Philadelphia (PA): AACR; Cancer Res 2014; 74(19 Suppl):Abstract nr 4573. doi:10.1158/1538-7445.AM2014-4573). Finally, specific combinations of two anti-DR5 antibodies recognizing different epitopes have shown enhanced agonistic efficacy in vitro and in vivo compared to combinations of two anti-DR5 antibodies recognizing overlapping or similar epitopes (WO2014/009358).
Above described approaches show enhanced efficacy compared to the conventional anti-DR5 antibodies in preclinical studies, however clinical data indicate that there is still a need for improving the DR5 agonists. Moreover, it is desirable for antibody-based formats to preserve a pharmacokinetic (PK) as well as other Fc-mediated effector functions of regular IgG, which usually is not the case with antibody fragment-based constructs. There is still a need for providing further DR5 agonists with improved properties.
Consequently, there is a need for providing improved anti-DR5 antibodies for the treatment of cancer, of infectious disease, autoimmune disease, cardiovascular anomalies and other diseases.
SUMMARY OF THE INVENTION
Surprisingly the inventors of the present invention have found that the introduction of a specific point mutation in the Fc region of an anti-DR5 antibody, which facilitates antibody clustering conditional on cell-surface antigen binding independent on secondary cross-linking, results in DR5 activation and significantly enhances the potency of the antibody in inducing apoptosis and cell death.
The objective of the present invention is to provide an improved anti-DR5 antibody for use in the treatment of cancer and other diseases. Such an improved antibody comprises a mutation in the Fc region. A further object of the present invention is to provide an improved composition for the treatment of cancer and other diseases comprising one or more anti-DR5 antibodies according the invention, e.g. wherein said antibodies bind to different epitopes on DR5. Such an improved composition as described herein comprises at least one anti-DR5 antibody according to the invention and more preferably the composition comprises two anti-DR5 antibodies binding to different regions on DR5, such as different non-competing epitopes on DR5.
The present invention provides an anti-DR5 antibody comprising an Fc region of a human immunoglobulin IgG and an antigen binding region binding to DR5, wherein the Fc region comprises a mutation at an amino acid position corresponding to position E430, E345 or S440 in human IgG1 according to EU numbering (Edelman et al., Proc Natl Acad Sci USA. 1969 May; 63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242).
That is, the inventors of the present invention have in a first aspect of the invention found that anti-DR5 antibodies of the invention comprising a mutation in the Fc region increase apoptosis of DR5 positive cells such as tumor cells compared to anti-DR5 antibodies without a mutation at an amino acid position corresponding to position E430, E345 or S440 in human IgG1, EU numbering. That is, the anti-DR5 antibody of the present invention is suitable for the treatment of DR5 positive or expressing tumors.
In one embodiment of the present invention the anti-DR5 antibody comprises a mutation at an amino acid position corresponding to E430 in human IgG1 according to EU numbering, wherein the mutation is selected form the group consisting of: E430G, E4305, E430F and E430T.
In one embodiment of the present invention the anti-DR5 antibody comprises an Fc region of a human immunoglobulin IgG and an antigen binding region binding to DR5, wherein the Fc region comprises the mutation E430G (glutamic acid at position 430 into glycine) or E345K (glutamic acid at position 345 into lysine) in human IgG1 according to EU numbering.
In one embodiment of the present invention the anti-DR5 antibody comprises an Fc region of a human IgG1 and an antigen binding region binding to DR5, wherein the Fc region comprises a E430G mutation.
In one embodiment of the present invention the anti-DR5 antibody comprises a mutation at an amino acid position corresponding to E345 in human IgG1 according to EU numbering, wherein the mutation is selected form the group consisting of: E345K, E345Q, E345R and E345Y.
In one embodiment of the present invention the anti-DR5 antibody comprises an Fc region of a human IgG1 and an antigen binding region binding to DR5, wherein the Fc region comprises an E345K mutation.
In one embodiment of the present invention the anti-DR5 antibody comprises a mutation at an amino acid position corresponding to S440 in human IgG1 according to EU numbering, wherein the mutation is selected form the group consisting of: S440W and S440Y.
In one embodiment of the present invention the anti-DR5 antibody comprises an Fc region of a human IgG1 and an antigen binding region binding to DR5, wherein the Fc region comprises a S440Y mutation.
In one aspect the invention provides a composition comprising one or more antibodies binding to DR5. In one embodiment the composition comprises one or more antibodies binding to different epitopes on DR5. Hereby are embodiments are provided where the antibodies bind different epitopes or require different amino acids within the DR5 sequence (SEQ ID NO 46) for binding to DR5. In one embodiment the composition comprises anti-DR5 antibodies which do not compete for binding to DR5, that is in one embodiment the anti-DR5 antibodies bind to non-overlapping epitopes.
In another aspect the invention provides a bispecific antibody comprising one or more antigen binding regions binding to DR5.
In one embodiment of the present invention the bispecific antibody comprises an Fc region comprising a first and a second heavy chain, wherein said first and second heavy chain comprises a mutation at an amino acid position corresponding to E430, E345 or S440 in human IgG1, EU numbering.
In one embodiment of the present invention the bispecific antibody comprises an Fc region comprising a first and a second heavy chain, wherein said first heavy chain comprises a mutation corresponding to position F405 and E430 and wherein said second heavy chain comprises a mutation corresponding to position K409 and E430, wherein the amino acid position is corresponding to human IgG1 according to EU numbering.
In one embodiment of the present invention the bispecific antibody comprises an Fc region comprising a first heavy chain with a F405L mutation and a second heavy chain with a K409R mutation in human IgG1 according to EU numbering. In another embodiment of the invention the bispecific antibody comprises an Fc region comprising a first heavy chain with a K409R mutation and a second heavy chain with a F405L mutation in human IgG1 according to EU numbering.
In yet another aspect the invention provides a method of treating a disease comprising administering to an individual in need thereof an effective amount of an antibody or composition as described herein. In one embodiment of the invention the disease is cancer.
In another aspect of the invention the anti-DR5 antibody, bispecific antibody or composition according to the present invention is for use as a medicament. In one embodiment the anti-DR5 antibody, bispecific antibody or composition is for use in treatment of a disease. In one embodiment the disease is a cancer or a tumor.
In another aspect the invention provides a kit of parts comprising an antibody or composition according to any one of the preceding claims, wherein said antibody or composition is in one or more containers such as a vial.
In another aspect the invention provides for the use of an antibody or a composition as described herein for the manufacture of a medicament for treatment of a disease. In one embodiment the invention provides the use of an antibody or a composition as described herein for the manufacture of a medicament for treatment of cancer.
The anti-DR5 antibodies and compositions comprising anti-DR5 antibodies described herein are directed against or specific for human DR5. The anti-DR5 antibodies and compositions described herein cross-react with rhesus and cynomolgus monkey DR5. In particular, in one embodiment of the invention the anti-DR5 antibodies and compositions bind specifically to the extracellular domain of human DR5. In one particular embodiment of the invention the antibodies and compositions comprising anti-DR5 antibodies bind to human DR5 at non-overlapping epitopes. That is in one embodiment the composition comprises at least one anti-DR5 antibody according to the invention. In one embodiment the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody according to the invention. That is a first anti-DR5 antibody described herein does not block binding of a second anti-DR5 antibody described herein. In one particular embodiment a composition described herein comprise a first and a second anti-DR5 antibody binding to human DR5 and the first anti-DR5 antibody does not block binding of the second anti-DR5 antibody to human DR5.
The anti-DR5 antibodies and compositions comprising anti-DR5 antibodies of the present invention i.e. anti-DR5 antibodies comprising an amino acid mutation in the Fc region can generally be used to modulate the activity of DR5. In one embodiment the anti-DR5 antibody or composition may trigger, activate and/or increase or enhance the signaling that is mediated by DR5. That anti-DR5 antibodies comprising an amino acid mutation in the Fc region according to the invention may increase or enhance the signaling that is mediated by DR5 is to be understood as when the signaling is compared to the same anti-DR5 antibody without said mutation in the Fc region. In one embodiment the anti-DR5 antibody or composition will have an agonistic effect on DR5 and in particular trigger or increase the downstream effects of DR5. That anti-DR5 antibodies comprising an amino acid mutation in the Fc region according to the invention may have an agonistic effect on DR5 is to be understood as when the agonistic effect is compared to the DR5 ligand TRAIL or the same anti-DR5 antibody i.e. having the same CDR sequences but without said mutation in the Fc region according to the invention. That is anti-DR5 antibodies or compositions of the present invention in able to induce apoptosis or cell death in cells, tumor mass or tissues expressing DR5, such as cancer cells or a tumor.
In one embodiment of the invention the anti-DR5 antibody or composition described herein induce, trigger, increase or enhance apoptosis, cell death or growth arrest in cells or tissues expressing DR5, such as cancer cells, tumor cells or a tumor compared to the same anti-DR5 antibody or composition without said mutation in the Fc region. In one embodiment the anti-DR5 antibodies or compositions described herein are capable of binding to DR5 on a cell surface, and in particular binding to DR5 in such a way that the signaling mediated by DR5 is induced, triggered, increased or enhanced compared to the same anti-DR5 antibody or composition without said mutation in the Fc region. In one embodiment the antibodies or compositions described herein may be such that they are capable of binding to DR5 in such a way that apoptosis or cell death is induced in cancer or tumor cells, tumors or tissues expressing DR5.
In one embodiment the antibodies or compositions of the present invention induce, trigger, increase or enhance apoptosis or cell death in cancer cells or cancer tissues expressing DR5. The increased or enhanced apoptosis or cell death can be measured by an increase or enhanced level of phosphatidylserine exposure on cells exposed to or treated with one or more anti-DR5 antibodies of the invention. Alternatively, the increase or enhanced apoptosis or cell death can be measured by measuring activation of caspase 3 or caspase 7 in cells that have been exposed to or treated with one or more anti-DR5 antibodies of the invention. Alternatively, the increase or enhanced apoptosis or cell death can be measured by a loss of viability in cell cultures that have been exposed to or treated with one or more anti-DR5 antibodies of the invention, compared to untreated cell cultures. Induction of caspase-mediated apoptosis can be assessed by demonstrating inhibition of the loss of viability after exposure to DR5 antibody by a caspase-inhibitor, for example ZVAD.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows an amino acid alignment of the four different human IgG1 Fc allotypes. The Fc sequence of the IgG1m(f), IgG1m(z), IgG1m(a), IgG1m(x) is specified in SEQ ID: 29, 30, 31 and 32 respectively.
FIG. 2 shows binding of humanized (hDR5) and chimeric (DR5) anti-DR5 antibodies to DR5-positive HCT 116 human colon cancer cells as measured by flow cytometry on FACS. Anti-gp120 antibody IgG1-b12 was used as a negative control. Binding is expressed as MFI (mean fluorescence intensity). Error bars indicate the standard deviation.
FIGS. 3A-3C show binding of anti-DR5 antibodies with and without hexamerization-enhancing mutations E430G or E345K to DR5-positive COLO 205 cells. Variants of the human-mouse chimeric antibodies IgG1-DR5-01-K409R (FIG. 3A), IgG1-DR5-05-F405L (FIG. 3B) and bispecific antibody IgG1-DR5-01-K409R×IgG1-DR5-05-F405L (BsAb IgG1-DR5-01-K409R×DR5-05-F405L) (FIG. 3C) were tested flowcytometric analysis on FACS for binding to COLO 205 cells. Binding is expressed as geometric mean of fluorescence intensity. Anti-gp120 antibody IgG1-b12 was used as negative control. Error bars indicate the standard deviation.
FIGS. 4A-4C show binding of anti-DR5 antibodies to human and rhesus monkey DR5. Human-mouse chimeric antibodies IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G were tested in flowcytometric analysis on FACS for binding to (FIG. 4A) mock-transfected CHO cells, (FIG. 4B) human DR5-transfected CHO cells and (FIG. 4C) Rhesus macaque DR5-transfected CHO cells. Binding is expressed as geometric mean of fluorescence intensity. Error bars indicate the standard deviation.
FIGS. 5A-5D show (FIG. 5A) Sequence alignment of part of the extracellular domains of human DR5 and mouse DR5 using EMBOSS Matcher (www.ebi.ac.uk/Tools/psa/emboss_matcher/); (.) similar amino acid; (:) identical amino acid. (FIG. 5B) Graphical representation of the domain-swapped DR5 extracellular domain (white: human DR5 sequences; black: mouse DR5 sequences). Amino acid number refer to the human sequence and domain swaps were made based on the alignment shown in panel A. (FIG. 5C) Binding of IgG1-hDR5-01-F405L and the isotype control antibody IgG1-b12 to a panel of human-mouse chimeric DR5 molecules, as assessed by flow cytometry. In each domain-swapped DR5 molecule, specific human amino acids have been replaced by the mouse sequence, as indicated on the x-axis. Error bars indicate the standard deviation of duplicate samples. (FIG. 5D) Binding of IgG1-hDR5-05-F405L to a panel of human-mouse chimeric DR5 molecules, as assessed by flow cytometry. In each domain-swapped DR5 molecule, specific human amino acids had been replaced by the mouse sequence, as indicated on the x-axis. IgG1-b12 was included an isotype control antibody. Error bars indicate the standard deviation of duplicate samples.
FIGS. 6A and 6B show crossblock ELISA with DR5-01 and DR5-05 antibodies. Graphs represent inhibition of binding of coated IgG1-hDR5-01-E430G (FIG. 6A) or IgG1-hDR5-05-E430G (FIG. 6B) to soluble DR5ECD-FcHisCtag in the presence of competing antibody IgG1-hDR5-01-E430G or IgG1-hDR5-05-E430G as measured by ELISA. Anti-gp120 antibody IgG1-b12 (b12) was used as negative control. DR5-01 is IgG1-hDR5-01-E430G; DR5-05 is IgG1-hDR5-05-E430G.
FIGS. 7A and 7B show a viability assay with variants of DR5-01 and DR5-05 antibodies. Introduction of the E430G hexamerization-enhancing mutation results in enhanced induction of killing of DR5-positive COLO 205 (FIG. 7A) and HCT 116 (FIG. 7B) colon cancer cells by the single human-mouse chimeric antibodies IgG1-DR5-01-K409R and IgG1-DR5-05-F405L used alone and by the combination thereof. Error bars indicate standard deviation.
FIGS. 8A-8C show (FIG. 8A) crossblock ELISA between IgG1-chTRA8-F405L and IgG1-DR5-01-K409R or IgG1-DR5-05-F405L, respectively. Combining the two non-crossblocking anti-DR5 antibodies IgG1-chTRA8-F405L-E430G and IgG1-DR5-01-K409R-E430G (FIG. 8B) resulted in enhanced induction of killing of HCT 116 colon cancer cells (decreased EC50), whereas combining the two crossblocking antibodies IgG1-chTRA8-F405L-E430G and IgG1-DR5-05-F405L-E430G (FIG. 8C) did not, as determined in a 3-days viability assay. Error bars indicate standard deviation.
FIGS. 9A and 9B that introduction of a hexamerization-enhancing mutation results in enhanced induction of killing of HCT 116 colon cancer cells by the combination of non-crossblocking antibodies IgG1-DR5-05-F405L-E345K+IgG1-CONA-K409R-E430G and BsAb IgG1-DR5-05-F405L-E345K×CONA-K409R-E430G. (FIG. 9A) crossblock ELISA with IgG1-CONA-K409R and IgG1-DR5-05-F405L. (FIG. 9B) 3-days viability assay. Error bars indicate standard deviation. RLU: Relative Luminescence Units.
FIG. 10 shows that the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G reduces the viability of a large panel of different human cancer cell lines, as determined in a 3-days viability assay. Graphs show the mean+/−standard deviation from duplicate samples. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 (One-way ANOVA with Tukey's multiple comparisons test).
FIG. 11 shows the potency of the combination of humanized IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G antibodies and of the combination of chimeric IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibodies as measured in a viability assay on B×PC-3 and PANC-1 pancreatic cancer cell lines. Graphs represent mean values of duplicate (B×PC-3) or triplicate (PANC-1) samples+/−standard deviation.
FIGS. 12A-12C show (FIG. 12A) Flowcytometric analysis using FACS analysis to study the effect of mimicking deamidation in humanized antibodies IgG1-hDR5-01-K409R and IgG1-hDR5-05-F405L on binding to HCT 116 human colon cancer cells. Introduction of the Asn deamidation-mimicking mutation N55D resulted in decreased binding of IgG1-hDR5-01-K409R, but had minimal effect on binding of IgG1-hDR5-05-F405L. (FIG. 12B) Flowcytometry analysis to study the effect of preventing deamidation in humanized antibody DR5-01 on binding to HCT 116 human colon cancer cells. Introduction of the amino acid substitution G56T in IgG1-hDR5-01-E430G had no effect on the binding of the antibody to HCT 116 cells. Binding is expressed as Geometric mean of fluorescence intensity. (FIG. 12C) Potency of the combination of humanized antibodies IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G as measured in a viability assay on B×PC-3 pancreatic cancer cells. Graphs represent mean values of duplicate samples+/−standard deviation.
FIGS. 13A and 13B viability assay with repulsing and complementary variants of IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G. Introduction of the same repulsing mutation (K439E or S440K) in both antibodies results in diminished induction of killing of B×PC-3 pancreatic (FIG. 13A) and HCT-15 colon cancer cells (FIG. 13B). By combining the two mutations (K439E and S440K) in both antibodies, repulsion is neutralized and killing restored. Error bars indicate standard deviation.
FIG. 14: Involvement of Fc interactions in the capacity of the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G with hexamerization-enhancing mutation to induce receptor clustering on the cell surface and induction of apoptosis. Induction of apoptosis is inhibited by the Fc-binding peptide DCAWHLGELVWCT as shown in a 3-days viability assay on B×PC-3 human cancer cells.
FIG. 15 shows the efficacy of different ratios of combinations of IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G (DR5-01:DR5-05) on adherent B×PC-3 human cancer cells as determined in a 3-days viability assay.
FIGS. 16A and 16B show efficacy of different ratios of IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G (DR5-01:DR5-05) on adherent B×PC-3 (FIG. 16A) and HCT-15 (FIG. 16B) human cancer cells as determined in a 3-days viability assay.
FIGS. 17A-17C show Caspase-dependent programmed cell death by the combination of humanized IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G antibodies as measured in a viability assay on PANC-1 (FIGS. 17A and 17B) and B×PC-3 (FIG. 17C) pancreatic cancer cells. 01-E430G is IgG1-hDR5-01-E430G; 05-E430G is IgG1-hDR5-05-E430G; ZVAD is pan-caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK).
FIGS. 18A-18E show cell death induction upon binding of anti-DR5 antibody or anti-DR5 antibody combinations on COLO 205 colon cancer cells. COLO 205 cells were incubated with antibody sample for 5 hours (FIGS. 18A-18C) and 24 hours (FIGS. 18D-18E). Different stages of cell death induction were analyzed by Annexin V/PI double staining and Active caspase-3 staining. Panels C and D show Annexin V/PI double staining at 5 and 24 hours respectively. Error bars indicate the standard deviation of 2 duplicate samples. 01 is IgG1-DR5-01-K409R, 05 is IgG1-DR5-05-F405L, 01-E430G is IgG1-DR5-01-K409R-E430G, 05-E430G is IgG1-DR5-05-F405L-E430G.
FIG. 19 shows the kinetics of Caspase-3/7 activation upon binding of DR5 antibodies on COLO 205 colon cancer cells. COLO 205 cells were incubated with antibody for 1, 2, 5 and 24 hours. Caspase-3/7 activation was analyzed in a homogenous luminescence assay. AU, arbitrary units. Error bars indicate the standard deviation of duplicate samples.
FIG. 20 shows efficacy of the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G in the presence or absence of Fc crosslinking by F(ab′)2 fragments of an anti-human IgG antibody and comparison to the anti-DR5 antibodies IgG1-DR5-CONA and IgG1-DR5-chTRA8-F405L in a 3-days viability assay on adherent COLO 205 colon cancer and B×PC-3 and PANC-1 pancreatic cancer cells. The non-target binding antibody IgG1-b12 was included as a negative control. Graphs show the mean+/−standard deviation from duplicate samples. * p<0.05, **p<0.01, *** p<0.001, **** p<0.0001 (One-way ANOVA with Bonferroni post-test for multiple comparisons).
FIG. 21 shows the potency of the combination of humanized IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G antibodies and of the combination of humanized IgG1-DR5-01-E430G+IgG1-DR5-05-E430G antibodies as measured in a viability assay on B×PC-3 pancreatic cancer cells. Graphs represent mean values of duplicate samples+/−standard deviation.
FIG. 22 shows the potency of the chimeric BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G antibody on different human cancer cell lines determined in a 3-days viability assay on adherent cells from COLO 205 colon, B×PC-3 pancreatic, SNU-5 gastric, SK-MES-1 lung, and A375 skin cancer cell lines. Graphs show the mean+/−standard deviation from duplicate samples. * p<0.05, *** p<0.001, **** p<0.0001 (One-way ANOVA with Bonferroni post-test for multiple comparisons). (01×05)-E430G is BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G.
FIG. 23 shows the efficacy of chimeric BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G in the presence or absence of Fc crosslinking by F(ab′)2 fragments of an anti-human IgG antibody in comparison with the anti-DR5 antibodies IgG1-DR5-CONA and IgG1-DR5-chTRA8-F405L in a 3-days viability assay on adherent B×PC-3 pancreatic and COLO 205 colon cancer cells. The non-target binding antibody IgG1-b12 was included as a negative control. Graphs show the mean+/−standard deviation from duplicate samples. * p<0.05, **p<0.01, *** p<0.001, **** p<0.0001 (One-way ANOVA with Bonferroni post-test for multiple comparisons). (01×05)-E430G is BsAb IgG1-DR5-01-K409R-E430G×IgG1-DR5-05-F405L-E430G
FIGS. 24A-24E show cell death induction upon binding of bispecific DR5 antibodies on COLO 205 colon cancer cells. COLO 205 cells were incubated with 1 μg/mL antibody for 5 hours (FIGS. 24A-24C) and 24 hours (FIGS. 24D-24E). Different stages of cell death induction were analyzed by Annexin V/PI double staining and Active caspase-3 staining. Error bars indicate the standard deviation of 2 duplicate samples. 01 is IgG1-DR5-01-K409R, 05 is IgG1-DR5-05-F405L, 01-E430G is IgG1-DR5-01-K409R-E430G, 05-E430G is IgG1-DR5-05-F405L-E430G, 01×05 is BsAb IgG1-DR5-01-K409R×DR5-05-F405L, 01-E430G×05-E430G is BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G.
FIGS. 25A-25C show evaluation of the in vivo efficacy of the combination of the chimeric IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibodies in a subcutaneous xenograft model with COLO 205 human colon cancer cells. Tumor size (mean & SEM) in mice treated with the indicated antibodies (5 mg/kg) is shown in time (FIG. 25A) and at day 23 (FIG. 25B). In (FIG. 25C) the percentage of mice with tumor sizes smaller than 750 mm3 is shown in a Kaplan-Meier plot.
FIGS. 26A-26C show evaluation of the in vivo efficacy of different doses of the IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibody combination and comparison to IgG1-CONA in a subcutaneous COLO 205 colon cancer xenograft. Tumor size (mean & SEM) in mice treated with the indicated antibody dose is shown in time (FIG. 26A) and on day 16 (FIG. 26B). In (FIG. 26C) the percentage of mice with tumor sizes smaller than 500 mm3 is shown in a Kaplan-Meier plot. * p<0.05, *** p<0.001.
FIGS. 27A-27C show evaluation of the in vivo efficacy of different doses of the IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibody combination and comparison to IgG1-CONA-F405L in a subcutaneous xenograft model with B×PC-3 human pancreatic cancer cells. Tumor size in mice treated with the indicated antibodies is shown in time (FIG. 27A, median tumor size) and at day 48 after tumor inoculation (FIG. 27B, mean tumor size & SEM). * p<0.05, ** p<0.01 (Unpaired t-test). In (FIG. 27C) the percentage of mice with tumor sizes smaller than 500 mm3 is shown in a Kaplan-Meier plot.
FIGS. 28A and 28B show evaluation of the in vivo efficacy of different doses of the IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibody combination and comparison to IgG1-CONA-F405L in a subcutaneous xenograft model with A375 human skin cancer cells. Tumor size in mice treated with the indicated antibodies is shown in time (FIG. 28A, median tumor size) and at day 29 after tumor inoculation (FIG. 28B, mean tumor size & SEM). * p<0.05, ** p<0.01, *** p<0.001 (Mann Whitney test).
FIGS. 29A-29C show evaluation of the in vivo efficacy of different doses of the IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibody combination and comparison to IgG1-CONA in a subcutaneous xenograft model with HCT-15 human colon cancer cells. Tumor size (mean & SEM) in mice treated with the indicated antibodies is shown in time (FIG. 29A) and at day 17 after start treatment (FIG. 29B). **** p<0.001 (Unpaired t test). In (FIG. 29C) the percentage of mice with tumor sizes smaller than 500 mm3 is shown in a Kaplan-Meier plot.
FIGS. 30A-30C show evaluation of the in vivo efficacy of different doses of the IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibody combination and comparison to IgG1-CONA in a subcutaneous xenograft model with SW480 human colon cancer cells. Tumor size (mean & SEM) in mice treated with the indicated antibodies is shown in time (FIG. 30A) and at day 28 after start treatment (FIG. 30B). * p<0.05, ** p<0.01 (Unpaired t-test). In (FIG. 30C) the percentage of mice with tumor sizes smaller than 500 mm3 is shown in a Kaplan-Meier plot.
FIG. 31A-31C show evaluation of the in vivo efficacy of different doses of the IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibody combination and comparison to IgG1-CONA in a subcutaneous xenograft model with SNU-5 human gastric cancer cells. Tumor size (mean & SEM) in mice treated with the indicated antibodies is shown in time (FIG. 31A) and at day 23 after start treatment (FIG. 31B). ** p<0.01, *** p<0.001 (Mann Whitney test). In (FIG. 31C) the percentage of mice with tumor sizes smaller than 500 mm3 is shown in a Kaplan-Meier plot.
FIGS. 32A-32C show evaluation of the in vivo efficacy of different doses of the IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G antibody combination and comparison to IgG1-CONA in a subcutaneous xenograft model with SK-MES-1 human lung cancer cells. Tumor size (mean & SEM) in mice treated with the indicated antibodies is shown in time (FIG. 32A) and at day 14 after start treatment (FIG. 32B). In (FIG. 32C) the percentage of mice with tumor sizes smaller than 1.000 mm3 is shown in a Kaplan-Meier plot.
FIG. 33 shows binding to DR5-positive HCT 116 human colon cancer cells by anti-DR5 antibodies IgG1-hDR5-01-G56T and IgG1-hDR5-05 with and without the E430G mutation as measured by flow cytometry. Anti-gp120 antibody IgG1-b12 was used as a negative control. Binding is expressed as geometric mean fluorescence intensity (FI). Error bars indicate the standard deviation. A representative example of seven experiments is shown.
FIG. 34 shows binding to DR5-positive HCT 116 human colon cancer cells by anti-DR5 antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G as measured by flow cytometry with directly labeled antibodies. Binding is expressed as Geometric mean Alexa 647 fluorescence intensity (FI). Error bars indicate the standard deviation.
FIGS. 35A and 35B show binding of anti-DR5 antibodies to human and cynomolgus monkey DR5. Antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G were tested by flow cytometry for binding to (FIG. 35A) human DR5-transfected CHO cells and (FIG. 35B) cynomolgus DR5-transfected CHO cells. Binding is expressed as geometric mean of fluorescence intensity (FI). Error bars indicate the standard deviation.
FIG. 36 shows a 3-days viability assay to show the effect of introducing the E430G mutation in the non-crossblocking antibodies IgG1-hDR5-01-G56T and IgG1-hDR5-05 on COLO 205 colon cancer cells. Error bars indicate standard deviation. A representative example of four experiments is shown.
FIGS. 37A and 37B show a viability assay with DR5 antibodies on COLO 205 human colon cancer cells. Introduction of the hexamerization-enhancing mutation S440Y resulted in induction of killing by the single antibodies IgG1-hDR5-01-G56T and IgG1-hDR5-05 (FIG. 37A) and increased efficacy of the antibody combination IgG1-hDR5-01-G56T+IgG1-hDR5-05 (FIG. 37B). Error bars indicate standard deviation.
FIGS. 38A and 38B show the efficacy of non-crossblocking antibodies IgG1-DR5-CONA-E430G+IgG1-DR5-chTRA8-E430G to induce killing of B×PC-3 human pancreatic cancer cells. (FIG. 38A) Crossblock ELISA between IgG1-DR5-CONA-K409R (CONA) and IgG1-DR5-chTRA8-F405L (chTRA8). (FIG. 38B) Introduction of the E430G hexamerization-enhancing mutation resulted in enhanced induction of killing of B×PC-3 cells by the combination of IgG1-DR5-CONA-C49W-E430G+IgG1-DR5-chTRA8-E430G as determined in a 3-days viability assay. Error bars indicate standard deviation.
FIG. 39 shows 3-days viability assays with 133 nM human recombinant TRAIL or 133 nM of the antibody combinations IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G (E430G) and IgG1-hDR5-01-G56T+IgG1-hDR5-05 (WT) on different human cancer cell lines. Graphs show the mean+/−standard deviation from duplicate samples. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 (One-way ANOVA with Tukey's multiple comparisons test).
FIGS. 40A and 40B show the percentage inhibition by (FIG. 40A) antibody (IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G) and (FIG. 40B) TRAIL therapy as determined in a 3-days viability assay screening of a cell line panel at Horizon, UK. Each data point represents an individual cell line of the indicated human cancer indication. Dotted lines indicate the 70% maximum response threshold value that was set to categorize cell lines as responders 70% inhibition) and non-responders (<70% inhibition).
FIGS. 41A and 41B show the efficacy of different antibody ratios in the combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G (indicated as 01-E430G:05-E430G) on adherent human (FIG. 41A) B×PC-3 pancreatic and (FIG. 41B) HCT-15 colon cancer cells as determined in a 3-days viability assay. Representative examples of two and three experiments are shown for HCT-15 and B×PC-3, respectively.
FIG. 42 shows Caspase-dependent programmed cell death by the combination of IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G antibodies, the parental WT combination without the E430G mutation and TRAIL as measured in a viability assay on B×PC-3 pancreatic cancer cells. ZVAD is pan-caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK).
FIG. 43 shows the kinetics of Caspase-3/7 activation upon binding of the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G on B×PC-3 pancreatic cancer cells, compared to the parental WT combination without the E430G mutation and TRAIL. B×PC-3 cells were incubated with antibody for 1, 2, 4 and 6 hours. Caspase-3/7 activation was analyzed in a homogenous luminescence assay. RLU, relative luminescence units. A representative example of four experiments is shown.
FIG. 44 shows efficacy of the combination of IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G in the presence or absence of Fc crosslinking by F(ab′)2 fragments of an anti-human IgG antibody and comparison to the anti-DR5 antibody IgG1-DR5-CONA and the combination of WT antibodies IgG1-hDR5-01-G56T+IgG1-hDR5-05 in a 3-days viability assay on adherent HCT-15 human colon cancer and B×PC-3 pancreatic cancer cells. The non-target binding antibody IgG1-b12 was included as a negative control. Graphs show the mean+/−standard deviation from duplicate samples. For both cell lines, a representative example of two experiments is shown.
FIGS. 45A-45D show the analysis of IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G to induce complement activation upon target cell binding on CHO cells transfected with human (FIG. 45A, FIG. 45C) or cynomolgus DR5 (FIG. 45B, FIG. 45D). (FIGS. 45A and 45B) In vitro CDC assay with antibody concentration series in the presence of 20% pooled normal human serum. CDC efficacy is presented as the percentage lysis determined by the percentage propidium iodide (PI)-positive cells. (FIGS. 45C and 45D) Deposition of complement activation products upon antibody binding in the presence of C5-depleted serum is expressed as geometric mean of fluorescence intensity. The IgG1-b12 mAb against HIV gp120 was used in as a non-binding isotype control antibody.
FIGS. 46A-46E show the effect of combining the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G with different therapeutic agents as determined in a viability assay on five different colon cancer cell lines. Five examples are shown from a synergy screen of 100 compounds from different therapeutic classes.
FIGS. 47A and 47B show evaluation of the in vivo efficacy of the antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G, both as single agents and as a combination in comparison to the parental antibodies without the E430G mutation in a subcutaneous xenograft model with COLO 205 human colon cancer cells. (FIG. 47A) Tumor size (mean & SEM) in mice treated with the indicated antibodies (0.5 mg/kg) as shown in time. (FIG. 47B) Kaplan-Meier plot of tumor progression, with a cutoff set at a tumor volume>500 mm3.
FIGS. 48A-48C shows the evaluation of the in vivo efficacy of the anti-DR5 antibody concentration IgG1-hDR5-01-G56T+IgG1-hDR5-05 with and without the hexamerization-enhancing mutation E430G in a subcutaneous xenograft model with HCT15 human colon cancer cells. Tumor size (mean & SEM) in mice treated with the 0.5 mg/kg antibodies is shown in time (FIG. 48A) and at day 21 after start treatment (FIG. 48B). **P<0.0011 (Mann Whitney test). In (FIG. 48C) the percentage of mice with tumor sizes smaller than 750 mm3 is shown in a Kaplan-Meier plot.
FIGS. 49A-49C show evaluation of the in vivo efficacy of the combination of IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-430G antibodies in combination with 15 mg/kg paclitaxel in a subcutaneous xenograft model with SK-MES-1 human lung cancer cells. (FIG. 49A) Tumor size (mean & SEM) in mice treated with the indicated compounds is shown in time. (FIG. 49B) Tumor volume per treatment group at day 16. (FIG. 49C) The percentage of mice with tumor sizes smaller than 500 mm3 is shown in a Kaplan-Meier plot.
FIGS. 50A and 50B show the clearance rate in SCID mice of 1 mg/kg i.v. administered IgG1-hDR5-01-G56T-E430G, IgG1-hDR5-05-E430G or the combination of the two antibodies in comparison to the parental WT antibodies without the E430G mutation. (FIG. 50A) Total human IgG in serum samples was determined by ELISA and plotted in a concentration versus time curve. Each data point represents the mean+/−standard deviation of four serial diluted samples. (FIG. 50B) Clearance until day 21 after administration of the antibody was determined following the formula D*1.000/AUC with D, injected dose and AUC, area under the curve of the concentration-time curve.
FIG. 51 shows a viability assays with DR5 antibodies IgG1-DR5-CONA and IgG1-DR5-CONA-E430G on attached COLO 205 human colon cancer cells. Introduction of the hexamerization-enhancing mutation E430G resulted in induction of killing. Data are presented as % viable cells calculated from the luminescence relative to samples incubated without antibody (no kill) and samples incubated with Staurosporine (maximal kill). Error bars indicate standard deviation.
DETAILED DESCRIPTION OF THE INVENTION
In describing the embodiments of the invention specific terminology will be resorted to for the sake of clarity. However, the invention is not intended to be limited to the specific terms so selected, and it is understood that each specific term includes all technical equivalents which operate in a similar manner to accomplish a similar purpose.
As described herein, surprisingly antibodies binding to DR5 and comprising a hexamerization enhancing mutation in the Fc region corresponding to position E430 or E345 of human IgG1 according to EU numbering, were found to be superior at inducing apoptosis in tumor cells expressing DR5 compared to antibodies binding DR5 without a mutation in one of the above mentioned positions. Furthermore, compositions comprising two anti-DR5 antibodies of the invention, which bind different epitopes on DR5, were found superior in in vitro and in vivo studies to compositions comprising the same anti-DR5 antibodies without the mutation. That is compositions with two antibodies of the present invention were superior at inducing apoptosis and/or inhibiting cell growth of tumor cells expressing DR5 compared to compositions comprising two DR5 antibodies without a mutation in the Fc region. By introducing specific mutations in the Fc region, hexamerization upon target binding on the cell surface can be enhanced, while the antibody molecules remain monomeric in solution WO2013/004842, WO2014/108198.
Definitions
The term “immunoglobulin” as used herein, refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four potentially inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. See for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region of IgG antibodies typically is comprised of three domains, CH1, CH2, and CH3. The heavy chains are inter-connected via disulfide bonds in the so-called “hinge region”. Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)). Unless otherwise stated or contradicted by context, reference to amino acid positions in the present invention is according to the EU-numbering (Edelman et al., Proc Natl Acad Sci USA. 1969 May; 63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242).
The term “hinge region” as used herein is intended to refer to the hinge region of an immunoglobulin heavy chain. Thus, for example the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to the EU numbering.
The term “CH2 region” or “CH2 domain” as used herein is intended to refer the CH2 region of an immunoglobulin heavy chain. Thus, for example the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the EU numbering. However, the CH2 region may also be any of the other isotypes or allotypes as described herein.
The term “CH3 region” or “CH3 domain” as used herein is intended to refer to the CH3 region of an immunoglobulin heavy chain. Thus, for example the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering. However, the CH3 region may also be any of the other isotypes or allotypes as described herein.
The term “fragment crystallizable region”, “Fc region”, “Fc fragment” or “Fc domain”, which may be used interchangeably herein, refers to an antibody region comprising, arranged from amino-terminus to carboxy-terminus, at least a hinge region, a CH2 domain and a CH3 domain. An Fc region of an IgG1 antibody can, for example, be generated by digestion of an IgG1 antibody with papain. The Fc region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q, the first component in the classical pathway of complement activation.
The term “Fab fragment” in the context of the present invention, refers to a fragment of an immunoglobulin molecule, which comprises the variable regions of the heavy chain and light chain as well as the constant region of the light chain and the CH1 region of the heavy chain of an immunoglobulin. The “CH1 region” refers e.g. to the region of a human IgG1 antibody corresponding to amino acids 118-215 according to the EU numbering. Thus, the Fab fragment comprises the binding region of an immunoglobulin.
The term “antibody” (Ab), as used herein refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof. The antibody of the present invention comprises an Fc-region of an immunoglobulin and an antigen-binding region. The Fc region generally contains two CH2-CH3 regions and a connecting region, e.g. a hinge region. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The term “antibody” as used herein, also refers to, unless otherwise specified or contradicted by the context, polyclonal antibodies, oligoclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures, recombinant polyclonal antibodies, chimeric antibodies, humanized antibodies and human antibodies. An antibody as generated can potentially possess any class or isotype.
The term “human antibody”, as used herein, refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another species, such as a mouse, have been grafted onto human framework sequences.
The term “chimeric antibody”, as used herein, refers to an antibody in which both chain types i.e. heavy chain and light chain are chimeric as a result of antibody engineering. A chimeric chain is a chain that contains a foreign variable domain (originating from a non-human species, or synthetic or engineered from any species including human) linked to a constant region of human origin.
The term “humanized antibody, as used herein, refers to an antibody in which both chain types are humanized as a result of antibody engineering. A humanized chain is typically a chain in which the complementarity determining regions (CDR) of the variable domains are foreign (originating from a species other than human, or synthetic) whereas the remainder of the chain is of human origin. Humanization assessment is based on the resulting amino acid sequence, and not on the methodology per se, which allows protocols other than grafting to be used.
The term “isotype”, as used herein, refers to the immunoglobulin class (for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgA2, IgE, or IgM) that is encoded by heavy chain constant region genes. To produce a canonical antibody, each heavy chain isotype is to be combined with either a kappa (κ) or lambda (λ) light chain.
The term “allotype”, as used herein, refers to the amino acid variation within one isotype class in the same species. The predominant allotype of an antibody isotype varies between ethnicity individuals. The known allotype variations within the IgG1 isotype of the heavy chain result from 4 amino acid substitutions in the antibody frame as illustrated in FIG. 1. In one embodiment the antibody of the invention is of the IgG1m(f) allotype as defined in SEQ ID NO 29. In one embodiment of the invention the antibody is of the IgG1m(z) allotype as defined in SEQ ID NO 30, the IgG1m(a) allotype as defined in SEQ ID NO 31, the IgG1m(x) allotype as defined in SEQ ID NO 32, or any allotype combination, such as IgG1m(z,a), IgG1m(z,a,x), IgG1m(f,a) (de Iange Exp Clin Immunogenet. 1989; 6(1):7-17).
The terms “monoclonal antibody”, “monoclonal Ab”, “monoclonal antibody composition”, “mAb”, or the like, as used herein refer to a preparation of Ab molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term “human monoclonal antibody” refers to Abs displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs may be generated by a hybridoma which includes a B cell obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene repertoire and a human light chain transgene repertoire, rearranged to produce a functional human antibody and fused to an immortalized cell. Alternatively, the human mAbs may be generated recombinantly.
The term “antibody mimetics” as used herein, refers to compounds that, like antibodies, can specifically bind antigens, but that are not structurally related to antibodies. They are usually artificial peptides, proteins, nucleic acids or small molecules.
The term “bispecific antibody” refers to an antibody having specificities for at least two different, typically non-overlapping, epitopes. Such epitopes may be on the same or different targets Examples of different classes of bispecific antibodies comprising an Fc region include but are not limited to: asymmetric bispecific molecules e.g. IgG-like molecules with complementary CH3 domains and symmetric bispecific molecules e.g. recombinant IgG-like dual targeting molecules wherein each antigen-binding region of the molecule binds at least two different epitopes.
Examples of bispecific molecules include but are not limited to Triomab® (Trion Pharma/Fresenius Biotech, WO/2002/020039), Knobs-into-Holes (Genentech, WO9850431), CrossMAbs (Roche, WO 2009/080251, WO 2009/080252, WO 2009/080253), electrostatically-matched Fc-heterodimeric molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), LUZ-Y (Genentech), DIG-body, PIG-body and TIG-body (Pharmabcine), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono, WO2007110205), Bispecific IgG1 and IgG2 (Pfizer/Rinat, WO11143545), Azymetric scaffold (Zymeworks/Merck, WO2012058768), mAb-Fv (Xencor, WO2011028952), XmAb (Xencor), Bivalent bispecific antibodies (Roche, WO2009/080254), Bispecific IgG (Eli Lilly), DuoBody® molecules (Genmab A/S, WO 2011/131746), DuetMab (Medimmune, US2014/0348839), Biclonics (Merus, WO 2013/157953), NovImmune (κλBodies, WO 2012/023053), FcΔAdp (Regeneron, WO 2010/151792), (DT)-Ig (GSK/Domantis), Two-in-one Antibody or Dual Action Fabs (Genentech, Adimab), mAb2 (F-Star, WO2008003116), Zybodies™ (Zyngenia), CovX-body (CovX/Pfizer), FynomAbs (Covagen/Janssen Cilag), DutaMab (Dutalys/Roche), iMab (Medimmune), Dual Variable Domain (DVD)-Ig™ (Abbott, U.S. Pat. No. 7,612,18), dual domain double head antibodies (Unilever; Sanofi Aventis, WO20100226923), Ts2Ab (Medimmune/AZ), BsAb (Zymogenetics), HERCULES (Biogen Idec, US007951918), scFv-fusions (Genentech/Roche, Novartis, Immunomedics, Changzhou Adam Biotech Inc, CN 102250246), TvAb (Roche, WO2012025525, WO2012025530), ScFv/Fc Fusions, SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS), Interceptor (Emergent), Dual Affinity Retargeting Technology (Fc-DART™) (MacroGenics, WO2008/157379, WO2010/080538), BEAT (Glenmark), Di-Diabody (Imclone/Eli Lilly) and chemically crosslinked mAbs (Karmanos Cancer Center), and covalently fused mAbs (AIMM therapeutics).
The term “full-length antibody” when used herein, refers to an antibody (e.g., a parent or variant antibody) which contains all heavy and light chain constant and variable domains corresponding to those that are normally found in a wild-type antibody of that class or isotype.
The term “oligomer” as used herein, refers to a molecule that consists of more than one but a limited number of monomer units (e.g. antibodies) in contrast to a polymer that, at least in principle, consists of an unlimited number of monomers. Exemplary oligomers are dimers, trimers, tetramers, pentamers and hexamers. Greek prefixes are often used to designate the number of monomer units in the oligomer, for example a tetramer being composed of four units and a hexamer of six units. Likewise, the term “oligomerization”, as used herein, is intended to refer to a process that converts molecules to a finite degree of polymerization. Herein, it is observed, that antibodies and/or other dimeric proteins comprising target-binding regions according to the invention can form oligomers, such as hexamers, via non-covalent association of Fc-regions after target binding, e.g., at a cell surface.
The term “antigen-binding region”, “antigen binding region”, “binding region” or antigen binding domain, as used herein, refers to a region of an antibody which is capable of binding to the antigen. This binding region is typically defined by the VH and VL domains of the antibody which may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). The antigen can be any molecule, such as a polypeptide, e.g. present on a cell, bacterium, or virion or in solution. The terms “antigen” and “target” may, unless contradicted by the context, be used interchangeably in the context of the present invention.
The term “target”, as used herein, refers to a molecule to which the antigen binding region of the antibody binds. The target includes any antigen towards which the raised antibody is directed. The term “antigen” and “target” may in relation to an antibody be used interchangeably and constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention.
The term “epitope” means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface groupings of building blocks such as amino acids, sugar side chains or a combination thereof and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The epitope may comprise amino acid residues directly involved in the binding and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen binding peptide (in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide).
The term “binding” as used herein refers to the binding of an antibody to a predetermined antigen or target, typically with a binding affinity corresponding to a KD of about 10−6 M or less, e.g. 10−7 M or less, such as about 10−8 M or less, such as about 10−9 M or less, about 10−10 M or less, or about 10−11 M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte or visa versa, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1,000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The amount with which the affinity is lower is dependent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is highly specific), then the degree with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000 fold. The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, and is obtained by dividing kd by ka.
The term “kd” (sec−1), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the koff value or off-rate.
The term “ka” (M−1×sec−1), as used herein, refers to the association rate constant of a particular antibody-antigen interaction. Said value is also referred to as the kon value or on-rate.
The term “KA” (M−1), as used herein, refers to the association equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing ka by kd.
As used herein, the term “affinity” is the strength of binding of one molecule, e.g. an antibody, to another, e.g. a target or antigen, at a single site, such as the monovalent binding of an individual antigen binding site of an antibody to an antigen.
As used herein, the term “avidity” refers to the combined strength of multiple binding sites between two structures, such as between multiple antigen binding sites of antibodies simultaneously interacting with a target. When more than one binding interactions are present, the two structures will only dissociate when all binding sites dissociate, and thus, the dissociation rate will be slower than for the individual binding sites, and thereby providing a greater effective total binding strength (avidity) compared to the strength of binding of the individual binding sites (affinity).
The term “hexamerization enhancing mutation”, as used herein, refers to a mutation of an amino acid position corresponding to E430, E345 or S440 in human IgG1 according to EU numbering. The hexamerization enhancing mutation strengthens Fc-Fc interactions between neighbouring IgG antibodies that are bound to a cell surface target, resulting in enhanced hexamer formation of the target-bound antibodies, while the antibody molecules remain monomeric in solution as described in WO2013/004842; WO2014/108198.
The term “repulsing mutation” or “self-repulsing mutation” or “hexamerization-inhibiting mutation”, as used herein, refers to a mutation of an amino acid position of human IgG1 that can result in charge repulsion between amino acids at the Fc-Fc interface, resulting in weakening of the Fc-Fc interaction between two adjacent Fc region containing polypeptides, and thus inhibiting hexamerization. Examples of such a repulsing mutation in human IgG1 are K439E and S440K. The repulsion in the Fc-Fc interaction between two adjacent Fc region containing polypeptides at the position of a repulsing mutation can be neutralized by introduction of a second mutation (complementary mutation) in the amino acid position that interacts with the position harboring the first mutation. This second mutation can be present either in the same antibody or in a second antibody. The combination of the first and second mutation results in neutralization of the repulsion and restoration of the Fc-Fc interactions and thus hexamerization. Examples of such first and second mutations are K439E (repulsing mutation) and S440K (neutralizing the repulsion by K439E), and vice versa S440K (repulsing mutation) and K439E (neutralizing the repulsion by S440K).
The term “complementary mutation”, as used herein, refers to a mutation of an amino acid position in a Fc region-containing polypeptide that relates to a first mutation in an adjacent Fc region containing polypeptide that preferably interacts with the Fc region-containing polypeptide containing the complementary mutation due to the combination of the two mutations in the two adjacent Fc region-containing polypeptides. The complementary mutation and the related first mutation can be present either in the same antibody (intramolecular) or in a second antibody (intermolecular). An example of intramolecular complementary mutations is the combination K409R and F405L that mediates preferential heterodimerization in a bispecific antibody according to WO 2011/131746. The combination of the K439E and S440K mutations that results in neutralization of repulsion and restoration of Fc-Fc interactions between two adjacent Fc region containing polypeptides and thus hexamerization is an example of complementary mutations that can be applied both inter- and intramolecularly.
The term “apoptosis”, as used herein refers to the process of programmed cell death (PCD) that may occur in a cell. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, phosphatidylserine exposure, loss of mitochondrial function, nuclear fragmentation, chromatin condensation, caspase activation, and chromosomal DNA fragmentation. In a particular embodiment, apoptosis by one or more agonistic anti-DR5 antibodies can be determined using methods such as, e.g., caspase-3/7 activation assays described in examples 19, 20, 25 and 45 or phosphatidylserine exposure described in examples 19 and 25. Anti-DR5 antibody at a fixed concentration of e.g. 1 μg/mL may be added to adhered cells and incubated for 1 to 24 hours. Caspase-3/7 activation can be determined by using special kits for this purpose, such as the PE Active Caspase-3 Apoptosis Kit of BD Pharmingen (Cat nr 550914) (example 19 and 25) or the Caspase-Glo 3/7 assay of Promega (Cat nr G8091) (examples 20 and 45). Phosphatidylserine exposure and cell death can be determined by using special kits for this purpose, such as the FITC Annexin V Apoptosis Detection Kit I from BD Pharmingen (Cat nr 556547) (examples 19 and 25).
The term “programmed cell-death” or “PCD”, as used herein refers to the death of a cell in any form mediated by an intracellular signaling, e.g. apoptosis, autophagy or necroptosis.
The term “Annexin V”, as used herein, refers to a protein of the annexin group that binds phosphatidylserine (PS) on the cell surface.
The term “caspase activation”, as used herein, refers to cleavage of inactive pro-forms of effector caspases by initiator caspases, leading to their conversion into effector caspases, which in turn cleave protein substrates within the cell to trigger apoptosis.
The term “caspase-dependent programmed cell death”, as used herein refers to any form of programmed cell death mediated by caspases. In a particular embodiment, caspase-dependent programmed cell death by one or more agonistic anti-DR5 antibodies can be determined by comparing the viability of a cell culture in the presence and absence of pan-caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK) as described in examples 18 and 44. Pan-caspase inhibitor Z-VAD-FMK (5 μM end concentration) may be added to adhered cells in 96-well flat bottom plates and incubated for one hour at 37° C. Next, antibody concentration dilution series (e.g. starting from e.g. 20,000 ng/mL to 0.05 ng/mL final concentration in 5-fold dilutions) may be added and incubated for 3 days at 37° C. Cell viability can be quantified using special kits for this purpose, such as the CellTiter-Glo luminescent cell viability assay of Promega (Cat nr G7571).
The term “cell viability”, as used herein refers to the presence of metabolically active cells. In a particular embodiment, cell viability after incubation with one or more agonistic anti-DR5 antibodies can be determined by quantifying the ATP present in the cells as described in examples 8-18, 21-24, 38-44, 46 and 48. Antibody concentration dilution series (e.g. starting from e.g. 20,000 ng/mL to 0.05 ng/mL final concentration in 5-fold dilutions) may be added to cells in 96-well flat bottom plates, medium may be used as negative control and 5 μM staurosporine may be used as positive control for the induction of cell death. After 3 days incubation cell viability may be quantified using special kits for this purpose, such as the CellTiter-Glo luminescent cell viability assay of Promega (Cat nr G7571). The percentage viable cells can be calculated using the following formula: % viable cells=[(luminescence antibody sample−luminescence staurosporine sample)/(luminescence no antibody sample−luminescence staurosporine sample)]*100.
The term “DR5”, as used herein, refers to death receptor 5, also known as CD262 and TRAILR2, which is a single-pass type I membrane protein with three extracellular cysteine-rich domains (CRD's), a transmembrane domain (TM) and a cytoplasmic domain containing a death domain (DD). In humans, the DR5 protein is encoded by a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO 46, (human DR5 protein: UniprotKB/Swissprot 014763).
The term “antibody binding DR5”, “anti-DR5 antibody” DR5-binding antibody”, “DR5-specific antibody”, “DR5 antibody” which may be used interchangeably herein, refers to any antibody binding an epitope on the extracellular part of DR5.”
The term “agonist” as used herein, refers to a molecule such as an anti-DR5 antibody that is able to trigger a response in a cell when bound to DR5, wherein the response may be programmed cell death. That the anti-DR5 antibody is agonistic is to be understood as that the antibody stimulates, activates or clusters DR5 as the result from anti-DR5 binding to DR5. That is an agonistic anti-DR5 antibody comprising an amino acid mutation in the Fc region according to the present invention bound to DR5 results in DR5 stimulation, clustering or activation of the same intracellular signaling pathways as TRAIL bound to DR5. In a particular embodiment, the agonistic activity of one or more antibodies can be determined by incubating target cells for 3 days with an antibody concentration dilution series (e.g. from 20,000 ng/mL to 0.05 ng/mL final concentration in 5-fold dilutions). The antibodies may be added directly when cells are seeded (described in examples 8, 9, 10, 39), or alternatively the cells are first allowed to adhere to 96-well flat-bottom plates before adding the antibody samples (described in examples 11, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24, 38, 40, 41, 42, 43, 44, 46, 48). The agonistic activity i.e. the agonistic effect can be quantified by measuring the amount of viable cells using special kits for this purpose, such as the CellTiter-Glo luminescent cell viability assay of Promega (Cat nr G7571).
The terms “DR5 positive” and “DR5 expressing” as used herein, refers to tissues or cell lines which show binding of a DR5-specific antibody which can be measured with e.g. flow cytometry or immunohistochemistry.
A “variant” or “antibody variant” of the present invention is an antibody molecule which comprises one or more mutations as compared to a “parent” antibody. Exemplary parent antibody formats include, without limitation, a wild-type antibody, a full-length antibody or Fc-containing antibody fragment, a bispecific antibody, a human antibody, humanized antibody, chimeric antibody or any combination thereof.
Exemplary mutations include amino acid deletions, insertions, and substitutions of amino acids in the parent amino acid sequence. Amino acid substitutions may exchange a native amino acid present in the wild-type protein for another naturally-occurring amino acid, or for a non-naturally-occurring amino acid derivative. The amino acid substitution may be conservative or non-conservative. In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected in one or more of the following three tables:
Amino Acid Residue Classes for Conservative Substitutions
Acidic Residues
Asp (D) and Glu (E)
Basic Residues
Lys (K), Arg (R), and His (H)
Hydrophilic Uncharged Residues
Ser (S), Thr (T), Asn (N), and Gln (Q)
Aliphatic Uncharged Residues
Gly (G), Ala (A), Val (V), Leu (L), and
Ile (I)
Non-polar Uncharged Residues
Cys (C), Met (M), and Pro (P)
Aromatic Residues
Phe (F), Tyr (Y), and Trp (W)
Alternative Conservative Amino Acid Residue Substitution Classes
1
A
S
T
2
D
E
3
N
Q
4
R
K
5
I
L
M
6
F
Y
W
Alternative Physical and Functional Classifications of Amino Acid Residues
Alcohol group-containing residues
S and T
Aliphatic residues
I, L, V, and M
Cycloalkenyl-associated residues
F, H, W, and Y
Hydrophobic residues
A, C, F, G, H, I, L, M, R, T, V,
W, and Y
Negatively charged residues
D and E
Polar residues
C, D, E, H, K, N, Q, R, S, and T
Positively charged residues
H, K, and R
Small residues
A, C, D, G, N, P, S, T, and V
Very small residues
A, G, and S
Residues involved in turn formation
A, C, D, E, G, H, K, N, Q, R, S,
P, and T
Flexible residues
Q, T, K, S, G, D, E, and R
In the context of the present invention, a substitution in a variant is indicated as:
Original amino acid−position−substituted amino acid;
The three letter code, or one letter code, are used, including the codes Xaa and X to indicate amino acid residue. Accordingly, the notation “E345R” or “GIu345Arg” means, that the variant comprises a substitution of Glutamic acid with Arginine in the variant amino acid position corresponding to the amino acid in position 345 in the parent antibody. Where a position as such is not present in an antibody, but the variant comprises an insertion of an amino acid, for example: Position−substituted amino acid; the notation, e.g., “448E” is used. Such notation is particular relevant in connection with modification(s) in a series of homologous polypeptides or antibodies. Similarly when the identity of the substitution amino acid residues(s) is immaterial: Original amino acid−position; or “E345”.
For a modification where the original amino acid(s) and/or substituted amino acid(s) may comprise more than one, but not all amino acid(s), the substitution of Glutamic acid for Arginine, Lysine or Tryptophan in position 345: “Glu345Arg,Lys,Trp” or “E345R,K,W” or “E345R/K/W” or “E345 to R, K or W” may be used interchangeably in the context of the invention. Furthermore, the term “a substitution” embraces a substitution into any one of the other nineteen natural amino acids, or into other amino acids, such as non-natural amino acids. For example, a substitution of amino acid E in position 345 includes each of the following substitutions: 345A, 345C, 345D, 345G, 345H, 345F, 345I, 345K, 345L, 345M, 345N, 345Q, 345R, 345S, 345T, 345V, 345W, and 345Y. This is, by the way, equivalent to the designation 345X, wherein the X designates any amino acid. These substitutions can also be designated E345A, E345C, etc, or E345A,C,ect, or E345A/C/ect. The same applies to analogy to each and every position mentioned herein, to specifically include herein any one of such substitutions.
For the purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the −nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment).
For the purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the −nobrief option) is used as the percent identity and is calculated as follows:
(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment).
The sequence of CDR variants may differ from the sequence of the CDR of the parent antibody sequences through mostly conservative, physical or functional amino acids substitutions at most 5 mutations or substitutions selected from conservative, physical or functional amino acids in total across the six CDR sequences of the antibody binding region, such as at most 4 mutations or substitutions selected from conservative, physical or functional amino acids, such as at most 3 mutations or substitutions selected from conservative, physical or functional amino acids, such as at most 2 mutations selected from conservative, physical or functional amino acids or substitutions, such as at most 1 mutation or substitution selected from a conservative, physical or functional amino acid, in total across the six CDR sequences of the antibody binding region. The conservative, physical or functional amino acids are selected from the 20 natural amino acids found i.e, Arg (R), His (H), Lys (K), Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln (Q), Cys (C), Gly (G), Pro (P), Ala (A), Ile (I), Leu (L), Met (M), Phe (F), Trp (W), Tyr (Y) and Val (V).
The sequence of CDR variants may differ from the sequence of the CDR of the parent antibody sequences through mostly conservative, physical or functional amino acids substitutions; for instance at least about 75%, about 80% or more, about 85% or more, about 90% or more, (e.g., about 75-95%, such as about 92%, 93% or 94%) of the substitutions in the variant are mutations or substitutions selected from conservative, physical or functional amino acids residue replacements.
The conservative, physical or functional amino acids are selected from the 20 natural amino acids found i.e, Arg (R), His (H), Lys (K), Asp (D), Glu (E), Ser (S), Thr (T), Asn (N), Gln (Q), Cys (C), Gly (G), Pro (P), Ala (A), Ile (I), Leu (L), Met (M), Phe (F), Trp (W), Tyr (Y) and Val (V).
An amino acid or segment in one sequence that “corresponds to” an amino acid or segment in another sequence is one that aligns with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW or similar, typically at default settings. Hence a standard sequence alignment program can be used to identify which amino acid in an e.g. immunoglobulin sequence corresponds to a specific amino acid in e.g. human IgG1. Further a standard sequence alignment program can be used to identify sequence identity e.g. a sequence identity to SEQ ID NO:29 of at least 80%, or 85%, 90%, or at least 95%. For example, the sequence alignments shown in FIG. 1 can be used to identify any amino acid in the Fc region of one IgG1 allotype that corresponds to a particular amino acid in another allotype of an IgG1 Fc sequence.
The term “vector,” as used herein, refers to a nucleic acid molecule capable of inducing transcription of a nucleic acid segment ligated into the vector. One type of vector is a “plasmid”, which is in the form of a circular double stranded DNA loop. Another type of vector is a viral vector, wherein the nucleic acid segment may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (for instance bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (such as non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (such as replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Recombinant host cells include, for example, transfectomas, such as CHO-S cells, HEK-293F cells, Expi293F cells, PER.C6, NSO cells, and lymphocytic cells, and prokaryotic cells such as E. coli and other eukaryotic hosts such as plant cells and fungi, as well as prokaryotic cells such as E. coli.
Specific Embodiments of the Invention
The present invention is based, at least in part, on the discovery that the ability of an anti-DR5 antibody to induce cell death in a target cell expressing DR5 can be greatly enhanced by introducing a specific mutation in the Fc region corresponding to amino acid positions E430, E345 or S440 in human IgG1 according to EU numbering. The invention is further based on the surprising finding that a combination of two antibodies binding to a first and a second epitope on DR5 and each comprising a mutation in the Fc region may form heterohexamers and show superior induction of cell death in a target cell compared to a combination of the two antibodies without the mutation in the Fc region.
In one aspect the present invention relates to an anti-DR5 antibody comprising an Fc region of a human immunoglobulin IgG and an antigen binding region binding to DR5, wherein the Fc region comprises a mutation at an amino acid position corresponding to position E430, E345 or S440 in human IgG1 according to EU numbering.
The positions corresponding to E430, E345 and S440 in human IgG1 according to EU numbering are located in the CH3 domain of the Fc region.
The anti-DR5 antibody according to the present invention comprises an Fc region comprising a first and a second heavy chain, wherein a mutation at a position according to the present invention corresponding to E430, E345 or S440 in human IgG1 according to EU numbering is present in both the first and the second heavy chain, or less preferred only be present in one of the heavy chains. In the context of the present invention the hexamerization enhancing mutation is an amino acid mutation at a position corresponding to E430, E345 or S440 in human IgG1 according to EU numbering. The hexamerixation enhancing mutation strengthens the Fc-Fc interactions between antibodies comprising the mutation when bound to the corresponding target on a cell surface.
By introducing specific mutations in the Fc region corresponding to at least one of the following positions E430, E345 and S440 in human IgG1 hexamerization upon target binding on the cell surface is enhanced, while the antibody molecules remain monomeric in solution (WO2013/004842; WO2014/108198).
In one embodiment of the present invention the Fc region of the anti-DR5 antibody comprises a mutation corresponding to E430G, E4305, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y or S440W in human IgG1, EU numbering. Thus the anti-DR5 antibody comprises a mutation selected from the group of: E430G, E4305, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in human IgG1, EU numbering. Hereby are embodiments provided that allow for enhanced hexamerization of antibodies upon cell-surface antigen binding. The anti-DR5 antibody comprises an Fc region comprising a first heavy chain and a second heavy chain, wherein one of the above mentioned hexamerization enhancing mutations may be present in the first and/or the second heavy chain.
In a preferred embodiment of the present invention the Fc region comprises a mutation corresponding to E430G or E345K in human IgG1 EU numbering. Thus the Fc region comprises a mutation selected from E430G and E345K.
In one embodiment of the present invention the anti-DR5 antibody comprises a mutation at an amino acid position corresponding to E430 in human IgG1 according to EU numbering, wherein the mutation is selected form the group consisting of: E430G, E4305, E430F and E430T.
In one embodiment of the present invention the Fc region comprises a mutation corresponding to E430G. Thus in one embodiment of the present invention the Fc region comprises an E430G mutation.
In one embodiment of the present invention the anti-DR5 antibody comprises a mutation at an amino acid position corresponding to E345 in human IgG1 according to EU numbering, wherein the mutation is selected form the group consisting of: E345K, E345Q, E345R and E345Y.
In one embodiment of the present invention the Fc region comprises a mutation corresponding to E345K. Thus in one embodiment of the present invention the Fc region comprises an E345K mutation.
In one embodiment of the present invention the anti-DR5 antibody comprises a mutation at an amino acid position corresponding to S440 in human IgG1 according to EU numbering, wherein the mutation is selected form the group consisting of: S440W and S440Y.
In one embodiment of the present invention the Fc region comprises a mutation corresponding to S440Y. Thus in one embodiment of the present invention the Fc region comprises an S440Y mutation.
In one embodiment of the present invention the Fc region comprises a further hexamerization-inhibiting mutation such as K439E or S440K in human IgG1, EU numbering. The hexamerization-inhibiting mutation such as K439E or S440K prevents Fc-Fc interaction with antibodies comprising the same hexamerization inhibiting mutation, but by combining antibodies with a K439E mutation and antibodies with a S440K mutation the inhibiting effect is neutralized and Fc-Fc interactions is restored. In one embodiment of the present invention the antibody comprises a further mutation at an amino acid position corresponding to one of the following positions S440 or K439 in human IgG1, EU numbering. In one embodiment of the invention the Fc region comprises a further mutation in a position corresponding to S440 or K439, with the proviso that the further mutation is not in position S440 if the hexamerization enhancing mutation is in S440. Antibodies comprising a mutation in a position corresponding to E430, E345 or S440 according to the present invention and a further mutation at an amino acid position corresponding to K439 such as a K439E mutation do not form oligomers with antibodies comprising a further mutation at an amino acid position corresponding to K439 such as a K439E mutation. However, antibodies comprising hexamerization enhancing mutation in E430, E345 or S440 and a further mutation in K439 such a K439E do form oligomers with antibodies comprising a hexamerization enhancing mutation in E430 or E345 and a further mutation in S440 such as S440K. Antibodies comprising a mutation in a position corresponding to E430 or E345 according to the present invention and a further mutation at an amino acid position corresponding to S440 such as a S440K mutation do not form oligomers with antibodies comprising a further mutation at an amino acid position corresponding to S440 such as a S440K mutation. However, antibodies comprising hexamerization enhancing mutation in E430 or E345 and a further mutation in S440 such a S440K do form oligomers with antibodies comprising a hexamerization enhancing mutation in E430 or E345 and a further mutation in K439 such as K439. In one embodiment of the present invention the Fc region comprises a hexamerization enhancing mutation such as E430G and a hexamerization inhibiting mutation such as K439E. In one embodiment of the present invention the Fc region comprises a hexamerization enhancing mutation such as E345K and a hexamerization inhibiting mutation such as K439E. In another embodiment of the present invention the Fc region comprises a hexamerization enhancing mutation such as E430G and a hexamerization inhibiting mutation such as S440K. In one embodiment of the present invention the Fc region comprises a hexamerization enhancing mutation such as E345K and a hexamerization inhibiting mutation such as S440K. In one embodiment of the present invention the Fc region comprises a hexamerization enhancing mutation such as S440Y and a hexamerization inhibiting mutation such as K439E Hereby embodiments are provided that allow for exclusive hexamerization between combinations of antibodies comprising a K439E mutation and antibodies comprising a S440K mutation.
The human DR5 molecule (Uniprot 014763) is comprised of 440 amino acids including a signaling peptide at the first 1-55 positions, followed by the extracellular domain at positions 56-210, a transmembrane domain at positions 211-231 and a cytoplasmic domain at positions 232-440. The extracellular domain is comprised of a 155 amino acid sequence. The isoform short of DR5 (Uniprot 014763-2) is missing 185-213 from the extracellular domain.
In one embodiment of the invention the anti-DR5 antibody comprises an antigen binding region binding to an epitope within the extracellular domain of DR5.
In one embodiment of the invention the antibody comprises an antigen binding region binding to the same binding site as TRAIL or a binding site overlapping with the binding site of TRAIL. The TRAIL binding motif is located in CRD2 and CRD3 based on a Crystal structure of TRAIL in complex with the DR5 ectodomain (Hymowitz et al., Mol Cell. 1999 October; 4(4):563-71). That is, in one embodiment of the invention the antibody comprises an antigen binding region binding to the same binding region on DR5 as TRAIL. Thus in one embodiment the DR5 antibody binds to CRD2 and/or CRD3 on DR5. In one embodiment of the invention the antibody comprises an antigen binding region that blocks TRAIL binding to DR5. In one embodiment of the invention the antibody comprises an antigen binding region that competes with TRAIL binding to DR5. In one embodiment of the invention the antibody blocks TRAIL induced mediated killing such as TRAIL induced apoptosis.
In another embodiment of the invention the antibody comprises an antigen binding region binding to an epitope on DR5 that is different from the binding site of TRAIL. In one embodiment of the invention the antibody comprises an antigen binding region binding to a different binding region on DR5 than TRAIL. In one embodiment of the invention the antibody does not block TRAIL induced mediated killing such as TRAIL induced apoptosis.
In an embodiment of the invention the antibody comprises an antigen binding region that binds to an epitope on DR5 comprising or requiring one or more amino acid residues located within amino acid residues 116-138 and one or more amino acid residues located within amino acid residues 139-166 of SEQ ID NO 46. That is the antigen binding region binds to or requires for binding to DR5 one or more amino acids located within positions 116-138 and one or more amino acids located within positions 139-166. That the antigen binding region binds to one or more amino acids comprised in a sequence is to be understood as the antigen binding region is in contact with or directly interacts with one or more amino acids within the sequence. That the antigen binding region requires one or more amino acids within a sequence means that no contact or direct interaction between antigen binding region and one or more amino acids in the sequence is needed, but that one or more amino acids are required for keeping the three-dimensional structure of the epitope.
In another preferred embodiment of the present invention the antibody comprises an antigen binding region that binds to an epitope on DR5 comprising or requiring one or more amino acid residues located within amino acid residues 79-138 of SEQ ID NO 46.
In one embodiment of the invention the anti-DR5 antibody comprises an antigen binding region comprising a variable heavy chain (VH) region comprising CDR1, CDR2 and CDR3 domains and a variable light chain (VL) region comprising CDR1, CDR2 and CDR3 domains having the amino acid sequences of:
a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6;
b) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6;
c) (VH) SEQ ID NOs 10, 2, 11 and (VL) SEQ ID NOs 13, RTS, 14;
d) (VH) SEQ ID NOs 16, 17, 18 and VL) SEQ ID NOs 21, GAS, 22 or
e) the (VH) CDR1, CDR2, CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in any of a) to d) above having one to five mutations e.g. substitutions in total across said six CDR sequences.
That is in one embodiment up to five mutations such as substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations e.g. substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations e.g. substitutions are made across the CDRs of the VH region but up to five mutations e.g. substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the invention, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises an antigen binding region comprising a variable heavy chain (VH) region comprising CDR1, CDR2 and CDR3 domains and a variable light chain (VL) region comprising CDR1, CDR2 and CDR3 domains, wherein said VH region and said VL region has at least 75%, 80%, 85% 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in the six CDR sequences selected from the group consisting of:
a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6;
b) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6;
c) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14; and
d) (VH) SEQ ID NOs: 16, 17, 18 and VL) SEQ ID NOs: 21, GAS, 22.
In one embodiment of the invention the anti-DR5 antibody comprises a variable heavy chain (VH) region comprising CDR1, CDR2 and CDR3 domains and a variable light chain (VL) region comprising CDR1, CDR2 and CDR3 domains having the CDR sequences selected from one of the groups consisting of:
a) (VH) SEQ ID NOs 1, 8, 3 and (VL) SEQ ID NOs 5, FAS, 6 or
b) (VH) SEQ ID NOs 10, 2, 11 and (VL) SEQ ID NOs 13, RTS, 14 or
c) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in any one of (a) or (b) above having one to five mutations in total across said six CDR sequences. That is in one embodiment up to five mutations such as substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations e.g. substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations e.g. substitutions are made across the CDRs of the VH region but up to five mutations e.g. substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the invention the anti-DR5 antibody comprises a variable heavy chain (VH) region comprising CDR1, CDR2 and CDR3 domains and a variable light chain (VL) region comprising CDR1, CDR2 and CDR3 domains having the CDR sequences selected from one of the groups consisting of:
a) (VH) SEQ ID NOs 1, 2, 3 and (VL) SEQ ID NOs 5, FAS, 6 or
b) (VH) SEQ ID NOs 10, 2, 11 and (VL) SEQ ID NOs 13, RTS, 14 or
c) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a) or (b) above having up to five mutations in total across said six CDR sequences.
That is in one embodiment up to five mutations such as substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions, such as one, two, three, four or five mutations e.g. substitutions are made across the three CDRs of the VH region and no mutations are made across the three CDRs or the VL region. In other embodiments no mutations e.g. substitutions are made across the three CDRs of the VH region but up to five mutations e.g. substitutions are made across the six CDRs of the VL region, wherein the mutations e.g. substitutions are conservative or concern amino acids with similar physical or functional properties and preferably do not modify binding affinity to DR5.
In one embodiment of the invention, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises an antigen binding region comprising a variable heavy chain (VH) region and a variable light chain (VL) region, wherein said VH region and said VL region has at least 75%, 80%, 85% 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in the VH and VL sequences selected from the group consisting of:
a) (VH) SEQ ID NO:4 and (VL) SEQ ID NO:7;
b) (VH) SEQ ID NO:9 and (VL) SEQ ID NO:7;
c) (VH) SEQ ID NO:12 and (VL) SEQ ID NO:15;
d) (VH) SEQ ID NO:19 and (VL) SEQ ID NO:23; and
e) (VH) SEQ ID NO:20 and (VL) SEQ ID NO:23.
In one embodiment of the invention the antibody comprises an antigen binding region comprising a variable heavy chain (VH) region and a variable light chain (VL) region having the amino acid sequences of:
a) (VH) SEQ ID NO:4 and (VL) SEQ ID NO:7;
b) (VH) SEQ ID NO:9 and (VL) SEQ ID NO:7;
c) (VH) SEQ ID NO:12 and (VL) SEQ ID NO:15;
d) (VH) SEQ ID NO:19 and (VL) SEQ ID NO:23;
e) (VH) SEQ ID NO:20 and (VL) SEQ ID NO:23 or
f) the (VH) and (VL) as defined in any one of a) to e) above having one to 10 mutations or substitutions in total across said (VH) and (VL) sequences. That is in one embodiment up to 10 mutations such as substitutions in total are allowed across the VH and VL regions defined by the VH and VL sequences. In some embodiments of the invention up to ten mutations e.g. substitutions, such as one, two, three, four, five, six, seven, eight, nine or ten mutations e.g. substitutions are made across the VH or VL sequences. In one embodiment of the invention up to10 mutations or substitutions are made in the VH sequence and no mutations are made in the VL sequence. In one embodiment of the invention no mutations are made in the VH sequence and up to ten mutations e.g. substitutions are made in the VL sequence. Hereby are embodiments provided that allow for up to 10 mutations such as substitutions across the VH and VL sequences, wherein the mutations such as substitutions are conservative or concern amino acids with similar physical or functional properties, thereby allowing mutations e.g. substitutions within the VH and VL sequence without modifying binding affinity or function of the anti-DR5 antibody.
In one embodiment of the present invention the antibody is a monoclonal antibody. In one embodiment of the present invention the antibody is of the IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgD or IgM isotype.
In a preferred embodiment of the invention the antibody is an IgG1 antibody.
In one embodiment of the present invention the antibody is an IgG1m(f), IgG1m(z), IgG1m(a) or an IgG1m(x) allotype, or any allotype combination, such as IgG1m(z,a), IgG1m(z,a,x), IgG1m(f,a).
In one embodiment of the present invention the antibody comprises an Fc region comprising an amino acid sequence of the group consisting of:
a) SEQ ID NO:29;
b) SEQ ID NO:30;
c) SEQ ID NO:31;
d) SEQ ID NO:32 or
e) an amino acid sequence defined in any one of a) to d) having one to five mutations e.g. substitutions in total across said sequence. That is in one embodiment up to five mutations e.g. substitutions in total are allowed across the Fc region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations e.g. substitutions, are allowed across the Fc region.
In one embodiment of the invention, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises a heavy chain (HC) and a light chain (LC), wherein the LC comprises the sequence of SEQ ID NO:39 and wherein the HC has at least 75%, 80%, 85%, 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in the HCs sequences selected from the group consisting of:
a) (HC) SEQ ID NO:33;
b) (HC) SEQ ID NO:34;
c) (HC) SEQ ID NO:35;
d) (HC) SEQ ID NO:36;
e) (HC) SEQ ID NO:37; and
f) (HC) SEQ ID NO:38.
In one embodiment of the invention, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises a heavy chain (HC) and a light chain (LC), wherein the LC has at least 75%, 80%, 85%, 90%, at 95%, at least 97%, or at least 99% amino acid sequence identity set forth in SEQ ID NO:39 and wherein the HC has the amino acid sequence as set forth in the HCs sequences selected from the group consisting of:
a) (HC) SEQ ID NO:33;
b) (HC) SEQ ID NO:34;
c) (HC) SEQ ID NO:35;
d) (HC) SEQ ID NO:36;
e) (HC) SEQ ID NO:37; and
f) (HC) SEQ ID NO:38.
In one embodiment according to the invention, the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the LC comprises the sequence of SEQ ID NO:39 and wherein the HC comprises of one of the sequences selected from the group consisting of:
a) (HC) SEQ ID NO:33;
b) (HC) SEQ ID NO:34;
c) (HC) SEQ ID NO:35;
d) (HC) SEQ ID NO:36;
e) (HC) SEQ ID NO:37; and
f) (HC) SEQ ID NO:38; or
g) the (HC) as defined in any one of a) to f) above having one to ten mutations in total across said (HC) sequence. That is in one embodiment up to 10 mutations such as substitutions in total are allowed across the heavy chain defined by the heavy chain sequence. In some embodiments of the invention up to ten mutations e.g. substitutions, such as one, two, three, four, five, six, seven, eight, nine or ten mutations e.g. substitutions are made across the heavy chain sequence. Hereby are embodiments provided that allow for up to 10 mutations such as substitutions across the heavy chain sequence, wherein the mutations such as substitutions are conservative or concern amino acids with similar physical or functional properties, thereby allowing mutations or substitutions within the heavy chain sequence without modifying binding affinity or function of the anti-DR5 antibody.
In one embodiment of the invention, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises a heavy chain (HC) and a light chain (LC), wherein the LC comprises the sequence of SEQ ID NO:43 and wherein the HC has at least 75%, 80%, 85%, 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequence as set forth in the HCs sequences selected from the group consisting of:
a) (HC) SEQ ID NO:40;
b) (HC) SEQ ID NO:41; and
c) (HC) SEQ ID NO:42.
In one embodiment of the invention, the anti-DR5 antibody as defined in any of the embodiments disclosed herein comprises a heavy chain (HC) and a light chain (LC), wherein the LC has at least 75%, 80%, 85%, 90%, at 95%, at least 97%, or at least 99% amino acid sequence identity set forth in SEQ ID NO:43 and wherein the HC has the amino acid sequence as set forth in the HCs sequences selected from the group consisting of:
a) (HC) SEQ ID NO:40;
b) (HC) SEQ ID NO:41; and
c) (HC) SEQ ID NO:42.
In one embodiment according to the invention the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the LC comprises the sequence of SEQ ID NO:43 and wherein the HC comprises of one of the sequences selected from the group consisting of:
a) (HC) SEQ ID NO:40;
b) (HC) SEQ ID NO:41;
c) (HC) SEQ ID NO:42; or
d) the (HC) as defined in any one of a) to c) above having one to ten mutations e.g. substitutions in total across said (HC) sequence.
That is in one embodiment up to 10 mutations such as substitutions in total are allowed across the heavy chain defined by the heavy chain sequence. In some embodiments of the invention up to ten mutations e.g. substitutions, such as one, two, three, four, five, six, seven, eight, nine or ten mutations e.g. substitutions are made across the heavy chain sequence. Hereby are embodiments provided that allow for up to 10 mutations such as substitutions across the heavy chain sequence, wherein the mutations such as substitutions are conservative or concern amino acids with similar physical or functional properties, thereby allowing mutations such as substitutions within the heavy chain sequence without modifying binding affinity or function of the anti-DR5 antibody.
In one embodiment the antibody is a human antibody, a chimeric antibody or a humanized antibody.
In one embodiment of the present invention the anti-DR5 antibody is agonistic. That the antibody is agonistic is to be understood as that the antibody clusters, stimulates or activates DR5. In one embodiment, an agonistic anti-DR5 antibody of the present invention bound to DR5 activates the same intracellular pathways as TRAIL bound to DR5. The agonistic activity of one or more antibodies can be determined by incubating target cells for 3 days with an antibody concentration dilution series (e.g. from 20,000 ng/mL to 0.05 ng/mL final concentration in 5-fold dilutions). The antibodies may be added directly when cells are seeded (described in examples 8, 9, 10, 39), or alternatively the cells are first allowed to adhere to 96-well flat-bottom plates before adding the antibody samples (described in examples 11, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24, 38, 40, 41, 42, 43, 44, 46, 48). The agonistic activity i.e. the agonistic effect can be quantified by measuring the amount of viable cells using special kits for this purpose, such as the CellTiter-Glo luminescent cell viability assay of Promega (Cat nr G7571).
In one embodiment of the present invention the anti-DR5 antibody has enhanced agonistic activity. That the anti-DR5 antibody has activity is to be understood as the antibody is able to cluster DR5 or activate at least the same intracellular pathways as TRAIL bound to DR5. That is anti-DR5 antibody with enhanced agonistic activity is able to induce increased level of apoptosis or programmed cell death in a cell or tissue expressing DR5 compared to TRAIL or a wild-type IgG1 antibody against DR5.
In one embodiment of the present invention the anti-DR5 antibody induces programmed cell death in a target cell. In one embodiment of the present invention the anti-DR5 antibody induces caspase-dependent cell death. Caspase-dependent cell death may be induced by activation of caspase-3 and/or caspase-7. In one embodiment of the invention the anti-DR5 antibody induces caspase-3 and/or caspase-7 dependent cell death. In one embodiment of the present invention the antibody induces apoptosis. Apoptosis by one or more agonistic anti-DR5 antibodies can be determined using methods such as, e.g., caspase-3/7 activation assays described in examples 19, 20, 25 and 45 or phosphatidylserine exposure described in examples 19 and 25. Anti-DR5 antibody at a fixed concentration of e.g. 1 μg/mL may be added to adhered cells and incubated for 1 to 24 hours. Caspase-3/7 activation can be determined by using special kits for this purpose, such as the PE Active Caspase-3 Apoptosis Kit of BD Pharmingen (Cat nr 550914) (example 19 and 25) or the Caspase-Glo 3/7 assay of Promega (Cat nr G8091) (examples 20 and 45). Phosphatidylserine exposure and cell death can be determined by using special kits for this purpose, such as the FITC Annexin V Apoptosis Detection Kit I from BD Pharmingen (Cat nr 556547) (examples 19 and 25).
In one embodiment of the present invention the anti-DR5 antibody induces phosphatidylserine (PS) exposure, which can be measured by Annexin-V binding. In one embodiment of the present invention anti-DR5 induces translocation of PS to the cell surface of the target cell. Therefore, Annexin-V binding correlates to programmed cell death and can be used to measure the anti-DR5 antibody's ability to induce cellular events leading to programmed cell death.
In a preferred embodiment of the present invention the anti-DR5 antibody induces apoptosis in a target cell expressing DR5, such as a tumor cell.
In one embodiment of the invention the anti-DR5 antibody reduces cell viability.
In one embodiment of the present invention the anti-DR5 antibody induces DR5 clustering. That the antibody can induce clustering and even enhance clustering leads to activation of at least the same intracellular signaling pathways as TRAIL bound to DR5.
In one embodiment the antibodies or compositions of the present invention induce, trigger, increase or enhance apoptosis or cell death in cancer cells or cancer tissues expressing DR5. The increased or enhanced apoptosis or cell death can be measured by an increase or enhanced level of phosphatidylserine exposure on cells exposed to or treated with one or more anti-DR5 antibodies of the invention. Alternatively, the increase or enhanced apoptosis or cell death can be measured by measuring activation of caspase 3 or caspase 7 in cells that have been exposed to or treated with one or more anti-DR5 antibodies of the invention. Alternatively, the increase or enhanced apoptosis or cell death can be measured by a loss of viability in cell cultures that have been exposed to or treated with one or more anti-DR5 antibodies of the invention, compared to untreated cell cultures. Induction of caspase-mediated apoptosis can be assessed by demonstrating inhibition of the loss of viability after exposure to DR5 antibody by a caspase-inhibitor, for example ZVAD.
In one embodiment of the present invention the anti-DR5 antibody engages into oligomerization such as hexamerization of antibodies on target cells expressing DR5. Oligomerization such as hexamerization is mediated through Fc-Fc interactions. One method for determining this is by inhibiting Fc-Fc interactions which indicate that antibodies oligomerizies e.g. hexamerizies. The Fc-Fc interactions can be inhibited by a peptide binding to the hydrophobic patch involved in Fc-Fc interactions such as DCAWHLGELVWCT as described in example 15.
Bispecific Antibodies
In another aspect, the present invention comprises a bispecific antibody comprising at least one antigen binding region as described herein.
In another aspect, the present invention comprises a bispecific antibody comprising one or more antigen binding regions as described herein.
In one embodiment of the invention the bispecific antibody comprises a first antigen binding region and a second antigen binding region as defined herein.
In one embodiment of the invention the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region and said second antigen binding region bind different epitopes on human DR5.
In one embodiment of the invention the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region binding to human DR5 does not block binding of said second antigen binding region binding to human DR5.
In one embodiment of the present invention the bispecific anti-DR5 antibody comprises a first and a second Fc region, wherein the first and/or second Fc region comprises a mutation of an amino acid position corresponding to E430, E345 or S440 in human IgG1, EU numbering according to the invention. In one embodiment of the present invention the bispecific anti-DR5 antibody comprises a first and a second Fc region, wherein the first and second Fc region comprises a mutation of an amino acid position corresponding to E430, E345 or S440 in human IgG1, EU numbering. In one embodiment of the present invention the bispecific anti-DR5 antibody comprises a first and a second Fc region, wherein the first Fc region comprises a mutation of an amino acid position corresponding to E430, E345 or S440 in human IgG1, EU numbering. In one embodiment of the present invention the bispecific anti-DR5 antibody comprises a first and a second Fc region, wherein the second Fc region comprises a mutation of an amino acid position corresponding to E430, E345 or
S440 in human IgG1, EU numbering.
In one embodiment of the invention the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second antigen binding region comprises the following six CDR sequences
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein the said first antigen binding region and said second antigen binding region comprises, c) the six CDR sequences defined in (a) or (b) above having one to five mutations or substitutions in total across said six CDR sequences respectively.
That is the one or more mutations or substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations or substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations or substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations or substitutions are made across the CDRs of the VH region but up to five mutations or substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the invention the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region comprises the following six CDR sequences,
a) said first antigen binding region comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second antigen binding region comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein said first antigen binding region and said second antigen binding region comprises, b) the six CDR sequences defined in (a) comprising one to five mutations e.g substitutions in total across said six CDR sequences of each first and second antigen binding region respectively. That is the one or more mutations e.g. substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations e.g. substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations e.g. substitutions are made across the CDRs of the VH region but up to five mutations e.g. substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the invention the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second antigen binding region comprises the following six CDR sequences
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NO:s 13, RTS, 14, or wherein the said first antigen binding region and said second antigen binding region comprises, c) the six CDR sequences defined in (a) or (b) above having one to five mutations or substitutions in total across said six CDR sequences respectively. That is the one or more mutations or substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations or substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations or substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations or substitutions are made across the CDRs of the VH region but up to five mutations or substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the invention the bispecific antibody comprises a first and a second antigen binding region wherein a) said first antigen binding region comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second antigen binding region comprises the following six CDR sequences(VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NO:s 13, RTS, 14, or wherein the said first antigen binding region and said second antigen binding region comprises b) the six CDR sequences defined in (a having one to five mutations or substitutions in total across said six CDR sequences of each antigen binding region respectively. That is the one or more mutations e.g. substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations e.g. substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations e.g. substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations or substitutions are made across the CDRs of the VH region but up to five mutations e.g. substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the invention the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 16, 17, 18 and (VL) SEQ ID NOs: 21, GAS, 6 and said second antigen binding region comprises the following six CDR sequences
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein the said first antigen binding region and said second antigen binding region comprises,
c) the six CDR sequences defined in a) or (b) above having one to five mutations or substitutions in total across said six CDR sequences. That is the one or more mutations or substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations or substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations or substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations or substitutions are made across the CDRs of the VH region but up to five mutations or substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the invention the bispecific antibody comprises a first and a second antigen binding region, wherein,
a) said first antigen binding region comprises the following six CDR sequences (VH) SEQ ID NOs: 16, 17, 18 and (VL) SEQ ID NOs: 21, GAS, 6 and said second antigen binding region comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or
b) said first antigen binding region and said second antigen binding region comprises the six CDR sequences defined in a) comprising one to five mutations e.g. substitutions in total across said six CDR sequences of each antigen binding region. That is the one or more mutations e.g. substitutions across the six CDR sequences of each antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations e.g. substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations e.g. substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations e.g. substitutions are made across the CDRs of the VH region but up to five mutations e.g. substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the invention the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region comprises the following sequences (a) (VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 8, CDR3 SEQ ID NO 3 and (VL) CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6, or b) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a) above having one to five mutations in total across said six CDR sequences and wherein said second antigen binding region comprises the following sequences (c) (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or (d) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (c) above having one to five mutations in total across said six CDR sequences.
In one embodiment of the invention the bispecific antibody comprises a first and a second antigen binding region, wherein (a) said first antigen binding region comprises the following sequences (VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 8, CDR3 SEQ ID NO 3 and (VL) CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6 and said second antigen binding region comprises the following sequences (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or b) said first antigen binding region or said second antigen binding region comprises one to five mutations in total across said six CDR sequences of each antigen binding region.
In one embodiment of the invention the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region comprises the following sequences (a) (VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 3 and (VL) CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6, or (b) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a) above having one to five mutations in total across said six CDR sequences and wherein said second antigen binding region comprises the following sequences (c) (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or (d) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (c) above having one to five mutations in total across said six CDR sequences.
In one embodiment of the invention the bispecific antibody comprises a first and a second antigen binding region, wherein (a) said first antigen binding region comprises the following sequences (VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 3 and (VL) CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6 and said second antigen binding region comprises the following sequences (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or b) said first antigen binding region or said second antigen binding region comprises one to five mutations in total across said six CDR sequences of each antigen binding region.
In one embodiment of the invention the bispecific antibody comprises a first and a second antigen binding region, wherein said first antigen binding region comprises the following sequences (a) (VH) CDR1 SEQ ID NO 16, CDR2 SEQ ID NO 17, CDR3 SEQ ID NO 18 and (VL) CDR1 SEQ ID NO 21, CDR2 GAS, CDR3 SEQ ID NO 22,or (b) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a) above having one to five mutations in total across said six CDR sequences and wherein said second antigen binding region comprises the following sequences (c) (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or (d) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (c) above having one to five mutations in total across said six CDR sequences.
In one embodiment of the invention the bispecific antibody comprises a first and a second antigen binding region, wherein (a) said first antigen binding region comprises the following sequences (VH) CDR1 SEQ ID NO 16, CDR2 SEQ ID NO 17, CDR3 SEQ ID NO 18 and (VL) CDR1 SEQ ID NO 21, CDR2 GAS, CDR3 SEQ ID NO 22 and said second antigen binding region comprises the following sequences (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or b) said first antigen binding region or said second antigen binding region comprises one to five mutations in total across said six CDR sequences of each antigen binding region.
If the antibody is a bispecific antibody that comprises an Fc region comprising a first and a second heavy chain, a mutation according to the present invention i.e. a mutation in a position corresponding to E430, E345 or S440 in IgG1, EU numbering, may in principle only be present in one of the heavy chains; i.e. in either the first or second heavy chain, although in a preferred embodiment according to the present invention, the mutation is present in both the first and second heavy chain of the bispecific antibody.
In a particular embodiment the antibody may be bispecific antibody such as the heterodimeric protein described in WO 11/131746, which is hereby incorporated herein by reference.
In one embodiment, the antibody is a bispecific antibody which comprises a first heavy chain comprising a first Fc region of an immunoglobulin and a first antigen-binding region, and a second heavy chain comprising a second Fc region of an immunoglobulin and a second antigen-binding region, wherein the first and second antigen-binding regions bind different epitopes on the same antigen or on different antigens. In a further embodiment said first heavy chain comprising a first Fc region comprises a further amino acid substitution at a position selected from those corresponding to K409, T366, L368, K370, D399, F405, and Y407 in the Fc region of a human IgG1 heavy chain; and wherein said second heavy chain comprising a second Fc region comprises a further amino acid substitution at a position selected from those corresponding to F405, T366, L368, K370, D399, Y407, and K409 in the Fc region of a human IgG1 heavy chain, and wherein said further amino acid substitution in the first heavy chain comprising a first Fcregion is different from the said further amino acid substitution in the second heavy chain comprising a second Fc region.
In a further embodiment said first heavy chain comprising a first Fc region comprises an amino acid substitution at a position corresponding to K409 in the Fc-region of a human IgG1 heavy chain; and said second heavy chain comprising a second Fc region comprises an amino acid substitution at a position corresponding to F405 in the Fc-region of a human IgG1 heavy chain.
In one embodiment of the invention the bispecific antibody comprises introducing a first and second Fc region comprising a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.
In a further embodiment the mutation in the first and second Fc region in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W, may be in the same amino acid residue position or a different position. In a further embodiment it may be the same or a different mutation in the same amino acid residue position in the first and second Fc region.
In another embodiment the bispecific antibody comprises a first or second CH2-CH3 region comprising a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.
In one embodiment of the invention the bispecific antibody comprises a first and a second heavy chain, wherein said first heavy chain comprises a mutation corresponding to F405L in human IgG1 according to EU numbering and said second heavy chain comprises a mutation corresponding to K409R in human IgG1 according EU numbering.
In one embodiment of the invention the bispecific antibody is comprised in a pharmaceutical composition.
Anti-DR5 Antibody Compositions
The anti-DR5 antibodies such as monoclonal antibodies or bispecific antibodies according to any aspect or embodiment of the present invention may be comprised in a composition, such as a pharmaceutical composition, diagnostic composition or any other composition.
In one aspect the invention relates to a composition comprising at least one anti-DR5 antibody according to any one of the embodiments described herein. In one aspect the invention relates to a composition comprising one or more anti-DR5 antibodies according to any one the embodiments described herein. The composition may comprise one, two or more anti-DR5 antibodies according to the invention as described herein that are not identical, such as a combination of two different monoclonal anti-DR5 antibodies.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody as described herein. That is in one embodiment of the present invention the composition comprises a first antibody as described herein and a second antibody as described herein, wherein the first and the second antibody are not identical.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody comprising a mutation in the position corresponding to E430 in human IgG1, EU numbering and a second anti-DR5 antibody comprising a mutation in the position corresponding to E430 in human IgG1, EU numbering, wherein the first and second antibody binds different epitopes on DR5.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody comprising a mutation in the position corresponding to E430 in human IgG1, EU numbering and a second anti-DR5 antibody comprising a mutation in the position corresponding to E430 in human IgG1, EU numbering, wherein the first antibody does not block binding of the second antibody to DR5.
In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody comprising a mutation in a position corresponding to E430 in human IgG1, EU numbering, such a mutation may be selected from the group consisting of: E430G, E4305 and E430T.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody comprising an E430G and a second anti-DR5 antibody comprising an E430G mutation, wherein the first and second antibody binds different epitopes on DR5.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein the first anti-DR5 antibody comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences,
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises a mutation in the position corresponding to E430.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises a mutation in the position corresponding to E430 in human IgG1, EU numbering.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein the first anti-DR5 antibody comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences,
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises an E430G mutation in the Fc region.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said first anti-DR5 antibody and said second anti-DR5 antibody comprises an E430G mutation in the Fc region.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein the first anti-DR5 antibody comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences,
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises a mutation in the position corresponding to E430.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said first anti-DR5 antibody and said second anti-DR5 antibody comprises a mutation in the position corresponding to E430 in human IgG1, EU numbering.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein the first anti-DR5 antibody comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences,
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises an E430G mutation in the Fc region.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said first anti-DR5 antibody and said second anti-DR5 antibody comprises an E430G mutation in the Fc region.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody comprising a mutation in the position corresponding to E345 in human IgG1, EU numbering and a second anti-DR5 antibody comprising a mutation in the position corresponding to E345 in human IgG1, EU numbering, wherein the first and second antibody binds different epitopes on DR5.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody comprising a mutation in the position corresponding to E345 in human IgG1, EU numbering and a second anti-DR5 antibody comprising a mutation in the position corresponding to E345 in human IgG1, EU numbering, wherein the first antibody does not block binding of the second antibody to DR5.
In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody comprising a mutation in a position corresponding to E345, such a mutation may be selected from the group consisting of: E345K, E345Q, E345R and E345Y.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody comprising an E345K and a second anti-DR5 antibody comprising an E345K mutation, wherein the first and second antibody binds different epitopes on DR5.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and second anti-DR5 antibody, wherein the first anti-DR5 antibody comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences,
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises a mutation in the position corresponding to E345.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises a mutation in the position corresponding to E345 in human IgG1, EU numbering.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein the first anti-DR5 antibody comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences,
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises an E345K mutation in the Fc region.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises an E345K mutation in the Fc region.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein the first anti-DR5 antibody comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences,
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises a mutation in the position corresponding to E345.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises a mutation in the position corresponding to E345 in human IgG1, EU numbering.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein the first anti-DR5 antibody comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences,
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises an E345K mutation in the Fc region.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises an E345K mutation in the Fc region.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody comprising a mutation in the position corresponding to S440 in human IgG1, EU numbering and a second anti-DR5 antibody comprising a mutation in the position corresponding to S440 in human IgG1, EU numbering, wherein the first and second antibody binds different epitopes on DR5.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody comprising a mutation in the position corresponding to S440 in human IgG1, EU numbering and a second anti-DR5 antibody comprising a mutation in the position corresponding to S440 in human IgG1, EU numbering, wherein the first antibody does not block binding of the second antibody to DR5.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody comprising an S440Y and a second anti-DR5 antibody comprising an S440Y mutation, wherein the first and second antibody binds different epitopes on DR5. In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody comprising a mutation in a position corresponding to S440 in human IgG1, EU numbering, such a mutation may be selected from the group consisting of: S440W and S440Y.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein the first anti-DR5 antibody comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences,
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises a mutation in the position corresponding to S440.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises a mutation in the position corresponding to S440 in human IgG1, EU numbering.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and second anti-DR5 antibody, wherein the first anti-DR5 antibody comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences,
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises an S440Y mutation in the Fc region.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises an S440Y mutation in the Fc region.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein the first anti-DR5 antibody comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences,
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises a mutation in the position corresponding to S440.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises a mutation in the position corresponding to S440 in human IgG1, EU numbering.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein the first anti-DR5 antibody comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences,
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises an S440Y mutation in the Fc region.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody, wherein said first anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second anti-DR5 antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, and wherein the said first anti-DR5 antibody and said second anti-DR5 antibody comprises an S440Y mutation in the Fc region.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody wherein the first and the second antibody comprises a further hexamerization-inhibiting mutation corresponding to K439E or S440K in human IgG1 EU numbering. In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody, wherein the first and second anti-DR5 antibody comprises a hexamerization enhancing mutation in an amino acid position corresponding to E430, E345 or S440 in human IgG1, EU numbering and wherein the first antibody comprises a further mutation in an amino acid position corresponding to K439 or and wherein the second antibody comprises a further mutation in an amino acid position corresponding to S440, with the proviso that the hexamerization enhancing mutation is not in S440 when the further mutation is in S440. That is in one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody, wherein the first anti-DR5 antibody comprises a hexamerization enhancing mutation such as E430G and K439E, and wherein the second anti-DR5 antibody comprises a hexamerization enhancing mutation such as E430G and S440K. That is in one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody, wherein the first anti-DR5 antibody comprises a hexamerization enhancing mutation such as E345K and K439E, and wherein the second anti-DR5 antibody comprises a hexamerization enhancing mutation such as E345K and S440K. Hereby are embodiments provided that allow compositions wherein hexamerization exclusively occur between combinations of antibodies comprising a K439E mutation and antibodies comprising a S440K mutation.
In one embodiment of the present invention the composition comprises a first anti-DR5 antibody and a second anti-DR5 antibody binding different epitopes on human DR5. In one embodiment of the present invention the composition comprises a first anti-DR5 antibody comprising an antigen binding region that binds to an epitope on DR5 comprising or requiring one or more amino acid residues located within amino acid residues 116-138 and one or more amino acid residues located within amino acid residues 139-166 of SEQ ID NO 46 and a second anti-DR5 antibody comprising an antigen binding region that binds to an epitope on DR5 comprising or requiring one or more amino acid residues located within amino acid residues 79-138 of SEQ ID NO 46.
In one embodiment of the present invention the composition comprises said first anti-DR5 antibody binding to DR5, which does not block binding of said second anti-DR5 antibody to DR5. That is in one embodiment of the invention the composition comprises a first anti-DR5 antibody binding to DR5 and a second anti-DR5 antibody binding to DR5, wherein the first and the second anti-DR5 antibody does not compete for binding to DR5. Thus it is to be understood in the context of the present invention that a first anti-DR5 antibody that does not block binding of a second anti-DR5 antibody may be the same as a first anti-DR5 antibody that does not compete with a second anti-DR5 antibody.
In one embodiment of the invention, the composition comprises a first and a second anti-DR5 antibody, wherein said first antibody comprises a VH region and a VL region comprising six CDR sequences, wherein the six CDR sequences in total have at least 75%, 80%, 85%, 90%, 95%, 97%, or at least 99% amino acid sequence identity to the CDR sequences as set forth in the following: a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6; and said second antibody comprises a VH region and a VL region comprising six CDR sequences, wherein the six CDR sequences in total have at least 75%, 80%, 85%, 90%, 95%, 97%, or at least 99% amino acid sequence identity to the CDR sequences as set forth in the following; b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14.
In one embodiment thereof the sequence identity of the six CDR sequences in total of said first antibody and said second antibody is at least 85%, 90%, 95%, 97%, or 99%.
In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody, wherein said first antibody comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second antibody comprises the following six CDR sequences,
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein the said first antibody and said second antibody comprises,
c) the six CDR sequences defined in (a) or (b) above having one to five mutations or substitutions in total across said six CDR sequences respectively. That is the one or more mutations or substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations or substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations or substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations or substitutions are made across the CDRs of the VH region but up to five mutations or substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody, wherein
a) said first antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 2, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein b) the said first antibody and said second antibody comprises the six CDR sequences of each antibody defined in (a) or comprising one to five mutations e.g. substitutions in total across said six CDR sequences respectively. That is the one or more mutations e.g. substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations e.g. substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations e.g. substitutions are made across the CDRs of the VH region but up to five mutations e.g. substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the invention, the composition comprises a first and a second anti-DR5 antibody, wherein said first antibody comprises a VH region and a VL region comprising six CDR sequences, wherein the six CDR sequences in total have at least 75%, 80%, 85%, 90%, 95%, 97%, or at least 99% amino acid sequence identity to the CDR sequences as set forth in the following: a) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS; and said second antibody comprises a VH region and a VL region comprising six CDR sequences, wherein the six CDR sequences in total have at least 75%, 80%, 85%, 90%, 95%, 97%, or at least 99% amino acid sequence identity to the CDR sequences as set forth in the following; b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14. In one embodiment thereof the sequence identity of the six CDR sequences in total of said first antibody and said second antibody is at least 85%, 90%, 95%, 97%, or 99%.
In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody, wherein said first antibody comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second antibody comprises the following six CDR sequences
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein the said first antibody and said second antibody comprises,
c) the six CDR sequences defined in (a) or (b) above having one to five mutations or substitutions in total across said six CDR sequences respectively.
That is the one or more mutations or substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations or substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations or substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations or substitutions are made across the CDRs of the VH region but up to five mutations or substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody, wherein
a) said first antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 1, 8, 3 and (VL) SEQ ID NOs: 5, FAS, 6 and said second antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein b) the said first antibody and said second antibody comprises the six CDR sequences of each antibody defined in (a) or comprising one to five mutations e.g. substitutions in total across said six CDR sequences respectively. That is the one or more mutations e.g. substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations e.g. substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations e.g. substitutions are made across the CDRs of the VH region but up to five mutations e.g. substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the invention, the composition comprises a first and a second anti-DR5 antibody, wherein said first antibody comprises a VH region and a VL region comprising six CDR sequences, wherein the six CDR sequences in total have at least 75%, 80%, 85%, 90%, 95%, 97%, or at least 99% amino acid sequence identity to the CDR sequences as set forth in the following: a) (VH) SEQ ID NOs: 16, 17, 18 and (VL) SEQ ID NOs: 21, GAS, 6; and said second antibody comprises a VH region and a VL region comprising six CDR sequences, wherein the six CDR sequences in total have at least 75%, 80%, 85%, 90%, 95%, 97%, or at least 99% amino acid sequence identity to the CDR sequences as set forth in the following; b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14. In one embodiment thereof the sequence identity of the six CDR sequences in total of said first antibody and said second antibody is at least 85%, 90%, 95%, 97%, or 99%.
In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody, wherein said first antibody comprises the following six CDR sequences,
a) (VH) SEQ ID NOs: 16, 17, 18 and (VL) SEQ ID NOs: 21, GAS, 6 and said second antibody comprises the following six CDR sequences
b) (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein the said first antibody and said second antibody comprises,
c) the six CDR sequences defined in (a) or (b) above having one to five mutations or substitutions in total across said six CDR sequences. That is the one or more mutations or substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations or substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations or substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations or substitutions are made across the CDRs of the VH region but up to five mutations or substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody, wherein
a) said first antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 16, 17, 18 and (VL) SEQ ID NOs: 21, GAS, 6 and said second antibody comprises the following six CDR sequences (VH) SEQ ID NOs: 10, 2, 11 and (VL) SEQ ID NOs: 13, RTS, 14, or wherein b) the said first antibody and said second antibody comprises the six CDR sequences of each antibody defined in (a) or comprising one to five mutations e.g. substitutions in total across said six CDR sequences respectively. That is the one or more mutations e.g. substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5. That is in one embodiment up to five mutations e.g. substitutions in total are allowed across the six CDRs comprising the antigen binding region. In some embodiments of the invention up to five mutations e.g. substitutions such as one, two, three, four or five mutations or substitutions, are made across the three CDRs of the VH region and no mutations are made across the CDRs of the VL region. In other embodiments no mutations e.g. substitutions are made across the CDRs of the VH region but up to five mutations e.g. substitutions, such as one, two, three, four or five are found across the CDRs of the VL region.
In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody as defined in any of the above embodiments wherein said first and second antibody further comprises a mutation in the Fc region corresponding to position K439 or S440 in human IgG1, EU numbering. In one embodiment of the invention the composition comprises a first antibody comprising a mutation corresponding to K439 such as K439E and a second antibody comprising a mutation corresponding to S440 such as S440K. In one embodiment of the invention the composition comprises a first antibody comprising a mutation corresponding to S440 such as S440K and a second antibody comprising a mutation corresponding to K439 such as K439E. Hereby embodiment are provided wherein the composition comprises a first antibody comprising at least two mutations such as E430G and K439E and a second antibody comprising at least two mutations such as E430G and S440K. In another embodiment of the present invention the composition comprises a first antibody comprising at least two mutations such as E345K and K439E and a second antibody comprising at least two mutation such as E345K and S440K. Hereby are embodiments provided that allow for hexamerization of antibodies with different specificities.
In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody, wherein said first antibody comprises the following sequences (a) (VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 8, CDR3 SEQ ID NO 3 and (VL) CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6 and said second antibody comprises the following sequences (b) (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or (c) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a) or (b) above having one to five mutations or substitutions in total across said six CDR sequences. That is the one or more mutations or substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5.
In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody, wherein said first and second antibody comprises the following CDR sequences (a) said first antibody comprises the following CDR sequences (VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 8, CDR3 SEQ ID NO 3 and (VL) CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6 and said second antibody comprises the following CDR sequences (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or (b) the CDR sequences described in (a) for each antibody comprising one to five mutations e.g. substitutions in total across said CDR sequences for each antibody. That is the one or more mutations e.g. substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5.
In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody, wherein said first antibody comprises the following sequences (a) (VH) CDR1 SEQ ID NOs 1, CDR2 2, CDR3 3 and (VL) CDR1 SEQ ID NOs 5, CDR2 FAS, CDR3 6 and said second antibody comprises the following sequences (b) (VH) CDR1 SEQ ID NOs 10, CDR2 2, CDR3 11 and (VL) SEQ ID NOs CDR1 13, CDR2 RTS, CDR3 14 or (c) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a) or (b) above having one to five mutations or substitutions in total across said six CDR sequences. That is the one or more mutations or substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5.
In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody, wherein said first and second antibody comprises the following CDR sequences (a) said first antibody comprises the following CDR sequences (VH) CDR1 SEQ ID NO 1, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 3 and (VL) CDR1 SEQ ID NO 5, CDR2 FAS, CDR3 SEQ ID NO 6 and said second antibody comprises the following CDR sequences (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or (b) the CDR sequences described in (a) for each antibody comprising one to five mutations e.g. substitutions in total across said CDR sequences for each antibody. That is the one or more mutations e.g. substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5.
In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody, wherein said first antibody comprises the following sequences (a) (VH) CDR1 SEQ ID NO 16, CDR2 SEQ ID NO 17, CDR3 SEQ ID NO 18 and (VL) CDR1 SEQ ID NO 21, CDR2 GAS, CDR3 SEQ ID NO 22 and said second antibody comprises the following sequences (b) (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or (c) the (VH) CDR1, CDR2 and CDR3 and (VL) CDR1, CDR2 and CDR3 as defined in (a) or (b) above having one to five mutations or substitutions in total across said six CDR sequences. That is the one or more mutations or substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5.
In one embodiment of the present invention the composition comprises a first and a second anti-DR5 antibody, wherein said first and second antibody comprises the following CDR sequences (a) said first antibody comprises the following CDR sequences (VH) CDR1 SEQ ID NO 16, CDR2 SEQ ID NO 17, CDR3 SEQ ID NO 18 and (VL) CDR1 SEQ ID NO 21, CDR2 GAS, CDR3 SEQ ID NO 22 and said second antibody comprises the following CDR sequences (VH) CDR1 SEQ ID NO 10, CDR2 SEQ ID NO 2, CDR3 SEQ ID NO 11 and (VL) CDR1 SEQ ID NO 13, CDR2 RTS, CDR3 SEQ ID NO 14 or (b) the CDR sequences described in (a) for each antibody comprising one to five mutations e.g. substitutions in total across said CDR sequences for each antibody. That is the one or more mutations e.g. substitutions across the six CDR sequences of the antigen binding region do not change the binding characteristics of said first or second antibody such as the agonistic properties, the DR5 epitope binding and/or the ability to induce apoptosis in a target cell expressing DR5.
In one embodiment of the invention the composition comprises a first and a second anti-DR5 antibody, wherein said first antibody and said second antibody are present in the composition at a 1:49 to 49:1 molar ratio, such as 1:1 molar ratio, a 1:2 molar ratio, a 1:3 molar ratio, a 1:4 molar ratio, a 1:5 molar ratio, a 1:6 molar ratio, a 1:7 molar ratio, a 1:8 molar ratio, a 1:9 molar ratio, a 1:10 molar ratio, a 1:15 molar ratio, a 1:20 molar ratio, a 1:25 molar ratio, a 1:30 molar ratio, a 1:35 molar ratio, a 1:40 molar ratio, a 1:45 molar ratio a 1:50 molar ratio, a 50:1 molar ratio, a 45:1 molar ratio, a 40:1 molar ratio, a 35:1 molar ratio, a 30:1 molar ratio a 25:1 molar ratio, a 20:1 molar ratio, a 15:1 molar ratio, a 10:1 molar ratio, a 9:1 molar ratio, a 8:1 molar ratio, a 7:1 molar ratio, a 6:1 molar ratio, a 5:1 molar ratio, a 4:1 molar ratio, a 3:1 molar ratio, a 2:1 molar ratio.
In one embodiment of the invention the composition comprises a first and a second anti-DR5 antibody, wherein said first antibody and said second antibody are present in the composition at a 1:9 to 9:1 molar ratio.
In one embodiment of the invention the composition comprises a first and a second anti-DR5 antibody, wherein said first antibody and said second antibody are present in the composition at approximately a 1:1 molar ratio.
In one embodiment of the invention the composition comprises a first and a second anti-DR5 antibody, wherein said first antibody and said second antibody are present in the composition at a 1:1 molar ratio.
In a preferred embodiment of the invention the composition comprises a first and a second anti-DR5 antibody, wherein said first antibody and second antibody and/or any additional antibodies are present in the composition at an equimolar ratio.
In one embodiment of the invention the composition is a pharmaceutical composition.
Pharmaceutical compositions of the present invention may comprise antibodies such as monoclonal antibodies or bispecific antibodies according to any aspect or embodiment of the present invention.
The pharmaceutical compositions may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in (Rowe et al., Handbook of Pharmaceutical Excipients, 2012 June, ISBN 9780857110275)
The pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients should be suitable for the antibody or bispecific antibody of the present invention and the chosen mode of administration. Suitability for carriers and other components of pharmaceutical compositions is determined based on the lack of significant negative impact on the desired biological properties of the chosen compound or pharmaceutical composition of the present invention (e.g., less than a substantial impact (10% or less relative inhibition, 5% or less relative inhibition, etc.) upon antigen binding).
A pharmaceutical composition of the present invention may also include diluents, fillers, salts, buffers, detergents (e. g., a nonionic detergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and/or other materials suitable for inclusion in a pharmaceutical composition.
The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
The pharmaceutical composition may be administered by any suitable route and mode. Suitable routes of administering a compound of the present invention in vivo and in vitro are well known in the art and may be selected by those of ordinary skill in the art.
In one embodiment, the pharmaceutical composition of the present invention is administered parenterally.
The terms “parenteral administration” and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-orbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion. In one embodiment, the pharmaceutical composition of the present invention is administered by intravenous or subcutaneous injection or infusion.
In one embodiment of the present invention the pharmaceutical composition comprises one or more antibodies according to the invention such as monoclonal antibodies or bispecific antibodies together with a pharmaceutical carrier.
Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption-delaying agents, and the like that are physiologically compatible with a compound of the present invention.
Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, saline, phosphate-buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethyl cellulose colloidal solutions, tragacanth gum and injectable organic esters, such as ethyl oleate, and/or various buffers. Other carriers are well known in the pharmaceutical arts.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the present invention is contemplated.
Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
Pharmaceutical compositions of the present invention may also comprise pharmaceutically acceptable antioxidants for instance (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Pharmaceutical compositions of the present invention may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the compositions.
The pharmaceutical compositions of the present invention may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the pharmaceutical composition. The compounds of the present invention may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and micro-encapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, poly-ortho-esters, and polylactic acid alone or with a wax, or other materials well known in the art. Methods for the preparation of such formulations are generally known to those skilled in the art. In one embodiment, the compounds of the present invention may be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the present invention is contemplated. Other active or therapeutic compounds may also be incorporated into the compositions.
Pharmaceutical compositions for injection or infusion must typically be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, micro-emulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier may be an aqueous or a non-aqueous solvent or dispersion medium containing for instance water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients e.g. as enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients e.g. from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum-drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum-drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The pharmaceutical composition of the present invention may contain one or more monoclonal antibodies or one or more bispecific antibodies of the present invention, a combination of an antibody or a bispecific antibody according to the invention with another therapeutic compound, or a combination of compounds of the present invention.
Therapeutic Applications
The antibodies such as monoclonal antibodies, bispecific antibodies or compositions according to any aspect or embodiment of the present invention may be used as a medicament, i.e. for therapeutic applications.
In one embodiment of the present invention the composition comprises one or more antibodies according to the invention such as monoclonal antibodies or bispecific antibodies for use as a medicament.
In another aspect, the present invention provides methods for treating or preventing a disorder involving cells expressing DR5 in a subject, which method comprises administration of a therapeutically effective amount of an anti-DR5 antibody, bispecific antibody or a composition comprising one or more antibodies of the present invention to a subject in need thereof. The method typically involves administering to a subject in need thereof an anti-DR5 antibody, a bispecific antibody or composition according to the present invention in an amount effective to treat or prevent the disorder.
The anti-DR5 antibodies of the present invention can be used in the treatment or prevention of disorders involving cells expressing DR5. For example, the antibodies may be administered to human subjects, e.g., in vivo, to treat or prevent disorders involving DR5-expressing cells. As used herein, the term “subject” is typically a human to whom the anti-DR5 antibody or bispecific antibody is administered. Subjects may for instance include human patients having disorders that may be corrected or ameliorated by modulating DR5 function or by killing of the DR5-expressing cell, directly or indirectly.
In one aspect, the present invention relates to an anti-DR5 antibody, bispecific antibody or composition as defined in any aspect or embodiment herein, for use in treatment or to ameliorate symptoms of a disease or disorder involving cells expressing DR5. In one embodiment of the present invention the composition comprising an anti-DR5 antibody or bispecific antibody according to any aspect or embodiment as disclosed herein, for use in treatment of infectious disease, autoimmune disease or cardiovascular anomalies.
In one aspect, the present invention relates to an anti-DR5 antibody, bispecific antibody or composition as defined in any aspect or embodiment herein, for use in treatment or to ameliorate symptoms of cancer and/or tumors.
In one embodiment of the present invention the composition comprising an anti-DR5 antibody or bispecific antibody according to any aspect or embodiment of the invention is for use in treatment of cancer and/or tumors.
The term “cancer” refers to or describes the physiological condition in mammals such as humans that is typically characterized by unregulated growth. Most cancers belong to one of two larger groups of cancers i.e., solid tumors and hematological tumors.
In a particular aspect, an anti-DR5 antibody, bispecific antibody or composition is administered prophylactically in order to reduce the risk of developing cancer, delay the onset of an event in cancer progression or reduce the risk of recurrence when a cancer is in remission and/or a primary tumor has been surgically removed. In the latter case, the anti-DR5 antibody, bispecific antibody or composition could, for example, be administered in association with (i.e., before, during, or after) the surgery. Prophylactic administration may also be useful in patients where it is difficult to locate a tumor that is believed to be present due to other biological factors.
In one embodiment the composition comprising one or more anti-DR5 antibodies or bispecific antibodies of the present invention is for use in treatment of solid tumors and/or hematological tumors
In one embodiment the composition comprising one or more anti-DR5 antibodies or bispecific antibodies of the present invention is for use in treatment of solid tumors such as, colorectal cancer, including colorectal carcinoma and colorectal adenocarcinoma, bladder cancer, osteosarcoma, chondrosarcoma, breast cancer, including triple-negative breast cancer, cancers of the central nervous system, including glioblastoma, astrocytoma, neuroblastoma, neural fibrosarcoma, neuroendocrine tumors, cervical cancer, endometrium cancer, gastric cancer, including gastric adenocarcinoma, head and neck cancer, kidney cancer, liver cancer, including hepatocellular carcinoma, lung cancer, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), ovarian cancer, pancreatic cancer, including pancreatic ductal carcinoma and pancreatic adenocarcinoma, sarcoma or skin cancer, including malignant melanoma and non-melanoma skin cancers.
In one embodiment of the invention the composition comprising one or more anti-DR5 antibodies or bispecific antibodies is for use in treatment of hematological tumors such as, leukemia, including chronic lymphocytic leukemia and myeloid leukemia, including acute myeloid leukemia and chronic myeloid leukemia, lymphoma, including Non-Hodgkin lymphoma or multiple myeloma, including Hodgkin Lymphoma, and including myelodysplastic syndromes.
In a particular embodiment of the present invention the composition comprising one or more anti-DR5 antibodies or bispecific antibodies is for use in treatment of a cancer selected from the following group of cancers; bladder cancer, bone cancer, colorectal cancer, sarcoma, endometrium cancer, fibroblast cancer, gastric cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, muscle cancer, neural tissue cancer, ovary cancer, pancreas cancer and skin cancer.
In one embodiment of the invention the composition comprising one or more anti-DR5 antibodies or bispecific antibodies is for use in inhibiting growth of DR5 positive or DR5 expressing tumors or cancers.
In the present invention DR5 positive tumors or cancers are to be understood as tumor cells and/or cancer cells expressing DR5 on the cell surface. Such DR5 expression may be detected by immunohistochemistry, flow cytometry, imaging or other suitable diagnostic method.
In one embodiment of the invention the composition comprising one or more anti-DR5 antibodies or bispecific antibodies is for use in inhibiting growth of DR5 expressing tumors or cancers. Tumors and cancer tissues that show heterogeneous expression of DR5 are also considered as DR5 positive tumors and cancers.
Tumors and/or cancers may express DR5 on some tumor and/or cancer cells and/or tissues showing DR5 expression, some tumor and/or cancers may show over-expression or aberrant expression of DR5, whereas other tumors and/or cancers show heterogeneous expression of DR5. Such tumors and/or cancers may all be suitable targets for treatment with anti-DR5 antibodies, bispecific antibodies and compositions comprising such antibodies according to the present invention.
In one embodiment of the invention the composition comprising one or more anti-DR5 antibodies or bispecific antibodies is for use in induction of apoptosis in DR5 expressing tumors.
Another aspect of the present invention comprises a method of treating an individual having a cancer comprising administering to said individual an effective amount of an anti-DR5 antibody, bispecific antibody or composition according to the invention.
In one embodiment of the invention the method of treating an individual having a cancer comprising administering to said individual an effective amount of an anti-DR5 antibody, bispecific antibody or composition according to the invention, further comprises administering an additional therapeutic agent to the said individual.
In one embodiment of the invention the additional therapeutic agent is a single agent or a combination of agents comprising an agent or regimen selected from the group chemotherapeutics (including but not limited to paclitaxel, temozolomide, cisplatin, carboplatin, oxaliplatin, irinotecan, doxorubicin, gemcitabine, 5-fluorouracil, pemetrexed), kinase inhibitors (including but not limited to sorafenib, sunitinib or everolimus), apoptosis-modulating agents (including but not limited to recombinant human TRAIL or birinapant), RAS inhibitors, proteasome inhibitors (including but not limited to bortezomib), histon deacetylase inhibitors (including but not limited to vorinostat), nutraceuticals, cytokines (including but not limited to IFN-γ), antibodies or antibody mimetics (including but not limited to anti-TF, anti-AXL, anti-EGFR, anti-IGF-1R, anti-VEGF, anti-CD20, anti-CD38, anti-HER2, anti-PD-1, anti-PD-L1, anti-CTLA4, anti-CD40, anti-CD137, anti-GITR, anti-VISTA (or other immunomodulatory targets) antibodies and antibody mimetics), and antibody-drug conjugates such as brentuximab vedotin, trastuzumab emtansine, HuMax-TF-ADC or HuMax-AXL-ADC.
In a further aspect, the invention comprises a kit of parts comprising an anti-DR5 antibody, bispecific antibody or composition according to the, wherein said antibody, bispecific antibody or composition is in one or more containers such as one or more vials.
In one embodiment of the invention the kit of parts comprising an anti-DR5 antibody, bispecific antibody or composition according to the invention is for simultaneous, separate or sequential use in therapy.
In a further embodiment the present invention is for use of an anti-DR5 antibody, bispecific antibody or a composition according to the invention for the manufacture of a medicament for treatment of cancer.
When describing the embodiments of the present invention, the combinations and permutations of all possible embodiments have not been explicitly described. Nevertheless, the mere fact that certain measures are recited in mutually different dependent claims or described in different embodiments does not indicate that a combination of these measures cannot be used to advantage. The present invention envisages all possible combinations and permutations of the described embodiments.
In another aspect of the present invention, the invention comprises a nucleic acid construct encoding an antibody according to amino acid sequences set forth in table 1. That is in one embodiment, the present invention comprises, a nucleic acid construct encoding an antibody corresponding to the amino acid sequences set forth in SEQ ID Nos: 1 to 23 or 29 to 43. In one embodiment of the present invention, the nucleic acid construct encodes an antibody according to any embodiments disclosed herein.
In a further aspect, the present invention relates to a nucleic acid encoding an antibody according to the present invention, wherein the Fc region comprises a mutation of an amino acids position corresponding to E430, E345 or S440 in a human IgG1, EU numbering. It is further contemplated that the nucleic acid encoding an antibody according to the invention comprises the amino acid substitutions in the specific amino acid positions herein described. Thus, in one embodiment, the nucleic acid encodes an antibody having the sequence according to SEQ ID NO: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 43.
In another aspect, the invention relates to nucleic acids encoding a sequence of a human, humanized or chimeric anti-DR5 antibody for use in the invention, to expression vectors encoding the sequences of such an antibody, to host cells comprising such expression vectors, to hybridomas which produce such antibodies, and to methods of producing such an antibody by culturing such host cells or hybridomas under appropriate conditions whereby the antibody is produced and, optionally, retrieved. Humanized anti-DR5 antibodies may also be denoted as “huDR5”.
In one embodiment, the invention provides an expression vector comprising a nucleotide sequence encoding one or more of the amino acid sequence according to SEQ ID Nos: 33 to 43
In one embodiment, the invention provides an expression vector comprising a nucleotide sequence encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 and 43., or any combination thereof. In another embodiment, the expression vector comprises a nucleotide sequence encoding any one or more of the VH CDR3 amino acid sequences selected from SEQ ID NOs: 3 and 11. In another embodiment, the expression vector comprises a nucleotide sequence encoding a VH amino acid sequence selected from SEQ ID NOs: 4, 9 and 12. In another embodiment, the expression vector comprises a nucleotide sequence encoding a VL amino acid sequence selected from SEQ ID NOs: 7, and 15. In another embodiment, the expression vector comprises a nucleotide sequence encoding the constant region of a human antibody light chain, of a human antibody heavy chain, or both. In another embodiment, the expression vector comprising a nucleotide sequence encoding the constant region of a human antibody heavy chain of selected from the group consisting of: SEQ ID NOs:58, 59, 60, 61, 62, 63, 64, 65, 66, 67 and 68.
In a particular embodiment, the expression vector comprises a nucleotide sequence encoding a variant of one or more of the above amino acid sequences, said variant having at most 25 amino acid modifications, such as at most 20, such as at most 15, 14, 13, 12, or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions, or at least 80% identity to any of said sequences, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity to any of the afore-mentioned amino acid sequences.
An expression vector in the context of the present invention may be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (a nucleic acid sequence comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, a humanized CD3 antibody-encoding nucleic acid is comprised in a naked DNA or RNA vector, including, for example, a linear expression element (as described in for instance Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a compacted nucleic acid vector (as described in for instance U.S. Pat. No. 6,077,835 and/or WO 00/70087), a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sized nucleic acid vector (as described in for instance Schakowski et al., Mol Ther 3, 793-800 (2001)), or as a precipitated nucleic acid vector construct, such as a CaPO4-precipitated construct (as described in for instance WO 00/46147, Benvenisty and Reshef, PNAS USA 83 9551-55 (1986), Wigler et al., Cell 14 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such nucleic acid vectors and the usage thereof are well known in the art (see for instance U.S. Pat. Nos. 5,589,466 and 5,973,972). In one embodiment, the vector is suitable for expression of the humanized anti-DR5 antibody, in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989)), pET vectors (Novagen, Madison, Wis.) and the like.
An expression vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be employed. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), and Grant et al., Methods in Enzymol 153, 516-544 (1987)).
A nucleic acid and/or vector may also comprise a nucleic acid sequence encoding a secretion/localization sequence, which can target a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or into cell culture media. Such sequences are known in the art, and include secretion leader or signal peptides, organelle-targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial transit sequences, chloroplast transit sequences), membrane localization/anchor sequences (e.g., stop transfer sequences, GPI anchor sequences), and the like.
In an expression vector of the invention, anti-DR5 antibody-encoding nucleic acids and the first and the second polypeptides nucleic acids may comprise or be associated with any suitable promoter, enhancer, and other expression-facilitating elements. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter/enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly (A) termination sequences, an origin of replication for plasmid product in E. coli, an antibiotic resistance gene as selectable marker, and/or a convenient cloning site (e.g., a polylinker). Nucleic acids may also comprise an inducible promoter as opposed to a constitutive promoter such as CMV IE (the skilled artisan will recognize that such terms are actually descriptors of a degree of gene expression under certain conditions).
In one embodiment, the anti-DR5 antibody-encoding expression is positioned in and/or delivered to the host cell or host animal via a viral vector.
Such expression vectors may be used for recombinant production of anti-DR5 antibodies. In one aspect, the anti-DR5 antibodies of any aspect or embodiment described herein are provided by use of recombinant eukaryotic or prokaryotic host cell which produces the antibody. Accordingly, the invention provides a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, which produces an anti-DR5 antibody as defined herein. Examples of host cells include yeast, bacterial and mammalian cells, such as CHO or HEK-293 cells. For example, in one embodiment, the host cell comprises a nucleic acid stably integrated into the cellular genome that comprises a sequence coding for expression of a anti-DR5 antibody described herein. In one embodiment, the host cell comprises a nucleic acid stably integrated into the cellular genome that comprise a sequence coding for expression of a first or a second polypeptide described herein. In another embodiment, the host cell comprises a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a sequence coding for expression of a anti-DR5 antibody, a first or a second polypeptide described herein.
The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK-293 cells, PER.C6, NSO cells, and lymphocytic cells, and prokaryotic cells such as E. coli and other eukaryotic hosts such as plant cells and fungi.
The term “transfectoma”, as used herein, includes recombinant eukaryotic host cells expressing the antibody or a target antigen, such as CHO cells, PER.C6, NSO cells, HEK-293 cells, plant cells, or fungi, including yeast cells.
In a further aspect, the invention relates to a method for producing an antibody of the invention, said method comprising the steps of
a) culturing a hybridoma or a host cell of the invention as described herein above, and
b) retrieving and/or purifying the antibody of the invention from the culture media.
In a further aspect, the nucleotide sequence encoding a sequence of an antibody further encodes a second moiety, such as a therapeutic polypeptide. Exemplary therapeutic antibodies are described elsewhere herein. In one embodiment, the invention relates to a method for producing an antibody fusion protein, said method comprising the steps of
a) culturing a host cell comprising an expression vector comprising such a nucleotide sequence, and
b) retrieving and/or purifying the antibody fusion protein from the culture media.
In one aspect of the present invention, the invention comprises an expression vector comprising on or more nucleic acid constructs encoding an antibody according to any embodiment disclosed herein.
In a further aspect of the invention, the invention comprises a host cell comprising an expression vector.
In one embodiment of the invention the host cell is a recombinant host cell, such as a recombinant prokaryotic cell, recombinant eukaryotic cell or recombinant microbial host cell.
SEQUENCE TABLE 1
SEQ ID NO:
Name
Sequence
Clone
SEQ ID NO: 1
VH hDR5-01 CDR1
GFNIKDTF
hDR5-01
SEQ ID NO: 2
VH hDR5-01 CDR2
IDPANGNT
SEQ ID NO: 3
VH hDR5-01 CDR3
VRGLYTYYFDY
SEQ ID NO: 4
VH hDR5-01
EVQLQQSGAEVVKPGA
SVKLSCKASGFNIKDTFI
HWVKQAPGQGLEWIG
RIDPANGNTKYDPKFQ
GKATITTDTSSNTAYME
LSSLRSEDTAVYYCVRGL
YTYYFDYWGQGTLVTV
SS
SEQ ID NO: 5
VL hDR5-01 CDR1
QSISNN
VL hDR5-01 CDR2
FAS
SEQ ID NO: 6
VL hDR5-01 CDR3
QQGNSWPYT
SEQ ID NO: 7
VL hDR5-01
EIVMTQSPATLSVSPGE
RATLSCRASQSISNNLH
WYQQKPGQAPRLLIKF
ASQSITGIPARFSGSGSG
TEFTLTISSLQSEDFAVY
YCQQGNSWPYTFGQG
TKLEIK
SEQ ID NO: 1
VH hDR5-01-G56T
GFNIKDTF
hDR5-01-G56T
CDR1
SEQ ID NO: 8
VH hDR5-01-G56T
IDPANTNT
CDR2
SEQ ID NO: 3
VH hDR5-01-G56T
VRGLYTYYFDY
CDR3
SEQ ID NO: 9
VH hDR5-01-G56T
EVQLQQSGAEVVKPGA
SVKLSCKASGFNIKDTFI
HWVKQAPGQGLEWIG
RIDPANTNTKYDPKFQG
KATITTDTSSNTAYMEL
SSLRSEDTAVYYCVRGL
YTYYFDYWGQGTLVTV
SS
SEQ ID NO: 5
VL hDR5-01-G56T
QSISNN
CDR1
VL hDR5-01-G56T
FAS
CDR2
SEQ ID NO: 6
VL hDR5-01-G56T
QQGNSWPYT
CDR3
SEQ ID NO: 7
VL hDR5-01-G56T
EIVMTQSPATLSVSPGE
RATLSCRASQSISNNLH
WYQQKPGQAPRLLIKF
ASQSITGIPARFSGSGSG
TEFTLTISSLQSEDFAVY
YCQQGNSWPYTFGQG
TKLEIK
SEQ ID NO: 10
VH hDR5-05 CDR1
GFNIKDTH
hDR5-05
SEQ ID NO: 2
VH hDR5-05 CDR2
IDPANGNT
SEQ ID NO: 11
VH hDR5-05 CDR3
ARWGTNVYFAY
SEQ ID NO: 12
VH hDR5-05
QVQLVQSGAEVKKPGA
SVKVSCKASGFNIKDTH
MHWVRQAPGQRLEWI
GRIDPANGNTEYDQKF
QGRVTITVDTSASTAYM
ELSSLRSEDTAVYYCAR
WGTNVYFAYWGQGTL
VTVSS
SEQ ID NO: 13
VL hDR5-05 CDR1
SSVSY
VL hDR5-05 CDR2
RTS
SEQ ID NO: 14
VL hDR5-05 CDR3
QQYHSYPPT
SEQ ID NO: 15
VL hDR5-05
DIQLTQSPSSLSASVGD
RVTITCSASSSVSYMYW
YQQKPGKAPKPWIYRT
SNLASGVPSRFSGSGSG
TDFTLTISSLQPEDFATY
YCQQYHSYPPTFGGGT
KVEIK
SEQ ID NO: 16
VH CONA-CDR1
GGSISSGDYF
Conatumumab
IgG1-DR5-CONA
SEQ ID NO: 17
VH CONA-CDR2
IHNSGTT
SEQ ID NO: 18
VH CONA-CDR3
ARDRGGDYYYGMDV
SEQ ID NO: 19
VH CONA
QVQLQESGPGLVKPSQ
TLSLTCTVSGGSISSGDY
FWSWIRQLPGKGLECIG
HIHNSGTTYYNPSLKSR
VTISVDTSKKQFSLRLSS
VTAADTAVYYCARDRG
GDYYYGMDVWGQGTT
VTVSS
SEQ ID NO: 20
VH CONA-C49W
QVQLQESGPGLVKPSQ
TLSLTCTVSGGSISSGDY
FWSWIRQLPGKGLEWI
GHIHNSGTTYYNPSLKS
RVTISVDTSKKQFSLRLS
SVTAADTAVYYCARDR
GGDYYYGMDVWGQG
TTVTVSS
SEQ ID NO: 21
VL CONA-CDR1
QGISRSY
VL CONA-CDR2
GAS
SEQ ID NO: 22
VL CONA-CDR3
QQFGSSPWT
SEQ ID NO: 23
VL CONA
EIVLTQSPGTLSLSPGER
ATLSCRASQGISRSYLA
WYQQKPGQAPSLLIYG
ASSRATGIPDRFSGSGS
GTDFTLTISRLEPEDFAV
YYCQQFGSSPWTFGQG
TKVEIK
SEQ ID NO: 24
Human DR5
MEQRGQNAPAASGA
RKRHGPGPREARGA
RPGPRVPKTLVLVVA
AVLLLVSAESALITQ
QDLAPQQRAAPQQK
RSSPSEGLCPPGHHI
SEDGRDCISCKYGQ
DYSTHWNDLLFCLR
CTRCDSGEVELSPCT
TTRNTVCQCEEGTFR
EEDSPEMCRKCRTG
CPRGMVKVGDCTPW
SDIECVHKESGTKH
SGEVPAVEETVTSSP
GTPASPCSLSGIIIGV
TVAAVVLIVAVFVCK
SLLWKKVLPYLKGIC
SGGGGDPERVDRSS
QRPGAEDNVLNEIVS
ILQPTQVPEQEMEVQ
EPAEPTGVNMLSPGE
SEHLLEPAEAERSQR
RRLLVPANEGDPTET
LRQCFDDFADLVPFD
SWEPLMRKLGLMDN
EIKVAKAEAAGHRDT
LYTMLIKWVNKTGR
DASVHTLLDALETLG
ERLAKKIEDHLLSSG
KFMYLEGNADSAMS
SEQ ID NO: 25
Rhesus monkey DR5
MGQLRQSAPAASGA
RKGRGPGPREARGA
RPGLRVLKTLVLVVA
AARVLVSADCAPITR
QSLDPQRRAAPQQK
RSSPTEGLCPPGHHI
SEDSRDCISCKYGQ
DYSTHWNDFLFCLR
CTKCDSGEVEVNSC
TTTRNTVCQCEEGTF
REEDSPEICRKCRTG
CPRGMVKVKDCTPW
SDIECVHKESGTKHT
GEVPAVEKTVTTSPG
TPASPCSLSGIIIGVI
VFVVIVVVAVIVWKT
SLWKKVLPYLKGVC
SGDGGDPERVDSSP
QRPGAEDNALNEIVS
IVQPSQVPEQEMEV
QEPAEQTDVNTLSP
GESEHLLEPAKAEGP
QRRGQLVPVNENDP
TETLRQCFDDFAAIV
PFDAWEPLVRQLGLT
NNEIKVAKAEAASSR
DTLYVMLIKWVNKT
GRAASVNTLLDALET
LEERLAKQKIQDRLL
SSGKFMYLEDNADS
ATS
SEQ ID NO: 26
Murine DR5
MEPPGPSTPTASAAA
RADHYTPGLRPLPKR
RLLYSFALLLAVLQAV
FVPVTANPAHNRPAG
LQRPEESPSRGPCLA
GQYLSEGNCKPCRE
GIDYTSHSNHSLDS
CILCTVCKEDKVVET
RCNITTNTVCRCKPG
TFEDKDSPEICQSCS
NCTDGEEELTSCTPR
ENRKCVSKTAWAS
WHKLGLWIGLLVPV
VLLIGALLVWKTGA
WRQWLLCIKRGCER
DPESANSVHSSLLD
RQTSSTTNDSNHNT
EPGKTQKTGKKLLVP
VNGNDSADDLKFIFE
YCSDIVPFDSWNRL
MRQLGLTDNQIQMV
KAETLVTREALYQML
LKWRHQTGRSASIN
HLLDALEAVEERDAM
EKIEDYAVKSGRFTY
QNAAAQPETGPGGS
QCV
SEQ ID NO: 27
DR5ECD-FcHistag
MEQRGQNAPAASGAR
KRHGPGPREARGARPG
LRVPKTLVLVVAAVLLLV
SAESALITQQDLAPQQR
VAPQQKRSSPSEGLCPP
GHHISEDGRDCISCKYG
QDYSTHWNDLLFCLRC
TRCDSGEVELSPCTTTR
NTVCQCEEGTFREEDSP
EMCRKCRTGCPRGMV
KVGDCTPWSDIECVHK
ESGTKHSGEAPAVEETV
TSSPGTPASPCSPKSCD
KTHTCPPCPAPEAEGAP
SVFLFPPKPKDTLMISRT
PEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNA
KTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPE
NNYKTAPPVLDSDGSFF
LYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYT
QKSLSLSPGKHHHHHH
HHEPEA
SEQ ID NO: 28
DR5ECDdelHis
MEQRGQNAPAASGA
RKRHGPGPREARGA
RPGPRVPKTLVLVVA
AVLLLVSAESALITQ
QDLAPQQRAAPQQK
RSSPSEGLCPPGHHI
SEDGRDCISCKYGQ
DYSTHWNDLLFCLR
CTRCDSGEVELSPCT
TTRNTVCQCEEGTFR
EEDSPEMCRKCRTG
CPRGMVKVGDCTPW
SDIECVHKESGHHH
HHHHH
SEQ ID NO: 29
Fc IgG1m(f)
STKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEP
VTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVT
VPSSSLGTQTYICNVNH
KPSNTKVDKRVEPKSCD
KTHTCPPCPAPELLGGP
SVFLFPPKPKDTLMISRT
PEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNA
KTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYT
QKSLSLSPGK
SEQ ID NO: 30
Fc IgG1m(z)
STKGPSVFPLAPSSK
STSGGTAALGCLVK
DYFPEPVTVSWNSG
ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS
SLGTQTYICNVNHKP
SNTKVDKKVEPKSC
DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT
LMISRTPEVTCVVVD
VSHEDPEVKFNWYV
DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV
LHQDWLNGKEYKCK
VSNKALPAPIEKTISK
AKGQPREPQVYTLPP
SREEMTKNQVSLTCL
VKGFYPSDIAVEWES
NGQPENNYKTTPPVL
DSDGSFFLYSKLTVD
KSRWQQGNVFSCS
VMHEALHNHYTQKS
LSLSPGK
SEQ ID NO: 31
Fc IgG1m(a)
STKGPSVFPLAPSSK
STSGGTAALGCLVK
DYFPEPVTVSWNSG
ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS
SLGTQTYICNVNHKP
SNTKVDKPVEPKSC
DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT
LMISRTPEVTCVVVD
VSHEDPEVKFNWYV
DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV
LHQDWLNGKEYKCK
VSNKALPAPIEKTISK
AKGQPREPQVYTLPP
SRDELTKNQVSLTCL
VKGFYPSDIAVEWES
NGQPENNYKTTPPVL
DSDGSFFLYSKLTVD
KSRWQQGNVFSCS
VMHEALHNHYTQKS
LSLSPGK
SEQ ID NO: 32
Fc IgG1m(x)
STKGPSVFPLAPSSK
STSGGTAALGCLVK
DYFPEPVTVSWNSG
ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS
SLGTQTYICNVNHKP
SNTKVDKPVEPKSC
DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT
LMISRTPEVTCVVVD
VSHEDPEVKFNWYV
DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV
LHQDWLNGKEYKCK
VSNKALPAPIEKTISK
AKGQPREPQVYTLPP
SREEMTKNQVSLTCL
VKGFYPSDIAVEWES
NGQPENNYKTTPPVL
DSDGSFFLYSKLTVD
KSRWQQGNVFSCS
VMHEGLHNHYTQKS
LSLSPGK
SEQ ID NO: 33
HC-hDR5-01
EVQLQQSGAEVVKPGA
SVKLSCKASGFNIKDTFI
HWVKQAPGQGLEWIG
RIDPANGNTKYDPKFQ
GKATITTDTSSNTAYME
LSSLRSEDTAVYYCVRGL
YTYYFDYWGQGTLVTV
SSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYF
PEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICN
VNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKT
ISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNG
QPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHN
HYTQKSLSLSPGK
SEQ ID NO: 34
HC-hDR5-01-E345K
EVQLQQSGAEVVKPGA
SVKLSCKASGFNIKDTFI
HWVKQAPGQGLEWIG
RIDPANGNTKYDPKFQ
GKATITTDTSSNTAYME
LSSLRSEDTAVYYCVRGL
YTYYFDYWGQGTLVTV
SSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYF
PEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICN
VNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKT
ISKAKGQPRKPQVYTLP
PSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNG
QPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHN
HYTQKSLSLSPGK
SEQ ID NO: 35
HC-hDR5-01-E430G
EVQLQQSGAEVVKPGA
SVKLSCKASGFNIKDTFI
HWVKQAPGQGLEWIG
RIDPANGNTKYDPKFQ
GKATITTDTSSNTAYME
LSSLRSEDTAVYYCVRGL
YTYYFDYWGQGTLVTV
SSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYF
PEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICN
VNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKT
ISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNG
QPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQ
QGNVFSCSVMHGALH
NHYTQKSLSLSPGK
SEQ ID NO: 36
HC-hDR5-01-G56T
EVQLQQSGAEVVKPGA
SVKLSCKASGFNIKDTFI
HWVKQAPGQGLEWIG
RIDPANTNTKYDPKFQG
KATITTDTSSNTAYMEL
SSLRSEDTAVYYCVRGL
YTYYFDYWGQGTLVTV
SSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYF
PEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICN
VNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKT
ISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNG
QPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHN
HYTQKSLSLSPGK
SEQ ID NO: 37
HC-hDR5-01-G56T-
EVQLQQSGAEVVKPGA
E345K
SVKLSCKASGFNIKDTFI
HWVKQAPGQGLEWIG
RIDPANTNTKYDPKFQG
KATITTDTSSNTAYMEL
SSLRSEDTAVYYCVRGL
YTYYFDYWGQGTLVTV
SSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYF
PEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICN
VNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKT
ISKAKGQPRKPQVYTLP
PSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNG
QPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHN
HYTQKSLSLSPGK
SEQ ID NO: 38
HC-hDR5-01-G56T-
EVQLQQSGAEVVKPGA
E430G
SVKLSCKASGFNIKDTFI
HWVKQAPGQGLEWIG
RIDPANTNTKYDPKFQG
KATITTDTSSNTAYMEL
SSLRSEDTAVYYCVRGL
YTYYFDYWGQGTLVTV
SSASTKGPSVFPLAPSSK
STSGGTAALGCLVKDYF
PEPVTVSWNSGALTSG
VHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICN
VNHKPSNTKVDKRVEP
KSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKT
ISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNG
QPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQ
QGNVFSCSVMHGALH
NHYTQKSLSLSPGK
SEQ ID NO: 39
LC-hDR5-01
EIVMTQSPATLSVSPGE
RATLSCRASQSISNNLH
WYQQKPGQAPRLLIKF
ASQSITGIPARFSGSGSG
TEFTLTISSLQSEDFAVY
YCQQGNSWPYTFGQG
TKLEIKRTVAAPSVFIFPP
SDEQLKSGTASVVCLLN
NFYPREAKVQWKVDN
ALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADY
EKHKVYACEVTHQGLSS
PVTKSFNRGEC
SEQ ID NO: 40
HC-hDR5-05
QVQLVQSGAEVKKPGA
SVKVSCKASGFNIKDTH
MHWVRQAPGQRLEWI
GRIDPANGNTEYDQKF
QGRVTITVDTSASTAYM
ELSSLRSEDTAVYYCAR
WGTNVYFAYWGQGTL
VTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKR
VEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPI
EKTISKAKGQPREPQVY
TLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEA
LHNHYTQKSLSLSPGK
SEQ ID NO: 41
HC-hDR5-05-E345K
QVQLVQSGAEVKKPGA
SVKVSCKASGFNIKDTH
MHWVRQAPGQRLEWI
GRIDPANGNTEYDQKF
QGRVTITVDTSASTAYM
ELSSLRSEDTAVYYCAR
WGTNVYFAYWGQGTL
VTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKR
VEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPI
EKTISKAKGQPRKPQVY
TLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEA
LHNHYTQKSLSLSPGK
SEQ ID NO: 42
HC-hDR5-05-E430G
QVQLVQSGAEVKKPGA
SVKVSCKASGFNIKDTH
MHWVRQAPGQRLEWI
GRIDPANGNTEYDQKF
QGRVTITVDTSASTAYM
ELSSLRSEDTAVYYCAR
WGTNVYFAYWGQGTL
VTVSSASTKGPSVFPLA
PSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKR
VEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLN
GKEYKCKVSNKALPAPI
EKTISKAKGQPREPQVY
TLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHG
ALHNHYTQKSLSLSPGK
SEQ ID NO: 43
LC-hDR5-05
DIQLTQSPSSLSASVGD
RVTITCSASSSVSYMYW
YQQKPGKAPKPWIYRT
SNLASGVPSRFSGSGSG
TDFTLTISSLQPEDFATY
YCQQYHSYPPTFGGGT
KVEIKRTVAAPSVFIFPP
SDEQLKSGTASVVCLLN
NFYPREAKVQWKVDN
ALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADY
EKHKVYACEVTHQGLSS
PVTKSFNRGEC
SEQ ID NO: 44
Human DR5, K415N
MEQRGQNAPAASGAR
Human DR5,
KRHGPGPREARGARPG
with
PRVPKTLVLVVAAVLLL
K415N mutation
VSAESALITQQDLAPQQ
RAAPQQKRSSPSEGLCP
PGHHISEDGRDCISCKY
GQDYSTHWNDLLFCLR
CTRCDSGEVELSPCTTT
RNTVCQCEEGTFREEDS
PEMCRKCRTGCPRGM
VKVGDCTPWSDIECVH
KESGTKHSGEVPAVEET
VTSSPGTPASPCSLSGIII
GVTVAAVVLIVAVFVCK
SLLWKKVLPYLKGICSG
GGGDPERVDRSSQRPG
AEDNVLNEIVSILQPTQ
VPEQEMEVQEPAEPTG
VNMLSPGESEHLLEPAE
AERSQRRRLLVPANEG
DPTETLRQCFDDFADLV
PFDSWEPLMRKLGLMD
NEIKVAKAEAAGHRDTL
YTMLIKWVNKTGRDAS
VHTLLDALETLGERLAN
QKIEDHLLSSGKFMYLE
GNADSAMS
SEQ ID NO: 45
Human DR5 (natural
MEQRGQNAPAASGA
variant)
RKRHGPGPREARGA
(Accession:
RPGLRVPKTLVLVVA
AAB70578)
AVLLLVSAESALITQ
QDLAPQQRVAPQQK
RSSPSEGLCPPGHHI
SEDGRDCISCKYGQ
DYSTHWNDLLFCLR
CTRCDSGEVELSPCT
TTRNTVCQCEEGTFR
EEDSPEMCRKCRTG
CPRGMVKVGDCTPW
SDIECVHKESGTKH
SGEAPAVEETVTSSP
GTPASPCSLSGIIIGV
TVAAVVLIVAVFVCK
SLLWKKVLPYLKGIC
SGGGGDPERVDRSS
QRPGAEDNVLNEIVS
ILQPTQVPEQEMEVQ
EPAEPTGVNMLSPGE
SEHLLEPAEAERSQR
RRLLVPANEGDPTET
LRQCFDDFADLVPFD
SWEPLMRKLGLMDN
EIKVAKAEAAGHRDT
LYTMLIKWVNKTGR
DASVHTLLDALETLG
ERLAKQKIEDHLLSS
GKFMYLEGNADSAM
S
SEQ ID NO: 46
Human DR5
MEQRGQNAPAASGA
(Uniprot O14763)
RKRHGPGPREARGA
RPGPRVPKTLVLVVA
AVLLLVSAESALITQ
QDLAPQQRAAPQQK
RSSPSEGLCPPGHHI
SEDGRDCISCKYGQ
DYSTHWNDLLFCLR
CTRCDSGEVELSPCT
TTRNTVCQCEEGTFR
EEDSPEMCRKCRTG
CPRGMVKVGDCTPW
SDIECVHKESGTKH
SGEVPAVEETVTSSP
GTPASPCSLSGIIIGV
TVAAVVLIVAVFVCK
SLLWKKVLPYLKGIC
SGGGGDPERVDRSS
QRPGAEDNVLNEIVS
ILQPTQVPEQEMEVQ
EPAEPTGVNMLSPGE
SEHLLEPAEAERSQR
RRLLVPANEGDPTET
LRQCFDDFADLVPFD
SWEPLMRKLGLMDN
EIKVAKAEAAGHRDT
LYTMLIKWVNKTGR
DASVHTLLDALETLG
ERLAKQKIEDHLLSS
GKFMYLEGNADSAM
S
SEQ ID NO: 47
Human DR5del-
MEQRGQNAPAASGA
K386N
RKRHGPGPREARGA
RPGPRVPKTLVLVVA
AVLLLVSAESALITQ
QDLAPQQRAAPQQK
RSSPSEGLCPPGHHI
SEDGRDCISCKYGQ
DYSTHWNDLLFCLR
CTRCDSGEVELSPCT
TTRNTVCQCEEGTFR
EEDSPEMCRKCRTG
CPRGMVKVGDCTPW
SDIECVHKESGIIIGV
TVAAVVLIVAVFVCK
SLLWKKVLPYLKGIC
SGGGGDPERVDRSS
QRPGAEDNVLNEIVS
ILQPTQVPEQEMEVQ
EPAEPTGVNMLSPGE
SEHLLEPAEAERSQR
RRLLVPANEGDPTET
LRQCFDDFADLVPFD
SWEPLMRKLGLMDN
EIKVAKAEAAGHRDT
LYTMLIKWVNKTGR
DASVHTLLDALETLG
ERLANQKIEDHLLSS
GKFMYLEGNADSAM
S
SEQ ID NO: 48
Cynomolgus DR5
MGQLRQSAPAASGA
(NCBI
RKGRGPGPREARGA
XP_005562887.1)
RPGLRVLKTLVLVVA
AARVLLSVSADCAPI
TRQSLDPQRRAAPQ
QKRSSPTEGLCPPG
HHISEDSRECISCKY
GQDYSTHWNDFLFC
LRCTKCDSGEVEVN
SCTTTRNTVCQCEE
GTFREEDSPEICRKC
RTGCPRGMVKVKDC
TPWSDIECVHKESG
TKHTGEVPAVEKTVT
TSPGTPASPCSLSGII
IGVIVLVVIVVVAVIV
WKTSLWKKVLPYLK
GVCSGGGGDPERVD
SSSHSPQRPGAEDN
ALNEIVSIVQPSQVP
EQEMEVQEPAEQTD
VNTLSPGESEHLLEP
AKAEGPQRRGQLVP
VNENDPTETLRQCFD
DFAAIVPFDAWEPLV
RQLGLTNNEIKVAKA
EAASSRDTLYVMLIK
WVNKTGRAASVNTL
LDALETLEERLAKQK
IQDRLLSSGKFMYLE
DNADSATS
SEQ ID NO: 49
Cynomolgus DR5-
MGQLRQSAPAASGA
K420N
RKGRGPGPREARGA
RPGLRVLKTLVLVVA
AARVLLSVSADCAPI
TRQSLDPQRRAAPQ
QKRSSPTEGLCPPG
HHISEDSRECISCKY
GQDYSTHWNDFLFC
LRCTKCDSGEVEVN
SCTTTRNTVCQCEE
GTFREEDSPEICRKC
RTGCPRGMVKVKDC
TPWSDIECVHKESG
TKHTGEVPAVEKTVT
TSPGTPASPCSLSGII
IGVIVLVVIVVVAVIV
WKTSLWKKVLPYLK
GVCSGGGGDPERVD
SSSHSPQRPGAEDN
ALNEIVSIVQPSQVP
EQEMEVQEPAEQTD
VNTLSPGESEHLLEP
AKAEGPQRRGQLVP
VNENDPTETLRQCFD
DFAAIVPFDAWEPLV
RQLGLTNNEIKVAKA
EAASSRDTLYVMLIK
WVNKTGRAASVNTL
LDALETLEERLANQK
IQDRLLSSGKFMYLE
DNADSATS
SEQ ID NO: 50
Cyno DR5Mfdel-
MGQLRQSAPAASGA
K420N
RKGRGPGPREARGA
RPGLRVLKTLVLVVA
AARVLLSVSADCAPI
TRQSLDPQRRAAPQ
QKRSSPTEGLCPPG
HHISEDSRECISCKY
GQDYSTHWNDFLFC
LRCTKCDSGEVEVN
SCTTTRNTVCQCEE
GTFREEDSPEICRKC
RTGCPRGMVKVKDC
TPWSDIECVHKESGI
IIGVIVLVVIVVVAVI
VWKTSLWKKVLPYL
KGVCSGGGGDPERV
DSSSHSPQRPGAED
NALNEIVSIVQPSQV
PEQEMEVQEPAEQT
DVNTLSPGESEHLLE
PAKAEGPQRRGQLV
PVNENDPTETLRQCF
DDFAAIVPFDAWEPL
VRQLGLTNNEIKVAK
AEAASSRDTLYVMLI
KWVNKTGRAASVNT
LLDALETLEERLANQ
KIQDRLLSSGKFMYL
EDNADSATS
SEQ ID NO: 51
VH chTRA8 CDR1
GFTFSSYV
SEQ ID NO: 52
VH chTRA8 CDR2
ISSGGSYT
SEQ ID NO: 53
VH chTRA8 CDR3
ARRGDSMITTDY
SEQ ID NO: 54
VL chTRA8 CDR1
QDVGTA
VL chTRA8 CDR2
WAS
SEQ ID NO: 55
VL chTRA8 CDR3
QQYSSYRT
SEQ ID NO: 56
HC-chTRA8
EVMLVESGGGLVKP
GGSLKLSCAASGFT
FSSYVMSWVRQTPE
KRLEWVATISSGGS
YTYYPDSVKGRFTIS
RDNAKNTLYLQMSS
LRSEDTAMYYCARR
GDSMITTDYWGQG
TTLTVSSASTKGPSV
FPLAPSSKSTSGGTA
ALGCLVKDYFPEPVT
VSWNSGALTSGVHT
FPAVLQSSGLYSLSS
VVTVPSSSLGTQTYI
CNVNHKPSNTKVDK
RVEPKSCDKTHTCPP
CPAPELLGGPSVFLF
PPKPKDTLMISRTPE
VTCVVVDVSHEDPE
VKFNWYVDGVEVHN
AKTKPREEQYNSTYR
VVSVLTVLHQDWLN
GKEYKCKVSNKALPA
PIEKTISKAKGQPRE
PQVYTLPPSREEMTK
NQVSLTCLVKGFYPS
DIAVEWESNGQPEN
NYKTTPPVLDSDGSF
FLYSKLTVDKSRWQ
QGNVFSCSVMHEAL
HNHYTQKSLSLSPG
K
SEQ ID NO: 57
LC-chTRA8
DIVMTQSHKFMSTS
VGDRVSITCKASQD
VGTAVAWYQQKPG
QSPKLLIYWASTRH
TGVPDRFTGSGSGT
DFTLTISNVQSEDLA
DYFCQQYSSYRTFG
GGTKLEIKRTVAAPS
VFIFPPSDEQLKSGT
ASVVCLLNNFYPREA
KVQWKVDNALQSG
NSQESVTEQDSKDS
TYSLSSTLTLSKADY
EKHKVYACEVTHQG
LSSPVTKSFNRGEC
SEQ ID NO: 58
Fc IgG1m(f)-E430G
STKGPSVFPLAPSSK
STSGGTAALGCLVK
DYFPEPVTVSWNSG
ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS
SLGTQTYICNVNHKP
SNTKVDKRVEPKSC
DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT
LMISRTPEVTCVVVD
VSHEDPEVKFNWYV
DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV
LHQDWLNGKEYKCK
VSNKALPAPIEKTISK
AKGQPREPQVYTLPP
SREEMTKNQVSLTCL
VKGFYPSDIAVEWES
NGQPENNYKTTPPVL
DSDGSFFLYSKLTVD
KSRWQQGNVFSCS
VMHGALHNHYTQKS
LSLSPGK
SEQ ID NO: 59
Fc IgG1m(f)-E345K
STKGPSVFPLAPSSK
STSGGTAALGCLVK
DYFPEPVTVSWNSG
ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS
SLGTQTYICNVNHKP
SNTKVDKRVEPKSC
DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT
LMISRTPEVTCVVVD
VSHEDPEVKFNWYV
DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV
LHQDWLNGKEYKCK
VSNKALPAPIEKTISK
AKGQPRKPQVYTLPP
SREEMTKNQVSLTCL
VKGFYPSDIAVEWES
NGQPENNYKTTPPVL
DSDGSFFLYSKLTVD
KSRWQQGNVFSCS
VMHEALHNHYTQKS
LSLSPGK
SEQ ID NO: 60
Fc IgG1m(f)-S440Y
STKGPSVFPLAPSSK
STSGGTAALGCLVK
DYFPEPVTVSWNSG
ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS
SLGTQTYICNVNHKP
SNTKVDKRVEPKSC
DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT
LMISRTPEVTCVVVD
VSHEDPEVKFNWYV
DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV
LHQDWLNGKEYKCK
VSNKALPAPIEKTISK
AKGQPREPQVYTLPP
SREEMTKNQVSLTCL
VKGFYPSDIAVEWES
NGQPENNYKTTPPVL
DSDGSFFLYSKLTVD
KSRWQQGNVFSCS
VMHEALHNHYTQKY
LSLSPGK
SEQ ID NO: 61
Fc IgG1m(f)-E430G-
STKGPSVFPLAPSSK
K439E
STSGGTAALGCLVK
DYFPEPVTVSWNSG
ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS
SLGTQTYICNVNHKP
SNTKVDKRVEPKSC
DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT
LMISRTPEVTCVVVD
VSHEDPEVKFNWYV
DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV
LHQDWLNGKEYKCK
VSNKALPAPIEKTISK
AKGQPREPQVYTLPP
SREEMTKNQVSLTCL
VKGFYPSDIAVEWES
NGQPENNYKTTPPVL
DSDGSFFLYSKLTVD
KSRWQQGNVFSCS
VMHGALHNHYTQES
LSLSPGK
SEQ ID NO: 62
Fc IgG1m(f)-E430G-
STKGPSVFPLAPSSK
S440K
STSGGTAALGCLVK
DYFPEPVTVSWNSG
ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS
SLGTQTYICNVNHKP
SNTKVDKRVEPKSC
DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT
LMISRTPEVTCVVVD
VSHEDPEVKFNWYV
DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV
LHQDWLNGKEYKCK
VSNKALPAPIEKTISK
AKGQPREPQVYTLPP
SREEMTKNQVSLTCL
VKGFYPSDIAVEWES
NGQPENNYKTTPPVL
DSDGSFFLYSKLTVD
KSRWQQGNVFSCS
VMHGALHNHYTQKK
LSLSPGK
SEQ ID NO: 63
Fc IgG1m(f)-K409R
STKGPSVFPLAPSSK
STSGGTAALGCLVK
DYFPEPVTVSWNSG
ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS
SLGTQTYICNVNHKP
SNTKVDKRVEPKSC
DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT
LMISRTPEVTCVVVD
VSHEDPEVKFNWYV
DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV
LHQDWLNGKEYKCK
VSNKALPAPIEKTISK
AKGQPREPQVYTLPP
SREEMTKNQVSLTCL
VKGFYPSDIAVEWES
NGQPENNYKTTPPVL
DSDGSFFLYSRLTVD
KSRWQQGNVFSCS
VMHEALHNHYTQKS
LSLSPGK
SEQ ID NO: 64
Fc IgG1m(f)-K409R-
STKGPSVFPLAPSSK
E345K
STSGGTAALGCLVK
DYFPEPVTVSWNSG
ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS
SLGTQTYICNVNHKP
SNTKVDKRVEPKSC
DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT
LMISRTPEVTCVVVD
VSHEDPEVKFNWYV
DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV
LHQDWLNGKEYKCK
VSNKALPAPIEKTISK
AKGQPRKPQVYTLPP
SREEMTKNQVSLTCL
VKGFYPSDIAVEWES
NGQPENNYKTTPPVL
DSDGSFFLYSRLTVD
KSRWQQGNVFSCS
VMHEALHNHYTQKS
LSLSPGK
SEQ ID NO: 65
Fc IgG1m(f)-K409R-
STKGPSVFPLAPSSK
E430G
STSGGTAALGCLVK
DYFPEPVTVSWNSG
ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS
SLGTQTYICNVNHKP
SNTKVDKRVEPKSC
DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT
LMISRTPEVTCVVVD
VSHEDPEVKFNWYV
DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV
LHQDWLNGKEYKCK
VSNKALPAPIEKTISK
AKGQPREPQVYTLPP
SREEMTKNQVSLTCL
VKGFYPSDIAVEWES
NGQPENNYKTTPPVL
DSDGSFFLYSRLTVD
KSRWQQGNVFSCS
VMHGALHNHYTQKS
LSLSPGK
SEQ ID NO: 66
Fc IgG1m(f)-F405L
STKGPSVFPLAPSSK
STSGGTAALGCLVK
DYFPEPVTVSWNSG
ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS
SLGTQTYICNVNHKP
SNTKVDKRVEPKSC
DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT
LMISRTPEVTCVVVD
VSHEDPEVKFNWYV
DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV
LHQDWLNGKEYKCK
VSNKALPAPIEKTISK
AKGQPREPQVYTLPP
SREEMTKNQVSLTCL
VKGFYPSDIAVEWES
NGQPENNYKTTPPVL
DSDGSFLLYSKLTVD
KSRWQQGNVFSCS
VMHEALHNHYTQKS
LSLSPGK
SEQ ID NO: 67
Fc IgG1m(f)-F405L-
STKGPSVFPLAPSSK
E345K
STSGGTAALGCLVK
DYFPEPVTVSWNSG
ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS
SLGTQTYICNVNHKP
SNTKVDKRVEPKSC
DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT
LMISRTPEVTCVVVD
VSHEDPEVKFNWYV
DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV
LHQDWLNGKEYKCK
VSNKALPAPIEKTISK
AKGQPRKPQVYTLPP
SREEMTKNQVSLTCL
VKGFYPSDIAVEWES
NGQPENNYKTTPPVL
DSDGSFLLYSKLTVD
KSRWQQGNVFSCS
VMHEALHNHYTQKS
LSLSPGK
SEQ ID NO: 68
Fc IgG1m(f)-F405L-
STKGPSVFPLAPSSK
E430G
STSGGTAALGCLVK
DYFPEPVTVSWNSG
ALTSGVHTFPAVLQS
SGLYSLSSVVTVPSS
SLGTQTYICNVNHKP
SNTKVDKRVEPKSC
DKTHTCPPCPAPELL
GGPSVFLFPPKPKDT
LMISRTPEVTCVVVD
VSHEDPEVKFNWYV
DGVEVHNAKTKPRE
EQYNSTYRVVSVLTV
LHQDWLNGKEYKCK
VSNKALPAPIEKTISK
AKGQPREPQVYTLPP
SREEMTKNQVSLTCL
VKGFYPSDIAVEWES
NGQPENNYKTTPPVL
DSDGSFLLYSKLTVD
KSRWQQGNVFSCS
VMHGALHNHYTQKS
LSLSPGK
EXAMPLES
Example 1: Antibody and Antigen Constructs
Expression Constructs for DR5
Codon-optimized constructs for expression of full-length DR5 proteins of human (SEQ ID NO 46), rhesus monkey (SEQ ID NO 25) and mouse (SEQ ID NO 26) were generated based on available sequences: human (Homo sapiens) DR5 (Genbank accession no. NP_003833, UniprotKB/Swiss-Prot 014763-1), Rhesus monkey (Macaca mulatta) DR5 (Genbank accession no. EHH28346), murine (Mus musculus) DR5 (UniprotKB/Swiss-Prot Q9QZM4). For mapping of the binding regions of the DR5 antibodies (as described in Example 6) the following chimeric human/mouse DR5 constructs were made; human DR5 in which, respectively, the following parts were replaced by the corresponding mouse DR5 sequence (numbers refer to human sequence), construct A aa 56-68, construct B aa 56-78, construct C aa 69-78, construct D aa 79-115, construct E 79-138, construct F aa 97-138, construct G aa 139-166, construct H aa 139-182, construct I aa 167-182, construct J 167-210, construct K aa 183-210. The loss-of-function mutation K415N was introduced in the human DR5 death domain (SEQ ID NO 44). In addition, codon-optimized construct for the extracellular domain (ECD) of human DR5 with a C-terminal His tag were generated: DR5ECD-FcHistag (SEQ ID NO 27) and DR5ECDdelHis (SEQ ID NO 28). All constructs contained suitable restriction sites for cloning and an optimal Kozak (GCCGCCACC) sequence. The constructs were cloned in the mammalian expression vector pcDNA3.3 (Invitrogen).
Expression Constructs for Antibodies
For antibody expression the VH and VL sequences, as earlier described, of the chimeric human/mouse DR5 antibodies DR5-01 and DR5-05 (based on EP2684896A1) and their humanized variants hDR5-01 and hDR5-05 (based on WO2014/009358) were cloned in expression vectors (pcDNA3.3) containing the relevant constant HC and LC regions. Desired mutations were introduced either by gene synthesis or site directed mutagenesis.
In some of the Examples, reference antibodies against DR5 were used that have been previously described. IgG1-CONA (based on U.S. Pat. No. 7,521,048 B2 and WO2010/138725) and IgG1-chTRA8 (based on EP1506285B1 and U.S. Pat. No. 7,244,429B2) were cloned in the relevant antibody expression vectors as supra.
In some of the examples the human IgG1 antibody IgG1-b12, a gp120-specific antibody was used as a negative control (Barbas et al., J Mol Biol. 1993 Apr. 5; 230(3):812-23).
Transient Expression
Antibodies were expressed as IgG1,K. Plasmid DNA mixtures encoding both heavy and light chains of antibodies were transiently transfected in Expi293F cells (Life technologies, USA) using 293fectin (Life technologies) essentially as described by Vink et al. (Vink et al., Methods, 65 (1), 5-10 2014).
Membrane proteins were expressed in Freestyle CHO-S cells (Life technologies), using the freestyle Max reagent, as described by the manufacturer.
Purification and Analysis of Proteins
Antibodies were purified by immobilized protein G chromatography. His-tagged recombinant protein was purified by immobilized metal affinity chromatography. Protein batches were analyzed by a number of bioanalytical assays including SDS-PAGE, size exclusion chromatography and measurement of endotoxin levels.
Generation of Bispecific Antibodies
Bispecific IgG1 antibodies were generated by Fab-arm-exchange under controlled reducing conditions. The basis for this method is the use of complementary CH3 domains, which promote the formation of heterodimers under specific assay conditions as described in WO2011/131746. The F405L and K409R (EU numbering) mutations were introduced in anti-DR5 IgG1 antibodies to create antibody pairs with complementary CH3 domains. The F405L mutation was introduced in IgG1-DR5-05 and IgG1-DR5-05-E430G; the K409R mutation was introduced in IgG1-DR5-01 and IgG1-DR5-01-E430G. To generate bispecific antibodies, the two parental complementary antibodies, each antibody at a final concentration of 0.5 mg/mL, were incubated with 75 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 μL TE at 31° C. for 5 hours. The reduction reaction was stopped by removing the reducing agent 2-MEA using spin columns (Microcon centrifugal filters, 30 k, Millipore) according to the manufacturer's protocol. In this way the bispecific antibodies IgG1-DR5-01-K409R×IgG1-DR5-05-F405L (BsAb DR5-01-K409R×DR5-05-F405L) and IgG1-DR5-01-K409R-E430G×IgG1-DR5-05-F405L-E430G (BsAb DR5-01-K409R-E430G×DR5-05-F405L-E430G) were generated.
The K409R mutation and/or the F405L mutation have no effect on the antibody's binding to the corresponding antigen. That is the K409R mutation and/or the F405L mutation have no effect of the anti-DR5 antibody's binding to DR5.
Example 2: DR5 Expression Levels on Different Human Cancer Cell Lines
DR5 density per cell was quantified for different human cancer cell lines by indirect immunofluorescence using QIFIKIT (DAKO, Cat nr K0078) with mouse monoclonal antibody B-K29 (Diaclone, Cat nr 854.860.000). Cells were harvested by trypsinization and passed through a cell strainer. Cells were pelleted by centrifugation for 5 minutes at 1,200 rpm, washed with PBS and resuspended at a concentration of 2×106 cells/mL. The next steps were performed at 4° C. 50 μL of the single cell suspensions (100,000 cells per well) were seeded in polystyrene 96-well round-bottom plates (Greiner Bio-One, Cat nr 650101). Cells were pelleted by centrifugation for 3 minutes at 300×g and resuspended in 50 μL antibody sample or mouse IgG1 isotype control sample (BD/Pharmingen, Cat nr 555746) at 10 μg/mL saturating concentrations. After an incubation of 30 minutes at 4° C., cells were pelleted and resuspended in 150 μL FACS buffer (PBS+0.1% (w/v) bovine serum albumin (BSA)+0.02% (w/v) sodium azide). Set-up and calibration beads were added to the plate according to the manufacturer's instructions. Cells and beads in parallel were washed two more times with 150 μL FACS buffer and resuspended in 50 μL FITC-conjugated goat-anti-mouse IgG (1/50; DAKO, Cat nr F0479). Secondary antibody was incubated for 30 minutes at 4° C. protected from light. Cells and beads were washed twice with 150 μL FACS buffer and resuspended in 150 μL FACS buffer. Immunofluorescence was measured on a FACS Canto II (BD Biosciences) by recording 10,000 events within the population of viable cells. The Geometric mean of fluorescence intensity of the calibration beads was used to calculate the calibration curve that was forced to go through zero intensity and zero concentration using GraphPad Prism software (GraphPad Software, San Diego, Calif., USA). For each cell line, the antibody binding capacity (ABC), an estimate for the number of DR5 molecules expressed on the plasma membrane, was calculated using the Geometric mean fluorescence intensity of the DR5-antibody-stained cells, based on the equation of the calibration curve (interpolation of unknowns from the standard curve, using GraphPad Software). Generally, DR5 cell surface expression was low to moderate on the cell lines assessed here. Based on these data, cell lines were categorized according to low DR5 expression (ABC<10,000) and moderate DR5 expression (ABC>10,000). HCT-15 (ATCC, CCL-225), HT-29 (ATCC, HTB-38) and SW480 (ATCC, CCL-228) colon cancer, B×PC-3 (ATCC, CRL-1687), HPAF-II (ATCC, CRL-1997) and PANC-1 (ATCC, CRL-1469) pancreatic cancer, and A549 (ATCC, CCL-185) and SK-MES-1 (ATCC, HTB-58) lung cancer cell lines were found to have low DR5 expression (QIFIKIT ABC range 3,081-8,411). COLO 205 (ATCC CCL-222™) and HCT 116 (ATCC CCL-247) colon cancer, A375 (ATCC, CRL-1619) skin cancer and SNU-5 (ATCC, CRL-5973) gastric cancer cell lines were found to have moderate DR5 expression (QIFIKIT ABC range 10,777-21,262).
Example 3: Binding of Humanized DR5-01 and DR5-05 Antibodies to HCT 116 Cells
The humanized antibodies hDR5-01 and hDR5-05 are described in patent application WO2014/009358. Binding of purified IgG1-hDR5-01-K409R and IgG1-hDR5-05-F405L to DR5-positive HCT 116 human colon cancer cells was analyzed and compared to binding of the chimeric antibodies IgG1-DR5-01-K409R and IgG1-DR5-05-F405L by FACS analysis. To prepare single cell suspensions, adherent HCT 116 cells were washed twice with PBS (B.Braun; Cat nr 3623140) before incubating with Trypsin 1×/EDTA 0.05% for 2 minutes at 37° C. 10 mL medium [McCoy's 5A medium with L-Glutamine and HEPES (Lonza; Cat nr BE12-168F)+10% Donor Bovine Serum with Iron (Life Technologies; Cat nr 10371-029)+100 Units Penicillin/100 Units Streptomycin (Lonza Cat nr DE17-603E)] was added before pelleting the cells by centrifugation for 5 minutes at 1200 rpm. Cells were resuspended in 10 mL medium, pelleted again by centrifugation for 5 minutes at 1200 rpm, and resuspended in FACS buffer at a concentration of 1.0×106 cells/mL. The next steps were performed at 4° C. 100 μL cell suspension samples (100,000 cells per well) were seeded in polystyrene 96-well round-bottom plates (Greiner Bio-One; Cat nr 650101) and pelleted by centrifugation at 300×g for 3 minutes at 4° C. Cells were resuspended in 100 μL samples of a serial dilution antibody preparation series (range 0 to 10 μg/mL in 5-fold dilutions) and incubated for 30 minutes at 4° C. Cells were pelleted by centrifugation at 300×g for 3 minutes at 4° C. and washed twice with 150 μL FACS buffer. Cells were incubated with 50 μL secondary antibody R-phycoerythrin (R-PE)-conjugated goat-anti-human IgG F(ab′)2 (Jackson ImmunoResearch; Cat nr 109-116-098; 1/100) for 30 minutes at 4° C., protected from light. Cells were washed twice with 150 μL FACS buffer, resuspended in 150 μL FACS buffer, and antibody binding was analyzed on a FACS Canto II (BD Biosciences) by recording 10,000 events. Binding curves were analyzed using non-linear regression analysis (sigmoidal dose-response with variable slope) using GraphPad Prism software.
As can be seen from FIG. 2 shows that the humanized antibodies IgG1-hDR5-01-K409R and IgG1-hDR5-05-F405L showed similar binding curves as their corresponding chimeric antibody IgG1-DR5-01-K409R or IgG1-DR5-05-F405L, respectively. Humanization had no effect on the binding of the DR5 antibodies.
Example 4: Introduction of a Hexamerization-Enhancing Mutation Does Not Affect Binding of Chimeric DR5-01 and DR5-05 Antibodies and Bispecific Antibody DR5-01×DR5-05 to DR5-Positive Human Colon Cancer Cells
Binding of purified antibody variants of IgG1-DR5-01-K409R, IgG1-DR5-05-F405L and bispecific antibody IgG1-DR5-01-K409R×IgG1-DR5-05-F405L (BsAb DR5-01-K409R×DR5-05-F405L) with and without a hexamerization-enhancing mutation (E430G or E345K) to human colon cancer cells COLO 205 was analyzed by FACS analysis. Cells were harvested by pooling the culture supernatant containing non-adherent cells and trypsinized adherent COLO 205 cells. Cells were centrifuged for 5 minutes at 1,200 rpm and resuspended in 10 mL culture medium [RPMI 1640 with 25 mM Hepes and L-Glutamine (Lonza Cat nr BE12-115F)+10% Donor Bovine Serum with Iron (Life Technologies Cat nr 10371-029)+50 Units Penicillin/50 Units Streptomycin (Lonza Cat nr DE17-603E)]. Cells were counted, centrifuged again and resuspended in FACS buffer at a concentration of 0.3×106 cells/mL. The next steps were performed at 4° C. 100 μL cell suspension samples (30,000 cells per well) were seeded in polystyrene 96-well round-bottom plates and pelleted by centrifugation at 300×g for 3 minutes at 4° C. Cells were resuspended in 50 μL samples of a serial dilution antibody preparation series (range 0 to 10 μg/mL final concentrations in 5-fold dilutions) and incubated for 30 minutes at 4° C. Plates were centrifuged at 300×g for 3 minutes at 4° C. and cells were washed twice with 150 μL FACS buffer. Cells were incubated with 50 μL secondary antibody R-PE-conjugated goat-anti-human IgG F(ab′)2 (Jackson ImmunoResearch; Cat nr 109-116-098; 1/100) for 30 minutes at 4° C. protected from light. Cells were washed twice with 150 μL FACS buffer, resuspended in 100 μL FACS buffer, and antibody binding was analyzed on a FACS Canto II (BD Biosciences) by recording 5,000 events. Binding curves were analyzed using non-linear regression analysis (sigmoidal dose-response with variable slope) using GraphPad Prism software.
FIG. 3A shows that the antibodies IgG1-DR5-01-K409R-E430G and IgG1-DR5-01-K409R-E345K showed similar dose-dependent binding to human colon cancer cells COLO 205 as IgG1-DR5-01-K409R. FIG. 3B shows that the antibodies IgG1-DR5-05-F405L-E430G and IgG1-DR5-05-F405L-E345K showed similar dose-dependent binding to COLO 205 cells as IgG1-DR5-05-F405L. FIG. 3C shows that BsAb DR5-01-K409R-E430G×DR5-05-F405L-E430G and BsAb DR5-01-K409R-E345K×DR5-05-F405L-E345K showed similar dose-dependent binding to COLO 205 cells as BsAb DR5-01-K409R×DR5-05-F405L. These data indicate that introduction of the hexamerization-enhancing mutations E430G or E345K did not affect binding of antibodies IgG1-DR5-01-K409R, IgG1-DR5-05-F405L and BsAb DR5-01-K409R×DR5-05-F405L on DR5-positive COLO 205 cells.
Example 5: Binding of Chimeric DR5-01 and DR5-05 Antibodies to Rhesus Macaque DR5
Binding of purified IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G to CHO cells expressing Rhesus macaque DR5 or human DR5 (described in Example 1) was analyzed by FACS analysis. One day before FACS analysis, CHO cells were transiently transfected with a vector encoding Rhesus macaque DR5, human DR5 or a non-coding vector (mock). To prepare single cell suspensions, cells were washed with PBS and resuspended in FACS buffer at a concentration of 1.0×106 cells/mL. The next steps were performed at 4° C. 75 μL cell suspension samples (75,000 cells per well) were seeded in polystyrene 96-well round-bottom plates and pelleted by centrifugation at 300×g for 3 minutes at 4° C. Cells were resuspended in 50 μL samples of a serial dilution antibody preparation series (range 10 to 0 μg/mL in 5-fold dilutions) and incubated for 30 minutes at 4° C. Plates were centrifuged at 300×g for 3 minutes at 4° C. and cells were washed twice with 150 μL FACS buffer. Cells were incubated with 50 μL secondary antibody R-PE-conjugated goat-anti-human IgG F(ab′)2 (Jackson ImmunoResearch; Cat nr 109-116-098; 1/100) for 30 minutes at 4° C. protected from light. Cells were washed twice with 150 μL FACS buffer, resuspended in 100 μL FACS buffer, and antibody binding was analyzed on a FACS Canto II (BD Biosciences) by recording 100,000 events. Binding curves were analyzed using non-linear regression analysis (sigmoidal dose-response with variable slope) using GraphPad Prism software. FIG. 4 shows that the antibodies IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G showed dose-dependent binding to Rhesus macaque DR5 expressed on CHO cells. Binding to CHO cells transfected with human DR5 and mock-transfected CHO cell was tested as positive and negative control, respectively. For both IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G, EC50 values for binding to human DR5 and Rhesus macaque DR5 were in the same range ([0.014-0.023 μg/mL] and [0.051-0.066 μg/mL], respectively), indicating that IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G show comparable binding to human and Rhesus macaque DR5.
Example 6: Mapping of Binding Regions of DR5-01 and DR5-05 Antibodies on Human DR5 Using Domain-Swapped DR5 Molecules
The amino acid sequences of the extracellular domains of human and murine DR5 show limited homology (FIG. 5A) and the humanized antibodies IgG1-hDR5-01-F405L and IgG1-hDR5-05-F405L do not bind murine DR5 (FIG. 5C, D). With the aim to identify amino acid stretches in the human DR5 extracellular domain that are involved in antibody binding, we developed eleven human-mouse chimeric DR5 molecules, in which specific human DR5 domains had been replaced by the mouse analogues (domain-swapped DR5 molecules described in Example 1) as visualized in FIG. 5B. The domain-swapped DR5 variants were transiently expressed on CHO cells. Loss of binding of the DR5 antibodies to domain-swapped DR5 molecules indicates that the swapped domain of human DR5 contains one or more amino acids that are crucial for binding. Vice versa, retention of binding of the DR5 antibodies to domain-swapped DR5 molecules indicates that the swapped domain of human DR5 does not contain amino acids that are crucial for binding. For the binding assay, 3×106 transfected cells were washed and resuspended in 3 mL FACS buffer. 100 μL cell suspension was added per well (100.000 cells per well) of 96-well round bottom plates (Greiner Bio-one; Cat nr 650101). The next steps were performed at 4° C. Cells were pelleted, resuspended in 50 μL DR5 antibody sample (10 μg/mL final concentration) and incubated for 30 minutes at 4° C. The cells were washed twice and incubated in 50 μL secondary antibody R-PE-conjugated goat-anti-human IgG F(ab′)2 (Jackson ImmunoResearch; Cat nr 109-116-098; 1/100) for 30 minutes at 4° C. protected from light. Cells were washed twice, resuspended in 120 μL FACS buffer, and analyzed on a FACS Canto II (BD Biosciences). The percentage of viable PE-positive cells was plotted using GraphPad Prism software. Surface expression was confirmed for each domain-swapped DR5 molecule using a panel of DR5 antibodies directed against different epitopes (not shown). The non-target binding antibody IgG1-b12 against gp120 was included as a negative control for binding. FIG. 5C shows that IgG1-hDR5-01-F405L showed loss of binding to constructs E (79-138), F (97-138), G (139-166) and H (139-182), whereas binding to constructs A-D (covering human DR5 sequence 56-115) and I-K (covering human DR5 sequence 167-210) was retained. Together, these data indicate that the amino acid regions 116-138 and 139-166 each contain one or more amino acids required for binding of IgG1-hDR5-01-F405L to human DR5. FIG. 5D shows that IgG1-hDR5-05-F405L showed loss of binding to constructs D (79-115), E (79-138) and F (97-138), whereas binding to constructs A-C (covering human DR5 sequence 56-78) and G-K (covering human DR5 sequence 139-210) was retained. Together, these data indicate that the amino acid region 79-138 contains one or more amino acids required for binding of IgG1-hDR5-05-F405L to human DR5.
Example 7: Crossblock ELISA with DR5-01 and DR5-05 Antibodies
The competition between humanized DR5-01 and DR5-05 antibodies for binding to the extracellular domain of DR5 was measured by sandwich binding assays in a sandwich enzyme-linked immunosorbent assay (ELISA) as described in this example and by Bio-Layer interferometry (BLI) using a ForteBio Octet® HTX system (data not shown). For the ELISA, 96-well flat bottom ELISA plates (Greiner bio-one; Cat nr 655092) were coated overnight at 4° C. with 2 μg/mL DR5 antibody (IgG1-hDR5-01-E430G or IgG1-hDR5-05-E430G) in 100 μL PBS. The wells were blocked by adding 200 μL PBSA [PBS/1% Bovine Serum Albumin (BSA; Roche Cat #10735086001)] and incubated for 1 hour at room temperature. The wells were washed three times with PBST [PBS/0.05% Tween-20 (Sigma-Aldrich; Cat nr 63158)]. Next, DR5ECD-FcHistag (SEQ ID 27) (0.2 μg/mL final concentration) and competing antibody (1 μg/mL final concentration) were added in a total volume of 100 μL PBSTA (PBST/0.2% BSA) and incubated for 1 hour at room temperature while shaking. After washing three times with PBST, wells were incubated on an ELISA shaker with 100 μL biotinylated anti-His tag antibody (R&D Systems; Cat nr BAM050; 1:2.000) in PBSTA for one hour at room temperature. After washing three times with PBST, wells were incubated with streptavidin-labelled Poly-HRP (Sanquin; Cat nr M2032; 1:10.000) in PBSTA for 20 minutes at room temperature on an ELISA shaker. After washing three times with PBST, the reaction was visualized through an incubation with 100 μL 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid [ABTS (Roche; Cat nr 11112597001)] for 30 minutes at RT protected from light. The substrate reaction was stopped by adding an equal volume of 2% oxalic acid. Fluorescence at 405 nm was measured on an ELISA reader (BioTek ELx808 Absorbance Microplate Reader). FIG. 6 shows binding competition expressed as percentage inhibition of DR5ECD-FcHisCtag binding to coated antibody in presence of competing antibody, relative to binding of DR5ECD-FcHisCtag in absence of competing antibody (% inhibition=100−[(binding in presence of competing antibody/binding in absence of competing antibody)]*100). Binding of DR5ECD-FcHistag to coated IgG1-hDR5-01-E430G was not inhibited in the presence of soluble IgG1-hDR5-05-E430G. Vice versa, binding of DR5ECD-FcHistag to coated IgG1-hDR5-05-E430G was also not inhibited in the presence of soluble IgG1-hDR5-01-E430G. These data indicate that IgG1-hDR5-01-E430G and IgG1-hDR5-05-E430G did not compete with each other for binding of DR5ECD-FcHisCtag, suggesting that they recognize distinct epitopes in the extracellular domain of human DR5. These data were confirmed by BLI using a classical sandwich assay, in which IgG1-hDR5-01-F405L or IgG1-hDR5-05-F405L (20 μg/ml in 10 mM Sodium Acetate pH 6.0, ForteBio Cat nr 18-1070) were immobilized on Amine-Reactive Second Generation biosensors (ForteBio Cat nr 18-5092). Subsequently, biosensors were incubated with DR5ECDdelHis (SEQ ID 28) (100 nM in Sample Diluent, ForteBio cat nr 18-1048) and binding of competing antibody (5 μg/mL in Sample Diluent) was analyzed (data not shown).
Example 8: Introduction of a Hexamerization-Enhancing Mutation Improves the Efficacy of Cell Death Induction by DR5-01 and DR5-05 Antibodies and of the Combination Thereof
A viability assay was performed to study the effect the hexamerization-enhancing mutation E430G in IgG1-DR5-01-K409R and IgG1-DR5-05-F405L on the capacity of the antibodies to kill human colon cancer cells COLO 205 and HCT 116. The antibodies were tested as single agent and as combinations of DR5-01 and DR5-05 antibodies. COLO 205 cells were harvested by pooling the culture supernatant containing non-adherent cells and trypsinized adherent cells. HCT 116 cells were harvested by trypsinization. Cells were passed through a cell strainer, pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 0.5×105 cells/mL. 100 μL of the single cell suspension (5,000 cells per well) was seeded in polystyrene 96-well flat-bottom plates (Greiner Bio-One, Cat nr 655182). 50 μL of a serial dilution antibody preparation series (range 0.05 to 20,000 ng/mL final concentrations in 5-fold dilutions) was added and incubated for 3 days at 37° C. In samples that were treated with a combination of two antibodies, the total antibody concentration in the assay was the same as in the samples that were treated with single antibodies. As a positive control, cells were incubated with 5 μM staurosporine (Sigma Aldrich, Cat nr S6942). The viability of the cultured cells was determined in a CellTiter-Glo luminescent cell viability assay (Promega, Cat nr G7571) that quantifies the ATP present, which is an indicator of metabolically active cells. From the kit, 20 μL luciferin solution reagent was added per well and mixed by shaking the plate for 2 minutes at 500 rpm. Next, plates were incubated for 1.5 hours at 37° C. 100 μL supernatant was transferred to a white OptiPlate-96 (Perkin Elmer, Cat nr 6005299) and luminescence was measured on an EnVision Multilabel Reader (PerkinElmer). Data were analyzed and plotted using non-linear regression (sigmoidal dose-response with variable slope) using GraphPad Prism software. FIG. 7 shows the percentage viable cells, as calculated using the following formula: % viable cells=[(luminescence antibody sample−luminescence staurosporine sample)/(luminescence no antibody sample−luminescence staurosporine sample)]*100.
FIG. 7 shows that introduction of the E430G mutation enhanced the potency of the chimeric antibodies IgG1-DR5-01-K409R and IgG1-DR5-05-F405L in both COLO 205 (A) and HCT 116 (B) cells. The combination of IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G was more potent than either antibody alone and more potent than the combination of the antibodies without the E430G mutation. The combination of IgG1-DR5-01-K409R and IgG1-DR5-05-F405L was more potent than either antibody alone. These data show that introduction of the hexamerization-enhancing mutation E430G resulted in enhanced induction of cell killing upon binding of the chimeric DR5 antibodies 01 and 05, both as single antibodies and in combination, with the combination being the most potent.
Example 9: Combining Two Non-Crossblocking DR5 Antibodies with Hexamerization-Enhancing Mutations Results in Enhanced Target Cell Killing
In Example 8 it is shown that combining the two non-crossblocking anti-DR5 antibodies IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G with hexamerization enhancing mutations resulted in enhanced killing on cancer cell lines compared to the efficacy of the single antibodies. Here, we compare the efficacy of two non-crossblocking versus two crossblocking anti-DR5 antibodies. A viability assay was performed to study the capacity of the combination of antibodies IgG1-chTRA8-F405L-E430G with either non-crossblocking antibody IgG1-DR5-01-K409R-E430G or crossblocking antibody IgG1-DR5-05-F405L-E430G to induce killing of HCT 116 colon cancer cells in comparison to the single antibodies. A crossblock ELISA for antibodies IgG1-chTRA8-F405L and IgG1-DR5-05-F405L was performed as described in Example 7 and confirmed by a sandwich binding assay on an Octet® HTX system (data not shown). The viability assay on HCT 116 cells was performed as described in Example 8 with a serial diluted antibody series ranging from 0.00005 to 20 μg/mL final concentrations in 5-fold dilutions. FIG. 8 shows that the efficacy of the single antibodies in killing of HCT116 cells was enhanced by combining the two non-crossblocking antibodies IgG1-chTRA8-F405L-E430G and IgG1-DR5-01-K409R-E430G (FIG. 8B) and not by combining the two crossblocking antibodies IgG1-chTRA8-F405L-E430G and IgG1-DR5-05-F405L-E430G (FIG. 8C).
Example 10: Capacity of the Combination of Non-Crossblocking Antibodies DR5-05+CONA and Bispecific Antibody DR5-05×CONA with Hexamerization-Enhancing Mutations to Induce Target Cell Killing
A viability assay was performed to study the capacity of another combination of two non-crossblocking antibodies (IgG1-CONA-K409R-E430G+IgG1-DR5-05-F405L-E345K) and its bispecific derivative BsAb IgG1-CONA-K409R-E430G×DR5-05-F405L-E345K to induce killing of HCT 116 colon cancer cells in comparison to the combination of antibodies and the bispecific antibody without hexamerization-enhancing mutation, respectively. A crossblock ELISA for antibodies IgG1-CONA-K409R and IgG1-DR5-05-F405L was performed as described in Example 7 and confirmed by a sandwich binding assay on an Octet® HTX system (data not shown). The viability assay on HCT 116 cells was performed as described in Example 8 with a serial diluted antibody series ranging from 0.01 to 20,000 ng/mL final concentrations in 5-fold dilutions. FIG. 9 shows that the combination of non-crossblocking antibodies IgG1-CONA-K409R-E430G+IgG1-DR5-05-F405L-E345K and BsAb IgG1-CONA-K409R-E430G×DR5-05-F405L-E345K with hexamerization-enhancing mutations showed enhanced efficacy in killing of HCT116 cells compared to these antibodies without the hexamerization-enhancing mutations E430G or E345K.
Example 11: Capacity of the DR5-01+DR5-05 Antibody Combination with E430G Hexamerization-Enhancing Mutation to Induce Target Cell Killing in Different Cancer Cell Lines
A viability assay was performed to study the capacity of the combination of human-mouse chimeric antibodies IgG1-DR5-01-K409R+IgG1-DR5-05-F405L with and without the hexamerization-enhancing mutation E430G to induce killing of COLO 205, HCT-15, HCT 116, HT-29 and SW480 colon cancer, B×PC-3, HPAF-II and PANC-1 pancreatic cancer, SNU-5 gastric cancer, A549 and SK-MES-1 lung cancer, and A375 skin cancer cells. Adherent cells were harvested by trypsinization and passed through a cell strainer. Cells were pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 0.5×105 cells/mL [COLO 205, HCT-15, SW480 and B×PC-3: RPMI 1640 with 25 mM Hepes and L-Glutamine (Lonza Cat nr BE12-115F)+10% DBSI (Life Technologies Cat nr 10371-029)+Pen/Strep (Lonza Cat nr DE17-603E); HCT116 and HT-29: McCoy's5A Medium with L-Glutamine and Hepes (Lonza, Cat nr BE12-168F)+10% DBSI+Pen/Strep; HPAF-II and SK-MES-1: Eagle's Minimum Essential Medium (EMEM, ATCC Cat nr 30-2003)+10% DBSI+Pen/Strep; PANC-1 and A375: DMEM 4.5 g/L Glucose without L-Gln with HEPES (Lonza Cat nr LO BE12-709F)+10% DBSI+1 mM L-Glutamine (Lonza Cat nr BE17-605E)+Pen/Strep; SNU-5: IMDM (Lonza Cat nr BE12-722F)+10% DBSI+Pen/Strep; A549: F-12K Medium (ATCC Cat nr 30-2004)+10% DBSI+1 mM L-Glutamine+Pen/Strep]. 100 μL of the single cell suspensions (5,000 cells per well) were seeded in polystyrene 96-well flat-bottom plates (Greiner Bio-One, Cat nr 655182) and incubated overnight at 37° C. Supernatant of the adherent cells was replaced by 150 μL antibody sample (final concentration 10 μg/mL) and incubated for 3 days at 37° C. As a positive control, cells were incubated with 5 μM staurosporine (Sigma Aldrich, Cat nr S6942). The viability of the cell cultures was determined in a CellTiter-Glo luminescent cell viability assay as described in Example 8. For all tested cell lines, the percentage viable cells was significant lower after incubation with 10 μg/mL of the antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G than after incubation with the non-target binding negative control antibody IgG1-b12 (FIG. 10). In all but two of the tested cell lines, the efficacy of the antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was significant better than for the combination IgG1-DR5-01-K409R+IgG1-DR5-05-F405L without hexamerization-enhancing mutation. These data indicate that the combination of chimeric DR5 antibodies with hexamerization-enhancing mutations IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was very effective in killing of cancer target cells of different origin, including colon, pancreatic, gastric, lung and skin cancer, without the requirement of a secondary cross-linking agent. There was no correlation between killing efficacy of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G and DR5 target expression levels (described in Example 2).
Example 12: Capacity of the Humanized DR5-01+DR5-05 Antibody Combination with E430G Hexamerization-Enhancing Mutation to Induce Target Cell Killing
A viability assay was performed to compare the potency of the combination of chimeric antibodies IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G with the potency of the combination of humanized antibodies IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G to induce killing of B×PC-3 and PANC-1 pancreatic cancer cells in vitro. Cells were harvested by trypsinization and passed through a cell strainer. Cells were pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 0.5×105 cells/mL. 100 μL of the single cell suspensions (5,000 cells per well) were seeded in polystyrene 96-well flat-bottom plates (Greiner Bio-One, Cat nr 655182) and incubated overnight at 37° C. Supernatant of the adherent cells was replaced by 150 μL antibody sample of a serial dilution antibody preparation series and incubated for 3 days at 37° C. As a positive control, cells were incubated with 5 μM staurosporine (Sigma Aldrich, Cat nr S6942). The viability of the cell cultures was determined in a CellTiter-Glo luminescent cell viability assay as described in Example 8. The combination of the humanized antibodies with hexamerization-enhancing mutation IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G showed similar dose-response curves as the combination of the corresponding chimeric antibodies IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G (FIG. 11).
Example 13: Optimization of Antibody IgG1-hDR5-01-E430G
Amino acid sequence N55-G56 was identified as a potential asparagine (Asn) deamidation motif in the CDR2 regions of the IgG1-hDR5-01 and IgG1-hDR5-05 heavy chains (SEQ ID NO:2). Deamidation at this position was mimicked by introduction of the N55D mutation in IgG1-hDR5-01-K409R and IgG1-hDR5-05-F405L to test the effect of deamidation on target binding. IgG1-hDR5-01-N55D-K409R and IgG1-hDR5-05-N55D-F405L were tested for binding to HCT 116 cells by FACS analysis as described in Example 3. FIG. 12A shows that mimicking deamidation by introduction of the N55D mutation resulted in strongly decreased binding of IgG1-hDR5-01-K409R on HCT 116 cells. In contrast, IgG1-hDR5-05-F405L and IgG1-hDR5-05-N55D-F405L showed comparable binding curves. To reduce the risk of Asn deamidation in the DR5-01 antibody, the G56T mutation was introduced in IgG1-hDR5-01-E430G and this antibody variant was tested for binding to HCT 116 cells by FACS analysis as described in Example 3. FIG. 12B shows that the mutation had no effect on the binding of IgG1-hDR5-01-E430G to HCT 116 cells.
A viability assay was performed to compare the capacity of the combination of humanized antibodies IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G with the capacity of the combination of humanized antibodies IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G to induce killing of B×PC-3 pancreatic cancer cells. Viability was assessed as described in Example 11 with 1,000 cells per well and antibody concentrations series ranging from 0.0001 to 10,000 ng/mL final concentrations in 4-fold dilutions in a total volume of 200 μL. FIG. 12C shows that introduction of the G56T mutation in IgG1-hDR5-01-E430G had no effect on the killing efficacy of the antibody in combination with IgG1-hDR5-05-E430G.
Example 14: Cell Death Induction by the Combination of Humanized Antibodies hDR5-01-G56T-E430G and hDR5-05-E430G Requires Fc:Fc Interactions to Form Hexamers
To analyse the requirement of antibody hexamer formation by IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G to induce cell death, we made use of the self-repulsing mutations K439E and S440K (Diebolder et al., Science. 2014 Mar. 14; 343(6176):1260-3). The Fc repulsion between antibodies that is introduced by the presence of either K439E or S440K in one IgG1 antibody or in a combination of antibodies results in inhibition of hexamerization, even in the presence of a hexamerization enhancing mutation such as E345K or E430G (WO2013/0044842). The repulsion by the K439E and S440K mutations is neutralized by combining both mutations in a mixture of two antibodies each harboring one or the other mutation, resulting in restoration of the Fc:Fc interactions and hexamerization. For both IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G, variants with either the K439E or S440K mutation were generated and tested in all different combinations. A viability assay was performed with serial dilution antibody preparation series ranging from 0.3 to 20,000 ng/mL total concentrations in 4-fold dilutions on B×PC-3 pancreatic and HCT-15 colon cancer cells as described in Example 11.
FIG. 13 shows that the combination of IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G variants that both harbor the same repulsion mutation (K439E or S440K) showed strongly diminished killing efficacy in B×PC-3 (A) and HCT-15 cells (B). Killing efficacy was restored when repulsion was neutralized by combining two antibodies each having one of the complementary mutations K439E or S440K. These data indicate that hexamerization by Fc-Fc interactions is required for the induction of cell death by IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G.
Example 15: Antibody Fc-Fc Interactions are Involved in DR5 Clustering and Induction of Apoptosis by the Antibody Combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G with Hexamerization Enhancing Mutations
To test the involvement of Fc-Fc-mediated antibody hexamerization in the induction of cell death by the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G, we made use of the 13-residue peptide DCAWHLGELVWCT (DeLano et al., Science 2000 Feb. 18; 287(5456):1279-83) that binds the Fc in a region containing the core amino acids in the hydrophobic patch that are involved in Fc-Fc interactions (Diebolder et al., Science. 2014 Mar. 14; 343(6176):1260-3). A viability assay on B×PC-3 cells was performed as described in Example 11 for the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G in presence or absence of the DCAWHLGELVWCT peptide. Briefly, after overnight incubation of the cells at 37° C., culture medium was removed and replaced by 100 μL culture medium containing a dilution series (range 0-100 μg/mL) of the Fc-binding DCAWHLGELVWCT peptide, a non-specific control peptide GWTVFQKRLDGSV, or no peptide. Next, 50 μL antibody samples (833 ng/mL final concentration) were added and incubated for 3 days at 37° C. The capacity of the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to induce killing of B×PC-3 cells was strongly inhibited by 100 μg/mL Fc-binding DCAWHLGELVWCT peptide (FIG. 14). These data indicate the involvement of Fc:Fc interactions in the capacity of the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G with hexamerization-enhancing mutations to induce DR5 clustering on the cell surface of cancer cells and induction of apoptosis.
Example 16: Capacity of Chimeric Antibody Combination DR5-01 and DR5-05 Antibodies with E430G Hexamerization Enhancing Mutation to Induce Cancer Cell Killing, at Different Combination Ratios
A viability assay was performed to study the capacity of the antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G to induce killing of B×PC-3 pancreatic cancer cells, when combined at different ratios of IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G. Cells were harvested by trypsinization and passed through a cell strainer. Cells were pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 0.5×105 cells/mL. 100 μL of the single cell suspensions (5,000 cells per well) were seeded in polystyrene 96-well flat-bottom plates (Greiner Bio-One, Cat nr 655182) and incubated overnight at 37° C. 50 μL antibody sample with different ratios of IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G (indicated as Ratio DR5-01:DR5-05 of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90 and 0:100 in serial dilution series ranging from 0.06 to 20 μg/mL final concentrations in 5-fold dilutions) was added and incubated for 3 days at 37° C. As a positive control, cells were incubated with 5 μM staurosporine (Sigma Aldrich, Cat nr S6942). The viability of the cell cultures was determined in a CellTiter-Glo luminescent cell viability assay as described in Example 8. At 20 μg/mL and 4 μg/mL total antibody concentrations, killing was equally effective at all tested antibody ratios containing both antibodies IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G. At 0.8 μg/mL and 0.16 μg/mL total antibody concentrations, all tested antibody ratios containing both antibodies IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G induced killing (FIG. 15).
Example 17: Capacity of the Combination of Humanized Antibodies DR5-01 and DR5-05 Antibodies with E430G Hexamerization Enhancing Mutation to Induce Cancer Cell Killing, at Different Combination Ratios
A viability assay was performed to study the capacity of the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to induce killing of B×PC-3 pancreatic and HCT-15 colon cancer cells, when combined at different antibody ratios. Generally, the experiments were performed as described in Example 16. Briefly, pre-attached cells (5,000 cells per well) were incubated for 3 days at 37° C. in 150 μL in polystyrene 96-well flat-bottom plates with different ratios of IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G (indicated in FIG. 16 as Ratio DR5-01:DR5-05 of 100:0, 98:2, 96:4, 94:6, 92:8, 90:10, 50:50, 10:90, 8:92, 6:94, 4:96, 2:98 and 0:100) at final antibody concentrations of 10 μg/mL for B×PC-3 and 20 μg/mL for HCT-15. The viability of the cell cultures was determined in a CellTiter-Glo luminescent cell viability assay as described in Example 8. Killing was equally effective at all tested antibody ratios containing both antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G (FIG. 16).
Example 18: The Combination of Humanized DR5-01+DR5-05 Antibodies with the E430G Hexamerization-Enhancing Mutation Induce Caspase-Dependent Cytotoxicity
A viability assay was performed to compare the cytotoxicity of the combination of humanized antibodies IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G in the presence and absence of a caspase inhibitor. PANC-1 and B×PC3 pancreatic cancer cells were harvested by trypsinization and passed through a cell strainer. Cells were pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 0.5×105 cells/mL. 100 μL of the single cell suspensions (5,000 cells per well) were seeded in polystyrene 96-well flat-bottom plates (Greiner Bio-One, Cat nr 655182) and incubated overnight at 37° C. 25 μL pan-caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK, 5 μM end concentration in 150 μL, Bachem, Cat nr 4026865.0005) was added to the cell cultures and incubated for one hour at 37° C. before adding 25 μL antibody sample of a serial dilution antibody preparation series (range 1 to 20 μg/mL final concentrations in 4-fold dilutions) and further incubation for 3 days at 37° C. As a positive control, cells were incubated with 5 μM staurosporine (Sigma Aldrich, Cat nr S6942). Recombinant human TRAIL/APO-2L (eBioscience, Cat nr BMS356) was used at 6 μg/mL final concentration. The viability of the cell cultures was determined in a CellTiter-Glo luminescent cell viability assay as described in Example 8. The combination of the humanized antibodies with hexamerization-enhancing mutation IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G was unable to reduce the viability of PANC-1 and B×PC3 pancreatic cancer cells in presence of the pan-caspase inhibitor Z-VAD-FMK, indicating that the combination of IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G induced caspase-dependent programmed cell death (FIG. 17). This was also shown for the natural DR5 ligand TRAIL.
Example 19: Cell Death Induction Upon Binding of the Combination of Chimeric DR5-01 and DR5-05 Antibodies on COLO 205 Colon Cancer Cells, as Assessed by Annexin V/Propidium Iodide and Active Caspase-3 Staining
The kinetics of cell death induction was analyzed by Annexin V/Propidium Iodide (PI) double staining and active caspase-3 staining. Annexin-V binds phosphatidylserine that is exposed on the cell surface after initiation of programmed cell death, which is a reversible process. PI is a dye that intercalates into double-stranded DNA and RNA when it enters cells. Because PI cannot pass intact plasma and nuclear membranes, it will not stain living cells but only enter and stain dying cells that have decreased membrane integrity. Due to these characteristics, the Annexin V/PI double staining can be applied to discriminate between initiation (Annexin V-positive/PI-negative) and irreversible (Annexin V-positive/PI-positive) programmed cell death. Caspase-3 is activated by both the extrinsic death receptor-induced and intrinsic mitochondrial cell death pathways. Therefore, active caspase-3 is also a marker for initiation of the death cascade. The induction of cell death upon binding of the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was analyzed in the DR5-positive COLO 205 colon cancer cells. Cells were harvested by pooling the culture supernatant containing non-adherent cells and trypsinized adherent cells. Cells were passed through a cell strainer, pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 0.2×106 cells/mL. 500 μL of the single cell suspensions (100,000 cells per well) were seeded in 24-wells flat-bottom culture plates (Greiner Bio-One, Cat nr 662160) and incubated for 16 hours at 37° C. 500 μL antibody sample was added (1 μg antibody final concentration) and incubated for 5 hours or 24 hours at 37° C. As a positive control, cells were incubated with 5 μM staurosporine (Sigma Aldrich, Cat nr S6942). Cells were washed once with 250 μL 1×PBS. Adherent cells were harvested by incubating with 100 μL 0.05% trypsin for 10 minutes at 37° C. 200 μL medium was added to the trypsinized cells and cells were transferred to a 96-wells round-bottom FACS plate (Greiner Bio-One, Cat nr 650101) and pooled with the non-adherent cells. Cells were pelleted by centrifugation for 5 minutes at 1,200 rpm, resuspended in 200 μL ice cold PBS and divided into two samples of 100 μL in 96-Wells round-bottom FACS plates for the Annexin V/PI and active caspase-3 staining, respectively.
Annexin V/PI double staining was performed using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, Cat nr 556547). Cells were washed once with ice cold PBS and incubated in 50 μL Annexin V/PI Staining Solution (Annexin V-FITC 1:00 and PI 1:25) for 15 minutes at 4° C. Cells were washed with 100 μL Binding Buffer, resuspended in 20 μL Binding Buffer and fluorescence was measured on an iQue Screener (IntelliCyt) within 1 hour. Data were analyzed and plotted using GraphPad Prism software.
Active caspase-3 staining was performed using the PE Active Caspase-3 Apoptosis Kit (BD Pharmingen, Cat nr 550914). Cells were washed once with ice cold PBS, resuspended in 100 μL Cytofix/Cytoperm Fixation and Permeabilization Solution and incubated for 20 minutes on ice. Cells were pelleted at room temperature, washed twice with 100 μL 1× Perm/Wash Buffer and resuspended in 100 μL PE Rabbit Anti-Active Caspase-3 (1:10) for an incubation of 30 minutes at room temperature. Cells were pelleted at room temperature, washed once with 100 μL 1× Perm/Wash Buffer and resuspended in 20 μL 1× Perm/Wash Buffer. Fluorescence was measured on an iQue Screener. Data were analyzed and plotted using GraphPad Prism software.
FIG. 18 shows that, after 5 hours of incubation, the combination of the chimeric antibodies IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G efficiently induced the early stages of cell death as indicated by an increase in the percentage of Annexin V-positive/PI-negative (A) and Active Caspase-3-positive cells (B), compared to the negative control antibody IgG1-b12. The percentage of Annexin V-positive/PI-negative and Active Caspase-3 positive cells was higher in cells treated with the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G compared to the combination of the DR5 antibodies without the E430G mutation (IgG1-DR5-01-K409R+IgG1-DR5-05-F405L) or any of the single antibodies. At the 5 hour time point, the percentage of AnnexinV/PI double-positive cells was comparable to background levels in all samples (C).
After 24 hours incubation, the percentage of Annexin V/PI double-positive cells (D) was enhanced in samples treated with IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G, indicating that the cells had entered the irreversible stages of cell death. Also at this stage, the effect of the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was stronger (larger increase in the percentage of Annexin V/PI double-positive cells (E)) than in samples treated with a combination of DR5 antibodies without the E430G mutation (IgG1-DR5-01-K409R+IgG1-DR5-05-F405L) or any of the single antibodies. At the same time point, the percentage of Active Caspase 3 positive cells was highest in cells treated with IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G.
These data indicate that the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G induces both the early and late stages of cell death in COLO 205 colon cancer cells, and does so more effectively than the combination of the antibodies without the E430G hexamerization enhancing mutation.
Example 20: Caspase-3 and -7 Activation Upon Binding of the Combination of Chimeric DR5-01 and DR5-05 Antibodies with Hexamerization-Enhancing Mutation on COLO 205 Colon Cancer Cells
In example 19 it was described that incubation with the combination of chimeric DR5 antibodies IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G induced caspase-3 activation in COLO 205 colon cancer cells. The percentage of active caspase-3-positive cells was higher after 5 hours than after 24 hours of incubation with the antibody combination. In this example, Caspase-3/7 activation was measured in time using the Caspase-Glo 3/7 assay (Promega, Cat nr G8091), in which a substrate with the Caspase-3/7 recognition motif DEVD releases aminoluciferin, a substrate of luciferase, upon cleavage. Cells were harvested by pooling the culture supernatant containing non-adherent cells and trypsinized adherent COLO 205. Cells were passed through a cell strainer, pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 0.8×105 cells/mL. 25 μL of the single cell suspensions (2,000 cells per well) were seeded in 384-wells culture plates (Perkin Elmer, Cat nr 6007680) and incubated for 16 hours at 37° C. 25 μL antibody sample was added (1 μg antibody final concentration) and incubated for 1, 2, 5 and 24 hours at 37° C. Plates were removed from the incubator to let the temperature decrease till room temperature. Cells were pelleted by centrifugation for three minutes at 300 g. 25 μL supernatant was removed and replaced by 25 μL Caspase-Glo 3/7 Substrate. After mixing by shaking for one minute at 500 rpm, the plates were incubated for one hour at room temperature. Luminescence was measured on an EnVision Multilabel Reader (PerkinElmer).
FIG. 19 shows that in the time course of 1, 2 to 5 hours, caspase-3/7 activation was induced by the antibody combinations IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G and IgG1-DR5-01-K409R+IgG1-DR5-05-F405L, and for the bispecific DR5 antibody BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G. After 24 hours, caspase-3/7 activation was almost reduced to baseline levels for all tested DR5 antibodies. After 1 hour, caspase-3/7 activation was already observed in cells that had been treated with the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G, whereas no caspase-3/7 activation was observed in cells that had been treated with the combination of IgG1-DR5-01-K409R+IgG1-DR5-05-F405L without the hexamerization-enhancing mutation.
Similarly, at 2 and 5 hours, the caspase-3/7 activation induced by the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was stronger than for the combination of IgG1-DR5-01-K409R+IgG1-DR5-05-F405L. These data indicate that the combination of chimeric DR5 antibodies with the hexamerization enhancing mutation IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G induced more rapid and more potent Caspase-3/7 activation than the combination of antibodies without the hexamerization enhancing mutation.
Example 21: The Potency of the Antibody Combination of Chimeric DR5-01 and DR5-05 with the E430G Hexamerization-Enhancing Mutation is Independent of the Presence of a Secondary Fc Crosslinker
A viability assay was performed to compare the capacity of the antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G in the absence and presence of secondary antibody crosslinker to induce killing of COLO 205 colorectal and B×PC-3 and PANC-1 pancreatic cancer cells. For comparison, two DR5 antibodies that are known to show enhanced killing in the presence of a secondary antibody crosslinker, IgG1-CONA and IgG1-chTRA8-F405L, were tested in the same settings. Cells were harvested by trypsinization and passed through a cell strainer. Cells were pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 0.5×105 cells/mL. 100 μL of the single cell suspensions (5,000 cells per well) were seeded in polystyrene 96-well flat-bottom plates (Greiner Bio-One, Cat nr 655182) and incubated overnight at 37° C. Supernatant of the adherent cells was replaced by 150 μL antibody sample (final concentration 10 μg/mL) in the absence or presence of F(ab′)2 fragments of a goat-anti-human IgG antibody (1/150; Jackson ImmunoResearch; Cat nr 109-006-098) and incubated for 3 days at 37° C. As a positive control for cell killing, cells were incubated with 5 μM staurosporine (Sigma Aldrich, Cat nr S6942). The viability of the cell cultures was determined in a CellTiter-Glo luminescent cell viability assay as described in Example 8. The antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G induced significant killing compared to the negative control of COLO 205, B×PC-3 and PANC-1 cancer cells, both in presence or absence of an Fc crosslinker (FIG. 20). In contrast, DR5 antibodies IgG1-DR5-CONA and IgG1-DR5-chTRA8-F405L did not induce target cell killing in the absence of an Fc crosslinker. Fc crosslinking induced killing by IgG1-DR5-CONA and IgG1-DR5-chTRA8-F405L in COLO 205 and B×PC-3 cells, although with significantly lower potency than the antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G in presence or absence of crosslinker. These data indicate that killing of COLO 205, B×PC-3 and PANC-1 cancer cells by the antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G is independent of the presence of a secondary Fc crosslinker and that this crosslinker-independent killing is more efficient than for Fc-crosslinked IgG1-DR5-CONA and IgG1-DR5-chTRA8-F405L.
Example 22: Introduction of the K409R Mutation in IgG1-hDR5-01-430G and the F4051 Mutation in IgG1-hDR5-05-E430G has No Effect on the Potency of the Combination of Humanized Antibodies IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G
In many of the experiments described in this application, the anti-DR5 antibodies IgG1-01 and IgG1-05 contain in the IgG Fc domain the K409R and F405L (EU numbering index) mutation, respectively. These mutations enable the generation of DR5 bispecific antibodies by Fab-arm-exchange reaction between IgG1-01-K409R and IgG1-05-F405L under controlled reducing conditions as described in WO2011/131746. Without Fab-arm exchange, human IgG1 antibodies bearing the K409R and F405L mutations are thought to show the same functional characteristics as wild type human IgG1 (Labrijn et al., Proc Natl Acad Sci USA. 2013 Mar. 26; 110(13):5145-50). Here we show that the presence of the K409R or F405L mutations has no effect on the capacity of the combination of the parental IgG1-01 and IgG1-05 antibodies to induce cell death in DR5-positive tumor cells in vitro. A viability assay was performed to compare the capacity of the combination of humanized antibodies IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G with the capacity of the combination of humanized antibodies IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G to induce killing of B×PC-3 pancreatic cancer cells. The viability assay on The B×PC-3 was performed as described in Example 11 with a serial diluted antibody series ranging from 0.001 to 20,000 ng/mL final concentrations in 4-fold dilutions. The B×PC-3 pancreatic cancer cell line showed similar viability curves after incubation with the combination of the humanized antibodies IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G as with the combination of the humanized antibodies IgG1-hDR5-01-E430G+IgG1-hDR5-05-E430G (FIG. 21). These data indicate that the K409R and F405L mutations had no effect on the potency of the combination of the humanized DR5-01 and DR5-05 antibodies with E430G hexamerization enhancing mutation.
Example 23: Chimeric Bispecific Antibody IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G Induces Killing of DR5-Positive Tumor Cells
A bispecific antibody targeting two different DR5 epitopes was generated by Fab-arm exchange between the chimeric antibodies IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G as described in Example 1. A viability assay was performed as described in Example 11 to test the capacity of 10 μg/mL of the chimeric BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G to induce killing of cancer cells of different tissue origin (COLO 205 colorectal cancer, A375 skin cancer, SK-MES-1 lung cancer, B×PC-3 pancreatic cancer and SNU-5 gastric cancer cell lines). For all tested cell lines, the percentage viable cells was significantly lower when incubated with 10 μg/mL of the chimeric BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G antibody compared to the non-target binding negative control antibody IgG1-b12 (FIG. 22). These data indicate that the bispecific anti-DR5×DR5′ antibody with hexamerization-enhancing mutation E430G induced killing of cancer cells of different origin, including colon, pancreatic, gastric, lung and skin cancer, without the requirement of a secondary crosslinker.
Example 24: The Potency of the Chimeric BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F4051-E430G is Independent of the Presence of a Secondary Fc Crosslinker
A viability assay was performed to compare the potency of the chimeric BsAb IgG1-DR5-01-K409R-E430G×IgG1-DR5-05-F405L-E430G in the absence and presence of a secondary antibody crosslinker to induce killing of B×PC-3 pancreatic and COLO 205 colon cancer cells as described in Example 21. For comparison, two DR5 antibodies that are known to show enhanced killing in the presence of a secondary antibody crosslinker, IgG1-CONA and IgG1-chTRA8-F405L, were tested in the same setting. The chimeric BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G showed significant killing compared to the negative control of COLO 205 and B×PC-3 cancer cells, both in presence or absence of an Fc crosslinker (FIG. 23). In contrast, DR5 antibodies IgG1-DR5-CONA and IgG1-DR5-chTRA8-F405L only induced killing in the presence of Fc crosslinker
Example 25: Cell Death Induction Upon Binding of the BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G on COLO 205 Colon Cancer Cells, as Assessed by Annexin V/Propidium Iodide and Active Caspase-3 Staining
The kinetics of cell death induction by 1 μg/mL BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G on COLO 205 cells was analyzed by Annexin V/Propidium Iodide (P1) double staining and active caspase-3 staining as described in Example 19.
FIG. 24 shows that, after 5 hours of incubation, BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G efficiently induced the early stages of cell death as indicated by an increase in the percentage of Annexin V-positive/PI-negative (A) and Active Caspase-3-positive cells (B), compared to the negative control antibody IgG1-b12. The percentage of Annexin V-positive/PI-negative and Active Caspase-3 positive cells was higher in cells that had been treated with BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G compared to the bispecific antibody without the E430G mutation (BsAb IgG1-DR5-01-K409R×DR5-05-F405L) or any of the monospecific antibodies. At the 5 hour time point, the percentage of AnnexinV/PI double positive cells was comparable to background levels in all samples (C). After 24 hours incubation, the percentage of Annexin V/PI double-positive cells (D) was enhanced in samples treated with BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G, indicating that the cells had entered the irreversible stages of cell death. Also at this stage, the effect of BsAb IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G was stronger (larger increase in the percentage of Annexin V/PI double-positive cells (E) than in samples treated with the bispecific antibody without the E430G mutation (BsAb IgG1-DR5-01-K409R×DR5-05-F405L) or any of the monospecific antibodies. At the same time point, the percentage of Active Caspase 3 positive cells was highest in cells treated with BsAB IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G.
These data indicate that BsAB IgG1-DR5-01-K409R-E430G×DR5-05-F405L-E430G induces both the early and late stages of cell death in COLO 205 colon cancer cells, and does so more effectively than the bispecific antibody without the E430G hexamerization enhancing mutation.
Example 26: In Vivo Efficacy of DR5-01 and DR5-05 Antibody Variants With and Without a Hexamerization-Enhancing Mutation in a Subcutaneous COLO 205 Colon Cancer Xenograft Model
The in vivo anti-tumor efficacy of different anti-DR5 antibodies and the combination of DR5-01+DR5-05 antibodies with hexamerization enhancing mutation was evaluated in a subcutaneous model with COLO 205 human colon cancer cells. At day 0, cells were harvested by pooling the culture supernatant containing non-adherent cells and trypsinized adherent cells. 3×106 cells were injected in a volume of 200 μL PBS into the flank of 6-11 weeks old female SCID mice (C.B-17/IcrHan® Hsd-Prkdcscid; Harlan). All experiments and animal handlings were approved by the local authorities, and were conducted according to all applicable international, national and local laws and guidelines. Tumor development was monitored at least twice per week by caliper (PLEXX) measurement as 0.52×(length)×(width)2. Tumors were measured until an endpoint tumor volume of 1,500 mm3, until tumors showed ulcerations, until serious clinical signs were observed, or until tumor growth blocked movements of the mouse. At day 6, the average tumor volume was ˜200 mm3 and the mice were sorted into groups with equal tumor size variance (Table 2 below). Mice were treated by intraperitoneal (i.p.) injection of 100 μg antibody in 200 μL PBS on day 6 and 13 (5 mg/kg per dose). To check for correct antibody administration, blood samples were obtained for IgG serum determination three days after the first dose. Three individual mice had no detectable human IgG plasma level and were excluded from statistical analysis (see Table 2 below). For the other mice, human antibody plasma concentrations were according to the expectations when assuming a 2-compartment model with Vcen=50 mL/kg, Vs=100 mL/kg and an elimination half-life of 11.6 days (data not shown). Tumors were measured until 16 weeks after tumor inoculation.
TABLE 2
Treatment groups and dosing
Dosing day after
# mice
# analyzed
Antibody
Total antibody dose
tumor inoculation
8
7
IgG1-DR5-01-K409R-E430G (50 μg)
100 μg (5 mg/kg)
6, 13
IgG1-DR5-05-F405L-E430G (50 μg)
7
7
IgG1-DR5-05-F405L (100 μg)
100 μg (5 mg/kg)
6, 13
8
8
IgG1-DR5-01-K409R-E430G (100 μg)
100 μg (5 mg/kg)
6, 13
8
8
IgG1-DR5-05-F405L-E430G (100 μg)
100 μg (5 mg/kg)
6, 13
8
8
IgG1-CONA (100 μg)
100 μg (5 mg/kg)
6, 13
8
7
BsAb DR5-01-K409R ×
100 μg (5 mg/kg)
6, 13
DR5-05-F405L (100 μg)
8
8
BsAb DR5-01-K409R-E430G ×
100 μg (5 mg/kg)
6, 13
DR5-05-F405L-E430G (100 μg)
8
7
IgG1-b12 (100 μg)
100 μg (5 mg/kg)
6, 13
FIG. 25A shows mean tumor volumes per treatment group in time. FIG. 25B represents mean tumor volumes on day 23 after tumor inoculation, when all groups were still intact. All anti-DR5 antibody samples inhibited tumor growth significantly compared to the negative control antibody IgG1-b12 (non-parametric ANOVA analysis (Kruskal-Wallis) followed by Dunn's multiple comparison test on day 23: p<0.0001). Complete tumor abrogation was observed for the combination of DR5-01+DR5-05 antibodies with hexamerization-enhancing mutation (IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G), for the bispecific antibodies with and without hexamerization-enhancing mutation (BsAb DR5-01-K409R×DR5-05-F405L and BsAb DR5-01-K409R-E430G×DR5-05-F405L-E430G), and for the anti-DR5 antibodies with hexamerization-enhancing mutation (IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G). IgG1-CONA and IgG1-DR5-05-F405L without hexamerization-enhancing mutation strongly inhibited tumor growth compared to IgG1-b12, but did not result in complete tumor abrogation.
FIG. 25C shows a Kaplan-Meier plot of tumor progression, with a cutoff set at a tumor volume>750 mm3. Compared to mice treated with negative control antibody IgG1-b12, tumor outgrowth was significantly delayed in all groups treated with anti-DR5 antibodies (Mantel-Cox analysis at tumor size cut-off 750 mm3: p<0.001). At the end of the study (day 112), the group of mice treated with the combination of DR5-01+DR5-05 antibodies with hexamerization enhancing mutation (IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G) showed significant less mice with tumor outgrowth than the conatumumab group (Fisher's exact contingency test p<0.01).
These data show that introduction of the E430G hexamerization-enhancing mutation in IgG1-DR5-05-F405L resulted in enhanced tumor inhibition in the subcutaneous COLO 205 colon cancer tumor model compared to IgG1-DR5-05-F405L without the hexamerization-enhancing mutation. Both DR5-01 and DR5-05 antibodies with hexamerization enhancing mutation (IgG1-DR5-01-K409R-E430G and IgG1-DR5-05-F405L-E430G), the bispecific antibodies with and without hexamerization enhancing mutation (BsAb DR5-01-K409R×DR5-05-F405L and BsAb DR5-01-K409R-E430G×DR5-05-F405L-E430G) and the combination of antibodies with hexamerization-enhancing mutation (IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G) showed better tumor inhibition as IgG1-CONA and IgG1-DR5-05-F405L without hexamerization-enhancing mutation.
Example 27: In Vivo Efficacy of Different Doses of the Antibody Combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G in a Subcutaneous COLO 205 Colon Cancer Xenograft Model
The in vivo anti-tumor efficacy of different doses IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and compared to an equivalent dosing of IgG1-CONA in the subcutaneous COLO 205 human colon cancer xenograft model. Tumor cell inoculation, mice handling, tumor outgrowth measurements and endpoint determination were performed as described in Example 26. At day 10, the average tumor volume was ˜400 mm3 and the mice were sorted into groups with equal tumor size variance (Table 3 below). Mice were treated by intravenous (i.v.) injection of 40 μg (2 mg/kg), 10 μg (0.5 mg/kg) or 2 μg (0.1 mg/kg) antibody in 100 μL PBS on day 10. Mice in the control group were treated with 40 μg (2 mg/kg) IgG1-b12. Tumors were measured until 17 weeks after tumor inoculation.
TABLE 3
Treatment groups and dosing
Dosing day after
# mice
Antibody
Total antibody dose
tumor inoculation
8
IgG1-DR5-01-K409R-E430G (20 μg)
40 μg
(2 mg/kg)
10
IgG1-DR5-05-F405L-E430G (20 μg)
8
IgG1-DR5-01-K409R-E430G (5 μg)
10 μg
(0.5 mg/kg)
10
IgG1-DR5-05-F405L-E430G (5 μg)
8
IgG1-DR5-01-K409R-E430G (1 μg)
2 μg
(0.1 mg/kg)
10
IgG1-DR5-05-F405L-E430G (1 μg)
8
IgG1-CONA (40 μg)
40 μg
(2 mg/kg)
10
8
IgG1-CONA (10 μg)
10 μg
(0.5 mg/kg)
10
8
IgG1-CONA (0.1 μg)
2 μg
(0.1 mg/kg)
10
8
IgG1-b12 (40 μg)
40 μg
(2 mg/kg)
10
FIG. 26A shows mean tumor volumes per treatment group. Treatment with a single dose of 0.5 mg/kg or 2 mg/kg of the antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G resulted in complete tumor regression until the study was stopped on day 126. Treatment with 0.5 mg/kg and 2 mg/kg IgG1-CONA also induced tumor regression, but the regression was incomplete with recurring tumor outgrowth in all mice or almost all (7/8) mice, respectively. At 0.1 mg/kg, neither IgG1-CONA nor the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G showed anti-tumor activity. FIG. 26B shows that on day 16 after tumor inoculation, tumor inhibition by 2 mg/kg and 0.5 mg/kg IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was significantly better compared to an equivalent dose IgG1-CONA (unpaired t-test).
FIG. 26C shows a Kaplan-Meier plot of tumor progression, with a cutoff set at a tumor volume>500 mm3. At a dose of 0.5 mg/kg and 2 mg/kg, the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G and IgG1-CONA significantly inhibited tumor growth progression compared to the negative control antibody IgG1-b12 (p<0.001 Mantel-Cox analysis at tumor size cut-off 500 mm3). At a dose of 0.5 mg/kg inhibition of tumor growth progression was significantly better for the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G to an equivalent dose IgG1-CONA.
These data indicate that the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G had stronger anti-tumor efficacy compared to IgG1-CONA, since dosed at 2 mg/kg the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G significantly reduced tumor load at day 16 compared to IgG1-CONA, and at 0.5 mg/kg the IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G combination significantly reduced tumor load at day 16 and prolonged progression free survival time (tumor size cut-off 500 mm3) compared to IgG1-CONA.
Example 28: In Vivo Efficacy of Different Doses of the Antibody Combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G in a Subcutaneous B×PC-3 Pancreatic Cancer Xenograft Model
The in vivo anti-tumor efficacy of different doses IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and compared to an equivalent dosing of IgG1-CONA-F405L in the subcutaneous B×PC-3 human pancreatic cancer xenograft model. At day 0, adherent cells were harvested by trypsinization. 5×106 cells were injected in a volume of 100 μL PBS into the flank of 6-11 weeks old female SCID mice (C.B-17/IcrHan® Hsd-Prkdcscid; Harlan). Mice handling, tumor outgrowth measurements and endpoint determination were performed as described in Example 26. At day 10, the average tumor volume was ˜250 mm3 and the mice were sorted into groups with equal tumor size variance (Table 4 below). Mice were treated by i.v. injection of 200 μg (10 mg/kg), 40 μg (2 mg/kg) or 10 μg (0.5 mg/kg) antibody in 200 μL PBS on day 20 and 28. Mice in the control group were treated with 200 μg (10 mg/kg) IgG1-b12. To check for correct antibody administration, blood samples were obtained for IgG serum determination one week after dosing. Tumors were measured until 10 weeks after tumor inoculation.
TABLE 4
Treatment groups and dosing
Dosing day after
# mice
Antibody
Total antibody per dose
tumor inoculation
8
IgG1-DR5-01-K409R-E430G (20 μg)
200 μg
(10 mg/kg)
20, 28
IgG1-DR5-05-F405L-E430G (20 μg)
8
IgG1-DR5-01-K409R-E430G (5 μg)
40 μg
(2 mg/kg)
20, 28
IgG1-DR5-05-F405L-E430G (5 μg)
8
IgG1-DR5-01-K409R-E430G (1 μg)
10 μg
(0.5 mg/kg)
20, 28
IgG1-DR5-05-F405L-E430G (1 μg)
8
IgG1-CONA-F405L (40 μg)
200 μg
(10 mg/kg)
20, 28
8
IgG1-CONA-F405L (10 μg)
40 μg
(2 mg/kg)
20, 28
8
IgG1-CONA-F405L (0.1 μg)
10 μg
(0.5 mg/kg)
20, 28
8
IgG1-b12 (40 μg)
200 μg
(10 mg/kg)
20, 28
FIG. 27A shows median tumor volumes per treatment group. All tested doses of the antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth compared to the negative control antibody IgG1-b12, whereas the IgG1-CONA-F405L treatment groups did not. FIG. 27B shows that on day 48 after tumor inoculation, tumor growth inhibition by the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was significantly better than equivalent doses IgG1-CONA-F405L (unpaired t-test, p<0.05).
FIG. 27C shows a Kaplan-Meier plot of tumor progression, with a cutoff set at a tumor volume>500 mm3. The combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G significantly inhibited tumor growth progression compared to the negative control antibody IgG1-b12 and compared to IgG1-CONA-F405L (Mantel-Cox analysis at tumor size cutoff 500 mm3: p<0.001).
These data indicate that the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and that anti-tumor efficacy was significantly better than for equivalent doses of IgG1-CONA-F405L in an in vivo B×PC-3 human pancreatic cancer xenograft model.
Example 29: In Vivo Efficacy of Different Doses of the Antibody Combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G in a Subcutaneous A375 Skin Cancer Xenograft Model
The in vivo anti-tumor efficacy of different doses IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and compared to an equivalent dosing of IgG1-CONA-F405L in the subcutaneous A375 human skin cancer xenograft model. At day 0, adherent cells were harvested by trypsinization. 5×106 cells were injected in a volume of 100 μL PBS into the flank of 6-11 weeks old female SCID mice (C.B-17/IcrHan® Hsd-Prkdcscid; Harlan). Mice handling, tumor outgrowth measurements and endpoint determination were performed as described in Example 26. At day 19, the average tumor volume was ˜250 mm3 and the mice were sorted into groups with equal tumor size variance (Table 5 below). Mice were treated by i.v. injection of 200 μg (10 mg/kg), 40 μg (2 mg/kg) or 10 μg (0.5 mg/kg) antibody in 200 μL PBS on day 19 and 26. Mice in the control group were treated with 200 μg (10 mg/kg) IgG1-b12. To check for correct antibody administration, blood samples were obtained for IgG serum determination one week after dosing. Tumor volumes were analyzed until 7 weeks after tumor inoculation.
TABLE 5
Treatment groups and dosing
Dosing day after
# mice
Antibody
Total antibody dose
tumor inoculation
8
IgG1-DR5-01-K409R-E430G (20 μg)
200 μg
(10 mg/kg)
19, 26
IgG1-DR5-05-F405L-E430G (20 μg)
8
IgG1-DR5-01-K409R-E430G (5 μg)
40 μg
(2 mg/kg)
19, 26
IgG1-DR5-05-F405L-E430G (5 μg)
8
IgG1-DR5-01-K409R-E430G (1 μg)
10 μg
(0.5 mg/kg)
19, 26
IgG1-DR5-05-F405L-E430G (1 μg)
8
IgG1-CONA-F405L (40 μg)
200 μg
(10 mg/kg)
19, 26
8
IgG1-CONA-F405L (10 μg)
40 μg
(2 mg/kg)
19, 26
8
IgG1-CONA-F405L (0.1 μg)
10 μg
(0.5 mg/kg)
19, 26
8
IgG1-b12 (40 μg)
200 μg
(10 mg/kg)
19, 26
FIG. 28A shows median tumor volumes per treatment group. All tested doses of the antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth compared to the negative control antibody IgG1-b12, whereas the IgG1-CONA-F405L treatment groups did not. FIG. 28B shows that on day 29 after tumor inoculation, the average tumor size in mice treated with the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was smaller than in mice treated with IgG1-b12 (p<0.05 for all dose levels, One-Way ANOVA with Dunnet's correction for multiple comparisons), and that the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was significantly more potent than IgG1-CONA-F405L (Mann Whitney test, p<0.05) at equivalent doses
These data indicate that the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and that anti-tumor efficacy was significantly better than for equivalent doses of IgG1-CONA-F405L in an in vivo A375 human skin cancer xenograft model.
Example 30: In Vivo Efficacy of Different Doses of the Antibody Combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G in a Subcutaneous HCT-15 Colon Cancer Xenograft Model
The in vivo anti-tumor efficacy of different doses IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and compared to an equivalent dosing of IgG1-CONA in the subcutaneous HCT-15 human colon cancer xenograft model at CrownBiosciences, Taicang, China. The cells were maintained in vitro as a monolayer culture in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37° C. in an atmosphere of 5% CO2 in air. Adherent cells in an exponential growth phase were harvested by trypsin-EDTA treatment. 5×106 cells were injected in a volume of 100 μL PBS into the flank of 6-8 weeks old female BALB/c nude mice (Shanghai Laboratory Animal Center). The care and use of animals during the study were conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Tumor volumes were measured twice weekly in two dimensions using a caliper, and the volume was expressed in mm3 using the formula: V=0.5 a×b2 where a and b are the long and short diameters of the tumor, respectively. Eleven days after tumor inoculation, the mean tumor size reached 186 mm3 and mice were assigned into groups using randomized block design and treatments were started. Mice were treated twice according to a Q7D regimen by i.v. injection of 200 μg (10 mg/kg), 40 μg (2 mg/kg) or 10 μg (0.5 mg/kg) antibody in 10 μL PBS per g body weight. Mice in the control group were treated in parallel with 200 μg (10 mg/kg) IgG1-b12. After tumor inoculation, welfare of the animals was checked daily and tumor volumes were measured twice weekly.
TABLE 6
Treatment groups and dosing, Example 30
Dosing day after
# mice
# analyzed
Antibody
Total antibody dose
tumor inoculation
8
8
IgG1-DR5-01-K409R-E430G (20 μg)
200 μg
(10 mg/kg)
11, 18
IgG1-DR5-05-F405L-E430G (20 μg)
8
8
IgG1-DR5-01-K409R-E430G (5 μg)
40 μg
(2 mg/kg)
11, 18
IgG1-DR5-05-F405L-E430G (5 μg)
8
8
IgG1-DR5-01-K409R-E430G (1 μg)
10 μg
(0.5 mg/kg)
11, 18
IgG1-DR5-05-F405L-E430G (1 μg)
8
8
IgG1-CONA (40 μg)
200 μg
(10 mg/kg)
11, 18
8
8
IgG1-CONA (10 μg)
40 μg
(2 mg/kg)
11, 18
8
8
IgG1-CONA (0.1 μg)
10 μg
(0.5 mg/kg)
11, 18
8
8
IgG1-b12 (40 μg)
200 μg
(10 mg/kg)
11, 18
FIG. 29A shows mean tumor volumes per treatment group. All tested doses of the antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth compared to the negative control antibody IgG1-b12, whereas IgG1-CONA did not. FIG. 29B shows that on day 17 after start of treatment, tumor growth inhibition by the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was significantly better than equivalent doses IgG1-CONA (Unpaired t test, p<0.05).
FIG. 29C shows a Kaplan-Meier plot of tumor progression, with a cutoff set at a tumor volume>500 mm3. The combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G significantly inhibited tumor growth progression compared to negative control antibody IgG1-b12 and compared to an equivalent dose IgG1-CONA (Mantel-Cox analysis at tumor size cutoff 500 mm3: p<0.001).
These data indicate that the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and that anti-tumor efficacy was significantly better than for equivalent doses of IgG1-CONA in an in vivo xenograft model with HCT-15 human colon cancer cells.
Example 31: In Vivo Efficacy of Different Doses of the Antibody Combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G in a Subcutaneous SW480 Colon Cancer Xenograft Model
The in vivo anti-tumor efficacy of different doses IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and compared to an equivalent dosing of IgG1-CONA in the subcutaneous SW480 human colon cancer xenograft model at CrownBiosciences, Taicang, China. The cells were maintained in vitro as a monolayer culture in L-15 medium supplemented with 10% fetal bovine serum at 37° C. in 100% air. Adherent cells in an exponential growth phase were harvested by trypsin-EDTA treatment. 1×107 cells were injected in a volume of 200 μL PBS with Matrigel (1:1) into the flank of 6-8 weeks old female NOD/SCID mice (Beijing HFK Bioscience). Mouse handling and tumor volume measurements were performed as described in Example 30. Ten days after tumor inoculation, the mean tumor size reached 175 mm3 and mice were assigned into groups using randomized block design and treatments were started. Mice were treated twice according to a Q7D regimen by i.v. injection of 200 μg (10 mg/kg), 40 μg (2 mg/kg) or 10 μg (0.5 mg/kg) antibody in 10 μL PBS per g body weight. Mice in the control group were treated in parallel with 200 μg (10 mg/kg) IgG1-b12. After tumor inoculation, welfare of the animals was checked daily and tumor volumes were measured twice weekly.
TABLE 7
Treatment groups and dosing, Example 31
Dosing day after
# mice
# analyzed
Antibody
Total antibody per dose
tumor inoculation
8
8
IgG1-DR5-01-K409R-E430G (20 μg)
200 μg
(10 mg/kg)
10, 17
IgG1-DR5-05-F405L-E430G (20 μg)
8
8
IgG1-DR5-01-K409R-E430G (5 μg)
40 μg
(2 mg/kg)
10, 17
IgG1-DR5-05-F405L-E430G (5 μg)
8
8
IgG1-DR5-01-K409R-E430G (1 μg)
10 μg
(0.5 mg/kg)
10, 17
IgG1-DR5-05-F405L-E430G (1 μg)
8
8
IgG1-CONA (40 μg)
200 μg
(10 mg/kg)
10, 17
8
8
IgG1-CONA (10 μg)
40 μg
(2 mg/kg)
10, 17
8
8
IgG1-CONA (0.1 μg)
10 μg
(0.5 mg/kg)
10, 17
8
8
IgG1-b12 (40 μg)
200 μg
(10 mg/kg)
10, 17
FIG. 30A shows mean tumor volumes per treatment group. All tested doses of the antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth compared to the negative control antibody IgG1-b12 (10 mg/kg p<0.0001; 2 mg/kg p<0.001; 0.5 mg/kg p<0.05). The IgG1-CONA treatment groups were only better than IgG1-b12 at the highest doses (10 mg/kg and 2 mg/kg p<0.01), but not at 0.5 mg/kg. FIG. 30B shows that on day 28 after start treatment, tumor growth inhibition by the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was significantly better than equivalent doses IgG1-CONA at 10 mg/kg and 0.5 mg/kg (Unpaired t test, p<0.05).
FIG. 30C shows a Kaplan-Meier plot of tumor progression, with a cutoff set at a tumor volume>500 mm3. The combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G dosed at 10 mg/kg significantly inhibited tumor growth progression compared to negative control antibody IgG1-b12 and compared to an equivalent dose IgG1-CONA (Mantel-Cox analysis at tumor size cutoff 500 mm3: p<0.001.
These data indicate that the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and that anti-tumor efficacy for doses of 10 mg/kg and 0.5 mg/kg was significantly better than for equivalent doses of IgG1-CONA in an in vivo SW480 human colon cancer xenograft model.
Example 32: In Vivo Efficacy of Different Doses of the Antibody Combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G in a Subcutaneous SNU-5 Gastric Cancer Xenograft Model
The in vivo anti-tumor efficacy of different doses IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G were evaluated and compared to an equivalent dosing of IgG1-CONA in the subcutaneous SNU-5 human gastric cancer xenograft model at CrownBiosciences, Taicang, China. The cells were maintained in vitro as a suspension culture in IMDM medium supplemented with 20% fetal bovine serum at 37° C. in an atmosphere of 5% CO2 in air. Cells in an exponential growth phase were harvested and 1×107 cells were injected in a volume of 200 μL PBS with Matrigel (1:1) into the flank of 6-8 weeks old female CB17/SCIDmice (Beijing HFK Bioscience). Mouse handling and tumor volume measurements were performed as described in Example 30. Eight days after tumor inoculation, the mean tumor size reached 169 mm3 and mice were assigned into groups using randomized block design and treatments were started. Mice were treated twice according to a Q7D regimen by i.v. injection of 200 μg (10 mg/kg), 40 μg (2 mg/kg) or 10 μg (0.5 mg/kg) antibody in 10 μL PBS per g body weight. Mice in the control group were treated in parallel with 200 μg (10 mg/kg) IgG1-b12. After tumor inoculation, welfare of the animals was checked daily and tumor volumes were measured twice weekly.
TABLE 8
Treatment groups and dosing, Example 32
Dosing day after
# mice
# analyzed
Antibody
Total antibody per dose
tumor inoculation
8
8
IgG1-DR5-01-K409R-E430G (20 μg)
200 μg
(10 mg/kg)
8, 15
IgG1-DR5-05-F405L-E430G (20 μg)
8
8
IgG1-DR5-01-K409R-E430G (5 μg)
40 μg
(2 mg/kg)
8, 15
IgG1-DR5-05-F405L-E430G (5 μg)
8
8
IgG1-DR5-01-K409R-E430G (1 μg)
10 μg
(0.5 mg/kg)
8, 15
IgG1-DR5-05-F405L-E430G (1 μg)
8
8
IgG1-CONA (40 μg)
200 μg
(10 mg/kg)
8, 15
8
8
IgG1-CONA (10 μg)
40 μg
(2 mg/kg)
8, 15
8
8
IgG1-CONA (0.1 μg)
10 μg
(0.5 mg/kg)
8, 15
8
8
IgG1-b12 (40 μg)
200 μg
(10 mg/kg)
8, 15
FIG. 31A shows mean tumor volumes per treatment group. All tested doses of the antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth compared to the negative control antibody IgG1-b12. At the 2 mg/kg and 10 mg/kg doses, the antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G resulted in complete tumor regression that lasted over the complete study time (7 weeks after start treatment). FIG. 31B shows that on day 23 after start treatment, tumor growth inhibition by the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was significant better than equivalent doses IgG1-CONA (Mann Whitney test, p<0.05).
FIG. 31C shows a Kaplan-Meier plot of tumor progression, with a cutoff set at a tumor volume>500 mm3. The combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G significantly inhibited tumor growth progression compared to negative control antibody IgG1-b12 and compared to an equivalent dose IgG1-CONA (Mantel-Cox analysis at tumor size cutoff 500 mm3: p<0.05).
These data indicate that the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and that anti-tumor efficacy was significantly better than for equivalent doses of IgG1-CONA in an in vivo SNU-5 human gastric cancer xenograft model.
Example 33: In Vivo Efficacy of Different Doses of the Antibody Combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G in a Subcutaneous SK-MES-1 Lung Cancer Xenograft Model
The in vivo anti-tumor efficacy of different doses IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was evaluated and compared to an equivalent dosing of IgG1-CONA in the subcutaneous SK-MES-1 human lung cancer xenograft model at CrownBiosciences, Taicang, China. The cells were maintained in vitro as a monolayer culture in MEM medium supplemented with 10% fetal bovine serum and 0.01 mM NEAA at 37° C. in an atmosphere of 5% CO2 in air. At day 0, adherent cells in an exponential growth phase were harvested by trypsin-EDTA treatment. 5×106 cells were injected in a volume of 100 μL PBS into the flank of 6-8 weeks old female BALB/c mice (Shanghai Laboratory Animal Center). Mouse handling and tumor volume measurements were performed as described in Example 30. Twenty-one days after tumor inoculation, the mean tumor size reached 161 mm3 and mice were assigned into groups using randomized block design and treatments were started. Mice were treated twice according to a Q7D regimen by i.v. injection of 200 μg (10 mg/kg), 40 μg (2 mg/kg) or 10 μg (0.5 mg/kg) antibody in 10 μL PBS per g body weight. Mice in the control group were treated in parallel with 200 μg (10 mg/kg) IgG1-b12. After tumor inoculation, welfare of the animals was checked daily and tumor volumes were measured twice weekly.
TABLE 9
Treatment groups and dosing, Example 33
Dosing day after
# mice
Antibody
Total antibody per dose
tumor inoculation
8
IgG1-DR5-01-K409R-E430G (20 μg)
200 μg
(10 mg/kg)
21, 28
IgG1-DR5-05-F405L-E430G (20 μg)
8
IgG1-DR5-01-K409R-E430G (5 μg)
40 μg
(2 mg/kg)
21, 28
IgG1-DR5-05-F405L-E430G (5 μg)
8
IgG1-DR5-01-K409R-E430G (1 μg)
10 μg
(0.5 mg/kg)
21, 28
IgG1-DR5-05-F405L-E430G (1 μg)
8
IgG1-CONA (40 μg)
200 μg
(10 mg/kg)
21, 28
8
IgG1-CONA (10 μg)
40 μg
(2 mg/kg)
21, 28
8
IgG1-CONA (0.1 μg)
10 μg
(0.5 mg/kg)
21, 28
8
IgG1-b12 (40 μg)
200 μg
(10 mg/kg)
21, 28
FIG. 32A shows mean tumor volumes per treatment group. All tested doses of the antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth significantly compared to the negative control antibody IgG1-b12 (p<0.0001), whereas IgG1-CONA only had a significant effect compared to IgG1-b12 at 10 mg/kg (p<0.01) and 2 mg/kg (p<0.05) but not at 0.5 mg/kg (one-way ANOVA followed by Dunnett's multiple comparisons test). FIG. 32B shows that on day 14 after start treatment, tumor growth inhibition by the combination of IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G was significant better than equivalent doses IgG1-CONA at 2 mg/kg and 0.5 mg/kg (unpaired t-test test, p<0.05 and p<0.01, respectively).
FIG. 32C shows a Kaplan-Meier plot of tumor progression, with a cutoff set at a tumor volume>1.000 mm3. The combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G significantly inhibited tumor growth progression compared to negative control antibody IgG1-b12 (Mantel-Cox analysis at tumor size cutoff 1.000 mm3: p≤0.001) and compared to an equivalent dose IgG1-CONA at 2 mg/kg and 0.5 mg/kg (Mantel-Cox analysis at tumor size cutoff 1.000 mm3: p<0.05).
These data indicate that the combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G inhibited tumor growth at different doses (0.5 mg/kg, 2 mg/kg and 10 mg/kg) and that anti-tumor efficacy was significantly better than for equivalent doses of IgG1-CONA at 0.5 mg/kg and 2 mg/kg in an in vivo SK-MES-1 human lung cancer xenograft model.
Example 34: DR5 Expression Levels on Different Human Cancer Cell Lines
DR5 density per cell was quantified for different human cancer cell lines by indirect immunofluorescence using QIFIKIT with mouse monoclonal antibody B-K29 as described in Example 2. The cell lines were categorized according to low DR5 expression (ABC<10,000) and moderate DR5 expression (ABC>10,000). The human cancer cell lines SK-MEL-5 (ATCC, HTB-070) malignant melanoma, Jurkat (ATCC, TIB-152) acute T cell leukemia and Daudi (ATCC, CCL-231) Burkitt's lymphoma were found to have low DR5 expression (QIFIKIT ABC range 3,500-6,500). The human colorectal carcinoma cell lines SNU-C2B (ATCC, CCL-250), LS411N (ATCC, CRL-2159) and DLD-1 (ATCC, CCL-221) were found to have moderate DR5 expression (QIFIKIT ABC range 12,000-44,500).
Example 35: Introduction of a Hexamerization-Enhancing Mutation Does Not Affect Binding of IgG1-hDR5-01-G56T and IgG1-hDR5-05 Antibodies to DR5-Positive Human Colon Cancer Cells
Binding to human colon cancer cells HCT 116 was analyzed by flow cytometry for purified antibody variants of IgG1-hDR5-01-G56T and IgG1-hDR5-05 with and without the E430G mutation. Single cell suspensions were prepared and binding was analyzed for serial dilution antibody preparation series (range 0.0006 to 10 μg/mL final concentrations in 4-fold dilutions) as described in Example 3. After incubation with the secondary antibody, cells were washed twice, resuspended in 100 μL FACS buffer, and antibody binding was analyzed on a BD LRSFFortessa cell analyzer (BD Biosciences). Binding curves were analyzed using non-linear regression analysis (sigmoidal dose-response with variable slope) using GraphPad Prism software.
FIG. 33 shows that the antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G showed similar dose-dependent binding to HCT 116 cells as their corresponding antibodies without the E430G mutation. Introduction of the E430G mutation had no effect on the binding of the DR5 antibodies. The EC50 values were calculated from six repeat experiments as 74.4 (+/−58.4) ng/mL for IgG1-hDR5-01-G56T-E430G and 101.2 (+/−52.6) ng/mL for IgG1-hDR5-05-E430G.
Example 36: Binding of IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G as Single Antibodies and as a Combination to DR5-Positive Human Cancer Cells
Antibody binding to HCT 116 human cancer cells with moderate DR5 expression was analyzed by flow cytometry for purified samples of Alexa 647-labeled IgG1-hDR5-01-G56T-E430G and Alexa 647-labeled IgG1-hDR5-05-E430G, both as single agents and as a combination of the two antibodies. 1 mg/mL IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G were labeled for 1 hour at room temperature with a 5 molar excess of Alexa Fluor® 647 carboxylic acid, succinimidyl ester (Molecular Probes; Cat #A-20006) in 0.1 M NaHCO3 conjugation buffer to reach a degree of labeling of three. Free excess Alexa 647 was removed on a PD 10 Column (Amersham Bioscience, Cat #17-0851-01). Single cell suspensions were prepared and binding was analyzed for serial dilution antibody preparation series (range 0.0019 to 30 μg/mL final concentrations in 5-fold dilutions) as described in Example 3. After antibody incubation, cells were washed twice, resuspended in 100 μL FACS buffer, and antibody binding was analyzed on a BD LRSFFortessa cell analyzer (BD Biosciences). Binding curves were analyzed using non-linear regression analysis (sigmoidal dose-response with variable slope) using GraphPad Prism software.
FIG. 34 shows that both the single antibodies and the combination of the non-crossblocking antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G showed dose-dependent binding on HCT 116 human cancer cells.
Example 37: Binding of Antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G to Cynomolgus Monkey DR5
Binding of purified IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G to CHO cells expressing the isoform short of human DR5 or cynomolgus monkey DR5 was analyzed by flow cytometry. Codon-optimized constructs for expression of the isoform short human DR5 protein with death domain loss-of-function mutation K386N (SEQ ID NO 47 based on Uniprot number 014763-2) and cynomolgus monkey DR5 protein with deletion of amino acids 185-213 and death domain loss-of-function mutation K420N (SEQ ID NO 50; based on NCBI accession number XP_005562887.1) were generated as described in Example 1. Binding to DR5-transfected CHO cells was analyzed, generally as described in Example 5. Transfected cells were stored in liquid nitrogen and quickly thawed at 37° C. and suspended in 10 mL medium. Cells were washed with PBS and resuspended in FACS buffer at a concentration of 1.0×106 cells/mL. 100 μL cell suspension samples (100,000 cells per well) were seeded in 96-well plates and pelleted by centrifugation at 300×g for 3 minutes at 4° C. 25 μL of serial dilution antibody preparation series (final concentrations 0 to 20 μg/mL in 6-fold dilutions) was added and incubated for 30 minutes at 4° C. Next, cells were washed and incubated with 50 μL secondary antibody R-PE-conjugated goat-anti-human IgG F(ab′)2 (Jackson ImmunoResearch; Cat nr 109-116-098; 1/100) for 30 minutes at 4° C. protected from light. Cells were washed twice with 150 μL FACS buffer, resuspended in 50 μL FACS buffer, and antibody binding was analyzed on a BD LRSFFortessa cell analyzer (BD Biosciences) by recording 10,000 events. Binding curves were analyzed using non-linear regression analysis (sigmoidal dose-response with variable slope) using GraphPad Prism software.
FIG. 35 shows that the antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G showed dose-dependent binding to human and cynomolgus DR5 expressed on CHO cells. For both IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G, EC50 values for binding to human DR5 and cynomolgus DR5 were in the same range based on four repeat experiments (Table 10).
TABLE 10
EC50 values for binding of IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-
05-E430G to human and cynomolgus DR5. Based on four experiments.
Human DR5-transfected CHO
Cynomolgus DR5-transfected CHO
EC50 (μg/mL)
SD
EC50 (μg/mL)
SD
IgG1-hDR5-01-G56T-E430G
0.13
0.034
0.27
0.175
IgG1-hDR5-05-E430G
0.12
0.027
0.17
0.084
Example 38: Introduction of the E430G Mutation Improves the Efficacy of Cell Death Induction by the Combination of Non-Crossblocking Antibodies IgG1-hDR5-01-G56T+IgG1-hDR5-05
A viability assay was performed to study the effect the hexamerization-enhancing mutation E430G in IgG1-hDR5-01-G56T and IgG1-hDR5-05 on the capacity of the antibodies to kill human colon cancer cells COLO 205. The antibodies with and without the E430G mutation were tested as single agent and as combinations of the two non-crossblocking antibodies. COLO 205 cells were harvested as described in Example 8. 100 μL of the single cell suspensions (5,000 cells per well) were seeded in polystyrene 96-well flat-bottom plates (Greiner Bio-One, Cat nr 655182) and allowed to adhere overnight at 37° C. Subsequently, 50 μL samples of antibody concentration series (range 0.3-20,000 ng/mL final concentration in 4-fold dilutions) were added and incubated for 3 days at 37° C. As a positive control, cells were incubated with 5 μM staurosporine (Sigma Aldrich, Cat nr S6942). The viability of the cell cultures was determined in a CellTiter-Glo luminescent cell viability assay as described in Example 8.
FIG. 36 shows that the combination of IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was more potent than either antibody alone and more potent than the combination of the antibodies without the E430G mutation. These data show that introduction of the hexamerization-enhancing mutation E430G resulted in enhanced induction of cell killing upon binding of the combination of the non-crossblocking antibodies IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to adherent COLO 205 colon cancer cells. In contrast to the experimental setup where antibodies were directly added when cells were seeded (Example 8), the single antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G did not show efficacy on COLO 205 cells in this experiment where the cells were first allowed to adhere to the 96-wells flat-bottom plate before adding the samples.
Example 39: Introduction of Hexamerization-Enhancing Mutation S440Y Improves the Efficacy of Anti-DR5 Antibodies to Induce Cell Death on Human Colon Cancer Cells
The effect of the hexamerization-enhancing mutation S440Y on the capacity of the single antibodies and the combination of IgG1-hDR5-01-G56T and IgG1-hDR5-05 to kill COLO 205 human colon cancer cells was studied in a viability assay. Cells were harvested and a CellTiter-Glo luminescent cell viability assay was performed as described in Example 8. Briefly, 100 μL single cell suspensions (5,000 cells per well) were seeded in 96-well plates and at the same time, 50 μL of serial dilution antibody preparation series (range 0.0003 to 20 μg/mL final concentrations in 4-fold dilutions) were added and incubated for 3 days at 37° C.
FIG. 37A shows that in the experimental setup where antibodies were directly added when cells were seeded, introduction of the hexamerization-enhancing mutation S440Y resulted in dose-dependent killing by the single antibodies IgG1-hDR5-01-G56T-S440Y and IgG1-hDR5-05-S440Y, whereas the parental wild type antibodies IgG1-hDR5-01-G56T and IgG1-hDR5-05 were not able to kill COLO 205 colon cancer cells. Also the efficacy of the combination of IgG1-hDR5-01-G56T+IgG1-hDR5-05 was improved by introduction of the S440Y mutation in both antibodies, represented by the decreased EC50 (FIG. 37B).
Example 40: Introduction of the Hexamerization-Enhancing Mutation E430G Improves the Efficacy of Cell Death Induction by the Combination of Anti-DR5 Antibodies IgG1-DR5-CONA+IgG1-DR5-chTRA8
A crossblock ELISA for antibodies IgG1-DR5-CONA-K409R and IgG1-DR5-chTRA8-F405L was performed as described in Example 7. The K409R and F405L mutations are not relevant here and were previously shown to have no effect on the potency of antibodies with an E430G mutation (Example 22). FIG. 38A shows binding competition expressed as percentage inhibition of DR5ECD-FcHisCtag binding to coated antibody in presence of competing antibody, relative to binding of DR5ECD-FcHisCtag in absence of competing antibody (% inhibition=100−[(binding in presence of competing antibody/binding in absence of competing antibody)]*100). Binding of DR5ECD-FcHisCtag to coated IgG1-DR5-CONA-K409R was not inhibited in the presence of soluble IgG1-DR5-chTRA8-F405L. Vice versa, binding of DR5ECD-FcHistag to coated IgG1-DR5-chTRA8-F405L was also not inhibited in the presence of soluble IgG1-DR5-CONA-K409R. These data indicate that IgG1-DR5-CONA-K409R and IgG1-DR5-chTRA8-F405L did not compete with each other for binding of DR5ECD-FcHisCtag.
Next, the effect of the hexamerization-enhancing mutation E430G on the capacity of the combination of the non-crossblocking anti-DR5 antibodies IgG1-DR5-CONA-C49W+IgG1-DR5-chTRA-8 to kill attached B×PC-3 human pancreatic cancer cells was studied in a viability assay as described in Example 11. FIG. 38B shows that the antibody combination IgG1-DR5-CONA-C49W-E430G+IgG1-DR5-chTRA8-E430G with hexamerization-enhancing mutations showed increased dose-dependent killing of B×PC-3 cells compared to the combination of the parental antibodies without the E430G hexamerization-enhancing mutation.
Example 41: Capacity of the Antibody Combination IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G to Induce Target Cell Killing in Different Cancer Cell Lines
The efficacy of the combination of the non-crossblocking antibodies IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to induce killing was analyzed on different human cancer cell lines and compared to the parental antibody combination without the E430G mutation and TRAIL. A viability assay on HCT-15, HCT 116, HT-29 and SW480 colon cancer, B×PC-3, HPAF-II and PANC-1 pancreatic cancer, SNU-5 gastric cancer, A549 and SK-MES-1 lung cancer, and A375 skin cancer cells was performed, essentially as described in Example 11. Briefly, 100 μL single cell suspensions (5,000 cells per well) were seeded in 96-well plates and incubated at 37° C. overnight. 50 μL of antibody sample (133 nM final concentration) or human recombinant TRAIL/APO-2L (eBioscience, Cat nr BMS356; 133 nM final concentration) was added and incubated for 3 days at 37° C.
Both TRAIL and the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G show killing of human cancer target cell lines originating from different indications (FIG. 39). Killing of the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was significant compared to the control antibody IgG1-b12 in 6 of the 11 tested cell lines. For these responding cell lines the percentage viable cells was significantly lower after incubation with the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G than after incubation with the antibody combination without the E430G mutation. There was no correlation between killing efficacy of IgG1-hDR5-01-K409R-E430G+IgG1-hDR5-05-F405L-E430G and DR5 target expression levels (described in Example 2).
Example 42: Screening of a Human Cancer Cell Line Panel for Cytotoxic Efficacy of the Antibody Combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G
The activity of the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was tested and compared to the activity of TRAIL in a panel of 235 cell lines representing 14 tumor lineages: kidney, neural tissue, colorectal, gastric, breast cancer (predominantly triple-negative breast cancer (TNBC)), non-small cell lung cancer (NSCLC), bladder, pancreatic, ovarian, melanoma, liver, endometrial, head and neck and small cell lung cancer (SCLC). A 72 hour ATPlite assay (except for DLD-1 and HCT116 cell lines, for which a 120 hour assay was performed) with growth inhibition analysis was performed in two parts at Horizon Discovery Ltd, UK. Samples were tested as four replicates in 384-well assay plates. Serial dilution series of antibody, starting from 0.072 μM final concentration was used for all tested cell lines. For TRAIL (Invitrogen; Cat #PHC1634) serial dilution series starting from 0.01 μM final concentration for the cell lines tested in the first part and 0.17 μM final concentration for the cell lines tested in the second part of the screening was used.
Percentage inhibition was calculated using the formulas: If T≥V(0) than percentage inhibition=100*[1−(T−V(0))/(V−V(0))]; If T<V(0) than percentage inhibition=100%, with T=luminescence of the test sample, V(0)=luminescence of the medium control sample on day 0 and V=luminescence of the medium control sample on day 3. Responder and non-responder cell lines were grouped by a maximum response threshold value categorizing cell lines showing 70% inhibition as responders and cell lines showing 69% inhibition as non-responders (FIG. 40; Table 11). Responder cell lines for both antibody (IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G) and TRAIL monotherapy were found for all tested tumor indications, except small cell lung cancer (SCLC).
TABLE 11
Results for antibody (IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G) and TRAIL monotherapy as determined in a 3-days
viability assay screening at Horizon Discovery Ltd, UK, for a panel of cell lines representing different human cancer
indications: kidney (A), neural tissue (B), colorectal (C), gastric (D), triple-negative breast cancer (TNBC) (E), non-
small cell lung cancer (NSCLC) (F), bladder (G), pancreatic (H), ovarian (I), melanoma (J), liver (K), endometrial (L),
head and neck (M) and small cell lung cancer (SCLC) (N). Tabulated are IC50 values and percentage maximal inhibition.
IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G
TRAIL
Cell Line
Screening #
IC50 (nM)
Max Inhibition (%)
IC50 (nM)
Max Inhibition (%)
A: Kidney cancer cell line screening results for antibody (IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G)
and TRAIL therapy as determined in a 3-days viability assay screening at Horizon, UK.
A704
1
0.475
99.7
3.443
77.1
A498
2
1.223
98.9
0.079
96.1
G-401
2
0.509
94.1
0.068
76.6
CAL-54
1
1.533
91.7
0.268
71.6
ACHN
2
0.843
89.9
0.356
32.4
CAKI-2
1
1.565
87.3
5.7
769-P
2
0.957
57.7
1.941
39.4
G-402
2
0.605
50.4
0.173
15.0
786-0
2
0.005
7.7
287.593
2.6
B: Neural tissue cancer cell line screening results for antibody (IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G)
and TRAIL therapy as determined in a 3-days viability assay screening at Horizon, UK.
A172
2
0.888
100.0
0.029
98.2
SF295
2
0.764
99.2
0.023
87.5
SF126
2
0.713
98.9
0.013
85.6
H4
2
0.459
98.8
0.007
98.9
YH-13
2
1.248
94.4
0.317
39.5
U-87 MG
2
1.784
87.8
0.053
8.0
DBTRG-05MG
2
0.971
46.8
0.087
36.0
KNS-81
2
13.008
30.6
0.013
−6.6
SNB-75
2
3.225
14.2
105.178
9.6
NMC-G1
2
0.005
13.8
17.591
15.2
C: Colorectal cancer cell line screening results for antibody (IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G)
and TRAIL therapy as determined in a 3-days viability assay screening at Horizon, UK.
CL-11
1
0.620
100.0
1.318
85.9
GP2D
1
0.738
100.0
0.003
100.0
HT-115
1
2.101
100.0
0.107
99.7
SNU-1197
1
1.076
100.0
0.053
100.0
COLO-205
1
0.360
99.9
2.269
83.5
COLO-206F
1
0.200
99.9
0.146
99.2
CL-34
1
0.380
99.8
0.024
98.8
HRT-186
1
0.433
99.3
9.240
52.5
HCT-15
1
0.813
98.8
0.129
98.6
SNU-407
1
0.836
98.5
0.098
96.1
MDST8
1
1.035
93.6
1.190
58.3
COLO-201
1
0.568
93.6
0.168
89.1
HT55
1
1.025
91.0
0.110
76.4
SNU-175
1
1.813
90.1
0.122
96.2
HCT-116_ARID1A (Q456*/Q456*)
1
0.235
86.2
10.502
50.2
DLD-1
1
0.938
83.1
3.954
55.1
SNU283
1
5.628
81.9
40.3
CL-40
1
1.555
79.9
1.975
57.3
HCT-116_KRAS (+/−)
1
0.855
77.7
0.253
88.7
SW837
1
1.399
76.6
0.940
83.8
LOVO
1
4.512
75.7
44.5
LS-411N
1
3.549
73.1
26.9
HT-29
1
2.966
65.5
12.2
SNU1033
1
4.446
60.9
10.057
51.7
SW480
1
1.093
60.2
2.037
55.2
COLO-678
1
3.670
58.6
10.1
DLD-1_BRCA2 (−/−)
1
1.826
58.3
0.516
60.0
HCT-116_PIK3A (+/−)KO mt H1047R
1
0.756
57.8
1.251
59.8
C2BBe1
1
49.5
8.8
SNU-C2B
1
43.0
0.713
71.3
SW1116
1
42.4
23.1
HCT-116_PAR-007
1
29.0
14.2
HCT-116_TP53 (−/−)
1
19.6
17.2
SW1417
1
19.1
12.2
RKO
1
14.5
10.5
HCT-116_PTEN (−/−)
1
11.9
38.0
COLO320DM
1
10.1
8.0
COLO-320
1
9.9
3.9
D: Gastric cancer cell line screening results for antibody (IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G)
and TRAIL therapy as determined in a 3-days viability assay screening at Horizon, UK.
SNU-620
1
0.809
100.0
0.045
99.9
SNU-668
1
0.370
99.9
0.041
99.6
SNU-719
1
1.483
98.9
1.132
85.4
SNU-601
1
0.520
98.6
0.284
85.2
NUGC-3
1
0.671
96.7
38.7
GSU
1
0.169
96.4
0.454
81.6
SNU5
1
0.729
95.9
0.109
91.2
SNU-216
1
5.484
84.0
49.6
NCC-StC-K140
1
1.059
77.8
9.8
KE-97
1
1.175
57.2
23.4
LMSU
1
3.563
56.6
9.0
RERF-GC-1B
1
45.1
14.0
MKN1
1
36.8
10.5
SH-10-TC
1
31.6
34.9
ECC12
1
22.7
27.4
GSS
1
11.8
15.4
ECC10
1
8.3
14.0
E: Breast cancer cell line screening results for antibody (IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G)
and TRAIL therapy as determined in a 3-days viability assay screening at Horizon, UK.
SUM159PT*
2
0.569
99.2
0.033
98.6
DU-4475*
2
1.102
94.5
0.079
87.0
HCC1806*
2
2.678
92.5
0.050
97.8
BT-549*
2
1.021
83.3
0.285
44.0
BT-20*
2
2.843
82.8
0.089
48.9
HCC1187*
2
1.521
82.8
0.066
99.4
MDA-MB-436*
2
0.762
77.7
0.053
76.3
HCC38*
2
0.903
70.6
0.080
96.9
HCC70*
2
18.703
69.3
346.839
3.6
HMC-1-8*
2
0.714
67.7
0.232
70.0
MDA-MB-231*
1
1.409
60.2
0.110
53.3
SK-BR-3
2
1.757
40.4
0.518
38.1
MDA-MB-468*
2
3.772
33.3
0.647
89.4
HCC1937*
2
8.548
28.0
0.647
28.5
T47D
2
2.557
17.1
346.732
8.4
MDA-MB-453*
2
0.723
13.0
34.643
20.7
MCF7
2
19.492
10.2
0.055
21.7
F: Non-small-cell lung cancer (NSCLC) cell line screening results for antibody (IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G)
and TRAIL therapy as determined in a 3-days viability assay screening at Horizon, UK.
DV-90
1
0.215
100.0
1.189
96.1
NCI-H820
1
0.857
99.7
0.023
100.0
LCLC-97TM1
1
1.202
96.6
4.6
COR-L23-CPR
1
0.707
96.0
5.482
58.4
LOU-NH91
1
0.529
94.8
5.382
63.0
LCLC-103H
1
0.548
92.2
8.018
54.2
T3M-10
1
1.058
90.1
0.495
83.7
LU-99
1
1.879
81.9
6.3
HOP-62
1
0.899
81.7
55.6
EPLC-272H
1
1.233
79.7
51.7
LUDLU-1
1
2.196
77.9
0.475
91.1
RERF-LC-KJ
1
2.771
71.4
0.633
68.9
LXF-289
1
3.582
62.7
12.7
COR-L105
1
2.990
57.5
35.7
LC-1sq
1
7.710
56.6
0.781
70.1
NCI-H460
1
15.034
53.3
0.853
86.1
LC1F
1
50.3
0.451
74.3
SW1573
1
46.1
13.9
LU-65
1
38.9
32.4
HLC-1
1
36.8
32.6
VMRC-LCD
1
21.6
19.2
LK-2
1
19.0
5.6
Calu-1
1
13.0
22.0
CAL-12T
1
10.4
11.9
COLO-699
1
7.7
2.8
BEN
1
7.5
8.1
G: Bladder cancer cell line screening results for antibody (IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G)
and TRAIL therapy as determined in a 3-days viability assay screening at Horizon, UK.
5637
2
0.828
99.3
0.060
99.1
SW780
1
0.421
98.5
0.016
97.9
RT-112
2
3.520
96.1
0.325
50.9
RT4
2
4.638
95.5
0.244
91.5
UM-UC-3
1
0.906
94.4
0.005
99.4
TCCSUP
2
1.367
69.6
0.048
51.5
T-24
2
1.300
63.0
0.166
20.5
HT-1197
2
0.782
40.9
0.167
31.8
SCaBER
1
3.768
29.6
0.082
25.8
J82
1
68.272
15.9
33.567
11.5
HT-1376
2
67.114
15.3
159.873
10.6
H: Pancreatic cancer cell line screening results for antibody (IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G)
and TRAIL therapy as determined in a 3-days viability assay screening at Horizon, UK.
HuP-T3
1
0.728
91.8
0.223
88.4
PSN1
1
0.655
91.6
0.205
86.9
Panc 02.13
1
2.288
85.9
1.905
60.5
BxPC-3
1
0.448
83.9
46.5
KP-4
1
1.853
80.0
23.6
CFPAC-1
1
13.635
57.2
13.2
HPAF-II
1
9.896
56.9
13.3
KP-2
1
10.251
54.2
3.9
KLM-1
1
41.0
12.7
KP-3
1
37.3
23.5
CAPAN-2
1
20.6
4.8
PK-1
1
6.4
0.548
88.3
I: Ovary cancer cell line screening results for antibody (IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G)
and TRAIL therapy as determined in a 3-days viability assay screening at Horizon, UK.
SNU119
1
0.681
99.3
0.082
83.7
59M
1
0.846
98.6
0.049
98.5
JHOM-2B
1
8.294
82.0
21.7
COV434
1
1.093
80.4
0.395
73.1
OVCAR-5
1
2.731
79.0
1.120
70.6
OVK18
1
0.865
73.2
0.230
80.0
JHOM-1
1
1.835
68.8
0.596
58.1
COV644
1
3.375
68.2
0.271
74.9
MCAS
1
13.877
57.2
56.779
48.4
JHOS-4
1
73.734
49.6
12.5
OV7
1
48.3
20.4
COV504
1
19.0
11.7
OVTOKO
1
18.9
28.0
OVISE
1
13.5
4.0
KURAMOCHI
1
10.1
13.4
JHOC-5
1
8.5
18.4
J: Melanoma cell line screening results for antibody (IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G)
and TRAIL therapy as determined in a 3-days viability assay screening at Horizon, UK.
COLO-679
1
0.524
99.5
24.9
COLO-783
1
0.503
98.2
0.224
80.9
COLO-800
1
0.365
95.7
33.5
Hs 294T
1
0.595
94.1
0.019
91.1
RVH-421
1
0.577
91.3
22.5
MEL-HO
1
0.760
89.2
16.7
WM-266-4
1
1.257
80.3
42.5
COLO858
1
0.567
68.1
12.6
MEL-JUSO
1
1.349
67.6
7.6
COLO-818
1
1.061
64.8
7.8
IGR-39
1
0.813
63.9
20.1
IGR-1
1
1.066
60.1
23.3
IGR-37
1
9.359
54.7
18.0
COLO-849
1
51.9
17.6
A375
1
51.2
47.1
Hs 936.T
1
41.0
20.7
SK-MEL-30
1
12.5
6.1
IPC-298
1
11.2
5.7
HMCB
1
7.0
1.0
K: Liver cancer cell line screening results for antibody (IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G)
and TRAIL therapy as determined in a 3-days viability assay screening at Horizon, UK.
SNU-878
1
0.709
99.5
36.2
SNU-308
1
1.521
98.8
0.937
72.4
HuH-28
1
0.903
96.7
17.5
SNU-478
1
2.097
82.2
0.516
83.3
HLE
1
0.315
81.9
8.360
56.0
SNU-869
1
1.842
68.8
2.951
58.6
Li-7
1
1.614
65.9
20.4
HuCCT1
1
8.034
55.6
7.5
SNU-1196
1
44.8
44.3
HUH-6-clone5
1
42.3
20.2
SNU-1079
1
40.3
24.9
HuH-1
1
30.7
47.7
RH-41
1
10.1
3.0
SNU-761
1
9.1
7.1
L: Endometrial cancer cell line screening results for antibody (IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G)
and TRAIL therapy as determined in a 3-days viability assay screening at Horizon, UK.
HEC-265
1
0.399
100.0
0.021
100.0
MES-SA
1
0.510
100.0
0.107
92.6
JHUEM-2
1
0.613
89.6
0.165
66.4
RL95-2
1
1.649
88.0
0.155
97.7
SNG-II
1
1.049
79.3
1.028
77.2
JHUEM-3
1
46.2
18.4
TEN
1
43.2
1.205
70.0
HEC-1-A
1
39.6
22.8
HEC-108
1
32.8
1.479
64.9
MFE-296
1
22.7
8.7
COLO-684
1
14.3
16.6
SK-UT-1
1
13.4
10.9
HEC-1
1
13.2
8.1
MFE-280
1
11.4
11.0
HEC-50B
1
5.5
16.6
M: Head and neck cancer cell line screening results for antibody (IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G)
and TRAIL therapy as determined in a 3-days viability assay screening at Horizon, UK.
YD-15
1
1.369
100.0
0.114
100.0
TE-4
1
0.879
99.9
0.107
81.0
KYM-1
1
0.438
99.4
30.2
FTC-238
1
0.426
93.8
3.922
86.7
KYSE-70
1
1.346
79.2
46.0
TE-10
1
3.602
68.0
24.4
TE-6
1
7.502
61.9
3.923
55.3
TE-9
1
1.139
60.3
0.300
73.6
TE-1
1
3.051
58.3
23.0
BICR 31
1
4.670
52.6
38.9
KYSE-410
1
51.0
10.6
CJM
1
48.8
12.8
BICR 22
1
42.0
13.6
KYSE-30
1
38.0
8.0
SCC-15
1
34.8
19.4
TE-8
1
33.4
39.6
PE-CA-PJ34-cl C12
1
28.9
4.7
EC-GI-10
1
25.0
29.6
TE-5
1
23.0
11.6
HSC-4
1
12.8
0.5
YD-8
1
10.8
3.4
KYSE-270
1
7.1
5.4
BICR 18
1
−0.1
3.4
N: Small cell lung cancer (SCLC) cancer cell line screening results for antibody (IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G)
and TRAIL therapy as determined in a 3-days viability assay screening at Horizon, UK.
LU-134-A
1
47.7
28.0
IST-SL2
1
24.4
21.0
NCI-H69
1
23.2
14.5
NCI-H345
1
19.2
16.4
LU-139
1
18.7
10.0
SHP-77
1
9.2
9.1
NCI-H446
1
7.3
11.7
LU-135
1
6.0
9.7
*TNBC
Example 43: Capacity of Antibody Combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to Induce Cancer Cell Killing at Different Combination Ratios
A viability assay was performed to study the capacity of the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to induce killing of B×PC-3 pancreatic cancer cells and HCT-15 colon cancer cells, when combined at different ratios of IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G. Antibody ratios of 1:0, 9:1, 3:1, 1:1, 1:3, 1:9 and 0:1 in serial dilution series (ranging from 0.006 to 20 μg/mL final concentrations in 5-fold dilutions) were tested in a CellTiter-Glo luminescent cell viability assay as described in Example 16.
At 20 μg/mL, 4 μg/mL and 0.8 μg/mL total antibody concentrations, killing of B×PC-3 (FIG. 41A) and HCT-15 (FIG. 41B) cells was equally effective at all tested antibody ratios containing both antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G. In contrast, the single antibodies (ratios 1:0 and 0:1) did not induce killing. At 0.16 μg/mL total antibody concentrations, the tested combinations of IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G induced killing, although to a lesser extent than the higher antibody concentrations and efficacy is impacted by the using different ratios.
Example 44: The Antibody Combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G Induce Caspase-Dependent Programmed Cell Death
A viability assay was performed to compare the cytotoxicity of the combination of antibody variants of IgG1-hDR5-01-G56T and IgG1-hDR5-05 with and without the hexamerization-enhancing mutation E430G in the presence and absence of a caspase inhibitor. A CellTiter-Glo luminescent cell viability assay with serial dilution series of antibody or TRAIL samples (range 0.002 to 133 nM final concentrations in 4-fold dilutions) was performed as described in Example 18.
The killing of B×PC-3 cells was inhibited in the presence of pan-caspase inhibitor Z-VAD-FMK for TRAIL and the antibody combinations IgG1-hDR5-01-G56T+IgG1-hDR5-05 and IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G (FIG. 42). These data indicate that, like TRAIL, the antibody combinations IgG1-hDR5-01-G56T+IgG1-hDR5-05 and IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G induced caspase-dependent programmed cell death.
Example 45: Caspase-3 and -7 Activation Upon Binding of the Antibody Combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G on Human Cancer Cells
Caspase-3/7 activation was measured in time using the Caspase-Glo 3/7 assay, essentially as described in Example 20. Briefly, cells were harvested by trypsinization, passed through a cell strainer, pelleted by centrifugation for 5 minutes at 1,200 rpm and resuspended in culture medium at a concentration of 1.6×105 cells/mL. 25 μL of the single cell suspensions (4,000 cells per well) were seeded in 384-wells culture plates (Perkin Elmer, Cat nr 6007680) and incubated overnight at 37° C. 25 μL sample was added (26.6 nM final concentrations) and incubated for 1, 2, 4 and 6 hours at 37° C. Plates were removed from the incubator to let the temperature decrease till room temperature. Cells were pelleted by centrifugation for three minutes at 300 g. 25 μL supernatant was removed and replaced by 25 μL Caspase-Glo 3/7 Substrate. After mixing by shaking for one minute at 500 rpm, the plates were incubated for one hour at room temperature. Luminescence was measured on an EnVision Multilabel Reader (PerkinElmer).
In the time course of 1, 2, 4 to 6 hours, both TRAIL and the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G induced more rapid and more potent caspase-3/7 activation on B×PC-3 cells compared to the WT antibody combination IgG1-hDR5-01-G56T+IgG1-hDR5-05 without the hexamerization enhancing mutation (FIG. 43).
Example 46: The In Vitro Potency of the Antibody Combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G Does Not Require the Presence of a Secondary Fc Crosslinker
A viability assay was performed to compare the capacity of the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G to induce killing of human HCT-15 colon cancer cells and B×PC-3 pancreatic cancer cells in the absence and presence of a secondary antibody crosslinker. IgG1-DR5-CONA, which is known to show enhanced killing in the presence of a secondary antibody crosslinker, was tested in the same assay for comparison. A viability assay in absence and presence of secondary crosslinker was performed, essentially as described in Example 21. Briefly, 100 μL of the single cell suspensions (5,000 cells per well) were seeded in 96-well plates and incubated overnight at 37° C. 50 μL antibody sample (final concentration 4 μg/mL) in the absence or presence of F(ab′)2 fragments of a goat-anti-human IgG antibody and incubated for 3 days at 37° C. As a positive control for cell killing, cells were incubated with 5 μM staurosporine. The viability of the cell cultures was determined in a CellTiter-Glo luminescent cell viability assay as described Example 8.
The combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G induced potent killing in B×PC-3 and HCT15 cells, and cytotoxicity was not further enhanced in the presence of a secondary crosslinker (FIG. 44). In contrast, the efficacy of IgG1-DR5-CONA and the wild type antibody combination IgG1-hDR5-01-G56T+IgG1-hDR5-05 was enhanced by the presence of a secondary crosslinker in both B×PC-3 and HCT15. These data indicate that killing of B×PC-3 and HCT15 cancer cells by the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G is independent of the presence of a secondary Fc crosslinker.
Example 47: Complement Activation Upon Binding of IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G to CHO Cells Transiently Transfected with Either Human or Cynomolgus DR5
To analyze the capacity of the antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G to activate complement, an in vitro complement-dependent cytotoxicity (CDC) assay and deposition of complement component C3c was measured on CHO cells that were transiently transfected with the isoform short of either human or monkey DR5. The DR5 constructs harbored the K386N (human) or K420N (cynomolgus monkey) mutation in their death domain to prevent killing by the induction of apoptosis upon binding of the agonistic antibodies. Transient transfections of CHO cells with human or monkey (Macaca fascicularis) DR5 were performed as described in Example 1.
For the CDC assay, 0.1×106 cells were pre-incubated in polystyrene round-bottom 96-well plates (Greiner bio-one Cat #650101) with concentration series of purified antibodies in a total volume of 80 μL for 15 min on a shaker at RT. Next, 20 μL normal human serum (NHS; Cat #M0008 Sanquin, Amsterdam, The Netherlands) was added as a source of complement and incubated in a 37° C. incubator for 45 min (20% final NHS concentration; 0.003-10.0 μg/mL final antibody concentrations in 3-fold dilutions). The reaction was stopped by putting the plates on ice before pelleting the cells by centrifugation and replacing the supernatant by 30 μL of 2 μg/mL propidium iodide solution (PI; Sigma Aldrich, Zwijnaarde, The Netherlands). The percentage of PI-positive cells was determined by flow cytrometry on an Intellicyt iQue™ screener (Westburg). The data were analyzed using best-fit values of a non-linear dose-response fit using log-transformed concentrations in GraphPad PRISM 5.
For the analysis of C3b deposition, 0.1×106 cells were pre-incubated in round-bottom 96-well plates with concentration series of purified antibodies (0.003-10.0 μg/mL final antibody concentrations in 3-fold dilutions) in a total volume of 80 μL for 15 min on a shaker at RT. Next, 20 μL C5-depleted serum (Quidel; Cat #A501) was added as a source of complement and incubated in a 37° C. incubator for 45 min (20% final NHS concentration). Cells were pelleted and subsequently incubated with 50 μL FITC-labeled polyclonal rabbit-anti-human C3c complement (Dako; Cat #F0201; 2 μg/mL) in FACS buffer for 30 minutes at 4° C. Cells were washed twice with FACS buffer and resuspended in 30 μL FACS buffer. The C3b-deposition on cells was determined by flow cytrometry on an Intellicyt iQue™ screener (Westburg). The data were analyzed using best-fit values of a non-linear dose-response fit using log-transformed concentrations in GraphPad PRISM 5.
Both complement-dependent killing (FIG. 45A-B) and C3b deposition (FIG. 45C-D) on DR5-transfected CHO cells was observed for IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G with dose-response curves for both the single antibodies and for the combination. These data indicate that the intrinsic capacity of the IgG1 antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G to induce complement activation upon target binding on the cell surface was preserved for both the single antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G and the combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G.
Example 48: Drug Combination Screen Analysis for Efficacy Enhancement of the Antibody Combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G with a Panel of Compounds on Human Colon Cancer Cell Lines
In order to identify clinically relevant compounds that display synergistic inhibitory effects in combination with the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G, 100 compounds representing different therapeutic classes were screened for potential synergy in colon cancer cell lines. A 72 hour (for LS-411N, SNU-C2B and SW480) or 120 hour (for DLD-1 and HCT 116) ATPlite assay with growth inhibition analysis was performed in a 6×6 optimized combination matrix in 384-well assay plates at Horizon Discovery Ltd, UK. All samples were tested in four replicates. Percentage growth inhibition was calculated using the formulas: If T≥V(0) than percentage growth inhibition=100*[1−(T−V(0))/(V−V(0))]; If T<V(0) than percentage growth inhibition=100*[1−(T−V(0))/V(0)], with T=luminescence of the test sample, V(0)=luminescence of the medium control sample on day 0 and V=luminescence of the medium control sample on day 3. In order to identify synergistic effects, mean self-cross activity was determined for each therapeutic class using representative compounds. To measure combination effects in excess of Loewe additivity, Horizon Discovery Ltd has devised a scalar measure to characterize the strength of synergistic interaction termed the Synergy Score. The Synergy Score equation integrates the experimentally-observed activity volume at each point in the matrix in excess of a model surface numerically derived from the activity of the component agents using the Loewe model for additivity. Additional terms in the Synergy Score equation are used to normalize for various dilution factors used for individual agents and to allow for comparison of synergy scores across an entire experiment. The inclusion of positive inhibition gating or an Idata multiplier removes noise near the zero effect level, and biases results for synergistic interactions that occur at high activity levels. The Synergy Score (S) was calculated using the formula: S=log fX log fY Σmax(0,Idata)(Idata−ILoewe) with fx,y=dilution factors used for each single agent. Synergy Scores greater than the mean self-cross plus 3σ were considered candidate synergies at the 99% confidence levels.
Table 12 shows the Synergy Scores for all 100 tested compounds. Synergy with the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was observed for one or more cell lines with compounds from the different therapeutic classes, including chemotherapeutics (including cytoskeletal regulators and DNA/RNA damaging agents), kinase inhibitors, PI3K pathway inhibitors, RAS inhibitors, apoptosis-modulating agents, proteasome inhibitors, epigenetic modulators (including HDAC inhibitors) and others. FIG. 46 shows five examples of the growth inhibition effect of tested compounds in combination with the antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G. Birinapant (FIG. 46C), oxaliplatin (FIG. 46A), irinotecan (FIG. 46B) and paclitaxel (FIG. 46E) are examples that enhanced the effect of IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G, while baricitinib (FIG. 46D) is an example that showed no effect on the activity of IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G.
TABLE 12
Table 12: synergy scores for 100 compounds of different therapeutic classes that were tested in combination with
IgG1-hDR5-01-G56T-E430G + IgG1-hDR5-05-E430G in viability assays on the colon cancer cell lines LS-411N, SNU-
C2B, SW480, DLD-1 and HCT 116. Synergistic effects (Synergy Scores > mean self-cross + 3σ) are indicated in italics.
Synergy Scores in italics > mean self-cross + 3σ
Therapeutic class
Compound
Target
DLD-1
HCT-116
LS-411N
SNU-C2B
SW480
Cytoskeletal regulator
vinblastine
TUBB2
17.7
14.6
36
12.6
16.8
Cytoskeletal regulator
docetaxel
TUBB1
17.6
7.9
36.2
15.8
19.5
Cytoskeletal regulator
paclitaxel
TUBB1
11.4
11.8
40.8
15.6
14.7
Cytoskeletal regulator
vincristine
TUBB2
17.3
13.9
33
17.2
12.1
Cytoskeletal regulator
vinorelbine
TUBB2
5.9
8.1
21.5
11
7.7
DNA/RNA damaging agents
Gemcitabine
Antimetabolite
20.3
12.2
5.7
18.5
31.1
DNA/RNA damaging agents
Cytarabine
Antimetabolite
20.8
10.7
11.4
24.6
19.6
DNA/RNA damaging agents
Daunorubicin
DNA Intercalator
13.8
5.5
10.4
17.5
16.7
DNA/RNA damaging agents
Cisplatin
DNA Alkylating Agent
8.8
6.7
15.4
12.9
18.9
DNA/RNA damaging agents
Carboplatin
DNA Alkylating Agent
11.3
5.8
12
11.6
19.5
DNA/RNA damaging agents
Oxaliplatin
DNA Alkylating Agent
2.8
3.1
10
13.1
5.8
DNA/RNA damaging agents
Chlorambucil
DNA Alkylating Agent
4.6
1
3.8
11.4
7.9
DNA/RNA damaging agents
Melphalan
DNA Alkylating Agent
6
1.1
3.4
9
7.6
DNA/RNA damaging agents
Methotrexate
Antimetabolite
0.1
0.5
6.6
1.7
2.5
DNA/RNA damaging agents
Dacarbazine
DNA Alkylating Agent
0.8
1.5
0.3
2
2.5
DNA/RNA damaging agents
Fludarabine
Antimetabolite
0.5
0.9
0.1
3.1
0.3
DNA/RNA damaging agents
Fluorouracil
Antimetabolite
1.2
1
1.1
0.4
0.8
DNA/RNA damaging agents
Bendamustine
DNA Alkylating Agent
0.1
0
0.3
3
0.6
DNA/RNA damaging agents
Temozolomide
DNA Alkylating Agent
0.1
0
0.1
0.6
0.8
DNA/RNA damaging agents
Ifosfamide
DNA Alkylating Agent
0.1
0
0.2
0.3
0.1
Epigenetic Modulators
Belinostat
HDAC
8.6
4.1
4.5
16.4
10.2
Epigenetic Modulators
(+)-JQ1
BET Bromodomain
8.2
2.6
13.4
10.7
5.8
Epigenetic Modulators
Decitabine
DNA Methyltransferase
1.1
2.8
0
4.8
3.4
PI3K Pathway Inhibitors
TIC10
Akt, ERK
15.5
2.2
23.3
13.1
3.4
PI3K Pathway Inhibitors
GDC-0941
PI3K
4.9
2
12.4
7.1
12.9
PI3K Pathway Inhibitors
AZD 8055
mTOR
3
1.7
10
12.2
3.9
PI3K Pathway Inhibitors
PIK-93
PI4K, PI3K
1
1.2
5.2
5.1
4.1
PI3K Pathway Inhibitors
BEZ235
mTOR, PI3K
2.4
1.4
1.1
5.4
3.2
PI3K Pathway Inhibitors
Temsirolimus
mTOR
0.7
0.6
0.6
4.5
1.6
PI3K Pathway Inhibitors
Everolimus
mTOR
0.4
0.4
0.3
3.2
1.6
PI3K Pathway Inhibitors
GSK1059615
mTOR, PI3K
0.4
0.6
0.4
0.8
1.7
PI3K Pathway Inhibitors
IPI-145
PI3K
0
0
0.2
1
0.1
PI3K Pathway Inhibitors
IC-87114
PI3K
0
0.1
0.2
0.1
0
Receptor Tyrosine Kinase
Crizotinib
c-Met, Alk
13.4
7.7
16.1
5.4
8.9
Inhibitors
Receptor Tyrosine Kinase
RAF265
RAF/VEGFR inhibitor
5.6
2.5
10.5
13.7
4.5
Inhibitors
Receptor Tyrosine Kinase
Dasatinib
Abl and Src Family
11.4
1.9
7.8
1
10.2
Inhibitors
Kinases
Receptor Tyrosine Kinase
BMS-754807
IGFR, InsR, c-Met, TrkB
11.3
2.1
10.8
1.3
5.9
Inhibitors
Receptor Tyrosine Kinase
Sunitinib
VEGFR, PDGFR
9.2
0.7
7.2
1.4
5.2
Inhibitors
Receptor Tyrosine Kinase
XL184
VEGFR, c-Met, Ret, c-
4.1
1
5.8
1.5
6
Inhibitors
Kit, Flt, Tie, AXL
Receptor Tyrosine Kinase
Lapatinib
EGFR, HER2
1
0.4
7.3
2.6
6.4
Inhibitors
Receptor Tyrosine Kinase
AP24534
Abl and Src Family
4.3
1.4
2.6
1.8
3.7
Inhibitors
Kinases
Receptor Tyrosine Kinase
GSK1904529A
IGF-1R
1.4
0.2
6
2.7
2.2
Inhibitors
Receptor Tyrosine Kinase
Erlotinib
EGFR
4.3
0.2
1.9
1.2
1
Inhibitors
Receptor Tyrosine Kinase
Gefitinib
EGFR
4.9
0.2
1
1.1
1.3
Inhibitors
Receptor Tyrosine Kinase
OSI-906
IGFR, InsR
0.5
0.1
1.7
1
3.5
Inhibitors
Receptor Tyrosine Kinase
Masitinib
c-Kit, PDGFR
0.6
0.3
1.4
1.7
1.9
Inhibitors
Receptor Tyrosine Kinase
BGJ398
FGFR
1
0.1
1.4
0.8
1.6
Inhibitors
Receptor Tyrosine Kinase
MGCD-265
c-Met, VEGFR
0.2
0.1
0.7
3.1
0.5
Inhibitors
Receptor Tyrosine Kinase
AST-1306
EGFR, HER2, HER4
0.3
0.1
1.8
0.7
0.9
Inhibitors
Receptor Tyrosine Kinase
Nilotinib
Abl and Src Family
0.1
0
0.2
1.7
0.4
Inhibitors
Kinases
Receptor Tyrosine Kinase
PCI-32765
BTK
0.5
0
0.3
0.6
0.4
Inhibitors
Receptor Tyrosine Kinase
Imatinib
Abl and Src Family
0.2
0.1
0.3
0.3
0.3
Inhibitors
Kinases
Receptor Tyrosine Kinase
INCB28060
c-Met
0.2
0.1
0
0.4
0
Inhibitors
Receptor Tyrosine Kinase
JNJ-38877605
c-Met
0.1
0
0.1
0.1
0
Inhibitors
Regulators of Apoptosis
Birinapant
XIAP, cIAP
5.8
16.4
61.2
37
54.5
Regulators of Apoptosis
TW-37
Bcl family
13.8
5.4
29.9
11.1
10.5
Regulators of Apoptosis
Obatoclax
Bcl family
6.1
1.6
22.8
4.7
11.8
Regulators of Apoptosis
YM155
Survivin
5.2
0.5
9
11.5
12.1
Regulators of Apoptosis
PAC 1
Caspase
10.6
4.2
2.9
6
3.9
Regulators of Apoptosis
ABT-263
Bcl family
3.4
0
7.6
1
8
Regulators of Apoptosis
ABT-737
Bcl family
1.5
0
4.2
1.1
7.5
Regulators of Apoptosis
SB 415286
GSK3
0.4
0.8
2.1
0.7
3.8
Regulators of Apoptosis
SB-216763
GSK3
0.5
0.1
1.1
0.3
1.4
Regulators of Apoptosis
TNF-related
TRAIL
0
0
0.1
0.2
0.9
apoptosis-inducing
ligand
Regulators of Apoptosis
ABT-199
Bcl family
0
0
0.1
0
0
Topoisomerase Inhibitors
Topotecan
Top1
20.3
5
29
21.2
30.5
Topoisomerase Inhibitors
Teniposide
Top2
18.8
6.2
29.3
19.8
25.1
Topoisomerase Inhibitors
10-
Top1
21.9
9.6
16.9
12.4
30.1
Hydroxycamptothecin
Topoisomerase Inhibitors
Doxorubicin
Top2
16.2
4.6
9.4
18
20.5
Topoisomerase Inhibitors
Irinotecan
Top1
15
4.4
6.8
13.4
24.7
Topoisomerase Inhibitors
Etoposide
Top2
17
2
11.1
14.2
19.4
Topoisomerase Inhibitors
Epirubicin
Top2
15.6
4.9
5.8
15.1
17.7
Tipifarnib
FTase
3.1
3.3
24.3
7.2
5
Idasanutlin
MDM2
6.3
3.2
16.3
10.4
6.1
Suberoylanilide
4.4
2.5
4.4
11
7.3
Hydroxamic Acid
Bortezomib
Proteasome
3
2.6
7.2
7.5
8.9
GSK429286A
4.9
2.6
9.8
5.3
5.3
GF 109203X
4.3
3
5.3
1.6
6.2
AZD6244
1.5
2.7
0.7
0.6
11.7
Trametinib
4.1
4
0.9
0.6
7.2
Sorafenib
2.6
0.5
6
2.9
3.9
Enzastaurin
0.9
0.6
5.6
0.5
6.3
Tamoxifen Citrate
0.3
0.3
8
0.7
2.9
Go 6976
2.9
0.9
1.9
1.7
3.1
Olaparib
0.7
1.8
2.7
2.5
1
SP 600125
3.3
0.6
0.5
2
0.9
Dabrafenib
2.2
0.7
0.5
0.5
3.1
GDC-0879
0.1
0.2
0.4
0.7
1.6
PLX-4032
0.6
0.1
0.6
0.5
1
Baricitinib
JAK inhibitors
0.1
0.1
1.1
0.6
0.1
JNK-IN-8
JNK inhibitor
0.2
0
0.1
1.1
0.4
Dexamethasone
0.1
0
0.4
0.8
0
ABT-888
0.2
0.2
0.7
0.1
0
Salirasib
0.1
0.1
0
0.4
0.4
CP-690550
JAK inhibitors
0.1
0
0.6
0.1
0
GDC-0449
0.1
0
0.2
0.3
0.1
Methylprednisolone
0
0
0
0.6
0
Pomalidomide
0.1
0
0
0.5
0
Prednisone
0.1
0
0.1
0.2
0
Lenalidomide
0
0
0.1
0.2
0
Example 49: In Vivo Efficacy of the Anti-DR5 Antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G in a Subcutaneous COLO 205 Colon Cancer Xenograft Model
The in vivo anti-tumor efficacy of antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G was evaluated for the single antibodies and the combination of both antibodies and compared to the parental antibodies without the E430G mutation in the subcutaneous COLO 205 human colon cancer xenograft model. Tumor cell inoculation, mice handling, tumor outgrowth measurements and endpoint determination were performed, essentially as described in Example 26. 3×106 cells were injected in a volume of 100 μL PBS into the flank of 5-8 weeks old female SCID mice (C.B-17/IcrHan® Hsd-Prkdcscid; Harlan). At day 9, the average tumor volume was measured and the mice were sorted into groups with equal tumor size variance. Mice were treated by intravenous (i.v.) injection of 10 μg (0.5 mg/kg) antibody in 200 μL PBS on day 9. Mice in the control group were treated with 10 μg (0.5 mg/kg) IgG1-b12.
TABLE 13
Treatment groups and dosing
Dosing day after
# mice
# analyzed
Antibody
Total antibody dose
tumor inoculation
8
8
IgG1-hDR5-01-G56T-E430G
0.5 mg/kg
9
8
8
IgG1-hDR5-05-E430G
0.5 mg/kg
9
8
8
IgG1-hDR5-01-G56T-E430G
0.5 mg/kg
9
IgG1-hDR5-05-E430G
8
8
IgG1-hDR5-01-G56T
0.5 mg/kg
9
8
8
IgG1-hDR5-05
0.5 mg/kg
9
8
8
IgG1-hDR5-01-G56T
0.5 mg/kg
9
IgG1-hDR5-05
8
8
IgG1-b12
0.5 mg/kg
9
FIG. 47A shows mean tumor volumes per treatment group in time. Introduction of the E430G mutation in the single antibodies IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G resulted in enhanced inhibition of tumor growth compared to the parental antibodies without the E430G mutation. Treatment with the antibody combinations induced complete tumor regression, both for IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G and for the combination of parental antibodies without the E430G mutation. At day 19 the average tumor size in all groups treated with DR5-antibodies was significantly smaller than in animals treated with the negative control antibody IgG1-b12 (Mann Whitney test (P<0.001))(data not shown). FIG. 47B shows a Kaplan-Meier plot of tumor progression, with a cutoff set at a tumor volume>500 mm3. Compared to mice treated with negative control antibody IgG1-b12, tumor outgrowth was significantly delayed in all groups treated with anti-DR5 antibodies (Mantel-Cox analysis at tumor size cut-off 500 mm3: p<0.0001). Mice treated with the single antibodies IgG1-hDR5-01-G56T and IgG1-hDR5-05 without the hexamerization-enhancing mutation E430G showed tumor outgrowth significantly earlier compared to the mice treated with the other tested anti-DR5 antibodies ((Mantel-Cox analysis at tumor size cut-off 500 mm3: p<0.0001).
Example 50: Effect of a Hexamerization-Enhancing Mutation on the In Vivo Efficacy of the Combination of Anti-DR5 Antibodies IgG1-hDR5-01-G56T+IgG1-hDR5-05 in a Subcutaneous HCT15 Colon Cancer Xenograft Model
The in vivo anti-tumor efficacy of the anti-DR5 antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was compared to that of IgG1-hDR5-01-G56T+IgG1-hDR5-05 without the E430G hexamerization-enhancing mutation in the subcutaneous HCT15 human colon cancer xenograft model at CrownBiosciences, Taicang, China. The cells were maintained in vitro as a monolayer culture in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37° C. in an atmosphere of 5% CO2 in air. Adherent cells in an exponential growth phase were harvested by trypsin-EDTA treatment. 5×106 cells were injected in a volume of 100 μL PBS into the flank of 7-9 weeks old female BALB/c nude mice. The care and use of animals during the study were conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Tumor volumes were measured twice weekly in two dimensions using a caliper, and the volume was expressed in mm3 using the formula: V=0.5 a×b2 where a and b are the long and short diameters of the tumor, respectively. Mice were assigned into groups using randomized block design and treatments were started when the mean tumor size reached 161 mm3 (8 mice per group). Mice were treated three times according to a Q7D regimen by i.v. injection of 0.5 mg/kg antibody (0.25 mg/kg of each antibody in the combination). Mice in the control group were treated in parallel with 0.5 mg/kg IgG1-b12.
FIG. 48A shows mean tumor volumes per treatment group. The antibody combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G showed better tumor growth inhibition than IgG1-hDR5-01-G56T+IgG1-hDR5-05. At day 21 the average tumor size in mice treated with the combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was significantly smaller than in mice treated with an equivalent dose IgG1-hDR5-01-G56T+IgG1-hDR5-05 (Mann Whitney test: P<0.0011) (FIG. 48B). FIG. 48C shows a Kaplan-Meier plot of tumor progression, with a cutoff set at a tumor volume>750 mm3. Tumor outgrowth in mice treated with the combination IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was significantly later than in mice treated with an equivalent dose IgG1-hDR5-01-G56T+IgG1-hDR5-05.
These data indicate that introduction of the E430G hexamerization-enhancing mutation in the anti-DR5 antibody combination IgG1-DR5-01-K409R-E430G+IgG1-DR5-05-F405L-E430G resulted in enhanced tumor growth inhibition in an in vivo xenograft model with HCT15 human colon cancer cells.
Example 51: In Vivo Efficacy of IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G in Combination with Paclitaxel in a Subcutaneous SK-MES-1 Human Lung Cancer Xenograft Model
The in vivo anti-tumor efficacy of IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G was evaluated in combination with paclitaxel in the subcutaneous SK-MES-1 human lung cancer xenograft model at CrownBiosciences, Taicang, China. Cell culturing, tumor cell inoculation, mice handling, tumor outgrowth measurements and endpoint determination were performed as described in Example 33. 21 days after tumor inoculation, the mean tumor size reached 167 mm3 and mice were assigned into groups using randomized block design and treatments were started. Mice were treated twice according to a Q7D regimen by i.v. injections of 2 mg/kg antibody and 15 mg/kg paclitaxel both dosed in 10 μL PBS per g body weight as indicated in Table 14.
TABLE 14
Treatment groups and dosing, Example 53
Dosing day after
# mice
Compound
Total per dose
randomization
8
IgG1-hDR5-01-G56T-E430G
2
mg/kg
0, 7
IgG1-hDR5-05-E430G
8
Paclitaxel
15
mg/kg
0, 7
8
IgG1-hDR5-01-G56T-E430G
2 mg/kg antibody +
0, 7
IgG1-hDR5-05-E430G
15 mg/kg paclitaxel
Paclitaxel
8
IgG1-b12
2
mg/kg
0, 7
FIG. 49A shows mean tumor volumes per treatment group. Antibody treatment alone (2 mg/kg IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G) or 2 mg/kg antibody treatment in combination with 15 mg/kg paclitaxel or 15 mg/kg paclitaxel alone all demonstrated anti-tumor efficacy compared to IgG1-b12. FIG. 49B shows tumor volume per treatment group at day 16. In all treatment groups, tumor load was significantly lower compared to IgG1-b12 (Mann-Whitney test, p<0.01). FIG. 49C shows a Kaplan-Meier plot of tumor progression, with a cutoff set at a tumor volume>500 mm3. The combination of 15 mg/kg paclitaxel with 2 mg/kg IgG1-hDR5-01-G56T-E430G+IgG1-hDR5-05-E430G antibody significantly prolonged progression-free survival compared to paclitaxel or antibody alone (Gehan-Breslow-Wilcoxon test, tumor size cut-off 500 mm3: p<0.05).
Example 52: Pharmacokinetic (PK) Analysis of IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G
The clearance rate of IgG1-hDR5-01-G56T-E430G and IgG1-hDR5-05-E430G was studied in a PK experiment in SCID mice for the single compounds and for the combination of the two antibodies in comparison to the parental antibodies without the E430G mutation.
7-10 weeks old female SCID (C.B-17/IcrHan@Hsd-Prkdc<scid, Harlan) mice (3 mice per group) were injected intravenously with 20 μg antibody (1 mg/kg) in a 200 μL injection volume. 50-100 μL blood samples were collected from the saphenous vein at 10 minutes, 4 hours, 1 day, 2 days, 7 days, 14 days and 21 days after antibody administration. Blood was collected into heparin-containing vials and centrifuged for 5 minutes at 10,000 g. Plasma samples were diluted 1:20 for the four first time points (15 μL sample in 285 μL PBSA (PBS supplemented with 0.2% bovine serum albumin (BSA)) and 1:10 for the last two time points (30 μL sample in 270 μL PBSA) and stored at −20° C. until determination of antibody concentrations.
Total human IgG concentrations were determined using a sandwich ELISA. Mouse anti-human IgG-kappa mAb clone MH16 (CLB Sanquin, Cat ##M1268) was used as capturing antibody and coated in 100 μL overnight at 4° C. to 96-well Microlon ELISA plates (Greiner, Germany) at a concentration of 2 μg/mL in PBS. Plates were blocked by incubating on a plate shaker for 1 h at RT with PBSA. After washing, 100 μL of serial diluted plasma samples (range 0.037-1 μg/mL in 3-fold dilutions) were added and incubated on a plate shaker for 1 h at RT. Plates were washed three times with 300 μL PBST (PBS supplemented with 0.05% Tween 20) and subsequently incubated on a plate shaker for 1 h at RT with 100 μL peroxidase-labeled goat anti-human IgG immunoglobulin (#109-035-098, Jackson, West Grace, Pa.; 1:10.000 in PBST supplemented with 0.2% BSA). Plates were washed again three times with 300 μL PBST before incubation for 15 minutes at RT with 100 μL substrate 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) [ABTS; Roche, Cat #11112 422001; 1 tablet in 50 mL ABTS buffer (Roche, Cat #11112 597001)] protected from light. The reaction was stopped by adding 100 μL2% oxalic acid and incubation for 10 minutes at RT. Absorbance was measured in a microplate reader (Biotek, Winooski, Vt.) at 405 nm. Concentration was calculated by using the injected material as a reference curve. As a plate control, purified human IgG1 (The binding site, Cat #BP078) was included. Human IgG concentrations (in μg/mL) were plotted (FIG. 50A) and Area under the curve (AUC) was calculated using Graphpad prism 6.0. Clearance until the last day of blood sampling (day 21) was determined by the formula D*1.000/AUC, in which D is the dose of injection (1 mg/kg) (FIG. 50B).
No difference in the plasma clearance rate was observed between IgG1-hDR5-01-G56T-E430G or IgG1-hDR5-05-E430G and their parental antibodies without the E430G mutation, both when injected as single agents or as the combinations of those (FIG. 50).
Example 53: Anti-DR5 Antibody IgG1-DR5-CONA with a Hexamerization-Enhancing Mutation E430G is Able to Kill Human Colon Cancer Cells
The present study illustrate the ability of the anti-DR5 antibody IgG1-DR5-CONA with the hexamerization-enhancing mutation E430G to kill attached human colon cancer cells COLO 205. COLO 205 cells were harvested as described in Example 8. 100 μL of the single cell suspensions (5,000 cells per well) were seeded in 96-well flat-bottom plates and incubated overnight at 37° C. 50 μL samples of antibody concentration series (range 0.04 to 10 μg/mL final concentrations in 4-fold dilutions) were added and incubated for 3 days at 37° C. As a positive control, cells were incubated with 5 μM staurosporine. The viability of the cell cultures was determined in a CellTiter-Glo luminescent cell viability assay as described in Example 8. Luminescence was measured on an EnVision Multilabel Reader (PerkinElmer). Data were analyzed and plotted using non-linear regression (sigmoidal dose-response with variable slope) using GraphPad Prism software. The percentage viable cells was calculated using the following formula: % viable cells=[(luminescence antibody sample−luminescence staurosporine sample)/(luminescence no antibody sample−luminescence staurosporine sample)]*100.
FIG. 51 shows that introduction of the hexamerization-enhancing mutation E430G resulted in dose-dependent killing by IgG1-DR5-CONA-E430G, whereas the parental wild type antibody IgG1-DR5-CONA was not able to kill attached COLO 205 colon cancer cells.
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16451714
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genmab b.v.
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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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Apr 1st, 2022 06:04PM
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Apr 1st, 2022 06:04PM
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Genmab A/S
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Health Care
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Pharmaceuticals & Biotechnology
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cph:gen
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Genmab A/S
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Sep 1st, 2020 12:00AM
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Jul 6th, 2012 12:00AM
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https://www.uspto.gov?id=US10759867-20200901
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Antibody variants and uses thereof
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Described herein are polypeptides and related antibodies comprising a variant Fc domain. The variant Fc domain provide for stabilized Fc:Fc interactions when the polypeptide(s), antibody or antibodies are bound to its target, antigen or antigens on the surface of a cell, thus providing for improved effector functions, such as CDC-response.
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10759867
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1. A method of increasing complement-dependent cytotoxicity (CDC) specificity of a mixture of a first IgG antibody and a second IgG antibody to cells which express a first antigen and a second antigen, wherein the first antibody binds to the first antigen and the second antibody binds to the second antigen, and wherein both the first and second antibodies comprise an Fc region, which method comprises
(i) introducing to the first antibody a K439E mutation in the Fc region; and
(ii) introducing to the second antibody a S440K mutation in the Fc region,
wherein the numbering is according to EU Index, and wherein the CDC specificity of the mixture of first antibody and second antibody is increased relative to the CDC specificity of the first antibody or second antibody alone.
2. The method according to claim 1, wherein the first antigen and second antigen are expressed on a cell membrane.
3. The method according to claim 2, wherein the first antigen and second antigen are tumor cell antigens.
4. A method of increasing complement-dependent cytotoxicity (CDC) specificity of a mixture of a first IgG antibody and a second IgG antibody to cells which express a first antigen and a second antigen, wherein the first antibody binds to the first antigen and the second antibody binds to the second antigen, and wherein both the first and second antibodies comprise an Fc region, which method comprises
(i) introducing to the first antibody a K439E mutation and a E345R mutation in the Fc region, and
(ii) introducing to the second antibody a S440K mutation and a E345R mutation in the Fc region,
wherein the numbering is according to EU Index, and wherein the CDC specificity of the mixture of the first antibody and second antibody is increased relative to the CDC specificity of the first antibody or second antibody alone.
5. The method according to claim 4, wherein the first antigen and second antigen are expressed on a cell membrane.
6. The method according to claim 5, wherein the first antigen and second antigen are tumor cell antigens.
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6
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RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national stage filing of International Application No. PCT/EP2012/063339, filed Jul. 6, 2012, which claims priority to U.S. Provisional Application No. 61/504,994, filed Jul. 6, 2011, Danish Patent Application No. PA201100519, filed Jul. 6, 2011, and Danish Patent Application No. PA201200371, filed May 30, 2012.
FIELD OF THE INVENTION
The present invention concerns polypeptides and related antibodies comprising a variant Fc domain. More particularly, the present invention concerns Fc domain-containing antibodies or polypeptides that have a modified effector function resulting from one or more amino acid modifications in the Fc-domain.
BACKGROUND OF THE INVENTION
The effector functions mediated by the Fc region of an antibody allow for the destruction of foreign entities, such as the killing of pathogens and the clearance and degradation of antigens. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) is initiated by binding of the Fc region to Fc receptor (FcR)-bearing cells, whereas complement-dependent cytotoxicity (CDC) is initiated by binding of the Fc region to C1q, which initiates the classical route of complement activation.
Each IgG antibody contains two binding sites for C1q, one in each heavy chain constant (Fc) region. A single molecule of IgG in solution, however, does not activate complement as the affinity of monomeric IgG for C1q is quite weak (Kd˜10−4 M) (Sledge et al., 1973 J. Biol. Chem. 248, 2818-13; Hughes-Jones et al., 1979 Mol. Immunol. 16, 697-701). Antigen-driven association of IgG can lead to much tighter binding of the multivalent C1q molecule (Kd˜10−8 M) and complement activation (Burton et al., 1990 Mol. Immunol. 22, 161-206). In contrast, IgM exists naturally in covalently bound penta- or hexamers, and upon binding of cellular expressed or immobilized antigen IgM pentamers and hexamers can efficiently elicit CDC. Antigen-binding is a requirement to induce a conformational change in IgM to expose the C1q binding sites (Feinstein et al., 1986, Immunology Today, 169-174).
It has been suggested that also IgG can achieve complement activation by the formation of hexameric ring structures, through interaction of the CH2/CH3 domains of the Fc region (Burton et al., 1990 Trends in Biochem. Sci. 15, 64-69). Evidence supporting the existence of such hexameric IgG structures has been found in two dimensional (Reidler et al., 1986 I Handbook of Experimental Immunology 4th edit. (Weir, D. M. ed.), pp 17.1-17.5. Blackwell, Edinburgh; Pinteric et al., 1971 Immunochem. 8, 1041-5) and three dimensional crystals, as well as for IgG1, IgG2a and IgG4 and human Fc in solution (Kuznetsov et al., 2000 J Struct. Biol. 131, 108-115). A hexameric ring formation was also observed in the crystal structure of the b12 human IgG1κ antibody directed against HIV-1 gp120 (1HZH in PDB) (Saphire et al., Science 2001 Aug. 10; 293(5532),1155-9). In the b12 hexamer ring, six accessible C1q binding sites were presented at the hexamer surface, one from each of the six antibodies, while the other six binding sites faced downwards.
C1q resembles a bunch of tulips with six globular heads, containing the antibody combining regions, tethered to six collagenous stalks [Perkins et al., 1985 Biochem J. 228, 13-26; Poon et al., 1983 J Mol Biol. 168, 563-77; Reid et al., 1983 Biochem Soc Trans 11, 1-12; Weiss et al., 1986 J. Mol. Biol. 189, 573-81]. C1q was found to fit onto the b12 hexameric assembly of the 1HZH crystal structure, so that each of the six globular heads were in contact with one of the six C1q binding sites (Parren, FASEB Summer Research Conference, Snowmass, Co., 5-10 Jul. 2010; “Crystal Structure of an intact human IgG: implications for HIV-1 neutralization and effector Function”, Thesis by Erica Ollmann Saphire, for the Scripps Research Institute, La Jolla, Calif. November 2000). Mutations in selected amino acids in the Fc interfaces observed between symmetry-related b12 antibodies in the crystal structure were observed to decrease the binding avidity of C1q, indicating the contribution of these amino acids to the intermolecular Fc:Fc interaction.
US 2011/0123440 describes altered antibody Fc-regions and the uses thereof. The alterated Fc-regions have one or more amino acid substitutions.
US 2008/0089892 describes polypeptide Fc-region variants and compositions comprising these Fc-region variants.
US 2010/0184959 describes methods of providing an Fc polypeptide variant with altered recognition of an Fc ligand and/or effector function.
US 2010/015133 describes methods of producing polypeptides by regulating polypeptide association.
US 2010/105873 describes integrated approach for generating multidomain protein therapeutics.
U.S. Pat. No. 6,737,056 describes polypeptide variants with a Itered effector function. Previous efforts have been made to identify antibody Fc-variants with an enhanced effector function or other modified properties. Such studies have focused on, e.g., exchanging segments between IgG isotypes to generate chimeric IgG molecules (Natsume et al., 2008 Cancer Res 68(10), 3863-72) or amino acid substitutions in the hinge region (Dall'Acqua et al., 2006 J Immunol 177, 1129-1138) or in or near the C1q-binding site in the CH2 domain, centered around residues D270, K322, P329, and P331 (Idusogie et al., 2001 J Immunol 166, 2571-2575; Michaelsen et al., 2009 Scand J Immunol 70, 553-564 and WO 99/51642). For example, Moore et al. (2010 mAbs 2(2), 181-189)) describes testing various combinations of S267E, H268F, S324T, S239D, I332E, G236A and I332E for enhanced effector function via CDC or ADCC. Other Fc mutations affecting binding to Fc-receptors (WO 2006/105062, WO 00/42072, U.S. Pat. Nos. 6,737,056 and 7,083,784) or physical properties of the antibodies (WO 2007/005612 A1) have also been suggested.
Despite these and other advances in the art, however, there remains a need for new and improved antibody-based therapeutics.
SUMMARY OF THE INVENTION
The present invention provides polypeptide and antibody variants having an enhanced effector function as compared to its parent polypeptide/antibody. Without being limited to theory, it is believed that the variants are capable of a more stable binding interaction between the Fc regions of two polypeptide/antibody molecules, thereby providing a more avid surface which leads to an enhanced effector function, such as an increased or more specific CDC response. Particular variants are also characterized by an improved ADCC response, ADCP response, and/or other enhanced effector functions. This subtle mechanism of polypeptide/antibody engineering can be applied, for instance, to increase the efficacy or specificity of antibody-based therapeutics, as described herein.
Thus in one aspect the present invention relatest to a variant of a parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region, wherein the variant comprises a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.
The invention also provides for the use of at least one such mutation to increase the effector function mediated by the polypeptide or antibody when bound to its antigen on, for example, the surface of an antigen-expressing cell, a cell membrane or a virion.
In one aspect, herein referred to as “single-mutant”, the variant has increased effector function as compared to the parent polypeptide or antibody.
In one aspect, herein referred to as “double-mutant”, the variant comprises at least two mutations in said segment, and has improved effector function as compared to a variant comprising only one of the two mutations, the parent polypeptide or antibody, or both.
In one aspect, herein referred to as “mixed-mutant”, the variant provides an increased effector function when used in combination with a second variant of the same or a different polypeptide or antibody comprising a mutation in a different amino acid residue in said segment, as compared to one or more of the variant, second variant, and the parent polypeptide or antibody alone.
Typically, the mutation is an amino acid substitution, such as a mutation exchanging a parent amino acid residue for one that has a different size and/or physicochemical property that promotes the formation of a new intermolecular Fc:Fc bond or increases the interaction strength of an existing pair. Exemplary amino acid residues for mutation according to the invention are shown in Tables 1 and 2A and B, along with exemplary amino acid substitutions. Non-limiting illustrations of different aspects of the invention are provided in FIG. 1.
These and other aspects of the invention, particularly various uses and therapeutic applications for the antibody variants, are described in further detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1C Schematic representation of IgG molecules in hexamer formation. The dotted circle illustrates two adjacent Fc:Fc interaction pairs of two neighbouring IgG molecules. The arrow in the box illustrates the direction from which the illustrations in FIGS. 1B, 1C and 1D are viewed: the two neighbouring Fc molecules are 90° rotated (in the plane of the drawing) and viewed from the Fab-arms in the direction of the CH3 domains. (FIG. 1B) Observed effect of oligomerization-enhancing mutations on CDC. Schematic representation illustrating Fc:Fc interaction pairs with increased efficacy according to the single mutant and double mutant aspects of the invention. (FIG. 1C) Observed effect of oligomerization-inhibiting mutations on CDC. Schematic representation illustrating how at least two oligomerization-inhibiting mutations that compensate each other can be, either combined into one molecule (double mutant aspect), or seperated over two molecules (mixed mutant aspect), to restore or increase Fc:Fc interaction according to the double mutant and mixed mutants aspects of the invention. Mixed mutants achieve specific effector function activation dependent on binding of both antibodies, which can recognize different targets. (FIG. 1D) Theoretical effect of C1q binding-inhibiting mutations on CDC. Schematic representation of Fc:C1q interactions, illustrating that if mutations inhibit C1q-binding, they cannot be combined or mixed to restore CDC activity, because C1q cannot compensate for the defect introduced in the antibody.
FIG. 2: Sequence alignment of the human IgG1, IgG1f, IgG2, IgG3 and IgG4 Fc segments corresponding to residues P247 to K447 in the IgG1 heavy chain, using Clustal 2.1 software, as numbered by the EU index as set forth in Kabat. The sequences shown represent residues 130 to 330 of the human IgG1 heavy chain constant region (SEQ ID NO:1; UniProt accession No. P01857) and of the allotypic variant IgG1m(f); residues 126 to 326 of the IgG2 heavy chain constant region (SEQ ID NO:2; UniProt accession No. P01859); and residues 177 to 377 of the IgG3 heavy chain constant region (SEQ ID NO:2; UniProt accession No. P01860); and residues 127 to 327 of the IgG4 heavy chain constant region (SEQ ID NO:4; UniProt accession No. P01861).
FIGS. 3A and 3B: Sequence alignment of anti-EGFr antibody 2F8 in an IgG1 (SEQ ID NO:3), IgG4 (SEQ ID NO:5) and (partial) IgG3 (SEQ ID NO:6) backbone. Amino acid numbering according to Kabat and according to the EU-index are depicted (both described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
FIG. 4: Detailed view of the K439/S440 interactions between the Fc of adjacent molecules (Fc and Fc′, respectively) in a multimeric (e.g., hexameric) arrangement, illustrating the interaction between wild-type, unmodified Fc and Fc′ molecules.
FIG. 5: Detailed view of the K439/S440 interactions between the Fc of adjacent molecules (Fc and Fc′, respectively) in a multimeric (e.g., hexameric) arrangement illustrating the interaction between variant Fc and Fc′ molecules comprising K439E and S440K mutations.
FIG. 6: C1q binding ELISA with 7D8 Fc:Fc mutants. Concentration series of the indicated antibodies were coated to the wells of a microtiter plate and incubated with a fixed concentration C1q. The efficiency to bind C1q was comparable to wild type 7D8 for all coated mutants, except I253D. A representative of at least 3 experiments is shown.
FIG. 7: CDC mediated by 7D8 variants on CD20-positive Raji cells. Raji cells were incubated with the 7D8 mutants (K439E, S440K, K439E/S440K Double mutant, K439E+S440K mix) and a concentration series of C1q to test the CDC efficacy by measuring cell lysis. A representative graph of repeated experiments is shown.
FIG. 8: CDC mediated by 7D8 mutants (7D8-WT, K439E, S440K, K439E/S440K double mutant, K439E+S440K mix) on CD20-positive Daudi cells. A concentration series of 7D8 mutants were tested for their efficacy to induce CDC.
FIGS. 9A-9D: CDC mediated by mutants of CD38 antibody HuMAb 005 on CD38-positive cells. (FIG. 9A) CDC efficacy on Daudi cells by a concentration series of 005 mutants. (FIG. 9B) CDC efficacy on Raji cells by a concentration series of HuMAb 005 mutants. (FIG. 9C) CDC efficacy of E345R mutant of HuMAb 005 with either 20% or 50% NHS on Wien133 cells. (FIG. 9D) CDC efficacy of E345R mutants of HuMAb 005 and 7D8 with either 20% or 50% NHS on Raji cells. Unpurified antibody samples isolated from transient transfections were tested. As a negative control, supernatant of mock-transfected cells was used.
FIGS. 10A and 10B: CDC by wild type and E345R mutants of CD38 antibody HuMAb 005, (FIG. 10A) and CD20 antibody HuMAb 7D8 (FIG. 10B) in a competition experiment with an Fc-binding peptide. Cell lysis was measured after CDC on antibody-opsonized Daudi-cells incubated with a concentration series of the Fc-binding DCAWHLGELVWCT peptide (SEQ ID NO:7). Unpurified antibody samples isolated from transient transfections were used. As a negative control, supernatant of mock-transfected cells was used.
FIG. 11: ADCC of CD38 expressing Daudi cells by wild type CD38 antibody HuMAb 005 and mutant IgG1-005-E345R. ADCC of PBMC of one donor is shown, depicted as % lysis.
FIGS. 12A-12C: Binding of wild type IgG1-7D8 and mutant IgG1-7D8-E345R to human, cynomolgus and mouse FcRn, as determined by ELISA at pH 6.
FIG. 13: Plasma concentrations of wild type IgG1-7D8 and -E354R, —S440K and K322A variants following intravenous injection in SCID mice.
FIGS. 14A-14D: CDC on CD20- and CD38-positive Wien133 cells.
FIGS. 15A and 15B: Evaluation of the in vivo efficacy of IgG1-7D8-E345R in a subcutaneous xenograft model with Raji-luc #2D1 cells.
FIGS. 16A and 16B: Evaluation of the in vivo efficacy of IgG1-005-E345R in a subcutaneous xenograft model with Raji-luc #2D1 cells.
FIG. 17: CDC on CD38-positive, EGFR-negative Wien133 cells by CD38/EGFR bispecific antibody with the E345R mutation.
FIGS. 18A and 18B: CDC on CD20-positive, CD38-negative Wien133 cells or Raji cells by CD20/CD38 bispecific antibody with and without the E345R mutation.
FIG. 19: CDC on EGFR-positive A431 cells by EGFR antibody 2F8 with the E345R mutation.
FIGS. 20A and 20B: CDC mediated by E345R mutant antibodies.
FIG. 21: Colocalization analysis of TF antibodies (FITC) with lysosomal marker LAMP1 (APC).
FIGS. 22A-22D: Introduction of E345R resulted in enhanced CDC-mediated killing compared to wild type rituximab tested on different B cell lines.
FIG. 22E: Introduction of E345R resulted in increased maximal CDC-mediated killing compared to wild type rituximab, independent of the expression levels of the complement regulatory proteins CD46 (A), CD55 (B) or CD59 (C) in different B cell lines with comparable CD20 expression levels.
FIGS. 23A-23D: CDC kinetics. E345R antibodies result in more rapid and more substantial target cell lysis by CDC than compared to wild type antibodies.
FIG. 24: CDC kinetics. Introduction of the E345R mutation in the bispecific CD38xCD20 antibody results in more rapid and more substantial CDC-mediated target cell lysis.
FIGS. 25: CDC kinetics. Introduction of the E345R mutation in bispecific antibody CD38xEGFR that bind monovalently to the EGFR-negative Raji cells, results in more rapid and more substantial CDC-mediated target cell lysis.
FIGS. 26A-26F: CDC on Wien133 cells by a combination of a wild type antibody with a mutant antibody containing (FIGS. 26A-26C) E345R and Q386K or (FIGS. 26D-26F) E345R, E430G and Q386K. IgG1-b12 mutants do not bind Wien133 cells and were used as negative control antibodies.
FIGS. 27A and 27B: CDC efficacy of IgG1, IgG2, IgG3 and IgG4 isotype antibodies containing the E345R mutation.
FIGS. 28A and 28B: Introduction of the Fc-Fc stabilizing E345R mutation in wild type CD38 antibody 005 results in enhanced killing of primary CLL cells in an ex vivo CDC assay (average ±standard error of the mean).
DETAILED DESCRIPTION OF THE INVENTION
As described herein, surprisingly, mutations in amino acids that are not directly involved in Fc:C1q binding can nevertheless increase the CDC of an antibody, and can also improve other Fc-mediated effector functions of the antibody. This supports the hypothesis that antibody molecules such as IgG1 antibodies can form oligomeric structures which are later bound by C1q. Further, while some mutations were found to decrease CDC-induction, some combinations of such mutations in the same or different antibody molecules resulted in restored CDC-induction, and showed further specificity for oligomerization of antibodies, and thereby promoting more specific CDC-induction. Particular mutations increasing the CDC-response were also characterized by an improved ADCC response, increased avidity, increased internalization and in vivo efficacy in a mouse tumor model system as shown in the Examples. These discoveries allow for novel antibody-based therapeutics with enhanced CDC-induction capability, more selective CDC-induction, and/or other improved effector functions.
The antibody variants of the invention all comprise an antigen-binding region and a full-length or partial Fc region comprising at least one mutation in the segment corresponding to amino acid residues P247 to K447 in IgG1. Without being limited to theory, it is believed that the identified mutations result in a more effective and/or more specific CDC-induction based on three different principles, schematically represented in FIG. 1, and herein referred to as “single mutant”, “double mutant” and “mixed mutants”.
The improved C1q and/or CDC effects from the variants of the invention are primarily only detectable in assays allowing antibody oligomers to form, such as in cell-based assays where the antigen is not fixed but present in a fluid membrane. Further, that these effects result from a more stable antibody oligomer and not from a modification of a direct binding site of C1q can be verified according to the principles shown in FIG. 1C.
Definitions
The term “single-mutant”, is to be understood as a variant of the present invention which has increased effector function as compared to the parent polypeptide or antibody.
The term “double-mutant”, is to be understood as a variant comprising at least two mutations in said segment, and has improved effector function as compared to a variant comprising only one of the two mutations, the parent polypeptide or antibody, or both.
The term “mixed-mutant”, is to be understood as a variant providing an increased effector function when used in combination with a second variant of the same or a different polypeptide or antibody comprising a mutation in a different amino acid residue in said segment, as compared to one or more of the variant, second variant, and the parent polypeptide or antibody alone.
The term “polypeptide comprising an Fc-domain of an immunoglobulin and a binding region” refers in the context of the present invention to a polypeptide which comprises an Fc-domain of an immunoglobulin and a binding region which is a capable of binding to any molecule, such as a polypeptide, e.g. present on a cell, bacterium, or virion. The Fc-domain of an immunoglobulin is defined as the fragment of an antibody which would be typically generated after digestion of an antibody with papain (which is known for someone skilled in the art) which includes the two CH2-CH3 regions of an immunoglobulin and a connecting region, e.g. a hinge region. The constant domain of an antibody heavy chain defines the antibody isotype, e.g. IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE. The Fc-domain mediates the effector functions of antibodies with cell surface receptors called Fc receptors and proteins of the complement system. The binding region may be a polypeptide sequence, such as a protein, protein ligand, receptor, an antigen-binding region, or a ligand-binding region capable to bind to a cell, bacterium, virion. If the binding region is e.g. a receptor the “polypeptide comprising an Fc-domain of an immunoglobulin and a binding region” may have been prepared as a fusion protein of Fc-domain of an immunoglobulin and said binding region. If the binding region is an antigen-binding region the “polypeptide comprising an Fc-domain of an immunoglobulin and a binding region” may be an antibody, like a human antibody or a heavy chain only antibody or a ScFv-Fc-fusion. The polypeptide comprising an Fc-domain of an immunoglobulin and a binding region may typically comprise a connecting region, e.g. a hinge region, and two CH2-CH3 region of the heavy chain of an immunoglobulin, thus the “polypeptide comprising a Fc-domain of an immunoglobulin and a binding region” may be a “polypeptide comprising at least an Fc-domain of an immunoglobulin and a binding region”. The term “Fc-domain of an immunoglobulin” means in the context of the present invention that a connecting region, e.g. hinge depending on the subtype of antibody, and the CH2 and CH3 region of an immunoglobulin are present, e.g. a human IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2 or IgE.
The term “CH2 region” or “CH2 domain” as used herein is intended to refer the CH2 region of an immunoglobulin. Thus for example the CH2 region of a human IgG1 antibody corresponds to amino acids 228-340 according to the EU numbering system. However, the CH2 region may also be any of the other subtypes as described herein.
The term “CH3 region” or “CH3 domain” as used herein is intended to refer the CH3 region of an immunoglobulin. Thus for example the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering system. However, the CH2 region may also be any of the other subtypes as described herein.
The term “immunoglobulin” refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four potentially inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. See for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region typically is comprised of three domains, CH1, CH2, and CH3. The heavy chains are inter-connected via disulfide bonds in the so-called “hinge region”. Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)). Unless otherwise stated or contradicted by context, the amino acids of the constant region sequences are herein numbered according to the EU-index (described in Kabat, E. A. et al., Sequences of proteins of immunological interest. 5th Edition—US Department of Health and Human Services, NIH publication No. 91-3242, pp 662,680,689 (1991)).
The term “antibody” (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions with a half life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about eight hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about three, four, five, six, seven or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity). The antibody of the present invention comprises an Fc-domain of an immunoglobulin and an antigen-binding region. An antibody generally contains two CH2-CH3 regions and a connecting region, e.g. a hinge region, e.g. at least an Fc-domain. Thus the antibody of the present invention may comprise an Fc region and an antigen-binding region. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant or “Fc” regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q, the first component in the classical pathway of complement activation. An antibody may also be a multispecific antibody, such as a bispecific antibody or similar molecule. The term “bispecific antibody” refers to antibody having specificities for at least two different, typically non-overlapping, epitopes. Such epitopes may be on the same or different targets. If the epitopes are on different targets, such targets may be on the same cell or different cells or cell types. As indicated above, unless otherwise stated or clearly contradicted by the context, the term antibody herein includes fragments of an antibody which comprise at least a portion of an Fc-region and which retain the ability to specifically bind to the antigen. Such fragments may be provided by any known technique, such as enzymatic cleavage, peptide synthesis and recombinant expression techniques. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “Ab” or “antibody” include, without limitation, monovalent antibodies (described in WO2007059782 by Genmab); heavy-chain antibodies, consisting only of two heavy chains and naturally occurring in e.g. camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMabs (Roche, WO2011069104), strand-exchange engineered domain (SEED or Seed-body) which are asymmetric and bispecific antibody-like molecules (Merck, WO2007110205); Triomab (Fresenius, Lindhofer et al. (1995 J Immunol 155:219); FcΔAdp (Regeneron, WO2010151792), Azymetric Scaffold (Zymeworks/Merck, WO2012/058768), mAb-Fv (Xencor, WO2011/028952), Dual variable domain immunoglobulin (Abbott, DVD-Ig, U.S. Pat. No. 7,612,181); Dual domain double head antibodies (Unilever; Sanofi Aventis, WO20100226923), Di-diabody (ImClone/Eli Lilly), Knobs-into-holes antibody formats (Genentech, WO9850431); DuoBody (Genmab, WO 2011/131746); Electrostatic steering antibody formats (Amgen, EP1870459 and WO 2009089004; Chugai, U5201000155133; Oncomed, WO2010129304A2); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545), CrossMAbs (Roche, WO2011117329), LUZ-Y (Genentech), Biclonic (Merus), Dual Targeting domain antibodies (GSK/Domantis), Two-in-one Antibodies recognizing two targets (Genentech, NovImmune), Cross-linked Mabs (Karmanos Cancer Center), CovX-body (CovX/Pfizer), IgG-like Bispecific (ImClone/Eli Lilly, Shen, J., et al. J Immunol Methods, 2007. 318(1-2): p. 65-74), and DIG-body and PIG-body (Pharmabcine), and Dual-affinity retargeting molecules (Fc-DART or Ig-DART, by Macrogenics, WO/2008/157379, WO/2010/080538), Zybodies (Zyngenia), approaches with common light chain (Crucell/Merus, U.S. Pat. No. 7,262,028) or common heavy chains (κλBodies by NovImmune), as well as fusion proteins comprising a polypeptide sequence fused to an antibody fragment containing an Fc-domain like scFv-fusions, like BsAb by ZymoGenetics/BMS), HERCULES by Biogen Idec (US007951918), SCORPIONS by Emergent BioSolutions/Trubion, Ts2Ab (MedImmune/AZ (Dimasi, N., et al. J Mol Biol, 2009. 393(3): p. 672-92), scFv fusion by Novartis, scFv fusion by Changzhou Adam Biotech Inc (CN 102250246), TvAb by Roche (WO 2012025525, WO 2012025530), mAb2 by f-Star (WO2008/003116), and dual scFv-fusions. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures (recombinant polyclonals) for instance generated by technologies exploited by Symphogen and Merus (Oligoclonics), and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies. An antibody as generated can potentially possess any isotype.
The term “full-length antibody” when used herein, refers to an antibody (e.g., a parent or variant antibody) which contains all heavy and light chain constant and variable domains corresponding to those that are normally found in a wild-type antibody of that isotype.
The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The terms “monoclonal antibody”, “monoclonal Ab”, “monoclonal antibody composition”, “mAb”, or the like, as used herein refer to a preparation of Ab molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term “human monoclonal antibody” refers to Abs displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs may be generated by a hybridoma which includes a B cell obtained from a transgenic or transchromosomal nonhuman animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene repertoire and a light chain transgene repertoire, rearranged to produce a functional human antibody and fused to an immortalized cell.
As used herein, “isotype” refers to the immunoglobulin class (for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE, or IgM or any allotypes thereof such as IgG1m(za) and IgG1m(f)) that is encoded by heavy chain constant region genes. Further, each heavy chain isotype can be combined with either a kappa (κ) or lambda (λ) light chain.
The term “monovalent antibody” means in the context of the present invention that an antibody molecule is capable of binding with only one of the binding domains of the antibody to an antigen, e.g. has a single antigen-antibody interaction, and thus is not able of antigen crosslinking.
As used herein, the term “target” is in the context of the present invention to be understood as a molecule to which the binding region of the polypeptide comprising an Fc domain and a binding region, when used in the context of the binding of an antibody includes any antigen towards which the raised antibody is directed. The term “antigen” and “target” may in relation to an antibody be used interchangeably and constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention.
As used herein, the term “binding” in the context of the binding of an antibody to a predetermined antigen typically is a binding with an affinity corresponding to a KD of about 10−6 M or less, e.g. 10−7 M or less, such as about 10−8 M or less, such as about 10−9 M or less, about 10−10 M or less, or about 10−11 M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1,000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The amount with which the affinity is lower is dependent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is highly specific), then the amount with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000 fold. The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.
A “variant” or “antibody variant” or “variant of a parent antibody” of the present invention is an antibody molecule or which comprises one or more mutations as compared to a “parent antibody”. Similarly, a “variant” or “a variant of a polypeptide comprising an Fc-domain of an immunoglobulin and a binding region” or “a variant of a parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region” of the present invention is a “polypeptide comprising an Fc-domain of an immunoglobulin and a binding region”, which comprises one or more mutations as compared to a “parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region”. The different terms may be used interchangeably and constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention. Exemplary parent antibody formats include, without limitation, a wild-type antibody, a full-length antibody or Fc-containing antibody fragment, a bispecific antibody, a human antibody, or any combination thereof. Exemplary mutations include amino acid deletions, insertions, and substitutions of amino acids in the parent amino acid sequence. Amino acid substitutions may exchange a native amino acid for another naturally-occurring amino acid, or for a non-naturally-occurring amino acid derivative. The amino acid substitution may be conservative or non-conservative. In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected in one or more of the following three tables:
Amino acid residue classes for conservative substitutions
Acidic Residues
Asp (D) and Glu (E)
Basic Residues
Lys (K), Arg (R), and His (H)
Hydrophilic Uncharged Residues
Ser (S), Thr (T), Asn (N), and
Gln (Q)
Aliphatic Uncharged Residues
Gly (G), Ala (A), Val (V), Leu (L),
and Ile (I)
Non-polar Uncharged Residues
Cys (C), Met (M), and Pro (P)
Aromatic Residues
Phe (F), Tyr (Y), and Trp (W)
Alternative conservative amino acid residue substitution classes
1
A
S
T
2
D
E
3
N
Q
4
R
K
5
I
L
M
6
F
Y
W
Alternative Physical and Functional Classifications of
Amino Acid Residues
Alcohol group-containing residues
S and T
Aliphatic residues
I, L, V, and M
Cycloalkenyl-associated residues
F, H, W, and Y
Hydrophobic residues
A, C, F, G, H, I, L, M, R, T, V, W,
and Y
Negatively charged residues
D and E
Polar residues
C, D, E, H, K, N, Q, R, S, and T
Positively charged residues
H, K, and R
Small residues
A, C, D, G, N, P, S, T, and V
Very small residues
A, G, and S
Residues involved in turn
A, C, D, E, G, H, K, N, Q, R, S, P,
formation
and T
Flexible residues
Q, T, K, S, G, P, D, E, and R
In the context of the present invention, a substitution in a variant is indicated as:
Original amino acid-position-substituted amino acid;
The three letter code, or one letter code, are used, including the codes Xaa and X to indicate amino acid residue. Accordingly, the notation “E345R” or “Glu345Arg” means, that the variant comprises a substitution of Glutamic acid with Arginine in the variant amino acid position corresponding to the amino acid in position 345 in the parent antibody, when the two are aligned as indicated below.
Where a position as such is not present in an antibody, but the variant comprises an insertion of an amino acid, for example:
Position—substituted amino acid; the notation, e.g., “448E” is used.
Such notation is particular relevant in connection with modification(s) in a series of homologous polypeptides or antibodies.
Similarly when the identity of the substitution amino acid residues(s) is immaterial:
Original amino acid—position; or “E345”.
For a modification where the original amino acid(s) and/or substituted amino acid(s) may comprise more than one, but not all amino acid(s), the substitution of Glutamic acid for Arginine, Lysine or Tryptophan in position 345:
“Glu345Arg,Lys,Trp” or “E345R,K,W” or “E345R/K/W” or “E345 to R, K or W” may be used interchangeably in the context of the invention.
Furthermore, the term “a substitution” embraces a substitution into any one of the other nineteen natural amino acids, or into other amino acids, such as non-natural amino acids. For example, a substitution of amino acid E in position 345 includes each of the following substitutions: 345A, 345C, 345D, 345G, 345H, 345F, 345I, 345K, 345L, 345M, 345N, 345Q, 345R, 345S, 345T, 345V, 345W, and 345Y. This is, by the way, equivalent to the designation 345X, wherein the X designates any amino acid. These substitutions can also be designated E345A, E345C, etc, or E345A,C,ect, or E345A/C/ect. The same applies to analogy to each and every position mentioned herein, to specifically include herein any one of such substitutions.
An amino acid or segment in one sequence that “corresponds to” an amino acid or segment in another sequence is one that (i) aligns with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW or similar, typically at default settings and (ii) has a sequence identity to SEQ ID NO:1 of at least 50%, at least 80%, at least 90%, or at least 95%. For example, the sequence alignments shown in FIGS. 2 and 3 can be used to identify any amino acid in the IgG2, IgG3 or IgG4 Fc sequence that corresponds to a particular amino acid in the IgG1 Fc sequence.
The present invention refers to variants, viz. parent antibodies, and/or variant antibodies, having a certain degree of identity to amino acids P247 to K447 of SEQ ID Nos:1, 2, 3, 4, and 5, such parent and/or variant antibodies being hereinafter designated “homologous antibodies”.
For purposes of the present invention the degree of identity between two amino acid sequences, as well as the degree of identity between two nucleotide sequences, is determined by the program “align” which is a Needleman-Wunsch alignment (i.e. a global alignment). The program is used for alignment of polypeptide, as well as nucleotide sequences. The default scoring matrix BLOSUM50 is used for polypeptide alignments, and the default identity matrix is used for nucleotide alignments, the penalty of the first residue of a gap is −12 for polypeptides and −16 for nucleotides. The penalties for further residues of a gap are −2 for polypeptides, and −4 for nucleotides.
“Align” is part of the FASTA package version v20u6 (see W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448, and W. R. Pearson (1990) “Rapid and Sensitive Sequence Comparison with FASTP and FASTA”, Methods in Enzymology 183:63-98). FASTA protein alignments use the Smith-Waterman algorithm with no limitation on gap size (see “Smith-Waterman algorithm”, T. F. Smith and M. S. Waterman (1981) J. Mol. Biolo. 147:195-197).
As used herein, the term “effector cell” refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, for instance lymphocytes (such as B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils. Some effector cells express Fc receptors (FcRs) or complement receptors and carry out specific immune functions. In some embodiments, an effector cell such as, e.g., a natural killer cell, is capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells and Kupffer cells which express FcRs, are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. In some embodiments the ADCC can be further enhanced by antibody driven classical complement activation resulting in the deposition of activated C3 fragments on the target cell. C3 cleavage products are ligands to complement receptors (CRs), such as CR3, expressoid on myeloid cells. The recognition of complement fragments by CRs on effector cells may promote enhanced Fc receptor-mediated ADCC. In some embodiments antibody driven classical complement activation leads to C3 fragments on the target cell. These C3 cleavage products may promote direct complement-dependent cellular cytotoxicity (CDCC). In some embodiments, an effector cell may phagocytose a target antigen, target particle or target cell. The expression of a particular FcR or complement receptor on an effector cell may be regulated by humoral factors such as cytokines. For example, expression of FcγRI has been found to be up-regulated by interferon γ (IFN γ) and/or G-CSF. This enhanced expression increases the cytotoxic activity of FcγRI-bearing cells against targets. An effector cell can phagocytose a target antigen or phagocytose or lyse a target cell. In some embodiments antibody driven classical complement activation leads to C3 fragments on the target cell. These C3 cleavage products may promote direct phagocytoses by effector cells or indirectly by enhancing antibody mediated phagocytosis.
The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of inducing transcription a nucleic acid segment ligated into the vector. One type of vector is a “plasmid”, which is in the form of a circular double stranded DNA loop. Another type of vector is a viral vector, wherein the nucleic acid segment may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (for instance bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (such as non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (such as replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK-293 cells, PER.C6, NS0 cells, and lymphocytic cells, and prokaryotic cells such as E. coli and other eukaryotic hosts such as plant cells and fungi.
The term “transfectoma”, as used herein, includes recombinant eukaryotic host cells expressing the Ab or a target antigen, such as CHO cells, PER.C6, NS0 cells, HEK-293 cells, plant cells, or fungi, including yeast cells.
The term “preparation” refers to preparations of antibody variants and mixtures of different antibody variants which can have an increased ability to form oligomers when interacting with antigen associated with a cell (e.g., an antigen expressed on the surface of the cell), a cell membrane, a virion or other structure, thereby enabling an increased C1q binding, complement activation, CDC, ADCC, ADCP, other Fc-mediated effector function, internalization, downmodulation, apoptosis, antibody-drug-conjugate (ADC) uptake, avidity or a combination of any thereof. Exemplary assays are provided in the Examples for, e.g., C1q-binding avidity (Example 4), CDC (Examples 5, 6 and 10, 16, 19, 22, 23, 24, 25); ADCC (Example 12) and in vivo efficacy (Example 20, 21). Variants according to the aspects herein referred to as “single-mutant”, “double-mutant”, and “mixed-mutants”, are described in further detail below, along with exemplary processes for their preparation and methods of use.
As used herein, the term “affinity” is the strength of binding of one molecule, e.g. an antibody, to another, e.g. a target or antigen, at a single site, such as the monovalent binding of an individual antigen binding site of an antibody to an antigen.
As used herein, the term “avidity” refers to the combined strength of multiple binding sites between two structures, such as between multiple antigen binding sites of antibodies simultaneously interacting with a target or e.g. between antibody and C1q. When more than one binding interactions are present, the two structures will only dissociate when all binding sites dissociate, and thus, the dissociation rate will be slower than for the individual binding sites, and thereby providing a greater effective total binding strength (avidity) compared to the strength of binding of the individual binding sites (affinity).
As used herein, the term “oligomer” refers to a molecule that consists of more than one but a limited number of monomer units (e.g. antibodies) in contrast to a polymer that, at least in principle, consists of an unlimited number of monomers. Exemplary oligomers are dimers, trimers, tetramers, pentamers and hexamers. Greek prefixes are often used to designate the number of monomer units in the oligomer, for example a tetramer being composed of four units and a hexamer of six units.
The term “oligomerization”, as used herein, is intended to refer to a process that converts monomers to a finite degree of polymerization. Herein, it is observed, that the oligomerization of Fc-domains takes place after target binding by Fc-domain containing polypeptides, such as antibodies, preferably but not limited to at a cell surface. The oligomerization of antibodies can be evaluated for example using a cell surface C1q-binding assay (as described in examples 4 and 9), C1q efficacy assay (as described in example 5) and complement dependent cytotoxicity described in Example 6, 10 and 19).
The term “C1q binding”, as used herein, is intended to refer to the binding of C1q in the context of the binding of C1q to an antibody bound to its antigen. The antibody bound to its antigen is to be understood as happening both in vivo and in vitro in the context described herein. C1q binding can be evaluated for example by using immobilized antibody on artificial surface (e.g. plastic in plates for ELISA, as described in example 3) or by using bound to a predetermined antigen on a cellular or virion surface (as described in examples 4 and 9). The binding of C1q to an antibody oligomer is to be understood herein as a multivalent interaction resulting in high avidity binding.
As used herein, the term “complement activation” refers to the activation of the classical complement pathway, which is triggered by the binding of complement component C1q to an antibody bound to its antigen. C1q is the first protein in the early events of the classical complement cascade that involves a series of cleavage reactions that culminate in the formation of an enzymatic activity called C3 convertase, which cleaves complement component C3 into C3b and C3a. C3b binds covalently to C5 on the membrane to form C5b that in turn triggers the late events of complement activation in which terminal complement components C5b, C6, C7, C8 and C9 assemble into the membrane attack complex (MAC). The complement cascade results in the creation of pores due to which causes cell lysis, also known as CDC. Complement activation can be evaluated by using C1q efficacy (as described in example 5), CDC kinetics (as described in examples 28, 29, and 30), CDC assays (as described in examples 6, 10, 19, 25, 27, and 33) or by the method Cellular deposition of C3b and C4b described in Beurskens et al Apr. 1, 2012 vol. 188 no. 7 3532-3541.
The term “complement-dependent cytotoxicity” (“CDC”), as used herein, is intended to refer to the process of antibody-mediated complement activation leading to lysis of the antibody bound to its target on a cell or virion as a result of pores in the membrane that are created by MAC assembly. CDC can be evaluated by in vitro assay such as a CDC assay in which normal human serum is used as a complement source, as described in example 6, 10, 19, 25, 27, and 33 or in a C1q efficacy assay, as described in example 5, in which normal human serum has been limited in C1q.
The term “antibody-dependent cell-mediated cytotoxicity” (“ADCC”) as used herein, is intended to refer to a mechanism of killing of antibody-coated target cells or virions by cells expressing Fc receptors that recognize the constant region of the bound antibody. ADCC can be determined using methods such as, e.g., the ADCC assay described in example 12.
The term “antibody-dependent cellular phagocytosis” (“ADCP”) as used herein is intended to refer to a mechanism of elimination of antibody-coated target cells or virions by internalization by phagocytes. The internalized antibody-coated target cells or virions is contained in a vesicle called a phagosome, which then fuses with one or more lysosomes to form a phagolysosome. ADCP may be evaluated by using an in vitro cytotoxicity assay with macrophages as effortor cells and video microscopy as described by van Bij et al. in Journal of Hepatology Volume 53, Issue 4, October 2010, Pages 677-685. Or as described in example 14 for e.g. S. aureus phagocytos by PMN.
The term “complement-dependent cellular cytotoxicity” (“CDCC”) as used herein is intended to refer to a mechanism of killing of target cells or virions by cells expressing complement receptors that recognize complement 3 (C3) cleavage products that are covalently bound to the target cells or virions as a result of antibody-mediated complement activation. CDCC may be evaluated in a similar manner as described for ADCC.
The term “downmodulation”, as used herein, is intended to refer a process that decreases the number of molecules, such as antigens or receptors, on a cellular surface, e.g. by binding of an antibody to a receptor.
The term “internalization”, as used herein, is intended to refer to any mechanism by which an antibody or Fc-containing polypeptide is internalized into a target-expressing cell from the cell-surface and/or from surrounding medium, e.g., via endocytosis. The internalization of an antibody can be evaluated using a direct assay measuring the amount of internalized antibody (such as, e.g., the lysosomal co-localization assay described in Example 26).
The term “antibody-drug conjugate”, as used herein refers to an antibody or Fc-containing polypeptide having specificity for at least one type of malignant cell, a drug, and a linker coupling the drug to e.g. the antibody. The linker is cleavable or non-cleavable in the presence of the malignant cell; wherein the antibody-drug conjugate kills the malignant cell.
The term “antibody-drug conjugate uptake”, as used herein refers to the process in which antibody-drug conjugates are bound to a target on a cell followed by uptake/engulfment by the cell membrane and thereby is drawn into the cell. Antibody-drug conjugate uptake may be evaluated as “antibody-mediated internalization and cell killing by anti-TF ADC in an in vitro killing assay” as described in WO 2011/157741.
The term “apoptosis”, as used herein refers to the process of programmed cell death (PCD) that may occur in a cell. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Binding of an antibody to a certain receptor may induce apoptosis.
Fc-receptor binding may be indirectly measured as described in Example 12.
The term “FcRn”, as used herein is intended to refer to neonatal Fc receptor which is an Fc receptor. It was first discovered in rodents as a unique receptor capable of transporting IgG from mother's milk across the epithelium of newborn rodent's gut into the newborn's bloodstream. Further studies revealed a similar receptor in humans. In humans, however, it is found in the placenta to help facilitate transport of mother's IgG to the growing fetus and it has also been shown to play a role in monitoring IgG turnover. FcRn binds IgG at acidic pH of 6.0-6.5 but not at neutral or higher pH. Therefore, FcRn can bind IgG from the intestinal lumen (the inside of the gut) at a slightly acidic pH and ensure efficient unidirectional transport to the basolateral side (inside the body) where the pH is neutral to basic (pH 7.0-7.5). This receptor also plays a role in adult salvage of IgG through its occurrence in the pathway of endocytosis in endothelial cells. FcRn receptors in the acidic endosomes bind to IgG internalized through pinocytosis, recycling it to the cell surface, releasing it at the basic pH of blood, thereby preventing it from undergoing lysosomal degradation. This mechanism may provide an explanation for the greater half-life of IgG in the blood compared to other isotypes. Example 13 describes an assay showing IgG binding to FcRn at pH 6.0 in ELISA.
The term “Protein A”, as used herein is intended to refer to a 56 kDa MSCRAMM surface protein originally found in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topology, cellular osmolarity, and a two-component system called ArIS-ArIR. It has found use in biochemical research because of its ability to bind immunoglobulins. It is composed of five homologous Ig-binding domains that fold into a three-helix bundle. Each domain is able to bind proteins from many of mammalian species, most notably IgGs. It binds the heavy chain Fc region of most immunoglobulins (overlapping the conserved binding site of FcRn receptors) and also interacts with the Fab region of the human VH3 family. Through these interactions in serum, IgG molecules bind the bacteria via their Fc region instead of solely via their Fab regions, by which the bacteria disrupts opsonization, complement activation and phagocytosis.
The term “Protein G”, as used herein is intended to refer to an immunoglobulin-binding protein expressed in group C and G Streptococcal bacteria much like Protein A but with differing specificities. It is a 65-kDa (G148 protein G) and a 58 kDa (C40 protein G) cell surface protein that has found application in purifying antibodies through its binding to the Fc region.
The term “CH2 region” or “CH2 domain” as used herein is intended to refer the CH2 region of an immunoglobulin. Thus for example the CH2 region of a human IgG1 antibody corresponds to amino acids 228-340 according to the EU numbering system.
The term “CH3 region” or “CH3 domain” as used herein is intended to refer the CH3 region of an immunoglobulin. Thus for example the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering system.
The term “allosteric mutations”, as used herein, is intended to refer to modifications, eg insertions, substitutions and deletions, of amino acids P247, and E430, in Fc-domain containing polypeptides, as numbered by the EU index as set forth in Kabat.
The term “hydrophobic knob mutations”, as used herein, is intended to refer to modifications, eg insertions, substitutions and deletions, of amino acids I253, and S254, and Q311, in Fc-domain containing polypeptides, as numbered by the EU index as set forth in Kabat. Hydrophobic knobs are described by Delano W L, et al., Science 287, (2000), pages 1279-1283, e.g. on page 1281.
The term “N-terminal CH3 helix mutations”, as used herein, is intended to refer to modifications, eg insertions, substitutions and deletions, of amino acids R355, and D356, and E356, and E357, and M358, and L358, and T359, more specifically of D356, and E356, and T359, in Fc-domain containing polypeptides, as numbered by the EU index as set forth in Kabat.
The term “C-terminal CH3 beta strand mutations”, as used herein, is intended to refer to modifications, eg insertions, substitutions and deletions, of amino acids Y436, and T437, and Q438, and K439, and S440, and L441, more specifically of Y436, and K439, and S440, in Fc-domain containing polypeptides, as numbered by the EU index as set forth in Kabat.
Methods of Affecting an Effector Function of an Antibody
It is to be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody are also to be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region.
In one aspect the present invention relates to a method of increasing an effector function of a parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region, which method comprises introducing a mutation to the parent polypeptide in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.
In one embodiment the parent polypeptide may be an antibody.
Thus the present invention relates to a method of increasing an effector function of a parent antibody, comprising introducing a mutation to the parent antibody in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.
The reference to “D/E356” refers in the present context to allotypic variants in the sequence of human IgG1. In the IgG1m(za) allotype of human IgG1 the amino acid in position 356 is D, while in the IgG1m(f) allotype of human IgG1 the amino acid in position 356 is E.
Introducing a mutation to a parent antibody according to a method or use of the present invention results in a variant or variant antibody. Thus the method(s) of the present invention may be performed so as to obtain any variant or variant antibody as described herein.
The variant antibody obtained from a method or use of the present invention has an increased effector function compared to the parent antibody. Typically, the effect of an antibody on an effector function may be determined by the EC50 value, which is the concentration of the antibody necessary to obtain half the value of the maximal lysis.
Maximal lysis is the lysis obtained when a saturating amount of the antibody is used, in which saturating is intended to refer to the amount of antibody at which all antigens for the antibody are bound by antibody.
The term “increasing an effector function” or “improving an effector function” refers in the context of the present invention that there is a decrease in the EC50 value of the variant antibody compared to the parent antibody. The decrease in the EC50 value may e.g. be at least or about 2-fold, such as at least or about 3-fold, or at least or about 5-fold, or at least or about 10-fold. Alternatively, “increasing an effector function” or “improving an effector function” means that there is an increase in the maximal amount of cells lysed (where the total amount of cells is set at 100%) by e.g. from 10% to 100% of all cells, such as by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100% under conditions where the parent antibody lyses less than 100% of all cells.
A variant could be tested for increased or improved effector function by cloning the variable domain of the IgG1-005 or IgG1-7D8 heavy chain into the variant and test its efficacy in CDC assays, such as described for Daudi (Example 6) and Wien (Example 10). Using an IgG1-7D8 HC variable domain and Daudi cells, an increase would be defined by a more than 2 fold lower EC50 than the EC50 of IgG1-7D8 under the studied condition, such as about 2-fold, about 3-fold, about 5-fold, about 10-fold or a more than 10-fold lower EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-005 HC variable domain and Daudi cells, an increase would be defined by a more than 2 fold lower EC50 than the EC50 of IgG1-005 under the studied condition, such as about 2-fold, about 3-fold, about 5-fold, about 10-fold or a more than 10-fold lower EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-7D8 HC variable domain and Wien133 cells, an increase would be defined by a more than 2 fold lower EC50 than the EC50 of IgG1-7D8 under the studied condition, such as about 2-fold, about 3-fold, about 5-fold, about 10-fold or a more than 10-fold lower EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-005 HC variable domain and Wien133 cells, an increase would be defined by an increase in the maximal lysis ranging from 10% to 100% of all cells, such as by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%. An increase in CDC efficacy could also be defined by a more than 2-fold lower EC50 than the EC50 of IgG1-005 under the studied condition, such as about 2-fold, about 3-fold, about 5-fold, about 10-fold or a more than 10-fold lower EC50 value, the concentration at which half-maximal lysis is observed under conditions where lysis of Wien133 cells is detectable.
The inventors of the present invention surprisingly found that mutations in these specific positions have an improved effect on effector functions of the variant antibody, which is obtained from introducing a mutation into a parent antibody according to a method of the present invention (e.g. as shown in Example 19). Without being bound by theory, it is believed that by substituting at least one amino acid from the above-mentioned group of positions oligomerization is stimulated. The antibodies bind with higher avidity (exemplified by example 2; direct labelling of IgG-7D8-E345R resulted in increased binding to Daudi cells in comparison to IgG-7D8-WT) which causes the antibodies to bind for a longer time to the cells and thereby different effector functions are enabled, e.g. increased C1q binding, C1q efficacy CDC, ADCC, internalization, ADCP, and/or in vivo efficacy. These effects have been exemplified by example 4 (C1q binding on cells), example 5 (C1q efficacy in a CDC assay), example 6, 7, 27, 28 and 29 (CDC assay), example 12 (ADCC), example 26 (internalization) and example 21 and 22 (in vivo efficacy). Thus the mutation of an amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain may also be referred to as “single mutant” aspect or “effector-enhancing mutations” in the context of the present invention.
In another aspect, the present invention also provides for the use of one or more mutations in Table 1, such as a mutation in an amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, to increase an effector function, e.g. one or more of (i) C1q-binding, (ii) complement activation, (iii) CDC, (iv) oligomer formation, (v) oligomer stability, (vi) antibody-dependent cell-mediated cytotoxity (ADCC), (vii) FcRn-binding, (viii) Fc-gamma receptor-binding, (ix) Protein A-binding, (x) Protein G-binding, (xi) antibody-dependent cellular phagocytosis (ADCP), (xii) complement-dependent cellular cytotoxicity (CDCC), (xiii) complement-enhanced cytotoxicity, (xiv) binding to complement receptor of an opsonized antibody mediated by the antibody, (xv) internalization, (xvi) downmodulation, (xvii) induction of apoptosis, (xviii) opsonisation and (xix) a combination of any of (i) to (xviii), of an antibody when bound to its antigen on a cell, on a cell membrane, on a virion, or on another particle. In one embodiment of (iv) or (v), the oligomer is a hexamer. In one embodiment, at least one other effector function of the antibody, such as C1q-binding, complement activation, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxity (ADCC), FcRn-binding, Fc-gamma receptor-binding, Protein A-binding, Protein G-binding, ADCP, complement-dependent cellular cytotoxicity (CDCC), complement-enhanced cytotoxicity, binding to complement receptor of an opsonized antibody mediated by the antibody, antibody mediated phagocytosis (ADCP), internalization, apoptosis, and/or binding to complement receptor of an opsonized antibody is also or alternatively increased, such as in particular FcRn binding, ADCC, Fc gamma receptor binding, Protein A binding, Protein G binding, ADCP, CDCC, complement enhanced cytotoxicity, opsonisation and any combinations thereof.
In one embodiment, the effector function of the parent antibody is increased when the parent antibody is bound to its antigen on an antigen-expressing cell, on a cell membrane, or on a virion.
The inventors of the present invention have also shown that introducing a mutation to a parent antibody in an amino acid residue corresponding to either K439 or S440 in the Fc region of a human IgG1 heavy chain decreases the effector function of the parent antibody (examples 5, 6 and 10).
In another aspect the present invention relates to a method of decreasing an effector function of a parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region, which method comprises introducing a mutation to the parent polypeptide in one amino acid residue selected from those corresponding to K439 and S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W, such as wherein the mutation in the position corresponding to K439 in the Fc-region of human IgG1 heavy chain is K439D/E, and/or the mutation in the position corresponding to S440 in the Fc-region of human IgG1 heavy chain is S440K/H/R.
In one embodiment the parent polypeptide may be an antibody.
Hence in another aspect, the present invention relates also to a method of decreasing an effector function of a parent antibody comprising introducing a mutation to the parent antibody in one amino acid residue selected from those corresponding to K439 and S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W, such as wherein the mutation in the position corresponding to K439 in the Fc-region of human IgG1 heavy chain is K439D/E, and/or the mutation in the position corresponding to S440 in the Fc-region of human IgG1 heavy chain is S440K/H/R.
As shown in Example 6, the amino acid substitution of position K439E or S440K as “single-mutants” decreased CDC as compared to any one of the first mutations according to the method of the present invention.
The variant antibody obtained from said method of decreasing an effector function has an decreased effector function compared to the parent antibody. Typically, the effect of an antibody on an effector function may be measured by the EC50 value, which is the concentration of the antibody necessary to obtain half the value of the maximal lysis.
Maximal lysis is the lysis obtained when a saturating amount of the antibody is used, in which saturating is intended to refer to the amount of antibody at which all antigens for the antibody are bound by antibody.
The term “decreasing an effector function” refers in the context of the present invention that there is a increase in the EC50 value of the variant antibody compared to the parent antibody. The increase in the EC50 value may e.g. be at least or about 2-fold, such as at least or about 3-fold, or at least or about 5-fold, or at least or about 10-fold. Alternatively, “decreasing an effector function” means that there is an decrease in the maximal amount of cells lysed by e.g. from 10% to 100% of all cells, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100% under conditions where the parent antibody lyses less than 100% of all cells.
A variant could be tested for decreased effector function by cloning the variable domain of the IgG1-005 or IgG1-7D8 heavy chain into the variant and test its efficacy in CDC assays, such as described for Daudi (Example 6) and Wien (Example 10). Using an IgG1-7D8 HC variable domain and Daudi cells, an decrease would be defined by a more than 2 fold lower EC50 than the EC50 of IgG1-7D8 under the studied condition, such as about 2-fold, about 3-fold, about 5-fold, about 10-fold or a more than 10-fold lower EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-005 HC variable domain and Daudi cells, an decrease would be defined by a more than 2 fold lower EC50 than the EC50 of IgG1-005 under the studied condition, such as about 2-fold, about 3-fold, about 5-fold, about 10-fold or a more than 10-fold lower EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-7D8 HC variable domain and Wien133 cells, an decrease would be defined by a more than 2 fold lower EC50 than the EC50 of IgG1-7D8 under the studied condition, such as about 2-fold, about 3-fold, about 5-fold, about 10-fold or a more than 10-fold lower EC50 value, the concentration at which half-maximal lysis is observed. Using an IgG1-005 HC variable domain and Wien133 cells, an decrease would be defined by an decrease in the maximal lysis ranging from 10% to 100% of all cells, such as by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, and about 100%. An decrease in CDC efficacy could also be defined by a more than 2-fold lower EC50 than the EC50 of IgG1-005 under the studied condition, such as about 2-fold, about 3-fold, about 5-fold, about 10-fold or a more than 10-fold lower EC50 value, the concentration at which half-maximal lysis is observed under conditions where lysis of Wien133 cells is detectable.
In one embodiment, the effector function is decreased, when the parent antibody is bound to its antigen on an antigen-expressing cell, on a cell membrane, or on a virion.
Thus in another aspect, the invention relates to use of at least a further mutation in an antibody variant comprising a mutation in one amino acid residue selected from those corresponding to K439 and S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W, to restore an effector function of the antibody variant when bound to its antigen on an antigen-expressing cell, on a cell membrane, or on a virion, wherein
the first mutation is in an amino acid residue corresponding to K439 in the Fc-region of a human IgG1 heavy chain and the second mutation is in an amino acid residue corresponding to S440 in the Fc-region of a human IgG1 heavy chain, or
the first mutation is in an amino acid residue corresponding to S440 in the Fc-region of a human IgG1 heavy chain and the second mutation is in an amino acid residue corresponding to K439 in the Fc-region of a human IgG1 heavy chain.
In one embodiment, the parent antibody is a monospecific, bispecific, or multispecific antibody.
If the parent antibody is a monospecific antibody comprising two CH2-CH3 regions, a mutation according to the present invention may in principle only be present in one of the CH2-CH3 regions, although for most practical purpose a mutation increasing or decreasing an effector function according to the present invention is present in both CH2-CH3 regions.
If the parent antibody is a bispecific antibody comprising two CH2-CH3 regions, a mutation according to the present invention may in principle only be present in one of the CH2-CH3 regions; i.e. in either the first or second CH2-CH3 region, although for most practical purpose a mutation increasing or decreasing an effector function according to the present invention is present in both the first and second CH2-CH3 regions of the bispecific antibody.
Suitable examples of monospecific, bispecific, or multispecific antibodies include any of those described herein.
In a particular embodiment the parent or first and/or second antibody may be bispecific antibody such as the heterodimeric protein described in WO 11/131,746, which is hereby incorporated herein by reference.
In one embodiment, the parent antibody is a bispecific antibody which comprises a first polypeptide comprising a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region, and a second polypeptide comprising a second CH2-CH3 region of an immunoglobulin and a second antigen-binding region, wherein the first and second antigen-binding regions bind different epitopes on the same antigen or on different antigens.
In a further embodiment said first CH2-CH3 region comprises a further amino acid substitution at a position selected from those corresponding to K409, T366, L368, K370, D399, F405, and Y407 in the Fc-region of a human IgG1 heavy chain; and wherein said second CH2-CH3 region comprises a further amino acid substitution at a position selected from those corresponding to F405, T366, L368, K370, D399, Y407, and K409 in the Fc-region of a human IgG1 heavy chain, and wherein said further amino acid substitution in the first CH2-CH3 region is different from the said further amino acid substitution in the second CH2-CH3 region.
In a further embodiment said first CH2-CH3 region comprises an amino acid substitution at a position corresponding to K409 in the Fc-region of a human IgG1 heavy chain; and said second CH2-CH3 region comprises an amino acid substitution at a position corresponding to F405 in the Fc-region of a human IgG1 heavy chain.
In one embodiment said method comprises introducing to each of the first and second CH2-CH3 regions a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.
In a further embodiment the mutation introduced in the first and second CH2-CH3 region in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W, may be in the same amino acid residue position or a different position. In a further embodiment it may be the same or a different mutation in the same amino acid residue position.
In another embodiment said method comprises introducing in the first or second CH2-CH3 region a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.
Any of the mutations listed in Table 1 may be introduced to the bispecific antibody. Example 24, shows that introducing the E345R mutation to a bispecific CD20xEGFR antibody enhances the CDC efficacy. Examples 23, 29 and 30 also describe some of the different of bispecific antibodies comprising a mutation according to the present invention.
In one embodiment said method comprises introducing the mutation in one or more positions other than S440 and K447, and further introducing a mutation
(i) in each of the amino acid residues corresponding to K439 and S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W,
(ii) in each of the amino acid residues corresponding to K447 and 448 in the Fc-region of a human IgG1 heavy chain, such as K447K/R/H and 448E/D in the Fc-region of a human IgG1 heavy chain, preferably K447K and 448E in the Fc-region of a human IgG1 heavy chain, or
(iii) in each of the amino acid residues corresponding to K447, 448 and 449 in the Fc-region of a human IgG1 heavy chain, such as K447D/E, 448K/R/H and 449P in the Fc-region of a human IgG1 heavy chain, preferably K447E, 448K and 449P in the Fc-region of a human IgG1 heavy chain.
In one embodiment, said method comprises introducing the mutation in one or more positions other than S440, and further introducing a mutation in each of the amino acid residues corresponding to K439 and S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the further mutation in S440 is not S440Y or S440W.
Introduction of mutations in both amino acid residues corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain in a parent antibody, with the proviso that the mutation in S440 is not S440Y or S440W is also referred herein to as the “double mutant” aspect. The S440Y and S440W mutations have as described elsewhere been found to increase an effector function when introduced into a parent antibody.
As also described elsewhere the inventors of the present invention have found that introducing an identified mutations in an amino acid residue corresponding to either K439 or S440 in the Fc region of a human IgG1 heavy chain results in a decrease in an effector function (examples 5, 6, 10). However, when inhibiting mutations in both of the amino acid residues corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain are introduced the decrease in effector function is restored, thereby making it similar to the effector function of the parent antibody without a mutation at the K439 and S440 mutations. However, the presence of the K439 and S440 mutations is, without being bound by any theory, believed to restrict the induction of effector functions to oligomeric complexes exclusively consisting of exclusively antibodies comprising both the K439 and the S440 mutations. Thus if the K439 and S440 mutations are included in a therapeutic antibody, it is believed, without being bound by any theory, that when such therapeutic antibodies are administered to a patient the induction of effector functions is limited to oligomeric antibody complexes containing the therapeutic antibodies comprising the K439/S440 mutations but not containing the patients own antibodies, which do not comprise the K439 and S440 mutations, thereby limiting any potential side-effects caused by interaction of a therapeutic antibody with the patients own antibodies.
When combining the mutations of position K439 and/or S440 with the first mutation, enhancement of CDC is obtained and the specificity of CDC is increased.
Thus in another aspect the present invention relates to a method of increasing the specificity of a combination of at least a first and a second parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region, comprising
A)
(i) introducing to the first parent polypeptide a mutation in an amino acid residue in the position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and
(ii) introducing to the second parent polypeptide a mutation in an amino acid residue in the position corresponding to S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W,
B)
(i) introducing to the first parent polypeptide a mutation in an amino acid residue in the position corresponding to K447D/E in the Fc region of a human IgG1 heavy chain; and
(ii) introducing to the second parent polypeptide a mutation in an amino acid residue in the position corresponding to K447K/R/H and 448P in the Fc-region of a human IgG1 heavy chain; or
C)
(i) introducing to the first parent polypeptide a mutation in an amino acid residue in the position corresponding to K447D/E in the Fc region of a human IgG1 heavy chain; and
(ii) introducing to the second parent polypeptide a mutation in an amino acid residue in the position corresponding to K447K/R/H, 448K/R/H and 449P in the Fc-region of a human IgG1 heavy chain.
In one embodiment the parent polypeptide, first parent polypeptide and second parent polypeptide may each be an antibody.
Thus in further aspect the present invention also relates to a method of increasing the specificity of a combination of at least a first and a second parent antibody, comprising
(i) introducing to the first parent antibody a mutation in an amino acid residue in the position corresponding to K439 in the Fc region of a human IgG1 heavy chain; and
(ii) introducing to the second parent antibody a mutation in an amino acid residue in the position corresponding to S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W.
The first and second variant antibodies will have preference for oligomerization with one another compared to any wildtype or naturally occurring antibody as shown in Example 10.
The increase in specificity is with respect to “induction of an effector function”. Thus said method is in one embodiment a method of increasing the specificity of induction of an effector function by a combination of at least a first and a second parent antibody.
By performing the method of increasing the specificity, or specificity of induction of an effector function, by a combination of at least a first and a second parent antibody, a combination of a first variant and a second variant antibody is obtained.
By introducing a mutation in either K439 or S440 of a parent antibody, the variant antibody thereby obtained has a decreased effector function compared to the parent antibody. However, as also described elsewhere herein, the mutation in K439 and S440 are able to complement each other or restore the effector function of an antibody comprising both mutations. This ability of the mutations in K439 and S440 to complement each other may similarly be utilized in two antibodies. Thus, when a mutation in K439 is introduced into a first parent antibody and a mutation in S440 is introduced into a second parent antibody, or vice versa, the decrease in effector function is no longer seen as the first and second variant antibody are used in combination. The term “increasing specificity” or “improving specificity” refers in this context to that an effector response induced by a combination of a first variant antibody comprising a mutation in K439 and a second variant antibody comprising a mutation in S440 is higher than the effector response induced by either the first variant antibody comprising a mutation in K439 or the second variant antibody comprising a mutation in S440.
By the introduction of both an amino acid substitution in a K439 and S440 the specificity of oligomerization is increased.
When combining the mutations of position K439 and/or S440 with the first mutation, enhancement of CDC is obtained and the specificity of CDC is increased.
In one embodiment the at least first and second parent antibodies bind to same epitope.
In one embodiment the at least first and second parent antibodies bind to different epitopes on the same antigen.
In one embodiment the at least first and second parent antibodies bind to different epitopes on different targets.
In one embodiment the first and second parent antibody have the same or different VL and VH sequences.
In one embodiment the combination of at least a first and a second parent antibody comprises one first parent antibody and one second antibody.
In one embodiment, the specificity is increased, when a combination of the first and second parent antibody is bound to its antigen on an antigen-expressing cell, on a cell membrane, or on a virion.
Hence in another aspect the present invention also relates to use of mutation in two or more amino acid residues of an antibody to increase the specificity of, e.g the effector function induced by, the antibody when bound to its antigen on an antigen-expressing cell, on a cell membrane, or on a virion, wherein
a first mutation is in an amino acid residue corresponding to K439 in the Fc-region of a human IgG1 heavy chain;
a second mutation is in an amino acid residue corresponding to S440 in the Fc-region of a human IgG1 heavy chain.
In a further aspect the present invention relates to a method of increasing an effector function of a combination of at least a first and a second parent polypeptide, wherein the at least first and second parent polypeptide each comprises an Fc-domain of an immunoglobulin and a binding region, wherein said method comprises
(i) introducing to the at least first and/or second parent polypeptide a mutation in one or more amino acid residues selected from the group consisting of:
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain.
In one embodiment the first and/or second parent polypeptide may each be an antibody.
Thus in one embodiment the present invention relates to a method of increasing an effector function of a combination of at least a first and a second parent antibody, wherein the at least first and second parent antibody each comprises a Fc-domain of an immunoglobulin and an antigen-binding region, wherein said method comprises
(i) introducing to the at least first and/or second parent antibody a mutation in one or more amino acid residues selected from the group consisting of:
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain.
By performing this method a combination of at least a first and second variant antibody is obtained. The at least of first and second variant antibody obtained by this method have when combined an increased effector function compared to a combination of the first and second parent antibody.
The term “increased effector function” is to be understood as described herein.
The first and/or second parent antibody may be any parent antibody as described herein.
The methods of increasing an effector function of a combination of a first and second antibody may in particular be performed so as to obtain a first and/or second variant antibody which has any of the features of a variant antibody as described herein. The inventors of the present invention have found that introducing a mutation into an amino acid residue selected from (a), (b), (c), (d) and/or (e) results in a combination of a first and second variant antibody with an increased effector function compared to a combination of the first and second parent antibody.
In one embodiment the at least first and second parent antibodies bind to the same epitope.
In one embodiment the at least first and second parent antibodies bind to different epitopes on the same antigen.
In one embodiment the at least first and second parent antibodies bind to different epitopes on different targets.
In one embodiment the first and second parent antibody have the same or different VL and VH sequences.
In one embodiment the combination of at least a first and a second parent antibody comprises one first parent antibody and one second antibody.
In one embodiment the combination of at least a first and a second parent antibody comprises further parent antibodies, such as a third, fourth or fifth parent antibody.
In one embodiment (a) an amino acid residue within the CH2-CH3 region providing allosteric mutations is an amino acid residue selected from those corresponding to P247 or E430 in the Fc-region of a human IgG1 heavy chain.
In one embodiment (b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region is an amino acid residue selected from those corresponding to I253, S254 and Q311 in the Fc-region of a human IgG1 heavy chain.
In one embodiment (c) an amino acid residue within the N-terminal CH3 helix is an amino acid residue selected from those corresponding to D/E356 and T359 in the Fc-region of a human IgG1 heavy chain.
In one embodiment (d) an amino acid residue within the C-terminal CH3 beta-strand is an amino acid residue selected from those corresponding to Y436 and S440.
The amino acid residues in (b), (c), (d) and (e) are amino acid residues which are located at the Fc:Fc interface of two antibodies, thus the Fc part of one antibody which can interact with the Fc part of another antibody the two antibodies are in proximity with each other.
Thus in a further embodiment the mutation in the at least first and/or second parent antibody is in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.
In one embodiment (i) comprises introducing a mutation in both the first and second parent antibodies.
In another embodiment said method comprises:
(i) introducing a mutation to the first parent antibody in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W,
(ii) providing the second parent antibody which does not comprise a mutation in an amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain.
In one embodiment said method comprises introducing the mutation in at least one amino residue other than S440, wherein said method further comprises the steps of introducing the mutation in one or more positions other than S440, and wherein said method further comprises the steps of
(i) introducing to the first parent antibody a second mutation in the amino acid residue corresponding to position K439 in the Fc-region of a human IgG1 heavy chain; and
(ii) introducing to the second parent antibody a second mutation in the amino acid residue corresponding to position S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W; wherein steps (ii) and (iii) may alternatively be
(i) introducing to the first parent antibody a second mutation in the amino acid residue corresponding to position S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W; and
(ii) introducing to the second parent antibody a second mutation in the amino acid residue corresponding to position K439 in the Fc-region of a human IgG1 heavy chain.
For those embodiments of the present invention wherein the second parent does not comprise a mutation in an amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, the term “second mutation” in step (ii) may be a first mutation, e.g. the second parent antibody may comprise no other mutations than the mutation introduced in step (ii). The term “second mutation” in steps (i) and (ii) are also not intended to limit the number of mutations that may be introduced into the first and/or second parent antibody.
In one embodiment the parent antibody, the first parent antibody and the second parent antibody may each be selected from the group consisting of but not limited to monospecific, bispecific and multispecific antibodies. The bispecific may e.g. be a heterodimeric protein.
In one embodiment the first and second parent antibodies are monospecific antibodies, which may e.g. bind to the same or different epitopes. If the first and second parent antibody bind to different epitopes it may on the same or different antigen.
In another embodiment the first parent antibody is a monospecific antibody and the second parent antibody is bispecific or multispecific antibody, or vice versa.
In another embodiment the first and second parent antibodies are bispecific or multispecific antibodies. In one embodiment the first and second bispecific or multispecific parent antibodies are the same or different antibodies. In one embodiment the first and second bispecific or multispecific parent antibodies bind to different epitopes on the same or different antigen. Thus in one embodiment said at least first and second parent antibodies are bispecific or multispecific antibodies which bind different epitopes on the same antigen or on different antigens.
In another embodiment the first parent antibody is a monospecific antibody and the second parent antibody is a bispecific antibody, or vice versa. The monospecific may bind the same epitope as the bispecific (one part of the bispecific) or the monospecific and the bispecific antibody may bind different epitopes on the same or different antigens. The bispecific antibody may bind to different epitopes on the same or different antigens.
In one embodiment said at least first and second parent antibodies are each a bispecific antibody which comprises a first polypeptide comprising a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region, and a second polypeptide comprising a second CH2-CH3 region of an immunoglobulin and a second antigen-binding region, wherein the first and second antigen-binding regions bind different epitopes on the same antigen or on different antigens, and wherein said first CH2-CH3 region comprises a further amino acid substitution at a position selected from those corresponding to K409, T366, L368, K370, D399, F405, and Y407 in the Fc-region of a human IgG1 heavy chain; and wherein said second CH2-CH3 region comprises a further amino acid substitution at a position selected from those corresponding to F405, T366, L368, K370, D399, Y407, and K409 in the Fc-region of a human IgG1 heavy chain, and wherein said further amino acid substitution in the first CH2-CH3 region is different from the said further amino acid substitution in the second CH2-CH3 region.
In a further embodiment said first CH2-CH3 region comprises an amino acid substitution at a position corresponding to K409 in the Fc-region of a human IgG1 heavy chain; and said second CH2-CH3 region comprises an amino acid substitution at a position corresponding to F405 in the Fc-region of a human IgG1 heavy chain.
In one embodiment of the methods and/or uses of the present invention the parent antibody, whether it is a parent antibody, a first parent antibody or a second parent antibody, may contain other mutations than those of the present invention which have been found to affect an effector function. Such other mutations may be introduced at the same time as the mutations of the present invention which affect an effector function or they may introduced sequentially, the methods or uses of the present invention are not limited to either simultaneous or sequential introduction of mutations. The bispecific antibody may be any bispecific antibody and the methods and uses of the present invention are not limited to any particular bispecific format as it is foreseen that different formats may be used.
The method of combining a first antibody which comprises one of said mutations capable of increasing an effector function with a second antibody which does not comprise such a mutation may as shown in Example 31 increase the effector function of the combination. Thus, without being bound by theory, it is believed that e.g. this method may be used to combine a therapeutic antibody, as a second antibody, which have been proven to be safe but not efficient enough with a first antibody comprising a mutation, and thereby resulting in a combination which is efficacious.
Thus in one embodiment the second parent antibody which does not comprise a mutation in an amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, is a therapeutic antibody. In a particular embodiment it is therapeutic antibody which has suitable safety profile. In one embodiment it may be a therapeutic antibody which has a suitable safety profile but which is not sufficiently efficacious.
Examples of suitable second antibodies which does not comprise a mutation in an amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, include but are not limited to any of the following; (90Y) clivatuzumab tetraxetan; (90Y) tacatuzumab tetraxetan; (99mTc) fanolesomab; (99mTc) nofetumomab Merpentan; (99mTc) pintumomab; 3F8; 8H9; abagovomab; abatacept; abciximab; Actoxumab; adalimumab; adecatumumab; afelimomab; aflibercept; Afutuzumab; alacizumab pegol; albiglutide; ALD518; alefacept; alemtuzumab; Alirocumab; altumomab; Altumomab pentetate; alvircept sudotox; amatuximab; AMG714/HuMax-IL15; anatumomab mafenatox; Anrukinzumab (=IMA-638); apolizumab; arcitumomab; aselizumab; atacicept; atinumab; Atlizumab (=tocilizumab); atorolimumab; baminercept; Bapineuzumab; basiliximab; bavituximab; bectumomab; belatacept; belimumab; benralizumab; bertilimumab; besilesomab; bevacizumab; Bezlotoxumab; biciromab; bifarcept; bivatuzumab; Bivatuzumab mertansine; blinatumomab; blosozumab; brentuximab vedotin; briakinumab; briobacept; brodalumab; canakinumab; cantuzumab mertansine; cantuzumab ravtansine; caplacizumab; capromab; Capromab pendetide; carlumab; catumaxomab; CC49; cedelizumab; certolizumab pegol; cetuximab; Ch.14.18; citatuzumab bogatox; cixutumumab; Clazakizumab; clenoliximab; Clivatuzumab tetraxetan; conatumumab; conbercept; CR6261; crenezumab; dacetuzumab; daclizumab; dalantercept; dalotuzumab; daratumumab; Demcizumab; denosumab; Detumomab; Dorlimomab aritox; drozitumab; dulaglutide; ecromeximab; eculizumab; edobacomab; edrecolomab; efalizumab; efungumab; elotuzumab; elsilimomab; enavatuzumab; enlimomab; enlimomab pegol; enokizumab; ensituximab; epitumomab; epitumomab cituxetan; epratuzumab; erlizumab; ertumaxomab; etanercept; etaracizumab; etrolizumab; exbivirumab; Fanolesomab; faralimomab; farletuzumab; Fasinumab; FBTA05; felvizumab; Fezakinumab; ficlatuzumab; figitumumab; flanvolumab; fontolizumab; foralumab; foravirumab; fresolimumab; fulranumab; galiximab; ganitumab; gantenerumab; gavilimomab; gemtuzumab; Gemtuzumab ozogamicin; gevokizumab; girentuximab; glembatumumab; Glembatumumab vedotin; golimumab; Gomiliximab; GS6624; anti-CD74 antibodies; anti-cMet antibodies as disclosed in WO 2011/110642; anti-Her2 antibodies as disclosed WO 2011/147986 or WO 2011/147982; anti-IL8 antibodies as disclosed in WO 2004/058797; anti-TAC antibodies as disclosed in WO 2004/045512; anti-tissue factor (TF) antibodies as disclosed in WO 2010/066803 or WO 2011/157741; ibalizumab; ibritumomab tiuxetan; icrucumab; igovomab; Imciromab; inclacumab; indatuximab ravtansine; infliximab; inolimomab; inotuzumab ozogamicin; intetumumab; iodine (124I) girentuximab; ipilimumab; iratumumab; itolizumab; ixekizumab; keliximab; labetuzumab; lebrikizumab; lemalesomab; lenercept; lerdelimumab; lexatumumab; libivirumab; lintuzumab; lorvotuzumab mertansine; lucatumumab; lumiliximab; mapatumumab; maslimomab; matuzumab; mavrilimumab; mepolizumab; metelimumab; milatuzumab; minretumomab; mirococept; mitumomab; mogamulizumab; morolimumab; motavizumab; moxetumomab; pasudotox; muromonab-CD3; nacolomab tafenatox; namilumab; naptumomab estafenatox; narnatumab; natalizumab; nebacumab; necitumumab; nerelimomab; nimotuzumab; Nivolumab; Nofetumomab; merpentan; obinutuzumab; Ocaratuzumab; ocrelizumab; odulimomab; ofatumumab; olaratumab; olokizumab; omalizumab; onartuzumab; onercept; oportuzumab monatox; oregovomab; otelixizumab; oxelumab; ozoralizumab; pagibaximab; palivizumab; panitumumab; panobacumab; pascolizumab; pateclizumab; patritumab; pegsunercept; Pemtumomab; pertuzumab; pexelizumab; Pintumomab; Placulumab; ponezumab; priliximab; pritumumab; PRO 140; quilizumab; racotumomab; radretumab; rafivirumab; ramucirumab; ranibizumab; raxibacumab; regavirumab; reslizumab; RG1507/HuMax-IGF1R; RG1512/HuMax-pSelectin; rilonacept; rilotumumab; rituximab; robatumumab; roledumab; romosozumab; rontalizumab; rovelizumab; ruplizumab; samalizumab; sarilumab; satumomab; Satumomab pendetide; secukinumab; sevirumab; sibrotuzumab; sifalimumab; siltuximab; siplizumab; sirukumab; solanezumab; solitomab; Sonepcizumab; sontuzumab; sotatercept; stamulumab; sulesomab; suvizumab; tabalumab; Tacatuzumab tetraxetan; tadocizumab; talizumab; tanezumab; taplitumomab paptox; tefibazumab; telimomab aritox; tenatumomab; teneliximab; teplizumab; teprotumumab; TGN1412; Ticilimumab (=tremelimumab); tigatuzumab; TNX-650; Tocilizumab (=atlizumab); toralizumab; torapsel; tositumomab; tralokinumab; trastuzumab; trastuzumab emtansine; TRBS07; trebananib; tregalizumab; tremelimumab; tucotuzumab celmoleukin; tuvirumab; ublituximab; urelumab; urtoxazumab; ustekinumab; vapaliximab; vatelizumab; vedolizumab; veltuzumab; vepalimomab; vesencumab; visilizumab; volociximab; Vorsetuzumab mafodotin; votumumab; zalutumumab; zanolimumab; ziralimumab; and zolimomab aritox.
In one embodiment of the methods and uses of the present invention the mutation in at least one amino acid residue, or in one or more amino acids residues, corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W, may be in any of the exemplary and preferred amino acid positions listed in Table 1. Hence each of the amino acid positions listed in Table 1 is a separate and non-limiting embodiment of a mutation in the at least one amino acid.
Any of the mutations or combinations thereof described herein may be introduced according to a method of the present invention.
Mutations selected from the exemplary or preferred amino acid substitutions can be tested in appropriate assays allowing for oligomer formation of antigen-bound antibodies and detecting enhanced C1q-binding, complement activation, CDC, ADCC and/or internalization, such as those described in the Examples. For example, C1q-binding avidity can be determined according to an assay similar to the one described in Example 4, using cells expressing the antigen for the antibody variant. Exemplary CDC assays are provided in Examples 5, 6, 10, 16, 19, 22, 23, 24, or 25. An exemplary ADCC assay is provided in Example 12. An exemplary internalization assay is provided in Example 26. Finally, to discriminate between mutations in amino acid residues directly involved in C1q-binding from mutations affecting oligomer formation, C1q-binding in an ELISA assay according to, e.g., Example 3 can be compared to C1q-binding in a cell-based assay according to, e.g., Example 4.
In a further embodiment said mutation is selected from those corresponding to E345, E430, S440 and Q386 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.
In an alternative embodiment the mutation in at least one amino acid residue, or one or more amino acid residues, is in an amino acid residue corresponding to E382 and H433 in the Fc region of a human IgG1 heavy chain.
In a particular embodiment one mutation is in the amino acid residue corresponding to E345 in the Fc region of a human IgG1 heavy chain.
In a particular embodiment one mutation is in the amino acid residue corresponding to E430 in the Fc region of a human IgG1 heavy chain.
In a particular embodiment one mutation is in the amino acid residue corresponding to S440 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation is S440Y or S440W.
In a particular embodiment one mutation is in the amino acid residue corresponding to Q386 in the Fc region of a human IgG1 heavy chain.
In an alternative embodiment one mutation is in the amino acid residue corresponding to E382 or H433 in the Fc region of a human IgG1 heavy chain.
In one embodiment the mutation in at least one amino acid residue may be an amino acid substitution, an amino acid deletion or an amino acid insertion.
In one embodiment the mutation in at least one amino acid residue is an amino acid deletion.
In one embodiment the mutation in at least one amino acid residue is an amino acid insertion.
In a particular embodiment mutation in at least one amino acid residue is an amino acid substitution.
In one embodiment the mutation in at least one amino acid residue may be selected from any of the amino acid substitutions, amino acid deletions listed in Table 1. Further, each preferred amino acid substitution in each specific amino acid residue listed in Table 1 is a separate and specific non-limiting embodiment for this use. Exemplary amino acid substitutions include exchanging an E residue for an R residue, and exchanging an H residue for an R residue.
In a further embodiment the mutation in at least one amino acid residue is an amino acid substitution selected from those corresponding to E345X, E430X, S440Y or W, and Q386K in the Fc-region of a human IgG1 heavy chain, wherein X refers to any amino acid, e.g. any natural amino acid or non-natural occurring amino acid. X may in particular refer to any of the 20 naturally occurring amino acids.
Thus in one embodiment the mutation is in at least one amino acid residue selected from those corresponding to E345, E430, S440 to Y or W, and Q386 in the Fc-region of a human IgG1 heavy chain, preferably wherein the mutation is at least one amino acid substitution of the following: E345 to R, Q, N, or K, E430 to T, S, or G, S440 to Y or W, or Q386 to K.
Thus in one embodiment E345X may be E345R, Q, N, K, Y, A, C, D, F, G, H, I, L, M, P, S, T, V, W, or Y; in particular E345A, D, G, H, K, N, Q, R, S, T, Y or W, or more particularly E345D, K, N, Q, R, or W; or even more particularly E345R, Q, N, K, or Y. In another further embodiment E430X may be E430T, S, G, F, H, A, C, D, I, K, L, M, N, P, Q, R, V, W, or Y; in particular E430T, S, G, F, or H. In a preferred embodiment the amino acid substitution is selected from the group comprising E345R, E345Q, E345N, E345K, E345Y, E430T, E430S, E430G, E430F, E430H, S440W and S440Y. In a further embodiment the mutation in at least one amino acid residue is selected from E345R and E430G. In a further embodiment the mutation in at least one amino acid residue is E345R. In a further embodiment the mutation in at least one amino acid residue is E430G.
In an alternative embodiment the mutation in at least one amino acid residue is selected from those corresponding to I253, H310, Q311, E382, G385, H433, N434, Y436, and Q438 in the Fc-region of a human IgG1 heavy chain, such as E382 or H433. In a further alternative embodiment the mutation in at least one amino acid residue may an amino acid substitution selected from those corresponding to I253E, N, Q, S or T, e.g. I253N or Q; H310N, Q, W or Y, e.g. H310Q; Q311E or R, E382D, H, K, R, N, Q, S, T, W or Y, e.g. E382D, Q, K, or R; G385E, H, K, N, Q, R, S, T, W or Y, e.g. G385D, E, K or R; H433R; N434D, E, H, K, Q, R, S, T, W or Y, e.g. N434H, K, Q or R; Y436A, E, F, H, I, K, L, M, N, Q, R, S, T or V, e.g. Y436N, Q, S or T; Q438A, E, G, H, K, N, Q, R, S, T, W or Y, or e.g. Q438 N, S or T.
Thus in an even further alternative embodiment the mutation in at least one amino acid residue may be an amino acid substitution selected from those corresponding to P247G, I253V, S254L/V, Q311L/W, D/E356G/R, T359R, E382L/V, and Y436I in the Fc-region of a human IgG1 heavy chain, e.g. in particular E382L, V, D, Q, K, or R or H433R. In a further alternative embodiment the mutation in at least one amino acid residue is selected from E382R and H433R. In an alternative embodiment, the mutation is E382R. In another alternative embodiment, the mutation is H433R.
In another embodiment, the mutation is not in an amino acid residue directly involved in C1q-binding, optionally as determined by comparing C1q-binding in an ELISA assay according to Example 3 with C1q-binding in a cell-based assay according to Example 4.
In one embodiment, the mutation is not in an amino acid residue corresponding to I253, N434, or Q311, and optionally not in an amino acid residue corresponding to H433, or the amino acid substitution is not H433A.
In one embodiment, the at least one mutation is one mutation, i.e. no more than one mutation is introduced to the parent antibody.
In another embodiment, the method or use according to the present invention comprises introducing a mutation in at least two, such as two, three, four, five, or more of the amino acids residues in Table 1.
Any of the combinations of mutations described herein may be introduced according to a method of the present invention.
In one embodiment the method or uses according to the present invention comprises introducing to the parent antibody a mutation in at least two amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.
In one embodiment the method or uses according to the present invention comprises introducing to the parent antibody a mutation in at least two amino acid residues selected from those corresponding to E345, E430, Q386, and S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W, such as wherein the mutation in the at least two amino acids are selected from the following: E345 to R, Q, N, or K, E430 to T, S, or G, S440 to Y or W, or Q386 to K.
In an alternative embodiment the positions of the first mutation may be selected from the group consisting of positions I253, H310, Q311, E345, E382, G385, H433, N434, Y436, and Q438.
In one embodiment, the method further comprises introducing to the antibody a further/third mutation in an amino acid residue corresponding to E345, E430, P247, I253, S254, Q311, D/E356, T359, E382, Q386, Y436, or K447 in both the first and/or the second Fc-regions.
For example more than one, such as two, three, four, or five, in particular two or three mutations are introduced to the parent antibody in amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain. For example, at least one of the amino acid residues corresponding to E345, E430 and S440 in the Fc region of a human IgG1 heavy chain, may be mutated, such as two or all of E345, E430 and S440, optionally in combination with a mutation in one or more other amino acids listed in Table 1. The at least two mutations may be any amino acid residue substitution of position E345 in combination with any amino acid residue substitution of position E430 or S440, or may be any amino acid substitution of position E430 in combination with any amino acid residue of position S440.
In a further embodiment the two or three mutations are introduced to the parent antibody in amino acid residues selected from those corresponding to E345, E430, S440 and Q386 in the Fc-region of a human IgG1 heavy chain.
In one embodiment the more than one mutation may in particular be amino acid substitutions.
Thus according to the present invention, the method or use, comprises introducing to the antibody at least one, such as one, two, three, four, five, or six, amino acid substitution selected from the following group consisting of P247G, I253V, S254L, Q311L/W, E345X, D/E356G/R, T359R, E382L/V, Q386K, E430X, Y436I, and S440Y/W. In the preferred embodiments, the amino acid substitution is selected from the group consisting of E345X, E430X, S440Y/W, and Q386K.
In an alternative embodiment, the at least two mutations, such as two, three, four or five mutations, are in amino acid residues selected from those corresponding to H310, G385, H433, N434, and Q438 in the Fc-region of a human IgG1 heavy chain.
In another alternative embodiment, the at least one mutation, optionally two or three mutations, are selected from the group consisting of E345R, E382R, and H433R. In another alternative embodiment, at least one of the amino acid residues corresponding to E382 and H433 in the Fc region of a human IgG1 heavy chain may be mutated, such as both, optionally in combination with a mutation in one or more other amino acids listed in Table 1.
In some embodiments of the methods and/or uses of the present invention a mutation in an amino acid residue corresponding to K439 and/or S440 is introduced in an antibody selected from the group consisting of a parent antibody, a first parent antibody, a second parent antibody and combinations thereof. As described above introducing a mutation in the amino acid residues corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain, is shown to limit the intermolecular interactions between antibodies to those comprising such mutations (Examples 4, 5, 6, 10). Depending on whether the K439 and S440 are introduced to the same parent antibody or in a first and second parent antibody, respectively, these aspects are also referred to as “double mutant” and “mixed mutant” aspect.
In one embodiment of the present invention, the mutation in an amino acid residue corresponding to K439 in the Fc region of a human IgG1 heavy chain is an amino acid substitution.
In one embodiment of the present invention, the mutation in an amino acid residue corresponding to S440 in the Fc region of a human IgG1 heavy chain is an amino acid substitution.
In all embodiments of the present invention wherein a mutation in a position corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain, whether in the same polypeptide or antibody, or in first and second polypeptide or antibody may be replaced by a mutation in:
(i) in each of the amino acid residues corresponding to K447 and 448 in the Fc-region of a human IgG1 heavy chain, such as K447K/R/H and 448E/D in the Fc-region of a human IgG1 heavy chain, preferably K447K and 448E in the Fc-region of a human IgG1 heavy chain, or
(ii) in each of the amino acid residues corresponding to K447, 448 and 449 in the Fc-region of a human IgG1 heavy chain, such as K447D/E, 448K/R/H and 449P in the Fc-region of a human IgG1 heavy chain, preferably K447E, 448K and 449P in the Fc-region of a human IgG1 heavy chain.
Thus combinations of such mutations include any of those described in Table 2A and 2B.
In one embodiment of the present invention, the mutations in an amino acid residue corresponding to K439 and S440 in the Fc region of a human IgG1 heavy chain are both an amino acid substitution.
In one embodiment, the mutation in an amino acid residue corresponding to K439 in the Fc region of a human IgG1 heavy chain is an amino acid substitution into an amino acid selected from E and D.
In another embodiment, the mutation is K439E.
In one embodiment, the mutation in an amino acid residue corresponding to S440 in the Fc region of a human IgG1 heavy chain is an amino acid substitution into an amino acid selected from K, R and H.
In another embodiment, the mutation is S440K.
Thus in a further embodiment the mutations introduced to the parent antibody in the amino acid residues corresponding K439 and S440 in the Fc region of a human IgG1 heavy chain are amino acid substitutions selected from K439E and D and S440K, R and H.
Thus in a further embodiment the mutations introduced to the parent antibody in the amino acid residues corresponding K439 and S440 in the Fc region of a human IgG1 heavy chain are the amino acid substitutions K439E and S440K.
Some methods and uses of the present invention comprise a first and a second parent antibody.
Thus in a further embodiment the mutation introduced to the first parent antibody in an amino acid residue corresponding K439 is an amino acid substitution selected from K439E and D, e.g. K439E and the mutation introduced to the second parent antibody in an amino acid residue corresponding S440 is an amino acid substitution selected from S440K, R and H, e.g. S440K. The mutations in the first and second parent antibody may be introduced vice versa, i.e. it may also be that the mutation in an amino acid residue corresponding to S440 is introduced in the first parent antibody, while the mutation in an amino acid residue corresponding to K439 is introduced in the second parent antibody wherein the mutations may be the preferred amino acid substitutions as described above.
In one embodiment of the methods or uses according to the present invention, the effector function is increased when the antibody is bound to its antigen.
In a further embodiment the effector function is increased when the antibody is bound to its antigen, wherein the antigen is on an antigen-expressing cell, cell membrane, or virion. In one embodiment, the Fc-region of an IgG1 heavy chain comprises the sequence of residues 130 to 330 of SEQ ID NO:1.
The parent antibody may be any parent antibody as described herein. The parent antibody in this context is intended to be also first parent and second parent antibodies.
In one embodiment, the parent antibody is a human IgG1, IgG2, IgG3 or IgG4, IgA1, IgA2, IgD or IgE antibody.
In one embodiment the parent antibody is human full-length antibody, such as a human full-length IgG1 antibody.
In one embodiment, the parent antibody, first parent antibody and second parent antibody is a human IgG1 antibody, e.g. the IgG1m(za) or IgG1m(f) allotype, optionally comprising an Fc-region comprising SEQ ID NO:1 or 5.
In one embodiment, the parent antibody is a human IgG2 antibody, optionally comprising an Fc-region comprising SEQ ID NO:2.
In one embodiment, the parent antibody is a human IgG3 antibody, optionally comprising an Fc-region comprising SEQ ID NO:3.
In one embodiment, the parent antibody is a human IgG4 antibody, optionally comprising an Fc-region comprising SEQ ID NO:4.
In one embodiment, the parent antibody is a bispecific antibody.
In one embodiment, the parent antibody is any antibody as described herein, e.g. an antibody fragment comprising at least part of an Fc-region, monovalent antibodies (described in WO2007059782 by Genmab); heavy-chain antibodies, consisting only of two heavy chains and naturally occurring in e.g. camelids (e.g., Hamers-Casterman (1993) Nature 363:446); ThioMabs (Roche, WO2011069104), strand-exchange engineered domain (SEED or Seed-body) which are asymmetric and bispecific antibody-like molecules (Merck, WO2007110205); Triomab (Fresenius, Lindhofer et al. (1995 J Immunol 155:219); FcΔAdp (Regeneron, WO2010151792), Azymetric Scaffold (Zymeworks/Merck, WO2012/058768), mAb-Fv (Xencor, WO2011/028952), Dual variable domain immunoglobulin (Abbott, DVD-Ig, U.S. Pat. No. 7,612,181); Dual domain double head antibodies (Unilever; Sanofi Aventis, WO20100226923), Di-diabody (ImClone/Eli Lilly), Knobs-into-holes antibody formats (Genentech, WO9850431); DuoBody (Genmab, WO 2011/131746); Electrostatic steering antibody formats (Amgen, EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO2010129304A2); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545), CrossMAbs (Roche, WO2011117329), LUZ-Y (Genentech), Biclonic (Merus), Dual Targeting domain antibodies (GSK/Domantis), Two-in-one Antibodies recognizing two targets (Genentech, NovImmune), Cross-linked Mabs (Karmanos Cancer Center), CovX-body (CovX/Pfizer), IgG-like Bispecific (ImClone/Eli Lilly, Shen, J., et al. J Immunol Methods, 2007. 318(1-2): p. 65-74), and DIG-body and PIG-body (Pharmabcine), and Dual-affinity retargeting molecules (Fc-DART or Ig-DART, by Macrogenics, WO/2008/157379, WO/2010/080538), Zybodies (Zyngenia), approaches with common light chain (Crucell/Merus, U.S. Pat. No. 7,262,028) or common heavy chains (κλBodies by NovImmune), as well as fusion proteins comprising a polypeptide sequence fused to an antibody fragment containing an Fc-domain like scFv-fusions, like BsAb by ZymoGenetics/BMS), HERCULES by Biogen Idec (U.S. Pat. No. 7,951,918), SCORPIONS by Emergent BioSolutions/Trubion, Ts2Ab (MedImmune/AZ (Dimasi, N., et al. J Mol Biol, 2009. 393(3): p. 672-92), scFv fusion by Novartis, scFv fusion by Changzhou Adam Biotech Inc (CN 102250246), TvAb by Roche (WO 2012025525, WO 2012025530), mAb2 by f-Star (WO2008/003116), and dual scFv-fusions. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures (recombinant polyclonals) for instance generated by technologies exploited by Symphogen and Merus (Oligoclonics), and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies. An antibody as generated can potentially possess any isotype.
optionally selected from the group consisting of a monovalent antibody, a heavy-chain antibody, a strand-exchange engineered domain (SEED), a triomab, a dual variable domain immunoglobulin (DVD-Ig), a knob-into-holes antibody, a mini-antibody, a dual-affinity retargeting molecule (Fc-DART or Ig-DART); a LUZ-Y antibody, a Biclonic antibody, a Dual Targeting (DT)-Ig antibody, a Two-in-one Antibody, a cross-linked Mab, a mAb2, a CovX-body, an IgG-like Bispecific antibody, a Ts2Ab, a BsAb, a HERCULES antibody, a TvAb, an ScFv/Fc Fusion antibody, a SCORPION, an scFv fragment fused to an Fc domain, and a dual scFv fragment fused to an Fc domain.
In another embodiment, the antigen is expressed on the surface of a cell.
In another embodiment, the cell is a human tumor cell.
In a further embodiment, the antigen is selected from the group consisting of erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD38, CD138, CXCR5, c-Met, HERV-envelop protein, periostin, Bigh3, SPARC, BCR, CD79, CD37, EGFrvIII, IGFr, L1-CAM, AXL, Tissue Factor (TF), CD74, EpCAM and MRP3.
In another embodiment, the antigen is associated with a cell membrane.
In another embodiment, the antigen is associated with a virion, optionally wherein the antigen is comprised in the protein coat or a lipid envelope of the virion.
In another embodiment, the antibody is a human antibody, optionally binding at least one antigen selected from CD20 and CD38.
In another embodiment, the antibody binds to the same epitope as at least one of 7D8 and 005, optionally comprising a variable heavy and/or variable light chain region of at least one of 7D8 and 005.
In any use according to the disclosed invention the antibody without any mutations of the present invention may be any parent antibody. Thus, the uses herein provides for any variants of such parent antibodies.
In a further embodiment of the present invention, the effector function is an Fc-mediated effector function selected from C1q-binding, complement activation, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxity (ADCC), FcRn-binding, Fc-receptor binding including Fc-gamma receptor-binding, Protein A-binding, Protein G-binding, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cellular cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonisation, Fc-containing polypeptide internalization, target downmodulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof.
In a particular embodiment the effector function is C1q-binding, complement activation (C1q efficacy)complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxity (ADCC), Fc-receptor binding, e.g Fc-gamma receptor-binding, Fc-containing polypeptide internalization or any combination thereof.
In one embodiment the effector function is C1q-binding.
In one embodiment the effector function is complement activation (C1q efficacy).
In one embodiment the effector function is complement dependent cytotoxicity (CDC).
In one embodiment the effector function is antibody-dependent cell-mediated cytotoxity (ADCC).
In one embodiment the effector function is Fc-receptor binding, e.g. including Fc-gamma receptor-binding.
In one embodiment the effector function is Fc-containing polypeptide internalization.
In one embodiment the effector function is a combination of complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxity (ADCC).
In another embodiment, the one or more mutations increase a further effector function selected from FcRn-binding, ADCC, Fc-gamma receptor-binding, Protein A-binding, Protein G-binding, ADCP, complement-dependent cellular cytotoxicity (CDCC), complement-enhanced cytotoxicity, binding to complement receptor of an opsonized antibody mediated by the antibody, and any combination thereof.
In another aspect, the invention relates to a method of increasing the avidity of a preparation of a parent antibody for C1q, comprising the step of mutating at least one amino acid in the Fc-region of the antibody, wherein the at least one amino acid is selected from the group consisting of E345, E430, S440, P247, I253, S254, Q311, D/E356, T359, E382, Q386, Y436, and K447.
As used herein, the term “C1q-binding”, when used in the context of a variant or antibody of a parent antibody includes any mechanism of the first component on the classical pathway of complement activation mediated by binding of the variant or antibody to host tissues or factors, including various cells of the immune system (such as effector cells). C1q-binding of an antibody can be evaluated using an ELISA (such as e.g. C1q-binding ELISA used in Examples 3 and 4), or the C1q efficacy can be evaluated by a CDC assay (such as e.g. the CDC assay used in Example 5). In a further embodiment, the C1q-binding avidity of the antibody is determined according to the assay described in Example 4.
In all the methods according to the disclosed invention the antibody without any mutations of the present invention may be any parent antibody. Thus, the methods herein provides for any variants of such parent antibodies.
The parent antibody, the first parent antibody, the second parent antibody, or the variants thereof obtained by the methods and/or uses of the present invention may bind to any target as described herein.
Examples of antigens or targets that the invention may be directed against are; 5T4; ADAM-10; ADAM-12; ADAM17; AFP; AXL; ANGPT2 anthrax antigen; BSG; CAIX; CAXII; CA 72-4; carcinoma associated antigen CTAA16.88; CCL11; CCL2; CCR4; CCR5; CCR6; CD2; CD3E; CD4; CD5; CD6; CD15; CD18; CD19; CD20; CD22; CD24; CD25; CD29; CD30; CD32B; CD33; CD37; CD38; CD40; CD40LG; CD44; CD47; CD52; CD56; CD66E; CD72; CD74; CD79a; CD79b; CD80; CD86; CD98; CD137; CD147; CD138; CD168; CD200; CD248; CD254; CD257; CDH3; CEA; CEACAM5; CEACAM6; CEACAM8; Claudin4; CS-1; CSF2RA; CSPG-4; CTLA4; Cripto; DLL4; ED-B; EFNA2; EGFR; Endothelin B receptor; ENPP3; EPCAM; ERBB2; ERBB3; FAP alpha; Fc gamma RI; FCER2; FGFR3; fibrin II beta chain; FLT1; FOLH1; FOLR1; FRP-1; GD3 ganglioside; GDF2; GLP1R; Glypican-3; GPNMB; HBV (hepatitis B virus); HCMV (human cytomegalovirus); heat shock protein 90 homolog [Candida albicans]; herpes simplex virus gD glycoprotein; HGF; HIV-1; HIV-1 IIIB gp120 V3 loop; HLA-DRB (HLA-DR beta); human respiratory syncytial virus, glycoprotein F; ICAM1; IFNA1; IFNA1; IFNB1 bispecific; IgE Fc; IGF1R; IGHE connecting region; IL12B; IL13; IL15; IL17A; IL1A; IL1B; IL2RA; IL4; IL5; IL5RA; IL6; IL6R; IL9; interleukin-2 receptor beta subunit; ITGA2; ITGA2B ITGB3; ITGA4 ITGB7; ITGA5; ITGAL; ITGAV_ITGB3; ITGB2; KDR; L1CAM; Lewis-y; lipid A, domain of lipopolyaccharide LPS; LTA; MET; MMP14; MMp15; MST1R; MSTN; MUC1; MUC4; MUC16; MUC5AC; NCA-90 granulocyte cell antigen; Nectin 4; NGF; NRP; NY-ESO-1; OX40L; PLAC-1; PLGF; PDGFRA; PD1; PDL1; PSCA; phosphatidylserine; PTK-7; Pseudomonas aeruginosa serotype IATS O11; RSV (human respiratory syncytial virus, glycoprotein F); ROR1; RTN4; SELL; SELP; STEAP1; Shiga-like toxin II B subunit [Escherichia coli]; SLAM7; SLC44A4; SOST; Staphylococcus epidermidis lipoteichoic acid; T cell receptor alpha_beta; TF; TGFB1; TGFB2; TMEFF2; TNC; TNF; TNFRSF10A; TNFRSF10B; TNFRSF12A; TNFSF13; TNFSF14; TNFSF2; TNFSF7; TRAILR2; TROP2; TYRP1; VAP-1; and Vimentin.
Methods of Inducing an Effector Response
It is to be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody are also to be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region.
In a further main aspect the present invention relates to a method of inducing an effector response, against a cell, cell membrane, or virion expressing a target to which a parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region binds, comprising
(i) providing a parent polypeptide or a combination of at least a first parent polypeptide and a second parent polypeptide which has been mutated according to any one of the claims 1 to 24; and
(ii) contacting a preparation of the mutated parent polypeptide of step (i) or the mutated combination of at least a first parent polypeptide and a second parent polypeptide of step (i) with the cell, cell membrane, or virion expressing an antigen in the presence of human complement or an effector cell.
In one embodiment any or all of the parent polypeptide, first parent polypeptide and second parent polypeptide may be an antibody.
Thus in one embodiment the present invention relates to methods of using the antibody variants described herein for inducing an effector response, e.g complement activation, CDC or other effector response against a cell, cell membrane, virion or other particle associated with the antigen or antigens. The present invention also relates to a method of inducing an effector response, against a cell, cell membrane, or virion expressing an antigen to which a parent antibody binds, comprising
(i) providing a parent antibody or a combination of at least a first parent antibody and a second parent antibody which has been mutated according to any of the methods described herein; and
(ii) contacting a preparation of the mutated parent antibody of step (i) or the mutated combination of at least a first parent antibody and a second parent antibody of step (i) with the cell, cell membrane, or virion expressing an antigen in the presence of human complement or an effector cell.
The parent antibody, the first parent antibody and the second parent antibody may each be selected from any parent antibody described herein, in particular any of those described above in relation to the methods of affecting an effector function of an antibody.
In one embodiment, the antigen is expressed on the surface of a cell.
In one embodiment, the cell is a human tumor cell.
In a further embodiment, the antigen is selected from the group consisting of erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD38, CD138, CXCR5, c-Met, HERV-envelop protein, periostin, Bigh3, SPARC, BCR, CD79, CD37, EGFrvIII, IGFr, L1-CAM, AXL, Tissue Factor (TF), CD74, EpCAM and MRP3.
In another embodiment, the antigen is associated with a cell membrane.
In another embodiment, the antigen is associated with a virion, optionally wherein the antigen is comprised in the protein coat or a lipid envelope of the virion.
In another embodiment, the antibody is a human antibody, optionally binding at least one antigen selected from CD20 and CD38.
In another embodiment, the antibody binds to the same epitope as at least one of 7D8 and 005, optionally comprising a variable heavy and/or variable light chain region of at least one of 7D8 and 005.
In a further embodiment of the present invention, the induced effector response is complement dependent cytotoxicity (CDC), an Fc-mediated effector response selected from an Fc-mediated effector response selected from C1q-binding, complement activation, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxity (ADCC), FcRn-binding, Fc-receptor binding including Fc-gamma receptor-binding, Protein A-binding, Protein G-binding, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cellular cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonisation, Fc-containing polypeptide internalization, target downmodulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof.
In a particular embodiment the effector response is C1q-binding, complement activation (C1q efficacy), complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxity (ADCC), Fc-receptor binding, e.g Fc-gamma receptor-binding, Fc-containing polypeptide internalization or any combination thereof.
In one embodiment the effector response is C1q-binding.
In one embodiment the effector response is complement activation (C1q efficacy).
In one embodiment the effector response is complement dependent cytotoxicity (CDC).
In one embodiment the effector response is antibody-dependent cell-mediated cytotoxity (ADCC).
In one embodiment the effector response is Fc-receptor binding, e.g. including Fc-gamma receptor-binding.
In one embodiment the effector response is Fc-containing polypeptide internalization.
In one embodiment the effector response is a combination of complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxity (ADCC).
In another embodiment, the method increases a further effector response selected from FcRn-binding, ADCC, Fc-gamma receptor-binding, Protein A-binding, Protein G-binding, ADCP, complement-dependent cellular cytotoxicity (CDCC), complement-enhanced cytotoxicity, binding to complement receptor of an opsonized antibody mediated by the antibody, and any combination thereof.
In another aspect, the invention relates to a method of increasing the avidity of a preparation of a parent antibody for C1q, comprising the step of mutating at least one amino acid in the Fc-region of the antibody, wherein the at least one amino acid is selected from the group consisting of E345, E430, S440, P247, I253, S254, Q311, D/E356, T359, E382, Q386, Y436, and K447.
Examples of antigens or targets that the invention may be directed against are; 5T4; ADAM-10; ADAM-12; ADAM17; AFP; AXL; ANGPT2 anthrax antigen; BSG; CAIX; CAXII; CA 72-4; carcinoma associated antigen CTAA16.88; CCL11; CCL2; CCR4; CCR5; CCR6; CD2; CD3E; CD4; CD5; CD6; CD15; CD18; CD19; CD20; CD22; CD24; CD25; CD29; CD30; CD32B; CD33; CD37; CD38; CD40; CD40LG; CD44; CD47; CD52; CD56; CD66E; CD72; CD74; CD79a; CD79b; CD80; CD86; CD98; CD137; CD147; CD138; CD168; CD200; CD248; CD254; CD257; CDH3; CEA; CEACAM5; CEACAM6; CEACAM8; Claudin4; CS-1; CSF2RA; CSPG-4; CTLA4; Cripto; DLL4; ED-B; EFNA2; EGFR; Endothelin B receptor; ENPP3; EPCAM; ERBB2; ERBB3; FAP alpha; Fc gamma RI; FCER2; FGFR3; fibrin II beta chain; FLT1; FOLH1; FOLR1; FRP-1; GD3 ganglioside; GDF2; GLP1R; Glypican-3; GPNMB; HBV (hepatitis B virus); HCMV (human cytomegalovirus); heat shock protein 90 homolog [Candida albicans]; herpes simplex virus gD glycoprotein; HGF; HIV-1; HIV-1 IIIB gp120 V3 loop; HLA-DRB (HLA-DR beta); human respiratory syncytial virus, glycoprotein F; ICAM1; IFNA1; IFNA1; IFNB1 bispecific; IgE Fc; IGF1R; IGHE connecting region; IL12B; IL13; IL15; IL17A; IL1A; IL1B; IL2RA; IL4; IL5; IL5RA; IL6; IL6R; IL9; interleukin-2 receptor beta subunit; ITGA2; ITGA2B ITGB3; ITGA4 ITGB7; ITGA5; ITGAL; ITGAV_ITGB3; ITGB2; KDR; L1CAM; Lewis-y; lipid A, domain of lipopolyaccharide LPS; LTA; MET; MMP14; MMp15; MST1R; MSTN; MUC1; MUC4; MUC16; MUC5AC; NCA-90 granulocyte cell antigen; Nectin 4; NGF; NRP; NY-ESO-1; OX40L; PLAC-1; PLGF; PDGFRA; PD1; PDL1; PSCA; phosphatidylserine; PTK-7; Pseudomonas aeruginosa serotype IATS O11; RSV (human respiratory syncytial virus, glycoprotein F); ROR1; RTN4; SELL; SELP; STEAP1; Shiga-like toxin II B subunit [Escherichia coli]; SLAM7; SLC44A4; SOST; Staphylococcus epidermidis lipoteichoic acid; T cell receptor alpha_beta; Tissue Factor (TF); TGFB1; TGFB2; TMEFF2; TNC; TNF; TNFRSF10A; TNFRSF10B; TNFRSF12A; TNFSF13; TNFSF14; TNFSF2; TNFSF7; TRAILR2; TROP2; TYRP1; VAP-1; and Vimentin.
In one embodiment, the cell is a human tumor cell or a bacterial cell.
In another embodiment, the antigen is selected from the group consisting of erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD25, CD32, CD37, CD38, CD74, CD138, CXCR5, c-Met, HERV-envelop protein, periostin, Bigh3, SPARC, BCR, CD79, EGFrvIII, IGFr, L1-CAM, EpCAM and MRP3.
In a further embodiment, the antigen is CD20 or CD38.
In another embodiment, the IgG1 parent antibody is a human IgG1 antibody.
In another embodiment, the parent antibody is selected from 7D8 and 005.
In one embodiment, the cell is a human tumor cell.
In another embodiment, the first and second antigens are separately selected from the group consisting of erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD25, CD32, CD37, CD38, CD74, CD138, CXCR5, c-Met, HERV-envelop protein, periostin, Bigh3, SPARC, BCR, CD79, EGFrvIII, IGFr, L1-CAM, AXL, Tissue Factor (TF), EpCAM and MRP3.
In another embodiment, the first and second parent antibodies are fully human, optionally wherein the first and second parent antibodies bind antigens separately selected from CD20 and CD38.
In a further embodiment, the first and second parent antibodies are separately selected from 7D8 and 005.
In an even further embodiment, the cell is a bacterial cell.
In another embodiment, the bacterial cell is selected from the group consisting of S. aureus, S. Epidermidis, S. pneumonia, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia, E. coli, Salmonella, Shigella, Yersinia, S. typhimurium, Neisseria meningitides and Mycobacterium tuberculosis.
In another embodiment, the first and/or second antigen is Lipoteichoic acid (LTA), optionally wherein at least one of the first and second parent antibody is pagibaximab.
In another embodiment, the antigen is expressed on a virion.
In another embodiment, the first and second antibody binds the same antigen.
In another embodiment, the first and second antibodies comprise the same VH sequence, VL sequence, or both VH and VL sequence.
For the purposes of the present invention, the target cell that expresses or is otherwise associated with an antigen can be any prokaryotic or eukaryotic cell. Exemplary antigen-expressing cells include, but are not limited to, mammalian cells, particularly human cells, such as human cancer cells; and unicellular organisms such as bacteria, protozoa, and unicellular fungi such as yeast cells. Cell membranes comprising or otherwise associated with an antigen include partial and/or disrupted cell membranes derived from an antigen-expressing cell. An antigen associated with a virion or virus particle may be comprised in or otherwise associated with the protein coat and/or a lipid envelope of the virion.
The target cell may, for example, be a human tumor cell. Suitable tumor antigens include any target or antigen described herein, but are not limited to, erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD25, CD32, CD37, CD38, CD74, CD138, CXCR5, c-Met, HERV-envelop protein, periostin, Bigh3, SPARC, BCR, CD79, EGFrvIII, IGFR, L1-CAM, AXL, Tissue Factor (TF), EpCAM and MRP3. Preferred antigens include CD20, CD38, HER2, EGFR, IGFR, CD25, CD74 and CD32. Exemplary antibodies include anti-CD20 antibody 7D8 as disclosed in WO 2004/035607, anti-CD38 antibody 005 as disclosed in WO 06/099875, anti-CD20 antibody 11B8 as disclosed in WO 2004/035607, anti-CD38 antibody 003 as disclosed in WO 06/099875, anti-EGFr antibody 2F8 as disclosed in WO 02/100348. Examples of other particular antibodies are provided herein.
Alternatively, the target cell can be a bacterial cell, such as, e.g., S. aureus, S. epidermidis, S. pneumonia, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia, E. coli, Salmonella, Shigella, Yersinia, S. typhimurium, Neisseria meningitides and Mycobacterium tuberculosis. Exemplary antigens include Lipoteichoic acid (LTA), and exemplary antibodies include pagibaximab.
Alternatively, the target may be present on the surface of a virus, fungal cell or other particle, such as, e.g., West Nile virus, Dengue virus, hepatitis C-virus (HCV), human immunodeficiency virus (HIV), human papillomavirus, Epstein-Barr virus, Herpesviruses, poxviruses, avian influenza virus, RVS, Aspergillus, Candida albicans, Cryptococcus, and Histoplasma.
In one embodiment, the contacting step (ii) takes place in vitro.
In one embodiment, the contacting step (ii) takes place in vivo.
In another embodiment, step (ii) comprises administering the variants to a subject.
In a further embodiment, the subject suffers from cancer, a bacterial infection, or a viral infection. The contacting step (ii) of the above-mentioned embodiments may take place in vitro or in vivo. In the latter case, step (ii) may further comprise administering the preparation or preparations to a subject, optionally a subject suffering from cancer or a bacterial infection. Further details on therapeutic applications are provided below.
The first and the second antibodies comprise antigen-binding regions which may bind to the same or different epitope. Such epitopes may be on the same or different target.
In an embodiment, the first and the second antibody binds different epitopes on different targets. Such targets may be expressed on the same cell or cell type, or may be expressed on different cells or cell types. In such an embodiment, the enhancement of an effector function is directed only towards cells or cell types expressing both the targets, and thereby reducing the risks of any collateral damage of cells or cell types which are not the cause of a disease to be treated.
Without being bound by any theory, it is believed that the enhancement of CDC can be restricted to target cells that express two specific targets/antigens simultaneously provided that the first and second antibody bind epitopes found on the same cell, thereby exploiting the combined expression of targets to improve selectivity of enhaved CDC induction.
In cases where the targets are expressed on different cells or cell types, it is believed without being bound by theory, that the administration in any order of the first and second antibody will improve CDC enhancement and possibly also other effector functions by “recruitment” of a second cell or cell type expressing the second target.
In one embodiment wherein a combination of a first and second antibody are used, step (ii) may be performed by simultaneously, separately, or sequentially contacting the cell with the mutated first and second parent antibodies in the presence of human complement and/or an effector cell.
In yet another aspect, the invention relates to a method of improving the CDC-inducing capability of a preparation of a parent antibody, comprising the step of mutating at least one amino acid in the Fc-region of the antibody, wherein the at least one amino acid is selected from the group consisting of E345, E430, S440, P247, I253, S254, Q311, D/E356, T359, E382, Q386, Y436, and K447.
In an alternative aspect, the present invention relates to a method of inducing an effector response, optionally a CDC-response, against a cell, cell membrane, or virion expressing an antigen to which an IgG1 parent antibody binds, comprising
(i) providing an antibody comprising a mutation in at least one amino acid residue selected from the group consisting of E345, E430, S440, P247, I253, S254, Q311, D/E356, T359, E382, Q386, Y436, and K447 in the Fc-region of an IgG1 heavy chain; and
(ii) contacting a preparation of the antibody with the cell, cell membrane, or virion in the presence of human complement or an effector cell.
In another alternative embodiment, the method further comprises administering a first antibody comprising a first mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, P247, I253, S254, Q311, D/E356, T359, E382, Q386, Y436, and K447 in the Fc-region of the first antibody;
administering a second antibody comprising a second mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, P247, I253, S254, Q311, D/E356, T359, E382, Q386, Y436, and K447 in the Fc-region of the second antibody;
wherein the first and second antibodies may be administered simultaneously, separately or sequentially. The first and second antibody may bind to the same or different epitope on the same or different target. The target(s) may be found on the same or different cell or cell types.
In another alternative aspect, the invention relates to a method of improving the CDC-inducing capability of a preparation of an IgG1 parent antibody, comprising mutating at least one amino acid in the Fc-region of the antibody, wherein the at least one amino acid is selected from the group consisting of E345, E382 and H433.
In another alternative aspect, the invention relates to a method of inducing an effector response, optionally a CDC-response, against a cell, cell membrane, or virion expressing an antigen to which an IgG1 parent antibody binds, comprising the steps of
(i) providing a variant of the parent antibody comprising a mutation in at least one amino acid in the Fc-region of the antibody, the at least one amino acid selected from the group consisting of E345, E382 and H433; and
(ii) contacting a preparation of the variant with the cell in the presence of human complement or an effector cell.
In another alternative aspect, the invention relates to a method of inducing an effector response, optionally a CDC-response, against a cell expressing an antigen to which an IgG1 parent antibody binds, comprising the steps of
(i) providing a variant of the parent antibody comprising K439E and a S440K mutations in the Fc-region of the antibody; and
(ii) contacting a preparation of the variant with the cell in the presence of human complement or an effector cell.
In another alternative aspect, the invention relates to a method of inducing a CDC-response against a cell, cell membrane or virion expressing a first antigen to which a first IgG1 parent antibody binds and a second antigen to which a second parent antibody binds, comprising the steps of
(i) providing a first variant of the first parent antibody comprising a K439E mutation and a second variant of the second parent antibody comprising a S440K mutation; and
(ii) simultaneously, separately or sequentially contacting the cell with the first and second variants in the presence of human complement and/or an effector cell.
In another alternative aspect, the invention provides for a method of inducing a CDC- or other effector response against a target cell, cell membrane, virion or other particle associated with an antigen to which an IgG1 or IgG3 antibody binds, comprising the steps of (i) providing a variant of the antibody comprising a mutation in at least one amino acid corresponding to E345, E430 or S440 in the Fc-region of an IgG1 antibody; and (ii) contacting a preparation of the variant with the cell in the presence of human complement and/or effector cells.
In further alternative aspect, the invention provides for a method of inducing ADCC or ADCP against, or phagocytosis of, a target cell, cell membrane, virion or other particle associated with an antigen to which an IgG1 or IgG3 antibody binds, comprising the steps of (i) providing a variant of the antibody comprising a mutation in at least one amino acid corresponding to E345, E430 or S440 in the Fc-region of an IgG1 antibody; and (ii) contacting a preparation of the variant with the cell in the presence of an effector cell.
The invention also provides for a method of inducing a CDC or other effector response against a target cell, cell membrane, virion or other particle associated with an antigen to which an IgG1 or IgG3 antibody binds, comprising the steps of (i) providing a variant of the antibody comprising a mutation in K439 which is K439E and a mutation in S440 which is S440K or S440R in the Fc-region of the antibody; and (ii) contacting a preparation of the variant with the cell in the presence of human complement and/or an effector cell
The invention also provides for a method of inducing a CDC or other effector response against a target cell, cell membrane or virion expressing a first antigen to which a first IgG1 antibody binds and a second antigen to which a second antibody binds, comprising the steps of (i) providing a first variant which is the first antibody comprising a K439E mutation and a second variant which is the second antibody comprising a S440K or S440R mutation; and (ii) simultaneously, separately or sequentially contacting the cell with preparations of the first and second variants in the presence of human complement or an effector cell.
In separate and specific embodiments, the first and second antibodies bind (i) different antigens; (ii) different epitopes on the same antigen, (iii) the same epitope on an antigen, and (iv) the same epitope on an antigen and comprise the same VH and/or VL sequences.
In one embodiment, the first and second antibodies further comprise a mutation in one or more of E345, E430 and S440, such as E345R. In one embodiment, the first and second antibodies further comprise a mutation in one or more of E345, E382 and H433, such as E345R.
Other Methods
In another main aspect, the invention relates to a method of identifying a mutation in an antibody which enhances the effector function of the antibody to bind C1q, comprising the steps of
(i) preparing at least one antibody comprising a mutation in at least one amino acid selected from the group consisting of E345, E430, S440, K439, P247, I253, S254, Q311, D/E356, T359, E382, Q386, Y436, and K447;
(ii) evaluating the C1q-activity of the antibody when bound to the surface of antigen-expressing cell as compared to the parent antibody; and
(iii) selecting the mutation of any variant having an increased C1q-avidity.
In one embodiment, the at least one antibody comprises at least one amino acid substitution selected from the group of E345R, E345Q, E345N, E345K, E345Y, E430T, E430S, E430G, E430F, E430H, S440W and S440Y.
In yet another main aspect, the invention relates to a method of identifying a mutation in a parent antibody which increases the ability of the antibody to induce a CDC-response, comprising the steps of
(i) preparing at least one variant of the parent antibody comprising a mutation in at least one amino acid selected from the group consisting of E345, E430, S440, K439, P247, I253, S254, Q311, D/E356, T359, E382, Q386, Y436, and K447;
(ii) evaluating the CDC-response induced by the variant when bound to the surface of an antigen-expressing cell, in the presence of effector cells or complement, as compared to the parent antibody; and
(iii) selecting the mutation of any variant having an increased CDC-response.
In one embodiment, the at least one amino acid is selected from E345, E382 and H433.
In one embodiment, the at least one antibody comprises at least one amino acid substitution selected from the group of E345R, E345Q, E345N, E345K, E345Y, E430T, E430S, E430G, E430F, E430H, S440W and S440Y.
In another aspect, the invention relates, to a method of increasing the avidity of a preparation of an IgG1 parent antibody for C1q, comprising mutating at least one amino acid in the Fc-region of the antibody, wherein the at least one amino acid is selected from the group consisting of E345, E382 and H433.
Antibodies of the Present Invention
Parent Antibodies
As described herein, the present invention inter alia relates to variants of parent antibodies comprising one or more mutations in the CH2 and/or CH3 region of an immunoglobin, e.g. in the antibody the heavy chain. The “parent” antibodies, which may be wild-type antibodies, to be used as starting material of the present invention before modification may e.g. be produced by the hybridoma method first described by Kohler et al., Nature 256, 495 (1975), or may be produced by recombinant DNA methods. Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352, 624 628 (1991) and Marks et al., J. Mol. Biol. 222, 581 597 (1991). Monoclonal antibodies may be obtained from any suitable source. Thus, for example, monoclonal antibodies may be obtained from hybridomas prepared from murine splenic B cells obtained from mice immunized with an antigen of interest, for instance in form of cells expressing the antigen on the surface, or a nucleic acid encoding an antigen of interest. Monoclonal antibodies may also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or non-human mammals such as rabbits, rats, dogs, primates, etc.
The parent antibodies may be e.g. chimeric or humanized antibodies. In another embodiment, the antibody is a human antibody. Human monoclonal antibodies may be generated using transgenic or transchromosomal mice, e.g. HuMAb mice, carrying parts of the human immune system rather than the mouse system. The HuMAb mouse contains a human immunoglobulin gene minilocus that encodes unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature 368, 856 859 (1994)). Accordingly, the mice exhibit reduced expression of mouse IgM or κ and in response to immunization, the introduced human heavy and light chain transgenes, undergo class switching and somatic mutation to generate high affinity human IgG,κ monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook of Experimental Pharmacology 113, 49 101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65 93 (1995) and Harding, F. and Lonberg, N. Ann. N.Y. Acad. Sci. 764 536 546 (1995)). The preparation of HuMAb mice is described in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287 6295 (1992), Chen, J. et al., International Immunology 5, 647 656 (1993), Tuaillon et al., J. Immunol. 152, 2912 2920 (1994), Taylor, L. et al., International Immunology 6, 579 591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845 851 (1996). See also U.S. Pat. Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, 5,770,429, 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187. Splenocytes from these transgenic mice may be used to generate hybridomas that secrete human monoclonal antibodies according to well known techniques.
Further, human antibodies of the present invention or antibodies of the present invention from other species may be identified through display-type technologies, including, without limitation, phage display, retroviral display, ribosomal display, mammalian display, yeast display and other techniques known in the art, and the resulting molecules may be subjected to additional maturation, such as affinity maturation, as such techniques are well known in the art. A particular strategy, described in Example 17, can be applied to any antibody to prepare and obtain a variant of the invention using phage-display.
The parent antibody is not limited to antibodies which have a natural, e.g. a human Fc domain but it may also be an antibody having other mutations than those of the present invention, such as e.g. mutations that affect glycosylation or enables the antibody to be a bispecific antibody. By the term “natural antibody” is meant any antibody which does not comprise any genetically introduced mutations. An antibody which comprises naturally occurred modifications, e.g. different allotypes, is thus to be understood as a “natural antibody” in the sense of the present invention, and can thereby be understood as a parent antibody. Such antibodies may serve as a template for the one or more mutations according to the present invention, and thereby providing the variant antibodies of the invention. An example of a parent antibody comprising other mutations than those of the present invention is the bispecific antibody as described in WO2011/131746 (Genmab), utilizing reducing conditions to promote half-molecule exchange of two antibodies comprising IgG4-like CH3 regions, thus forming bispecific antibodies without concomitant formation of aggregates. Other examples of parent antibodies include but are not limited to bispecific antibodies such as heterodimeric bispecifics: Triomabs (Fresenius); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation); FcΔAdp (Regeneron); Knobs-into-holes (Genentech); Electrostatic steering (Amgen, Chugai, Oncomed); SEEDbodies (Merck); Azymetric scaffold (Zymeworks); mAb-Fv (Xencor); and LUZ-Y (Genentch). Other exemplary parent antibody formats include, without limitation, a wild-type antibody, a full-length antibody or Fc-containing antibody fragment, a human antibody, or any combination thereof.
The parent antibody may bind any target, examples of such targets or antigens the invention may be, and is not limited to, directed against are; 5T4; ADAM-10; ADAM-12; ADAM17; AFP; AXL; ANGPT2 anthrax antigen; BSG; CAIX; CAXII; CA 72-4; carcinoma associated antigen CTAA16.88; CCL11; CCL2; CCR4; CCR5; CCR6; CD2; CD3E; CD4; CD5; CD6; CD15; CD18; CD19; CD20; CD22; CD24; CD25; CD29; CD30; CD32B; CD33; CD37; CD38; CD40; CD40LG; CD44; CD47; CD52; CD56; CD66E; CD72; CD74; CD79a; CD79b; CD80; CD86; CD98; CD137; CD147; CD138; CD168; CD200; CD248; CD254; CD257; CDH3; CEA; CEACAM5; CEACAM6; CEACAM8; Claudin4; CS-1; CSF2RA; CSPG-4; CTLA4; Cripto; DLL4; ED-B; EFNA2; EGFR; Endothelin B receptor; ENPP3; EPCAM; ERBB2; ERBB3; FAP alpha; Fc gamma RI; FCER2; FGFR3; fibrin II beta chain; FLT1; FOLH1; FOLR1; FRP-1; GD3 ganglioside; GDF2; GLP1R; Glypican-3; GPNMB; HBV (hepatitis B virus); HCMV (human cytomegalovirus); heat shock protein 90 homolog [Candida albicans]; herpes simplex virus gD glycoprotein; HGF; HIV-1; HIV-1 IIIB gp120 V3 loop; HLA-DRB (HLA-DR beta); human respiratory syncytial virus, glycoprotein F; ICAM1; IFNA1; IFNA1; IFNB1 bispecific; IgE Fc; IGF1R; IGHE connecting region; IL12B; IL13; IL15; IL17A; IL1A; IL1B; IL2RA; IL4; IL5; IL5RA; IL6; IL6R; IL9; interleukin-2 receptor beta subunit; ITGA2; ITGA2B ITGB3; ITGA4 ITGB7; ITGA5; ITGAL; ITGAV_ITGB3; ITGB2; KDR; L1CAM; Lewis-y; lipid A, domain of lipopolyaccharide LPS; LTA; MET; MMP14; MMp15; MST1R; MSTN; MUC1; MUC4; MUC16; MUC5AC; NCA-90 granulocyte cell antigen; Nectin 4; NGF; NRP; NY-ESO-1; OX40L; PLAC-1; PLGF; PDGFRA; PD1; PDL1; PSCA; phosphatidylserine; PTK-7; Pseudomonas aeruginosa serotype IATS O11; RSV (human respiratory syncytial virus, glycoprotein F); ROR1; RTN4; SELL; SELP; STEAP1; Shiga-like toxin II B subunit [Escherichia coli]; SLAM7; SLC44A4; SOST; Staphylococcus epidermidis lipoteichoic acid; T cell receptor alpha_beta; TF; TGFB1; TGFB2; TMEFF2; TNC; TNF; TNFRSF10A; TNFRSF10B; TNFRSF12A; TNFSF13; TNFSF14; TNFSF2; TNFSF7; TRAILR2; TROP2; TYRP1; VAP-1; and Vimentin.
The parent antibody may be any human antibody of any isotype, e.g. IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, and IgD, optionally a human full-length antibody, such as a human full-length IgG1 antibody. The parent antibody may comprise a sequence according to any of SEQ ID NOs: 1, 2, 3, 4, and 5.
Monoclonal antibodies, such as the parent and/or variants, for use in the present invention, may be produced, e.g., by the hybridoma method first described by Kohler et al., Nature 256, 495 (1975), or may be produced by recombinant DNA methods. Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies may be obtained from any suitable source. Thus, for example, monoclonal antibodies may be obtained from hybridomas prepared from murine splenic B cells obtained from mice immunized with an antigen of interest, for instance in form of cells expressing the antigen on the surface, or a nucleic acid encoding an antigen of interest. Monoclonal antibodies may also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or non-human mammals such as rats, dogs, primates, etc.
In one embodiment, the antibody is a human antibody. Human monoclonal antibodies directed against any antigen may be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. Such transgenic and transchromosomic mice include mice referred to herein as HuMAb® mice and KM mice, respectively, and are collectively referred to herein as “transgenic mice”.
The HuMAb® mouse contains a human immunoglobulin gene miniloci that encodes unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature 368, 856-859 (1994)). Accordingly, the mice exhibit reduced expression of mouse IgM or κ and in response to immunization, the introduced human heavy and light chain transgenes, undergo class switching and somatic mutation to generate high affinity human IgG,κ monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook of Experimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65-93 (1995) and Harding, F. and Lonberg, N. Ann. N.Y. Acad. Sci. 764 536-546 (1995)). The preparation of HuMAb® mice is described in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994), Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845-851 (1996). See also U.S. Pat. Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, 5,770,429, 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.
The HCo7, HCo12, HCo17 and HCo20 mice have a JKD disruption in their endogenous light chain (kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)), a CMD disruption in their endogenous heavy chain genes (as described in Example 1 of WO 01/14424), and a KCo5 human kappa light chain transgene (as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)). Additionally, the Hco7 mice have a HCo7 human heavy chain transgene (as described in U.S. Pat. No. 5,770,429), the HCo12 mice have a HCo12 human heavy chain transgene (as described in Example 2 of WO 01/14424), the HCo17 mice have a HCo17 human heavy chain transgene (as described in Example 2 of WO 01/09187) and the HCo20 mice have a HCo20 human heavy chain transgene. The resulting mice express human immunoglobulin heavy and kappa light chain transgenes in a background homozygous for disruption of the endogenous mouse heavy and kappa light chain loci.
In the KM mouse strain, the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al., EMBO J. 12, 811-820 (1993) and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of WO 01/09187. This mouse strain carries a human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also carries a human heavy chain transchromosome composed of chromosome 14 fragment hCF (SC20) as described in WO 02/43478. HCo12-Balb/C mice can be generated by crossing HCo12 to KCo5[J/K](Balb) as described in WO/2009/097006. Splenocytes from these transgenic mice may be used to generate hybridomas that secrete human monoclonal antibodies according to well known techniques.
Further, any antigen-binding regions may be obtained from human antibodies or antibodies from other species identified through display-type technologies, including, without limitation, phage display, retroviral display, ribosomal display, and other techniques, using techniques well known in the art and the resulting molecules may be subjected to additional maturation, such as affinity maturation, as such techniques are well known in the art (see for instance Hoogenboom et al., J. Mol. Biol. 227, 381 (1991) (phage display), Vaughan et al., Nature Biotech 14, 309 (1996) (phage display), Hanes and Plucthau, PNAS USA 94, 4937-4942 (1997) (ribosomal display), Parmley and Smith, Gene 73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245 (1992), Cwirla et al., PNAS USA 87, 6378-6382 (1990), Russel et al., Nucl. Acids Research 21, 1081-1085 (1993), Hogenboom et al., Immunol. Reviews 130, 43-68 (1992), Chiswell and McCafferty TIBTECH 10, 80-84 (1992), and U.S. Pat. No. 5,733,743). If display technologies are utilized to produce antibodies that are not human, such antibodies may be humanized.
In another aspect, the invention relates to a parent polypeptide comprising a Fc domain and a binding region. It is understood in the context of the present invention all embodiments relating to parent antibody similarly applies to a “parent polypeptide”.
A mutation according to the present invention may be, but is not limited to, a deletion, insertion or substitution of one or more amino acids. Such a substitution of amino acids may be with any naturally occurring or non-naturally amino acid.
“Single-Mutants”
It is to be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody are also to be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region.
Antibody or polypeptide variants according to the “single-mutant” aspect of the present invention comprise a mutation, typically an amino acid substitution, in at least one amino acid residue shown in Table 1, which lists each amino acid residue, numbered according to the EU index in a human IgG1 antibody, along with the amino acid in the corresponding position in an IgG2, IgG3, and IgG4 parent antibody and “Exemplary” and “Preferred” amino acid substitutions. The IgG2 segment corresponding to residues P247 to K447, the IgG3 Fc-segment corresponding to residues P247 to K447 and the IgG4 segment corresponding to residues P247 to K447 in IgG1 are shown in FIG. 2.
TABLE 1
Exemplary mutation sites and amino acid substitutions for the “single-mutant” aspect
Amino
Amino
Amino
Amino acid
acid
acid
acid
Preferred
(IgG1)
(IgG2)
(IgG3)
(IgG4)
Exemplary substitutions
substitutions
P247
P247
P247
P247
ACDFGHIKLMNRSTVW
G
I253
I253
I253
I253
ADKLMNRSV, alternatively
LV, alternatively QN
EQT
S254
S254
S254
S254
EFGHIKLPTVW
L
H310
H310
H310
H310
AGFKLPRTVW, alternatively
PW, alternatively Q
NQY
Q311
Q311
Q311
Q311
ACEGHFIKLNPRSTWY
LW, alternatively ER
E345
E345
E345
E345
ACDGHFIKLMNPQRSTVWY
ADGHFIKLMNPQRSTVWY
D356/E356
E356
E356
E356
GILRTV
R
T359
T359
T359
T359
GNPR
R
E382
E382
E382
E382
FKLMPVW, alternatively
LV, alternatively DQKR
DHNQSTY
G385
G385
G385
G385
ADHILNPQRSTV, alternatively
NR, alternatively DEKR
EKWY
Q386
Q386
Q386
Q386
ACDEGHFIKLNPRSTVWY
K
E430
E430
E430
E430
ACDFGHIKLMNPQRSTVWY
ADGHFIKLMNPQRSTVWY
H433
H433
H433
H433
R
R
N434
N434
N434
N434
DEGKRSVW, alternatively
W, alternatively QHKR
HQTY
Y436
Y436
F436
Y436
IKLRSTVW, alternatively
IV, alternatively NQST
AEFHMNQ
Q438
Q438
Q438
Q438
CEIKLSTVWY, alternatively
CL, alternatively NST
AGHNQR
K439
K439
K439
K439
ADEHLPRTY, alternatively QW
DEHR, alternatively Q
S440
S440
S440
S440
ACDEGHFIKLMNPQRTVWY
WY, alternatively DEQ
K447
K447
K447
K447
DENQ, deletion
DENQ, deletion
As seen in Table 1, the amino acid substitutions which resulted in an increase of cell lysis of Wien133 cells in Example 19 are included as “Preferred substitutions”.
In one aspect the present invention relates to a variant of a parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region, wherein the variant comprises a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.
In one embodiment the variant polypeptide may be a variant antibody.
Thus in another aspect, the invention relates to a variant of a parent antibody comprising an antigen-binding region and Fc-domain of an immunoglobulin, wherein the variant comprises a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W. Alternatively, the amino acid residue is selected from those corresponding to H310, G385, H433, N434, Q438, and K439 in the Fc-region of a human IgG1 heavy chain.
Each of the amino acid residues corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain may be grouped according to the following as described above:
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain.
A mutation according to the present invention may be, but is not limited to, a deletion, insertion or substitution of one or more amino acids. Such a substitution of amino acids may be with any naturally occurring or non-naturally amino acid. Thus, in one embodiment, the mutation in at least one amino acid residue is a deletion. In another embodiment, the mutation in at least one amino acid residue is an insertion. In another embodiment, the mutation in at least one amino acid residue is a substitution.
In one embodiment, the mutation in at least one amino acid residue is selected from those corresponding to E345X, E430X, S440W/Y, Q386K, P247G, I253V, S254L, Q311L/W, D/E356R, E382V, Y436I, and K447D/E/deletion in the Fc region of a human IgG1 heavy chain, wherein X is any amino acid, such as a natural occurring amino acid.
In one specific embodiment, the antibody variant comprises a mutation in at least one amino acid residue selected from E345, E430, S440, and Q386 in the Fc-region of a human IgG1 heavy chain.
In a further embodiment, the mutation in at least one amino acid residue is an amino acid substitution selected from those corresponding to E345X, E430X, S440W/Y, Q386K, in the Fc region of a human IgG1 heavy chain, wherein X is any amino acid, such as a natural occurring amino acid.
In a preferred embodiment, the mutation in at least one amino acid residue is an amino acid substitution selected from those corresponding to E345R,Q,N,K,A,C,D,F,G,H,I,L,M,P,S,T,V,W,Y; E430T,S,G,A,C,D,F,H,I,L,K,M,N,P,Q,R,V,W,Y; S440W,Y, and Q386K in the Fc region of a human IgG1 heavy chain.
In a further preferred embodiment, the mutation in at least one amino acid residue is an amino acid substitution selected from those corresponding to E345R/Q/N/K, E430T/S/G, S440Y/W, and Q386K in the Fc-region of a human IgG1 heavy chain.
Alternatively, the at least one amino acid residue is selected from E382 and H433. Particular alternatively amino acid substitutions include E345Y,D,W; and E430F,H. Alternatively, E382D,Q,K,R; and H433R.
In one specific embodiment, the amino acid substitution is E345R. In an alternative embodiment, the mutation is selected from the group consisting of I253 to E, N, Q, S or T; H310 to N, Q, W or Y; Q311 to E or R; E382 to D, H, K, R, N, Q, S, T, W or Y; G385 to E, H, K, N, Q, R, S, T, W or Y; H433 to R; N434 to D, E, H, K, Q, R, S, T, W or Y; Y436 to A, E, F, H, I, K, L, M, N, Q, R, S, T or V; Q438 to A, E, G, H, K, N, Q, R, S, T, W or Y; K439 to D, H, Q, R, W or Y; and S440 to D, E, H, F, N, Q, W or Y.
In another alternative embodiment, the mutation is selected from the group consisting of I253 to N or Q; H310 to Q; Q311 to E or R; E382 to D, Q, K, or R; G385 to D, E, K or R; H433 to R; N434 to H, K, Q or R; Y436 to N, Q, S or T; Q438 to N, S or T; K439 to Q; and S440 to D, E or Q.
In another alternative embodiment, the mutation is selected from the group consisting of E382 to D, Q, K, or R; and H433 to R.
In one embodiment, the variant comprises a E382R mutation.
In one embodiment, the variant comprises a H433R mutation.
As shown in the Examples, variants of CD38 antibody HuMab-005 and -003 and/or CD20 antibody HuMab-7D8 and -11B8 and rituximab and/or EGFR antibody HuMab-2F8 comprising one of these amino acid substitutions had higher C1q-binding, complement activation and/or CDC than wild-type HuMab 005 and 7D8, respectively.
It is to be understood that the variant may also only comprise one mutation of the “Exemplary substitutions” listed in Table 1. The variant may also comprise more than one mutation, such as two, three, four, five or six of any the mutations listed in Table 1.
A preferred embodiment of the present invention, thus, provides a variant comprising one mutation in an amino acid residue selected from those listed in the aspect above. Particular amino acid mutations may be an amino acid substitution corresponding to any of the group consisting of P247G, I253V, S254L, Q311L, Q311W, E345A, E345C, E345D, E345F, E345G, E345H, E345I, E345K, E345L, E345M, E345N, E345P, E345Q, E345R, E345S, E345T, E345V, E345W, E345Y, D/E356G, D/E356R, T359R, E382L, E382V, Q386K, E430A, E430C, E430D, E430F, E430G, E430H, E430I, E430K, E430L, E430M, E430N, E430P, E430Q, E430R, E430S, E430T, E430V, E430W, E430Y, Y436I, S440Y and S440W. These have an increased cell lysis (>39% on Wien133 cells) as shown in Example 19, Table 17.
In an alternative embodiment, the variant comprises a mutation in one amino acid residue selected from those corresponding to E382R, H433R, H435R, and H435A.
Besides the indicated mutations, the variant may have any of the features as described for the parent antibody. In particular, it may be a human antibody. The variant may further be, besides the mutations, of any IgG1 subtype.
When bound to its antigen on the surface of an antigen-expressing cell, on a cell membrane, on a virion, or on another particle, or the antigen is associated with a virion, optionally wherein the antigen is comprised in the protein coat or a lipid envelope of the virion, such an antibody variant can have compared to the parent antibody at least one of an increased (i) C1q-binding, (ii) complement activation mediated by the antibody, (iii) CDC mediated by the antibody, (iv) oligomer formation, (v) oligomer stability, or a combination of any of (i) to (v). In one embodiment of (iv) or (v), the oligomer is a hexamer. In one embodiment, the variant also or alternatively has a retained or improved other effector function, such as C1q-binding, complement activation, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxity (ADCC), FcRn-binding, Fc-receptor binding including Fc-gamma receptor-binding, Protein A-binding, Protein G-binding, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cellular cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonisation, Fc-containing polypeptide internalization, target downmodulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof.
Without being limited to any specific theory, the effect caused by substituting amino acids at the indicated positions, with the amino acid residues in Table 1 may, for example, cause the effect itself, be involved in contacting the Fc domain of another molecule directly, or may be mutated to interact with another Fc domain directly or indirectly affect the intermolecular Fc:Fc interaction. Thus, substitutions are believed to, without being bound by theory, directly or indirectly enhance the binding strength between the antibody molecules in the oligomeric form, enhancing the stability of the oligomer structure, such as a hexameric, pentameric, tetrameric, trimeric, or dimeric structure. For example, the amino acid substitution can be one that promotes or strengthens the formation of new intermolecular Fc:Fc bonds, such as, but not limited to, Van der Waals interactions, hydrogen bonds, charge-charge interactions, or aromatic stacking interactions, or one that promotes increased entropy upon Fc:Fc interaction by release of water molecules. With reference to Table 1, “Exemplary substitutions” may be selected based on size and physicochemical properties engaging in or promoting intermolecular Fc:Fc interactions or intramolecular interactions (allosteric mutations). “Preferred substitutions” may be selected based on size and physicochemical properties optimal for engaging in or stimulating intermolecular Fc:Fc interactions or intramolecular interactions (allosteric mutations).
“Exemplary substitutions” of amino acids listed in Table 1, include exchanging an E residue for an R residue, and exchanging an H residue for an R residue. Each “Exemplary substitution” of amino acids in each specific amino acid residue listed in Table 1 is a separate and specific non-limiting embodiment according to the invention. Further, each “Preferred substitution” in each specific amino acid residue listed in Table 1 is a separate and specific non-limiting embodiment according to the invention.
In another aspect the present invention relates to a variant of a parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region, wherein the variant comprises a mutation in at least two amino acid residues selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain,
wherein the at least two amino acid mutations are different.
In one embodiment the parent polypeptide and thereby also the variant thereof, may be an antibody.
Thus the present invention also relates to a variant of a parent antibody comprising an antigen-binding region and a Fc-domain, wherein the variant comprises a mutation in at least two amino acid residues selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain,
wherein the at least two amino acid mutations are different.
Thus, a variant of the embodiment above may comprise a mutation in at least two, such as two, three, four, five, or more amino acids in Table 1.
In any embodiments where such a mutation in at least two amino acids is comprised in the variant, it may be present in each of the heavy chains of the variant, or one of the two may be comprised in one of the heavy chains and the other may be comprised in the other heavy chain, respectively, or vice versa.
In one embodiment, the variant comprises a mutation in at least two amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W. Alternatively, the variant further comprises a mutation in at least one residue selected from the group consisting of H310, G385, H433, N434, Q438, and K439.
In one embodiment, the variant comprises a mutation in at least two amino acid residues selected from those corresponding to E345X, E430X, S440W/Y, Q386K, P247G, I253V, S254L, Q311L/W, D/E356R, E382V, and Y436I in the Fc region of a human IgG1 heavy chain, wherein X is any amino acid, such as a natural occurring amino acid.
For example, the antibody variant may comprise a mutation in at least one of E345, E430, S440, and Q386, alternatively E382 and H433, such as two or all of E345, E430, S440, and Q386, alternatively E382 and H433, optionally further comprising a mutation in one or more other amino acids listed in Table 1. Thus, in a further embodiment, the variant comprises a mutation in at least two amino acid residues selected from the group of corresponding to those of E345X, E430X, S440W/Y, and Q386K in the Fc-region of a human IgG1 heavy chain, wherein X is any amino acid, such as a natural occurring amino acid.
Exemplary combinations of a mutation in at least two amino acid residues are E345X/E430X, E345X/S440Y or W, E345X/Q386K, E430X/S440Y or W, and E430X/Q386K.
In one embodiment, the mutation in at least two amino acid residues is a deletion, insertion or substitution. Such a substitution of amino acids may be with any naturally occurring or artificially amino acids.
In a particular embodiment, the mutation in at least two amino acid residues may be an amino acid substitution corresponding to any of the group consisting of P247G, I253V, S254L, Q311L, Q311W, E345A, E345C, E345D, E345F, E345G, E345H, E345I, E345K, E345L, E345M, E345N, E345P, E345Q, E345R, E345S, E345T, E345V, E345W, E345Y, D/E356G, D/E356R, T359R, E382L, E382V, Q386K, E430A, E430C, E430D, E430F, E430G, E430H, E430I, E430K E430L, E430M, E430N, E430P, E430Q, E430R, E430S, E430T, E430V, E430W, E430Y, Y436I, S440Y and S440W.
In a preferred embodiment, the variant comprises a mutation in at least two amino acid residues are amino acid substitutions selected from those corresponding to E345R,Q,N,K,A,C,D,F,G,H,I,L,M,P,S,T,V,W,Y; E430T,S,G,A,C,D,F,H,I,L,K,M,N,P,Q,R,V,W,Y; S440W,Y; and Q386K, in the Fc region of a human IgG1 heavy chain.
Alternatively the further mutation is selected from those corresponding to I253E,N,Q,S,T; H310N,Q,W,Y; Q311E,R; E382D,H,K,R,N,Q,S,T,W,Y; G385E,H,K,N,Q,R,S,T,W,Y; H433R; N434D,E,H,K,Q,R,S,T,W,Y; Y436,A,E,F,H,I,K,L,M,N,Q,R,S,T,V; Q438A,E,G,H,K,N,Q,R,S,T,W,Y; K439D,H,Q,R,W,Y; and S440D,E,H,F,N,Q
In a preferred embodiment, the mutation in at least two amino acid residues are is amino acid substitutions selected from those corresponding to E345R/Q/N/K, E430T/S/G, S440Y/W, and Q386K in the Fc-region of a human IgG1 heavy chain. Alternatively the further mutation is selected from those corresponding to I253N,Q; H310Q; Q311E,R; E382D,Q,K,R; G385D,E,K,R; H433R; N434H,K,Q,R; Y436N,Q,S,T; Q438N,S,T; K439Q; and S440D,E,Q.
Exemplary specific combinations of a mutation in at least two amino acid residues are E345R/E430T, E345R/S440Y, E345R/S440W, E345R/Q386K, E345R/E430G, E345Q/E430T, E345Q/S440Y, E345Q/S440W, E430T/S440Y, E430T/S440W, E430T/Q386K, and S440Y/Q386K.
In one specific embodiment, the mutation is not in an amino acid residue corresponding to I253, N434, or Q311. In one additional or alternative embodiment, the mutation is not in H433, or the amino acid substitution is not H433A.
In one embodiment, the present invention relates to a variant comprising a mutation in at least three amino acid residues selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that it does not comprise a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain.
wherein the at least three amino acid mutations are different.
In one particular embodiment, the variant comprises a mutation in the amino acid residues, which are amino acid substitutions corresponding to E345R, Q396K and E430G, which may be in either one or both the heavy chains of the variant.
The mutation in the at least three amino acid residues may be individually selected from the substitutions listed in Table 1. Non-limiting examples of variants comprising at least three mutations are; E345R/E430G/S440Y, E345R/E430G/S440W, E345K/E430G/S440Y, E345K/E430G/S440W, E345Q/E430G/S440Y, E345Q/E430G/S440W, E345N/E430G/S440Y, E345N/E430G/S440W, E345R/E430T/S440Y, E345R/E430T/S440W, E345K/E430T/S440Y, E345K/E430T/S440W, E345Q/E430T/S440Y, E345Q/E430T/S440W, E345N/E430T/S440Y, E345N/E430T/S440W, E345R/E430S/S440Y, E345R/E430S/S440W, E345K/E430S/S440Y, E345K/E430S/S440W, E345Q/E430S/S440Y, E345Q/E430S/S440W, E345N/E430S/S440Y, E345N/E430S/S440W, E345R/E430F/S440Y, E345R/E430F/S440W, E345K/E430F/S440Y, E345K/E430F/S440W, E345Q/E430F/S440Y, E345Q/E430F/S440W, E345N/E430F/S440Y, and E345N/E430F/S440W.
Apart from mutations in one or more amino acids in Tables 1 or 2A and B, the IgG heavy chain may comprise additional mutations known in the art, e.g., mutations that further improve effector functions. Such additional mutations include known mutations enhancing CDC, Fc-gamma receptor binding or FcRn-binding and/or improving Fc-gamma receptor-mediated effector functions.
In one embodiment, a variant according to the invention further comprises a known CDC enhancing modification e.g., an exchange of segments between IgG isotypes to generate chimeric IgG molecules (Natsume et al., 2008 Cancer Res 68(10), 3863-72); one or more amino acid substitutions in the hinge region (Dall'Acqua et al., 2006 J Immunol 177, 1129-1138), and/or one or more amino acid substitutions in or near the C1q-binding site in the CH2 domain, centered around residues D270, K322, P329, and P331 (Idusogie et al., 2001 J Immunol 166, 2571-2575; Michaelsen et al., 2009 Scand J Immunol 70, 553-564 and WO 99/51642). For example, in one embodiment, a variant according to the invention further comprises a combination of any of the amino acid substitutions S267E, H268F, S324T, S239D, G236A and I332E, providing enhanced effector function via CDC or ADCC (Moore et al., 2010 mAbs 2(2), 181-189)). Other Fc mutations affecting binding to Fc-receptors (described in WO 2006/105062, WO 00/42072, U.S. Pat. Nos. 6,737,056 and 7,083,784) or physical properties of the antibodies (described in WO 2007/005612 A1) can also be used in the variants of the invention.
In one embodiment, a variant according to the invention further comprises modifications enhancing Fc-gamma receptor binding and/or Fc-gamma receptor-mediated effector function. Such modifications include (i) reducing the amount of fucose in the CH2 attached glycosylation (glyco-engineering) (Umana P, et al., Nat Biotechnol 1999; 17: 176-80; Niwa R, et al., Clin Cancer Res 2004; 10: 6248-55.)), and (ii) site-directed mutagenesis of amino acids in the hinge or CH2 regions of antibodies (protein-engineering) (Lazar G A, et al., Proc Natl Acad Sci USA 2006; 103: 4005-10).
In one embodiment, a variant according to the invention is further engineered in the FcRn binding site, e.g., to extend the half-life (t1/2) of IgG antibodies. Such modifications include (i) N434A and T307A/E380A/N434A mutations (Petcova et al. Int Immunol. 2006 December; 18(12):1759); (ii) a substitution of one or more of Pro238, Thr256, Thr307, Gln311, Asp312, Glu380, G1u382, and Asn434 into an alanine residue improving FcRn binding (Shields R L, et al. J. Biol. Chem. 2001; 276:6591); and (iii) an amino acid substitution or combination of amino acid substitutions selected from M252Y/S254T/T256E, M252W, M252Y, M252Y/T256Q, M252F/T256D, V308T/L309P/Q311S, G385D/Q386P/N389S, G385R/Q386T/P387R/N389P, H433K/N434F/Y436H, N434F/Y436H, H433R/N434Y/Y436H, M252Y/S254T/T256E-H433K/N434F/Y436H or M252Y/S254T/T256E-G385R/Q386T/P387R/N389P in IgG1, increasing the affinity for FcRn (Dall'Acqua et al., supra).
“Double-Mutant”
It is to be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody are also to be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region.
As described above and further below, the present invention also relates to a “double-mutant” aspect, wherein two mutations individually each decrease an effector function but together restores the effector function to the level of the parent antibody. When used together the specificity of the variant is increased. Antibody variants according to the “double-mutant” aspect comprise two mutations, typically amino acid substitutions, in the specific amino acid residue interaction pair K439 and S440.
Thus in one aspect the present invention relates to a variant of a parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region, wherein the variant comprises a mutation
(i) in at least one amino acid residue selected from those corresponding to K439 and S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W, such as wherein the mutation in the position corresponding to K439 in the Fc-region of human IgG1 heavy chain is K439D/E, and/or the mutation in the position corresponding to S440 in the Fc-region of human IgG1 heavy chain is S440K/H/R;
(ii) in at least one amino acid residue corresponding to K447D/E or corresponding to K447K/R/H and 448P in the Fc region of a human IgG1 heavy chain; or
(iii) in at least one amino acid residue corresponding to K447D/E or corresponding to K447K/R/H and 448K/R/H and 449P in the Fc region of a human IgG1 heavy chain.
In one embodiment the parent polypeptide, and thereby also the variant thereof, may be an antibody.
Thus, in one aspect, the present invention relates to a variant of a parent antibody comprising an antigen-binding region and Fc-domain of an immunoglobulin, wherein the variant comprises a mutation in at least one amino acid residue selected from those corresponding to K439 and S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W, such as wherein the mutation in the position corresponding to K439 in the Fc-region of human IgG1 heavy chain is K439D/E, and/or the mutation in the position corresponding to S440 in the Fc-region of human IgG1 heavy chain is S440K/H/R. Table 2A and B shows “Exemplary” and “Preferred substitutions” for the “double-mutant” (Table A) and “mixed-mutant” (Table 2B) aspects.
TABLE 2A
Exemplary mutation sites and amino acid substitutions
for “double-mutant” aspects
Amino acid pair
Exemplary
Preferred
(IgG1, 2, 3, 4)
substitutions
substitutions
K439/S440
K439ED, alternatively
K439E/S440K
R/S440KR, alternatively ED
K447/448/449
K447ED/448KRH/449P
K447E/448K/449P
K447/448
K447KRH/448ED
K447K/448E
TABLE 2B
Exemplary mutation sites and amino acid substitutions for
“mixed-mutants” aspect (Ab1 + Ab2)
Amino acid pair
Exemplary
Preferred
(IgG1)
substitutions
substitutions
K439 + S440
K439DER + S440DEKR
K439E + S440K
K447 + K447/448
K447DE + K447KRH/448P
K447E + K447/448P
K447 +
K447DE +
K447E +
K447/448/449
K447KRH/448KRH/449P
K447/448K/449P
In one embodiment the of the variant, wherein the mutation is on position(s) other than S440 and K447, and wherein the variant further comprises a mutation
(i) in at least one amino acid residue corresponding to K439 or S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440W or S440Y;
(ii) in at least one amino acid residue corresponding to K447D/E or corresponding to K447K/R/H and 448P in the Fc region of a human IgG1 heavy chain; or
(iii) in at least one amino acid residue corresponding to K447D/E or corresponding to K447K/R/H and 448K/R/H and 449P in the Fc region of a human IgG1 heavy chain.
Accordingly, the invention provides a variant of an antibody comprising a first mutation in a residue in the CH2 and/or CH3 region of a human IgG1 heavy chain corresponding to K439, and a second mutation in a residue in the CH2 and/or CH3 region of a human IgG1 heavy chain corresponding to S440.
It is contemplated by the present invention that the variant may also comprise only one of the amino acid residue substitutions, such as either K439E or S440K, such as the variant comprises a mutation in K439, optionally with no mutation in S440.
In one embodiment, the invention relates to the variant, wherein the mutation in K439 is an amino acid substitution into an amino acid selected from E and D, such as K439E.
In another embodiment, the variant comprises a mutation in S440, optionally with no mutation in K439.
In one embodiment, the invention relates to the variant, wherein the mutation in S440 is an amino acid substitution into an amino acid selected from K, R and H, such as S440K.
In one embodiment, the variant comprises mutations in both K439 and S440.
In another embodiment, the mutation in K439 is selected from K439 to D, E or R, and the mutation in S440 is selected from S440 to D, E, K, H and R.
In another embodiment, the mutation in K439 is selected from K439D and K439E, and the mutation in S440 is selected from S440K, S440R, and S440H.
In another embodiment, the variant comprises K439E and S440K mutations.
As described in the Examples 4-6, antibody variants comprising only one of the K439E and S440K mutations had a drastically increased KD for C1q, reflecting a decreased complement activation and/or CDC capability. Surprisingly, it was found that antibody variants of HuMAb 7D8 or 005 comprising both mutations had a restored or increased C1q-binding or CDC. Without being bound by any specific theory, the underlying mechanism could perhaps be explained by the respective mutations sterically compensating for each other, as illustrated in FIGS. 4 and 5.
Any “double-mutant” as described herein may also be used in combination with a mutation which by itself is capable of increasing an effector function. Thus, the “double-mutant” aspect may be combined with the “single-mutant” aspect, e.g. the variant may further comprise a mutation in any of the amino acid positions listed in Table 1 or any other embodiments described for the “single-mutant” aspect above. Thus, in one embodiment, the mutation is on position(s) other than S440, and wherein the variant further comprises a mutation in at least one amino acid residue corresponding to K439 or S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440W or S440Y.
In one aspect the present invention relates to a variant of a parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region, wherein the variant comprises a mutation in at least one amino acid residues selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that it does not comprise a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain, and
wherein the variant comprises a further mutation
(i) in at least one amino acid residue corresponding to K439 or S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440W or S440Y;
(ii) in at least one amino acid residue corresponding to K447D/E or corresponding to K447K/R/H and 448P in the Fc region of a human IgG1 heavy chain; or
(iii) in at least one amino acid residue corresponding to K447D/E or corresponding to K447K/R/H and 448K/R/H and 449P in the Fc region of a human IgG1 heavy chain.
In one embodiment the parent polypeptide, and thereby the variant thereof, may be an antibody.
Thus in one aspect, the present invention relates to a variant of a parent antibody comprising an antigen-binding region and a Fc-domain of an immunoglobulin, wherein the variant comprises a mutation in at least one amino acid residues selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that it does not comprise a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain, and
wherein the variant comprises a further mutation
(i) in at least one amino acid residue corresponding to K439 or S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440W or S440Y,
(ii) in at least one amino acid residue corresponding to K447D/E or corresponding to K447K/R/H and 448P in the Fc region of a human IgG1 heavy chain; or
(iii) in at least one amino acid residue corresponding to K447D/E or corresponding to K447K/R/H and 448K/R/H and 449P in the Fc region of a human IgG1 heavy chain.
In one embodiment, the variant comprises a mutation in at least an amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W, and the variant comprises a further mutation in at least one amino acid residue corresponding to K439 or S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440W or S440Y.
In a further embodiment, the variant comprises a mutation in at least one amino acid residue is an amino acid substitution selected from those corresponding to E345X, E430X, S440W/Y, Q386K, in the Fc region of a human IgG1 heavy chain, wherein X is any amino acid, such as a natural occurring amino acid, and the variant comprises a further mutation in at least one amino acid residue corresponding to K439 or S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440W or S440Y.
In one embodiment, the variant comprises an amino acid mutation in both of the positions corresponding to K439 and S440 in the Fc-region of an IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W.
In a further embodiment, the mutation in the position corresponding to K439 in the Fc-region of human IgG1 heavy chain is K439D/E, and/or the mutation in the position corresponding to S440 in the Fc-region of human IgG1 heavy chain is S440K/H/R.
In a further embodiment, the first mutation is in an amino acid residues selected from those corresponding to E345, E430, Q386, and S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W; and the second and third mutation is an amino acid substitution in position K439E or S440K.
In one embodiment, the first mutation is a deletion, insertion or substitution. Such a substitution may be any naturally occurring or non-naturally amino acid.
In a further embodiment, the first mutation is selected from the group of E345R,Q,N,K,A,F,G,H,I,L,M,P,S,T,V,W,Y,C,D; E430T,S,G,A,F,H,L,P,R,V,C,D,I,K,M,N,Q,W,Y; and S440W,Y,D; and the second and third mutation is an amino acid substitution in position K439E or S440K.
In a preferred embodiment, the one mutation is selected from the group of E345R,Q,N,K,Y; E430T,S,G,F,H; S440W,Y; and Q386K.
Another example, in one embodiment of the present invention, the variant comprises E345R, K439E and S440K mutations, thus providing for both increased and more specific mediation of a CDC-response.
In one embodiment, the variant comprises a mutation in at least two amino acid residues selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W, and the variant comprises a further mutation in at least one amino acid residue corresponding to K439 or S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440W or S440Y.
In a further embodiment, the variant comprises a mutation in at least two amino acid residues is an amino acid substitution selected from those corresponding to E345X, E430X, S440W/Y, Q386K, in the Fc region of a human IgG1 heavy chain, wherein X is any amino acid, such as a natural occurring amino acid, and the variant comprises a further mutation in at least one amino acid residue corresponding to K439 or S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440W or S440Y.
In one embodiment, the variant comprises an amino acid mutation in both of the positions corresponding to K439 and S440 in the Fc-region of an IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W.
In a further embodiment, the mutation in the position corresponding to K439 in the Fc-region of human IgG1 heavy chain is K439D/E, and/or the mutation in the position corresponding to S440 in the Fc-region of human IgG1 heavy chain is S440K/H/R.
In a further embodiment, the first and second mutation is in an amino acid residues selected from those corresponding to E345, E430, Q386, and S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W; and the third and fourth mutation is an amino acid substitution in position K439E or S440K.
In another embodiment, the variant comprising a mutation in both positions K439 and S440 as described herein has an increase in an Fc-mediated effector function selected from C1q-binding, complement activation, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxity (ADCC), FcRn-binding, Fc-receptor binding including Fc-gamma receptor-binding, Protein A-binding, Protein G-binding, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cellular cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonisation, Fc-containing polypeptide internalization, target downmodulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof, as compared to parent antibody or an antibody variant comprising a mutation in only one of K439 and S440.
The invention also provides for the use of the K439E and S440K mutations in an antibody to restore one or more of (i) C1q-binding avidity, (ii) complement activation mediated by the antibody, (iii) CDC mediated by the antibody, (iv) oligomer formation, (v) oligomer stability, or a combination of any of (i) to (v), as compared to parent antibody, which may, e.g., be a wild-type antibody or an antibody variant comprising only one of the K439E or S440K mutations. In one embodiment of (iv) or (v), the oligomer is a hexamer.
In one embodiment, the variant is selected from a monospecific antibody, bispecific antibody or multispecific antibody.
Mixed Mutants
It is to be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody are also to be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region.
As described above, the inventors of the present invention have also found that there are mutations which by itself decreases an effector function but when used together the effector function is restored, e.g. the mutations in positions K439 and S440 of in the Fc-region of a human IgG1 heavy chain. This concept may also be used to ensure pairing of two different antibodies, thus, by introducing K439 in one antibody and S440 in the other. Thus, antibody variants according to the “mixed-mutant” aspect comprise a mutation, but one that typically leads to a reduced or much reduced Fc:Fc interaction between identical Fc-molecules. However, as the “mixed-mutant” antibody variants of the invention are capable of pairing with each other; providing a restored or even increased C1q-binding, complement activation, CDC, oligomer formation, and/or oligomer stability for the specific antibody variant pair, as compared to, e.g., each variant alone or a mix of the parent antibody or parent antibodies. In one embodiment of the invention, the oligomer is a hexamer. In one embodiment, the antibody variant pair also or alternatively has a retained or improved other effector function, such as C1q-binding, complement activation, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxity (ADCC), FcRn-binding, Fc-receptor binding including Fc-gamma receptor-binding, Protein A-binding, Protein G-binding, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cellular cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonisation, Fc-containing polypeptide internalization, target downmodulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest, and any combination thereof. This aspect of the invention provides for a number of applications where not only the strength but also the selectivity in the C1q-binding, complement activation, CDC or other effector function can be regulated.
Exemplary mutation sites for each antibody variant in a “mixed-mutant” pair are shown in Table 2. Specifically, the invention provides a variant of an antibody comprising an antigen-binding region and an Fc-domain of an immunoglobulin, which variant comprises a mutation in a residue in the Fc-region of a human IgG1 heavy chain corresponding to one of K439 and S440. In one embodiment, the mutation is in K439, and is an amino acid substitution into an amino acid selected from E or D, such as K439E. In one embodiment, the mutation is in S440, and is an amino acid substitution into an amino acid selected from K, R or H, such as S440K.
Thus in one embodiment the present invention also relates to a variant comprising a mutation in at least one amino acid residue selected from:
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain;
and in an amino acid residue corresponding to K439 in the Fc region of a human IgG1 heavy chain.
In another embodiment the present invention also relates to a variant comprising a mutation in at least one amino acid residue selected from:
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain;
and in an amino acid residue corresponding to S440 in the Fc region of a human IgG1 heavy chain.
In one embodiment, the two above described embodiments may be combined in the “mixed-mutant” pair aspect according to the present invention.
Each variant in a “mixed-mutant” pair may further comprise a mutation in an amino acid listed in Table 1.
In one embodiment of the present invention, the “mixed-mutant” pair comprises a first variant of a parent antibody and a second variant of a parent antibody,
wherein the first variant comprises a first Fc-domain of an immunoglobulin and an antigen-binding region, wherein said first variant comprises (i) a first mutation in at least one amino acid residue other than a mutation in K439 selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within that C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain, and
(ii) a second mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain; and
wherein the second variant comprises a second Fc-domain of an immunoglobulin and an antigen-binding region, wherein said second variant comprises (i) a first mutation in at least one amino acid residue other than a mutation in S440 selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within that C-terminal CH3 beta-strand, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain,
and (ii) a second mutation in the position corresponding to S440 in the Fc region of an IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W.
Other exemplary “mixed-mutant” pairs may further comprise, and is not limited to, any of the following pairs; a first variant comprising the mutation K447E and a second variant comprising the mutation K447/P448; a first variant comprising the mutation K447E and a second variant comprising the mutation K447/K448/P449.
In one embodiment, the mutation is a deletion, insertion or substitution. Such a substitution of amino acids may be with any naturally occurring or non-naturally amino acids. In one embodiment, the mutation is a deletion. In another embodiment, the mutation is an insertion. In another embodiment, the mutation is a substitution of an amino acid.
In a particular embodiment, the first variant and/or second variant comprises a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.
In a particular embodiment, the first variant and/or the second variant comprises a mutation in at least one amino acid residues may be an amino acid substitution corresponding to any of the group consisting of P247G, I253V, S254L, Q311L, Q311W, E345A, E345C, E345D, E345F, E345G, E345H, E345I, E345K, E345L, E345M, E345N, E345P, E345Q, E345R, E345S, E345T, E345V, E345W, E345Y, D/E356G, D/E356R, T359R, E382L, E382V, Q386K, E430A, E430C, E430D, E430F, E430G, E430H, E430I, E430K, E430L, E430M, E430N, E430P, E430Q, E430R, E430S, E430T, E430V, E430W, E430Y, Y436I, S440Y and S440W, and the first variant comprises a second mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain; and the second variant comprises a second mutation in the position corresponding to S440 in the Fc-region of a human IgG1 heavy chain.
For example, in one embodiment, one variant in a “mixed-mutant” pair comprises E345R and K439E mutations, while the other variant comprises E345R and S440K mutations, thus providing for both increased and more specific C1q-binding avidity, complement activation, CDC, oligomer formation, oligomer stability, and/or other effector-related function such as FcRn-binding, ADCC, Fc-gamma receptor-binding, Protein A-binding, Protein G-binding, ADCP, CDCC, complement-enhanced cytotoxicity, antibody mediated phagocytosis, internalization, apoptosis, binding to complement receptor of an opsonized antibody, and/or combinations thereof.
The “mixed-mutant” aspect, may also comprise two variants comprising each more than one mutations listed in Table 1, in the Fc-region of a human IgG1 heavy chain, such as a first variant comprising the mutations S440K/K447E, and a second variant comprising the mutation K439E/K447/P448; such as a first variant comprising the mutations K439E/K447E, and a second variant comprising the mutation S440K/K447/P448.
The variants in a “mixed-mutant” pair as described herein may derive from the same or from different parent antibodies. Further, the “mixed-mutant” aspect can also be employed in bispecific or asymmetrical antibodies. Further, the first, second and third antibody may bind different epitopes, on the same or different targets.
Further, the “mixed-mutant” aspect can provide for a CDC or other effector response that is more specifically directed to tumor cells expressing two specific tumor antigens, by utilizing a first antibody against the first antigen with a K439E mutation and a second antibody against the second antigen with a S440K or S440R mutation. By utilizing the “mixed-mutant” aspect comprising three variants, optionally being bispecific antibodies, may provide for a CDC or other effector response that is more specifically directed to tumor cells expressing at least two, such as two, three, four, five or six, specific tumor antigens.
In one embodiment of any of the “single-mutant”, “double-mutant” and “mixed-mutant” aspects, the variant is selected from a monospecific antibody, bispecific antibody or multispecific antibody.
In any embodiment of the “mixed-mutant” aspect, the first, second and/or third variant may comprise the same or different mutation of any of the amino acid substitutions listed in Table 1.
Multispecific Antibodies
It is to be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody are also to be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region.
It is to be understood that any embodiment of the “single-mutant”, “double-mutant” and “mixed-mutant” aspects described herein may be used in the multispecific antibody aspect described below.
In one main aspect, the invention relates to a variant, which is a bispecific antibody comprising a first polypeptide comprising a first CH2-CH3 region of an immunoglobulin and a first antigen-binding region, and a second polypeptide comprising a second CH2-CH3 region of an immunoglobulin and a second antigen-binding region, wherein the first and second antigen-binding regions bind different epitopes on the same antigen or on different antigens, and wherein the first and second CH2-CH3 region each comprises a first mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.
In one embodiment, the mutation is a deletion, insertion or substitution. Such a substitution of amino acids may be with any naturally occurring or non-naturally acids.
The bispecific antibody of the present invention is not limited to a particular format and it may be any of those described above and herein.
In one embodiment of the present invention, the first and second polypeptide comprises one first mutation in an amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain.
In a particular embodiment, the mutation in at least one amino acid residues may be an amino acid substitution corresponding to any of the group consisting of P247G, I253V, S254L, Q311L, Q311W, E345A, E345C, E345D, E345F, E345G, E345H, E345I, E345K, E345L, E345M, E345N, E345P, E345Q, E345R, E345S, E345T, E345V, E345W, E345Y, D/E356G, D/E356R, T359R, E382L, E382V, Q386K, E430A, E430C, E430D, E430F, E430G, E430H, E430I, E430L, E430M, E430N, E430P, E430Q, E430R, E430S, E430T, E430V, E430W, E430Y, Y436I, S440Y and S440W.
In a particular embodiment, the bispecific antibody has the format described in WO 2011/131746. Thus, in one embodiment, the variant which is a bispecific antibody, wherein the first polypeptide comprises a further mutation in an amino acid residue selected from those corresponding to K409, T366, L368, K370, D399, F405, and Y407 in the Fc-region of a human IgG1 heavy chain; and
the second polypeptide comprises a further mutation in an amino acid residue selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc-region of a human IgG1 heavy chain, and wherein the said further mutation in the first polypeptide is different from the said further mutation in the second polypeptide.
In a particular embodiment, the bispecific antibody has a first polypeptide comprises the further mutation in the amino acid residue corresponding to K409 in the Fc-region of a human IgG1 heavy chain, and the second polypeptide comprises the further mutation in the amino acid residue corresponding to F405 in the Fc-region of a human IgG1 heavy chain. Such bispecific antibodies according to the invention can be generated as described in Example 22. Furthermore, the effect on CDC killing by the generated heterodimeric proteins can be tested by using an assay as used in Example 23.
In a particular embodiment, the bispecific antibody comprising a first and a second polypeptide, wherein the first polypeptide comprises a mutation in the amino acid residue corresponding to K409 in the Fc-region of a human IgG1 heavy chain; the second polypeptide comprises a mutation in the amino acid residue corresponding to F405 in the Fc-region of a human IgG1 heavy chain; and the first and/or second polypeptide comprises further a mutation in the amino acid residue corresponding to the amino acid substitution E345R in the Fc-region of a human IgG1 heavy chain.
In a particular embodiment, the bispecific antibody comprising a first and a second polypeptide, wherein the first polypeptide comprises a mutation in the amino acid residue corresponding to K409 in the Fc-region of a human IgG1 heavy chain; the second polypeptide comprises a mutation in the amino acid residue corresponding to F405 in the Fc-region of a human IgG1 heavy chain; the first and/or the second polypeptide comprises each further a mutation in the amino acid residues corresponding to the amino acid substitution E345R and Q386K in the Fc-region of a human IgG1 heavy chain. Said further mutations may be both in the first and second polypeptide, or E345R may be in the first polypeptide and Q386K in the second polypeptide; or vice versa.
In a particular embodiment, the bispecific antibody comprising a first and a second polypeptide, wherein the first polypeptide comprises a mutation in the amino acid residue corresponding to K409 in the Fc-region of a human IgG1 heavy chain; the second polypeptide comprises a mutation in the amino acid residue corresponding to F405 in the Fc-region of a human IgG1 heavy chain; and the first and/or second polypeptide comprises each further a mutation in the amino acid residues corresponding to the amino acid substitution E345R, Q386K, and E430G in the Fc-region of a human IgG1 heavy chain. Said mutations may be in both the first and second polypeptide, or the first polypeptide may comprise the mutations E345R and E430G, and the second polypeptide may comprise the mutation Q386K; or vice versa.
The bispecific antibody may, for example, comprise an antigen-binding region of a CD20 antibody and an antigen-binding region of a CD38 antibody, and an amino acid substitution in one or more amino acids listed in Tables 1 and/or 2. Exemplary CD20-binding regions include those of ofatumumab (2F2), 7D8 and 11B8, described in WO2004/035607, which is hereby incorporated by reference in its entirety, and rituximab (WO 2005/103081). Exemplary CD38-binding regions include those of 003 and daratumumab (005), described in WO2006/099875, which is hereby incorporated by reference in its entirety.
In one embodiment, the bispecific antibody binds different epitopes on the same or different target.
In another embodiment, the first mutation in the first and second polypeptide may be the same or different.
In one embodiment of the “single-mutant”, “double-mutant”, “mixed-mutant” and multispecific antibody aspect, the variant is a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD or IgE antibody, optionally a human full-length antibody, such as a human full-length IgG1 antibody.
In any “single-mutant”, “double-mutant”, “mixed-mutant” aspect, and the multispecific antibody aspects the C1q-binding of the antibody is determined according to the assay described in Example 4, the CDC is determined according to the assay described in Example 5, 6 or 10, the mutation is not in an amino acid residue directly involved in C1q-binding, optionally as determined by comparing C1q-binding in an ELISA assay according to Example 3 with C1q-binding in a cell-based assay according to Example 4, and the ADCC is determined according to the assay described in Example 12.
Additionally, the invention provides for a preparation of a variant of any “single-mutant”, “double-mutant”, “mixed-mutant” and multispecific antibody aspect or embodiment described above. The invention also provides for a composition comprising a variant of any “double-mutant” aspect and embodiment described above, e.g., a pharmaceutical compositions. The invention also provides for the use of any such variant, preparation, or composition as a medicament.
The above “single-mutant”, “double-mutant”, “mixed mutant” and multispecific antibody aspects of the invention are particularly applicable to human antibody molecules having an IgG1 heavy chain comprising the relevant segment, P247 to K447, corresponding to the underlined residues 130 to 330 of the human IgG1 heavy chain constant region (UniProt accession No. P01857; SEQ ID NO:1):
1
astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv
51
htfpavlqss glyslssvvt vpssslgtqt yicnvnhkps ntkvdkkvep
101
kscdkthtcp pcpapellgg psvflfppkp kdtlmisrtp evtcvvvdvs
151
hedpevkfnw yvdgvevhna ktkpreeqyn styrvvsvlt vlhqdwlnqk
201
eykckvsnka lpapiektis kakgqprepq vytlppsrde ltknqvsltc
251
lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw
301
qqgnvfscsv mhealhnhyt qkslslspgk
The present invention can also be applied to antibody molecules having a human IgG2 heavy chain portion. Amino acid residues P247 to K447 of the IgG1 heavy chain correspond to the underlined residues 126 to 326 of the IgG2 heavy chain constant region (accession number P01859; SEQ ID NO:2)
1
astkgpsvfp lapcsrstse staalgclvk dyfpepvtvs wnsgaltsgv
51
htfpavlqss glyslssvvt vpssnfgtqt ytcnvdhkps ntkvdktver
101
kccvecppcp appvagpsvf lfppkpkdtl misrtpevtc vvvdvshedp
151
evqfnwyvdg vevhnaktkp reeqfnstfr vvsvltvvhq dwlngkeykc
201
kvsnkglpap iektisktkg qprepqvytl ppsreemtkn qvsltclvkg
251
fypsdiavew esngqpenny kttppmldsd gsfflysklt vdksrwqqgn
301
vfscsvmhea lhnhytqksl slspgk
The present invention can also be applied to antibody molecules having a human IgG3 heavy chain portion. Amino acid residues P247 to K447 of the IgG1 heavy chain correspond to residues 177 to 377 of the IgG3 heavy chain constant region (UniProt accession No. P01860, SEQ ID NO:3), underlined in the following:
1
astkgpsvfp lapcsrstsg gtaalgclvk dyfpepvtvs wnsgaltsgv
51
htfpavlqss glyslssvvt vpssslgtqt ytcnvnhkps ntkvdkrvel
101
ktplgdttht cprcpepksc dtpppcprcp epkscdtppp cprcpepksc
151
dtpppcprcp apellggpsv flfppkpkdt lmisrtpevt cvvvdvshed
201
pevqfkwyvd gvevhnaktk preeqynstf rvvsvltvlh qdwlngkeyk
251
ckvsnkalpa piektisktk gqprepqvyt lppsreemtk nqvsltclvk
301
gfypsdiave wessgqpenn ynttppmlds dgsfflyskl tvdksrwqqg
351
nifscsvmhe alhnrftqks lslspgk
The present invention can also be applied to antibody molecules having a human IgG4 heavy chain portion. Amino acid residues P247 to K447 of the IgG1 heavy chain correspond to the underlined residues 127 to 327 of the IgG4 heavy chain constant region (accession number P01859, SEQ ID NO:4)
1
astkgpsvfp lapcsrstse staalgclvk dyfpepvtvs wnsgaltsgv
51
htfpavlqss glyslssvvt vpssslgtkt ytcnvdhkps ntkvdkrves
101
kygppcpscp apeflggpsv flfppkpkdt lmisrtpevt cvvvdvsqed
151
pevqfnwyvd gvevhnaktk preeqfnsty rvvsvltvlh qdwlnqkeyk
201
ckvsnkglps siektiskak gqprepqvyt lppsqeemtk nqvsltclvk
251
gfypsdiave wesngqpenn ykttppvlds dgsfflysrl tvdksrwqeg
301
nvfscsvmhe alhnhytqks lslslgk
The present invention can also be applied to an antibody having a human IgG1m(f) allotype heavy chain portion. The amino acid sequence of the IgG1m(f) allotype (the CH3 sequence is underlined)—SEQ ID NO:5
1
astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv
51
htfpavlqss glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep
101
kscdkthtcp pcpapellgg psvflfppkp kdtlmisrtp evtcvvvdvs
151
hedpevkfnw yvdgvevhna ktkpreeqyn styrvvsvlt vlhqdwlngk
201
eykckvsnka lpapiektis kakgqprepq vytlppsree mtknqvsltc
251
lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw
301
qqgnvfscsv mhealhnhyt qkslslspgk
An alignment of the respective segments of the IgG1, IgG2, IgG3, IgG4, and IgG1m(f) constant regions is shown in FIG. 2. Accordingly, any mutation in an amino acid described in Table 1 or Table 2A and B can be introduced at its equivalent position in IgG2, IgG3, IgG4, and/or IgG1m(f) as defined by the alignment to obtain a variant according to the invention.
In one embodiment, the invention provides a variant of a full-length IgG1, IgG2, IgG3, or IgG4 antibody, comprising one or more amino acid substitutions according to any aspect described above.
In any “single-mutant”, “double-mutant”, “mixed-mutant” aspects and multispecific antibody, the Fc-region of an IgG1 heavy chain may comprise the sequence of residues 130 to 330 of SEQ ID NO:1, residues 126 to 326 of SEQ ID NO:2, residues 177 to 377 of SEQ ID NO:3, or residues 127 to 327 of SEQ ID NO:4.
In one embodiment, a parent antibody comprises a sequence selected from SEQ ID No.: 1-5, such as SEQ ID No.:1, SEQ ID No.:2, SEQ ID No.:3, SEQ ID No.:4, or SEQ ID No.:5.
In one embodiment, the Fc-region of an IgG1 heavy chain comprises the sequence of residues 130 to 330 of SEQ ID NO:1.
The parent antibody may be any parent antibody as described herein. The parent antibody in this context is intended to be also first parent and second parent antibodies.
In one embodiment, the parent antibody is a human IgG1, IgG2, IgG3 or IgG4, IgA1, IgA2, IgD or IgE antibody.
In one embodiment the parent antibody is human full-length antibody, such as a human full-length IgG1 antibody.
In one embodiment, the parent antibody, first parent antibody and second parent antibody is a human IgG1 antibody, e.g. the IgG1m(za) or IgG1m(f) allotype, optionally comprising an Fc-region comprising SEQ ID NO:1 or 5.
In one embodiment, the parent antibody is a human IgG2 antibody, optionally comprising an Fc-region comprising SEQ ID NO:2.
In one embodiment, the parent antibody is a human IgG3 antibody, optionally comprising an Fc-region comprising SEQ ID NO:3.
In one embodiment, the parent antibody is a human IgG4 antibody, optionally comprising an Fc-region comprising SEQ ID NO:4.
In particular embodiments of any of the “single-mutant”, “double-mutant”, “mixed-mutant” and multispecific antibody aspects, the variant comprises an amino acid sequence which has a degree of identity to amino acids P247 to K447 of SEQ ID Nos: 1, 2, 3, 4, and 5 of at least 70%, 72%, 74%, 76%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or of at least about 99%, except for the mutations introduced according to the present invention.
Thus, the variant may comprise a sequence according to SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No: 4, or SEQ ID No:5 except for any mutation defined herein.
In any of the above “single-mutant”, “double-mutant”, “mixed-mutant” and multispecific aspects according to the present invention may be understood to include the following embodiments.
In one embodiment, the first and/or second parent antibody is an antibody fragment, optionally selected from the group consisting of a monovalent antibody, a heavy-chain antibody, a strand-exchange engineered domain (SEED), a triomab, a dual variable domain immunoglobulin (DVD-Ig), a knob-into-holes antibody, a mini-antibody, a dual-affinity retargeting molecule (Fc-DART or Ig-DART); a LUZ-Y antibody, a Biclonic antibody, a Dual Targeting (DT)-Ig antibody, a Two-in-one Antibody, a cross-linked Mab, a mAb2, a CovX-body, an IgG-like Bispecific antibody, a Ts2Ab, a BsAb, a HERCULES antibody, a TvAb, an ScFv/Fc Fusion antibody, a SCORPION, an scFv fragment fused to an Fc domain, and a dual scFv fragment fused to an Fc domain.
In a further embodiment, both the first and the second parent antibody bind an antigen expressed on the surface of a human tumor cell.
In a further embodiment, the antigens for the first and second parent antibody are separately selected from the group consisting of erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD38, CD138, CXCR5, c-Met, HERV-envelop protein, periostin, Bigh3, SPARC, BCR, CD79, CD37, EGFrvIII, L1-CAM, AXL, Tissue Factor (TF), CD74, EpCAM and MRP3.
In a further embodiment, the first and second parent antibodies are fully human.
In a further embodiment, the antigens for the first and second parent antibody are, in any order, selected from CD20 and CD38, optionally wherein the first and second parent antibodies are, in any order, selected from 7D8 and 005.
In a further embodiment, both the first antibody and the second antibody bind antigens expressed on the surface of a bacterial cell or a virion.
In another embodiment, the bacterial cell is selected from the group consisting of S. aureus, S. epidermidis, S. pneumonia, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia trachomatis, E. coli, Salmonella, Shigella, Yersinia, S. typhimurium, Neisseria meningitides, and Mycobacterium tuberculosis.
In a further embodiment, the first and second parent antibody binds the same antigen.
In another embodiment, the first and second parent antibodies are the same antibody.
In another embodiment, the parent antibody is selected from 7D8 and 005.
Compositions
It is to be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody are also to be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region.
The invention also relates to compositions comprising variants and parent antibodies may be any variant and parent antibody as described herein. Specific aspects and embodiments will be described below. Furthermore, such variants may be obtained according to any method described herein.
In one aspect the present invention relates to a composition comprising a first variant of a parent polypeptide and a second variant of a parent polypeptide, wherein the first variant comprises a first Fc-domain of an immunoglobulin and a binding region, wherein the second variant comprises a second Fc-domain of an immunoglobulin and a binding region, and wherein
(i) said first variant comprises a mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain, and said second variant comprises a mutation in the position corresponding to S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W,
(ii) said first variant comprises a mutation in the position corresponding to K447D/E in the Fc region of a human IgG1 heavy chain; and said second variant comprises a mutation in the position corresponding to K447K/R/H and 448P in the Fc-region of a human IgG1 heavy chain, or
(iii) said first variant comprises a mutation in the position corresponding to K447D/E in the Fc region of a human IgG1 heavy chain; and said second variant comprises a mutation in the position corresponding to K447K/R/H, 448K/R/H and 449P in the Fc-region of a human IgG1 heavy chain.
In one embodiment the first one or both of the variant of a parent polypeptide and the second variant of a parent polypeptide may be an antibody.
Thus in one an aspect, the invention relates to a composition comprising a first variant of a parent antibody and a second variant of a parent antibody, wherein the first variant comprises a first Fc-domain of an immunoglobulin and an antigen-binding region, wherein the second variant comprises a second Fc-domain of an immunoglobulin and an antigen-binding region, and wherein
(i) said first variant comprises a mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain, and said second variant comprises a mutation in the position corresponding to S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W,
(ii) said first variant comprises a mutation in the position corresponding to K447D/E in the Fc region of a human IgG1 heavy chain; and said second variant comprises a mutation in the position corresponding to K447K/R/H and 448P in the Fc-region of a human IgG1 heavy chain, or
(iii) said first variant comprises a mutation in the position corresponding to K447D/E in the Fc region of a human IgG1 heavy chain; and said second variant comprises a mutation in the position corresponding to K447K/R/H, 448K/R/H and 449P in the Fc-region of a human IgG1 heavy chain.
In one embodiment the composition comprising a first variant of a parent antibody and a second variant of a parent antibody, wherein the first variant comprises a first Fc-domain of an immunoglobulin and an antigen-binding region, wherein the second variant comprises a second Fc-domain of an immunoglobulin and an antigen-binding region, and wherein said first variant comprises a mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain, and wherein the second variant comprises a mutation in the position corresponding to S440, with the proviso that the mutation in S440 is not S440Y or S440W.
In one embodiment, the composition comprising the first variant of a parent antibody and the second variant of a parent antibody,
wherein the first variant comprises a first Fc-domain of an immunoglobulin and an antigen-binding region, wherein said first variant comprises (i) a first mutation in at least one amino acid residue other than a mutation in K439 selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain, and
(ii) a second mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain; and
wherein the second variant comprises a second Fc-domain of an immunoglobulin and an antigen-binding region, wherein said second variant comprises (i) a first mutation in at least one amino acid residue other than a mutation in S440 selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain, and
(ii) a second mutation in the position corresponding to S440 in the Fc region of an IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W.
In another embodiment, the composition comprising the first variant of an antibody and the second variant of a parent antibody, wherein the first variant comprises (i) a first mutation in at least one amino acid residue other than a mutation in K439 selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain, and
(ii) a second mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain, and
wherein the second variant comprises a mutation in the position corresponding to S440 in the Fc-region of a human IgG1 heavy chain, which is not is not S440Y or S440W.
In another embodiment, the composition comprising the first variant of a parent antibody and the second variant of a parent antibody,
wherein the first variant comprises a mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain; and
wherein the second variant comprises a second Fc-domain of an immunoglobulin and an antigen-binding region, wherein said second variant comprises (i) a first mutation in at least one amino acid residue other than a mutation in S440 selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain,
and (ii) a second mutation in the position corresponding to S440 in the Fc region of an IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W.
In one embodiment, the mutation in the position corresponding to K439 in the Fc-region of human IgG1 heavy chain is K439D/E, and/or the mutation in the position corresponding to S440 in the Fc-region of human IgG1 heavy chain is S440K/H/R.
In another aspect the present invention relates to a composition comprising a first variant of an parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region and a second variant of a parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region, wherein
the first variant comprises a first Fc-domain of an immunoglobulin and a first antigen-binding region, wherein the first variant comprises a first mutation in at least an amino acid residue selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain, and wherein the second variant does not comprise a mutation in an amino acid residue selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain.
In one embodiment the first and/or second parent polypeptide may be an antibody.
The present invention also relates to an embodiment of the composition, wherein the first variant comprises (i) a first mutation in at least one amino acid residue other than a mutation in K439 selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain, and
(ii) a second mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain, and
wherein the second variant comprises a mutation in the position corresponding to S440 in the Fc-region of a human IgG1 heavy chain, which is not is not S440Y or S440W.
The present invention also relates to an embodiment of the composition, wherein the first variant comprises a mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain; and
wherein the second variant comprises a second Fc-domain of an immunoglobulin and an antigen-binding region, wherein said second variant comprises (i) a first mutation in at least one amino acid residue other than a mutation in S440 selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain,
and (ii) a second mutation in the position corresponding to S440 in the Fc region of an IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W.
In another aspect, the present invention relates to a composition comprising a first variant of an antibody and a second variant of a parent antibody, wherein
the first variant comprises a first Fc-domain of an immunoglobulin and a first antigen-binding region, wherein the first variant comprises a first mutation in at least an amino acid residue selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain, and
wherein the second variant comprises a second Fc-domain of an immunoglobulin and a second antigen-binding region, wherein said second variant does not comprise a mutation in an amino acid residue selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain.
In the embodiments, wherein the second variant does not comprise any of the listed mutations herein described, such second variant may include any of the suitable second antibody examples listed above in relation to the methods of effector functions.
In one embodiment, the first and second variant comprise a first mutation in at least one amino acid residue selected from those corresponding to E345, E430, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain.
In one embodiment, the first variant comprises a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.
In one particular embodiment, the first variant comprises a mutation in the amino acid residues corresponding to E345R and Q386K in the Fc-region of a human IgG1 heavy chain, and the second variant does not comprise such mutations.
In one particular embodiment, the first variant comprises a mutation in the amino acid residues corresponding to E345R, Q386K and E430G in the Fc-region of a human IgG1 heavy chain, and the second variant does not comprise such mutations.
In one embodiment, the at least one first mutation in the first and second variants are different.
In one embodiment, the first variant and second variant is each a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD or IgE antibody, optionally each a human full-length antibody, such as each a human full-length IgG1 antibody.
In one embodiment, the first variant and second variant is selected from a monospecific antibody, bispecific antibody or multispecific antibody.
In a further embodiment, the first and the second variant bind different epitopes on the same antigen or on different antigens. Thus, in the embodiment, wherein the first and second antibody are bispecific antibodies may be binding each two different epitopes. The at least two bispecific antibodies may be the same or different. If the bispecific antibodies are different, the composition, thus, comprises targeting up to four different epitopes on either the same or different targets.
In a further embodiment, one or both of the first variant and second variant is conjugated to a drug, toxin or radiolabel, such as wherein one or both of the first variant and second variant is conjugated to a toxin via a linker.
In a further embodiment, one or both of the first variant and second variant is part of a fusion protein.
In another aspect, the invention relates to a composition comprising any variant, any bispecific antibody or any composition described here and a pharmaceutically acceptable carrier.
It is contemplated that any of the embodiments according to the “mixed-mutant” aspect also may be comprised in any of the composition embodiments.
In one embodiment, the variants of the first and second parent antibodies bind to antigens expressed on the same cell.
In another embodiment, the variant of the first parent antibody comprises an amino acid substitution of K439 into an amino acid selected from E and D.
In another embodiment, the amino acid substitution in the variant of the first parent antibody is K439E.
In another embodiment, the variant of the second parent antibody comprises an amino acid substitution of S440 into an amino acid selected from K, R and H.
In another embodiment, the amino acid substitution in the variant of the second parent antibody variant is S440K.
In an alternative embodiment, the variant of the first and/or second antibody further comprises a mutation in a residue selected from the group consisting of H310, G385, H433, N434, and Q438.
In a further alternative embodiment, the variant of the first and/or second parent antibody further comprise a mutation selected from E345 to D, K, N, Q, R, or W; E382 to D, Q, K, or R; and H433 to R.
In a further embodiment, the variants of the first and second parent antibodies further comprise a mutation selected from E345R, E382R and H433R, such as E345R.
In another aspect, the invention relates to a pharmaceutical composition comprising the variant of the first parent antibody and the variant of the second parent antibody of any one of embodiments listed above.
The pharmaceutical compositions may be formulated in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995. A pharmaceutical composition of the present invention may e.g. include diluents, fillers, salts, buffers, detergents (e.g., a nonionic detergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, isotonicity agents, antioxidants, tissue fixatives, solubilizers, and/or other materials suitable for inclusion in a pharmaceutical composition. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol).
The pharmaceutical composition may be administered by any suitable route and mode. In one embodiment, a pharmaceutical composition of the present invention is administered parenterally. The term “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion.
Kit-of-Parts
It is to be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody are also to be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region.
The invention also relates to kit-of-parts for simultaneous, separate or sequential use in therapy comprising variants and parent antibodies, wherein any variant and parent antibody may be as described herein. Specific aspects and embodiments will be described below. Furthermore, such variants may be obtained according to any method described herein.
The invention also relates to kit-of-parts for simultaneous, separate or sequential use in therapy comprising variants and parent antibodies may be any variant and parent antibody as described herein. Specific aspects and embodiments will be described below. Furthermore, such variants may be obtained according to any method described herein.
In one aspect the present invention relates to a kit-of-parts for simultaneous, separate or sequential use in therapy comprising a first variant of a parent polypeptide and a second variant of a parent polypeptide, wherein the first variant comprises a first Fc-domain of an immunoglobulin and a binding region, wherein the second variant comprises a second Fc-domain of an immunoglobulin and a binding region, and wherein
(i) said first variant comprises a mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain, and said second variant comprises a mutation in the position corresponding to S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W,
(ii) said first variant comprises a mutation in the position corresponding to K447D/E in the Fc region of a human IgG1 heavy chain; and said second variant comprises a mutation in the position corresponding to K447K/R/H and 448P in the Fc-region of a human IgG1 heavy chain, or
(iii) said first variant comprises a mutation in the position corresponding to K447D/E in the Fc region of a human IgG1 heavy chain; and said second variant comprises a mutation in the position corresponding to K447K/R/H, 448K/R/H and 449P in the Fc-region of a human IgG1 heavy chain.
In one embodiment the first one or both of the variant of a parent polypeptide and the second variant of a parent polypeptide may be an antibody.
Thus in one an aspect, the invention relates to a kit-of-parts for simultaneous, separate or sequential use in therapy, comprising a first variant of a parent antibody and a second variant of a parent antibody, wherein the first variant comprises a first Fc-domain of an immunoglobulin and an antigen-binding region, wherein the second variant comprises a second Fc-domain of an immunoglobulin and an antigen-binding region, and wherein
(i) said first variant comprises a mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain, and said second variant comprises a mutation in the position corresponding to S440 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W,
(ii) said first variant comprises a mutation in the position corresponding to K447D/E in the Fc region of a human IgG1 heavy chain; and said second variant comprises a mutation in the position corresponding to K447K/R/H and 448P in the Fc-region of a human IgG1 heavy chain, or
(iii) said first variant comprises a mutation in the position corresponding to K447D/E in the Fc region of a human IgG1 heavy chain; and said second variant comprises a mutation in the position corresponding to K447K/R/H, 448K/R/H and 449P in the Fc-region of a human IgG1 heavy chain.
In one embodiment the kit-of-parts for simultaneous, separate or sequential use in therapy, comprising a first variant of a parent antibody and a second variant of a parent antibody, wherein the first variant comprises a first Fc-domain of an immunoglobulin and an antigen-binding region, wherein the second variant comprises a second Fc-domain of an immunoglobulin and an antigen-binding region, and wherein said first variant comprises a mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain, and wherein the second variant comprises a mutation in the position corresponding to S440, with the proviso that the mutation in S440 is not S440Y or S440W.
In one embodiment, the kit-of-parts for simultaneous, separate or sequential use in therapy, comprising the first variant of a parent antibody and the second variant of a parent antibody,
wherein the first variant comprises a first Fc-domain of an immunoglobulin and an antigen-binding region, wherein said first variant comprises (i) a first mutation in at least one amino acid residue other than a mutation in K439 selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain, and
(ii) a second mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain; and
wherein the second variant comprises a second Fc-domain of an immunoglobulin and an antigen-binding region, wherein said second variant comprises (i) a first mutation in at least one amino acid residue other than a mutation in S440 selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain, and
(ii) a second mutation in the position corresponding to S440 in the Fc region of an IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W.
In another embodiment, the kit-of-parts for simultaneous, separate or sequential use in therapy, comprising the first variant of an antibody and the second variant of a parent antibody, wherein the first variant comprises (i) a first mutation in at least one amino acid residue other than a mutation in K439 selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain, and
(ii) a second mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain, and
wherein the second variant comprises a mutation in the position corresponding to S440 in the Fc-region of a human IgG1 heavy chain, which is not is not S440Y or S440W.
In another embodiment, the kit-of-parts for simultaneous, separate or sequential use in therapy, comprising the first variant of a parent antibody and the second variant of a parent antibody,
wherein the first variant comprises a mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain; and
wherein the second variant comprises a second Fc-domain of an immunoglobulin and an antigen-binding region, wherein said second variant comprises (i) a first mutation in at least one amino acid residue other than a mutation in S440 selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain,
and (ii) a second mutation in the position corresponding to S440 in the Fc region of an IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W.
In one embodiment, the mutation in the position corresponding to K439 in the Fc-region of human IgG1 heavy chain is K439D/E, and/or the mutation in the position corresponding to S440 in the Fc-region of human IgG1 heavy chain is S440K/H/R.
In another aspect the present invention relates to a kit-of-parts for simultaneous, separate or sequential use in therapy, comprising a first variant of an parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region and a second variant of a parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region, wherein
the first variant comprises a first Fc-domain of an immunoglobulin and a first antigen-binding region, wherein the first variant comprises a first mutation in at least an amino acid residue selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain, and wherein the second variant does not comprise a mutation in an amino acid residue selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain.
In one embodiment the first and/or second parent polypeptide may be an antibody.
The present invention also relates to an embodiment of the kit-of-parts for simultaneous, separate or sequential use in therapy, wherein the first variant comprises (i) a first mutation in at least one amino acid residue other than a mutation in K439 selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain, and
(ii) a second mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain, and
wherein the second variant comprises a mutation in the position corresponding to S440 in the Fc-region of a human IgG1 heavy chain, which is not is not S440Y or S440W.
The present invention also relates to an embodiment of the kit-of-parts for simultaneous, separate or sequential use in therapy, wherein the first variant comprises a mutation in the position corresponding to K439 in the Fc-region of a human IgG1 heavy chain; and
wherein the second variant comprises a second Fc-domain of an immunoglobulin and an antigen-binding region, wherein said second variant comprises (i) a first mutation in at least one amino acid residue other than a mutation in S440 selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain,
and (ii) a second mutation in the position corresponding to S440 in the Fc region of an IgG1 heavy chain, with the proviso that the mutation in S440 is not S440Y or S440W.
In another aspect, the present invention relates to a kit-of-parts for simultaneous, separate or sequential use in therapy, comprising a first variant of an antibody and a second variant of a parent antibody, wherein the first variant comprises a first Fc-domain of an immunoglobulin and a first antigen-binding region, wherein the first variant comprises a first mutation in at least an amino acid residue selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain, and
wherein the second variant comprises a second Fc-domain of an immunoglobulin and a second antigen-binding region, wherein said second variant does not comprise a mutation in an amino acid residue selected from the group of
(a) an amino acid residue within the CH2-CH3 region providing allosteric mutations,
(b) an amino acid residue within the hydrophobic knobs of the CH2-CH3 region,
(c) an amino acid residue within the N-terminal CH3 helix,
(d) an amino acid residue within the C-terminal CH3 beta-strand, with the proviso that in case of a mutation corresponding to S440 in the Fc-region of a human IgG1 heavy chain the mutation is S440Y or S440W, and
(e) an amino acid residue corresponding to E345, E382 or Q386 in the Fc-region of a human IgG1 heavy chain.
In the embodiments, wherein the second variant does not comprise any of the listed mutations herein described, such second variant may include any of the suitable second antibody examples listed above in relation to the methods of effector functions.
In one embodiment, the first and second variant comprise a first mutation in at least one amino acid residue selected from those corresponding to E345, E430, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain.
In one embodiment, the first variant comprises a mutation in at least one amino acid residue selected from those corresponding to E345, E430, S440, Q386, P247, I253, S254, Q311, D/E356, T359, E382, Y436, and K447 in the Fc-region of a human IgG1 heavy chain, with the proviso that the mutation in S440 is S440Y or S440W.
In one particular embodiment, the first variant comprises a mutation in the amino acid residues corresponding to E345R and Q386K in the Fc-region of a human IgG1 heavy chain, and the second variant does not comprise such mutations.
In one particular embodiment, the first variant comprises a mutation in the amino acid residues corresponding to E345R, Q386K and E430G in the Fc-region of a human IgG1 heavy chain, and the second variant does not comprise such mutations.
In one embodiment, the at least one first mutation in the first and second variants are different.
In one embodiment, the first variant and second variant is each a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD or IgE antibody, optionally each a human full-length antibody, such as each a human full-length IgG1 antibody.
In one embodiment, the first variant and second variant is selected from a monospecific antibody, bispecific antibody or multispecific antibody.
In a further embodiment, the first and the second variant bind different epitopes on the same antigen or on different antigens. Thus, in the embodiment, wherein the first and second antibody are bispecific antibodies may be binding each two different epitopes. The at least two bispecific antibodies may be the same or different. If the bispecific antibodies are different, the kit-of-parts for simultaneous, separate or sequential use in therapy, thus, comprises targeting up to four different epitopes on either the same or different targets.
In a further embodiment, one or both of the first variant and second variant is conjugated to a drug, toxin or radiolabel, such as wherein one or both of the first variant and second variant is conjugated to a toxin via a linker.
In a further embodiment, one or both of the first variant and second variant is part of a fusion protein.
It is contemplated that any of the embodiments according to the “mixed-mutant” aspect also may be comprised in any of the kit-of-parts for simultaneous, separate or sequential use in therapy, embodiments.
In one embodiment, the variants of the first and second parent antibodies bind to antigens expressed on the same cell.
In another embodiment, the variant of the first parent antibody comprises an amino acid substitution of K439 into an amino acid selected from E and D.
In another embodiment, the amino acid substitution in the variant of the first parent antibody is K439E.
In another embodiment, the variant of the second parent antibody comprises an amino acid substitution of S440 into an amino acid selected from K, R and H.
In another embodiment, the amino acid substitution in the variant of the second parent antibody variant is S440K.
In an alternative embodiment, the variant of the first and/or second antibody further comprises a mutation in a residue selected from the group consisting of H310, G385, H433, N434, and Q438.
In a further alternative embodiment, the variant of the first and/or second parent antibody further comprise a mutation selected from E345 to D, K, N, Q, R, or W; E382 to D, Q, K, or R; and H433 to R.
In a further embodiment, the variants of the first and second parent antibodies further comprise a mutation selected from E345R, E382R and H433R, such as E345R.
In another aspect, the invention relates to a pharmaceutical kit-of-parts for simultaneous, separate or sequential use in therapy, comprising the variant of the first parent antibody and the variant of the second parent antibody of any one of embodiments listed above.
The pharmaceutical kit-of-parts for simultaneous, separate or sequential use in therapy, may be administered by any suitable route and mode. In one embodiment, a pharmaceutical kit-of-parts for simultaneous, separate or sequential use in therapy, of the present invention is administered parenterally. The term “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion.
Combinations
It is to be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody are also to be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region.
Additionally, the invention provides for a preparation of a variant of any “single mutant” aspect or embodiment described above, i.e., preparations comprising multiple copies of the variant. The invention also provides for a composition comprising a variant of any “single-mutant” aspect and embodiment described above, e.g., a pharmaceutical composition. The invention also provides for the use of any such “single-mutant” variant, preparation, or composition as a medicament.
The invention also provides for combinations of variants, wherein one variant comprises at least one mutation independently selected from those in Table 1 and one variant comprises at least one other mutation independently selected from those in Table 1, as well as preparations and pharmaceutical compositions of such variant combinations and their use as a medicament. Preferably, the two variants bind the same antigen or to different antigens typically expressed on the surface of the same cell, cell membrane, virion and/or other particle.
Conjugates
It is to be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody are also to be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region.
In one aspect, the present invention relates to a variant, wherein said variant is conjugated to a drug, toxin or radiolabel, such as wherein the variant is conjugated to a toxin via a linker.
In one embodiment said variant is part of a fusion protein.
In another aspect, the variant of the invention is not conjugated at the C-terminus to another molecule, such as a toxin or label. In one embodiment, the variant is conjugated to another molecule at another site, typically at a site which does not interfere with oligomer formation. For example, the antibody variant may, at the other site, be linked to a compound selected from the group consisting of a toxin (including a radioisotope) a prodrug or a drug. Such a compound may make killing of target cells more effective, e.g. in cancer therapy. The resulting variant is thus an immunoconjugate.
Thus, in a further aspect, the present invention provides an antibody linked or conjugated to one or more therapeutic moieties, such as a cytotoxin, a chemotherapeutic drug, a cytokine, an immunosuppressant, and/or a radioisotope. Such conjugates are referred to herein as “immunoconjugates” or “drug conjugates”. Immunoconjugates which include one or more cytotoxins are referred to as “immunotoxins”.
A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Suitable therapeutic agents for forming immunoconjugates of the present invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, maytansine or an analog or derivative thereof, enediyene antitumor antibiotics including neocarzinostatin, calicheamycins, esperamicins, dynemicins, lidamycin, kedarcidin or analogs or derivatives thereof, anthracyclins, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin; as well as duocarmycin A, duocarmycin SA, CC-1065 (a.k.a. rachelmycin), or analogs or derivatives of CC-1065), dolastatin, pyrrolo[2,1-c][1,4]benzodiazepins (PDBs) or analogues thereof, antibiotics (such as dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), anti-mitotic agents (e.g., tubulin-inhibitors) such as monomethyl auristatin E, monomethyl auristatin F, or other analogs or derivatives of dolastatin 10; Histone deacetylase inhibitors such as the hydroxamic acids trichostatin A, vorinostat (SAHA), belinostat, LAQ824, and panobinostat as well as the benzamides, entinostat, CI994, mocetinostat and aliphatic acid compounds such as phenylbutyrate and valproic acid, proteasome inhibitors such as Danoprevir, bortezomib, amatoxins such as α-amantin, diphtheria toxin and related molecules (such as diphtheria A chain and active fragments thereof and hybrid molecules); ricin toxin (such as ricin A or a deglycosylated ricin A chain toxin), cholera toxin, a Shiga-like toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica Charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins. Other suitable conjugated molecules include antimicrobial/lytic peptides such as CLIP, Magainin 2, mellitin, Cecropin, and P18; ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, diphtherin toxin, and Pseudomonas endotoxin. See, for example, Pastan et al., Cell 47, 641 (1986) and Goldenberg, Calif. A Cancer Journal for Clinicians 44, 43 (1994). Therapeutic agents that may be administered in combination with an antibody of the present invention as described elsewhere herein, such as, e.g., anti-cancer cytokines or chemokines, are also candidates for therapeutic moieties useful for conjugation to an antibody of the present invention.
In one embodiment, the drug conjugates of the present invention comprise an antibody as disclosed herein conjugated to auristatins or auristatin peptide analogs and derivates (U.S. Pat. Nos. 5,635,483; 5,780,588). Auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis and nuclear and cellular division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584) and have anti-cancer (U.S. Pat. No. 5,663,149) and anti-fungal activity (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42:2961-2965. The auristatin drug moiety may be attached to the antibody via a linker, through the N (amino) terminus or the C (terminus) of the peptidic drug moiety.
Exemplary auristatin embodiments include the N-terminus-linked monomethyl auristatin drug moieties DE and DF, disclosed in Senter et al., Proceedings of the American Association for Cancer Research. Volume 45, abstract number 623, presented Mar. 28, 2004 and described in US 2005/0238649).
An exemplary auristatin embodiment is MMAE (monomethyl auristatin E). Another exemplary auristatin embodiment is MMAF (monomethyl auristatin F).
In one embodiment, an antibody of the present invention comprises a conjugated nucleic acid or nucleic acid-associated molecule. In one such embodiment, the conjugated nucleic acid is a cytotoxic ribonuclease, an antisense nucleic acid, an inhibitory RNA molecule (e.g., a siRNA molecule) or an immunostimulatory nucleic acid (e.g., an immunostimulatory CpG motif-containing DNA molecule). In another embodiment, an antibody of the present invention is conjugated to an aptamer or a ribozyme.
In one embodiment, antibodies comprising one or more radiolabeled amino acids are provided. A radiolabeled variant may be used for both diagnostic and therapeutic purposes (conjugation to radiolabeled molecules is another possible feature). Non-limiting examples of labels for polypeptides include 3H, 14C, 15N, 35S, 90Y, 99Tc, and 125I, 131I, and 186Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art, (see, for instance Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2nd Ed., Chafner and Longo, eds., Lippincott Raven (1996)) and U.S. Pat. Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (U.S. RE35,500), U.S. Pat. No. 5,648,471 and U.S. Pat. No. 5,697,902. For example, a radioisotope may be conjugated by the chloramine-T method.
In one embodiment, the variant of the present invention is conjugated to a radioisotope or to a radioisotope-containing chelate. For example, the variant can be conjugated to a chelator linker, e.g. DOTA, DTPA or tiuxetan, which allows for the antibody to be complexed with a radioisotope. The variant may also or alternatively comprise or be conjugated to one or more radiolabeled amino acids or other radiolabeled molecule. A radiolabeled variant may be used for both diagnostic and therapeutic purposes. In one embodiment the variant of the present invention is conjugated to an alpha-emitter. Non-limiting examples of radioisotopes include 3H, 14C, 15N, 35S, 90Y, 99Tc, 125I, 111In, 131I, 186Re, 213Bs, 225Ac and 227Th.
In one embodiment the variant of the present invention may be conjugated to a cytokine selected from the group consisting of IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα, IFNβ, IFNγ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFα.
Variants of the present invention may also be chemically modified by covalent conjugation to a polymer to for instance increase their circulating half-life. Exemplary polymers, and methods to attach them to peptides, are illustrated in for instance U.S. Pat. Nos. 4,766,106, 4,179,337, 4,495,285 and 4,609,546. Additional polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., a PEG with a molecular weight of between about 1,000 and about 40,000, such as between about 2,000 and about 20,000).
Any method known in the art for conjugating the variant of the present invention to the conjugated molecule(s), such as those described above, may be employed, including the methods described by Hunter et al., Nature 144, 945 (1962), David et al., Biochemistry 13, 1014 (1974), Pain et al., J. Immunol. Meth. 40, 219 (1981) and Nygren, J. Histochem. and Cytochem. 30, 407 (1982). Such variants may be produced by chemically conjugating the other moiety to the N-terminal side or C-terminal side of the variant or fragment thereof (e.g., an antibody H or L chain) (see, e.g., Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). Such conjugated variant derivatives may also be generated by conjugation at internal residues or sugars, where appropriate.
The agents may be coupled either directly or indirectly to a variant of the present invention. One example of indirect coupling of a second agent is coupling via a spacer or linker moiety to cysteine or lysine residues in the bispecific antibody. In one embodiment, an variant is conjugated to a prodrug molecule that can be activated in vivo to a therapeutic drug via a spacer or linker. In some embodiments, the linker is cleavable under intracellular conditions, such that the cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In some embodiments, the linker is cleavable by a cleavable agent that is present in the intracellular environment (e.g. within a lysosome or endosome or caveola). For example, the spacers or linkers may be cleaveable by tumor-cell associated enzymes or other tumor-specific conditions, by which the active drug is formed. Examples of such prodrug technologies and linkers are described in WO02083180, WO2004043493, WO2007018431, WO2007089149, WO2009017394 and WO201062171 by Syntarga B V, et al. Suitable antibody-prodrug technology and duocarmycin analogs can also be found in U.S. Pat. No. 6,989,452 (Medarex), incorporated herein by reference. The linker can also or alternatively be, e.g. a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleaving agents can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside the target cells (see e.g. Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit (valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine) linker (see e.g. U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the Val-Cit linker and different examples of Phe-Lys linkers). Examples of the structures of a Val-Cit and a Phe-Lys linker include but are not limited to MC-vc-PAB described below, MC-vc-GABA, MC-Phe-Lys-PAB or MC-Phe-Lys-GABA, wherein MC is an abbreviation for maleimido caproyl, vc is an abbreviation for Val-Cit, PAB is an abbreviation for p-aminobenzylcarbamate and GABA is an abbreviation for γ-aminobutyric acid. An advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high.
In yet another embodiment, the linker unit is not cleavable and the drug is released by antibody degradation (see US 2005/0238649). Typically, such a linker is not substantially sensitive to the extracellular environment. As used herein, “not substantially sensitive to the extracellular environment” in the context of a linker means that no more than 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linkers, in a sample of variant antibody drug conjugate compound, are cleaved when the variant antibody drug conjugate compound presents in an extracellular environment (e.g. plasma). Whether a linker is not substantially sensitive to the extracellular environment can be determined for example by incubating the variant antibody drug conjugate compound with plasma for a predetermined time period (e.g. 2, 4, 8, 16 or 24 hours) and then quantitating the amount of free drug present in the plasma. Exemplary embodiments comprising MMAE or MMAF and various linker components have the following structures (wherein Ab means antibody and p, representing the drug-loading (or average number of cytostatic or cytotoxic drugs per antibody molecule), is 1 to about 8, e.g. p may be from 4-6, such as from 3-5, or p may be 1, 2, 3, 4, 5, 6, 7 or 8).
Examples where a cleavable linker is combined with an auristatin include MC-vc-PAB-MMAF (also designated as vcMMAF) and MC-vc-PAB-MMAF (also designated as vcMMAE), wherein MC is an abbreviation for maleimido caproyl, vc is an abbreviation for the Val-Cit (valine-citruline) based linker, and PAB is an abbreviation for p-aminobenzylcarbamate.
Other examples include auristatins combined with a non-cleavable linker, such as mcMMAF (mc (MC is the same as mc in this context) is an abbreviation of maleimido caproyl).
In one embodiment, the drug linker moiety is vcMMAE. The vcMMAE drug linker moiety and conjugation methods are disclosed in WO2004010957, U.S. Pat. Nos. 7,659,241, 7,829,531, 7,851,437 and U.S. Ser. No. 11/833,028 (Seattle Genetics, Inc.), (which are incorporated herein by reference), and the vcMMAE drug linker moiety is bound to the antibodies at the cysteines using a method similar to those disclosed in therein.
In one embodiment, the drug linker moiety is mcMMAF. The mcMMAF drug linker moiety and conjugation methods are disclosed in U.S. Pat. No. 7,498,298, U.S. Ser. No. 11/833,954, and WO2005081711 (Seattle Genetics, Inc.), (which are incorporated herein by reference), and the mcMMAF drug linker moiety is bound to the variants at the cysteines using a method similar to those disclosed in therein.
In one embodiment, the variant of the present invention is attached to a chelator linker, e.g. tiuxetan, which allows for the bispecific antibody to be conjugated to a radioisotope.
In one embodiment, each arm (or Fab-arm) of the variant is coupled directly or indirectly to the same one or more therapeutic moieties.
In one embodiment, only one arm of the variant is coupled directly or indirectly to one or more therapeutic moieties.
In one embodiment, each arm of the variant is coupled directly or indirectly to different therapeutic moieties. For example, in embodiments where the variant is a bispecific antibody and is prepared by controlled Fab-arm exchange of two different monospecific antibodies, e.g. a first and second antibody, as described herein, such bispecific antibodies can be obtained by using monospecific antibodies which are conjugated or associated with different therapeutic moieties.
Further Uses
It is to be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody are also to be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region.
In a further aspect, the invention relates to a variant of the invention as described above for use as a medicament, in particular for use as a medicament for the treatment of diseases or disorders, wherein CDC-mediated killing of a target cell (e.g., a tumor, bacterial or fungal cell) or target organism (e.g., a virus) is desired or a bacterial or virus infected cell. Examples of such diseases and disorders include, without limitation, cancer and bacterial, viral or fungal infections.
In another aspect, the present invention relates to the variants, bispecific antibodies, compositions and kit-of-parts described herein, for treatment of a disease, such as cancer.
In another aspect, the present invention relates to a method for treatment of a human comprising administration of a variant, a composition or a kit-of-parts described herein.
In another aspect, the present invention relates to a method for treatment of cancer in a human comprising administration of a variant, a composition or a kit-of-parts
“Treatment” refers to the administration of an effective amount of a therapeutically active compound of the present invention with the purpose of easing, ameliorating, arresting or eradicating (curing) symptoms or disease states.
An “effective amount” or “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of an antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.
Without being bound by theory, the effective amount of a therapeutically active compound may be decreased when any “single-mutant” aspect or embodiment according to the present invention is introduced to such a therapeutically active compound.
Suitable antigens for cancer antibodies may be the same as described herein. Examples 15 to 18 describe specific applications for providing an enhanced and/or more specific complement activation or CDC of tumor cells. For example, an anti-tumor antibody according to the “single-mutant” aspect, comprising, e.g., a E345R mutation, can provide for an enhanced CDC or ADCC, ADCP response of tumor cells. Further, in a variant of this method, a mutation according to the “single-mutant” aspect, such as, e.g., E345R, E430, S440, or Q386, alternatively E382 or H433R, or any other mutation as listed in Table 1, can be added to each antibody, thus providing for an enhanced CDC and/or ADCC response specifically directed to tumor cells expressing at least two antigens.
Suitable antibodies for bacterial infections include, without limitation, those targeting S. aureus, such as the chimeric monoclonal IgG1 pagibaximab (BSYX-A110; Biosynexus), targeting Lipoteichoic acid (LTA) that is embedded in the cell wall of staphylococci, and described in Baker (Nat Biotechnol. 2006 December; 24(12):1491-3) and Weisman et al. (Int Immunopharmacol. 2009 May; 9(5):639-44), both of which are incorporated by reference in their entirety. Example 14 describes a specific embodiment using S. aureus antibody variants comprising an E345R mutation. However, other mutations in Table 1, including but not limited to E430G and S440W, alternatively E382R and H433R, can be applied in a similar manner to enhance the CDC-mediating capability of an antibody against a bacterial antigen.
Suitable antigens for viral or fungal infections may be any of the herein described.
In one embodiment, the antigen to which the variant binds is not human EphA2. In another embodiment, the variant is not derived from human EphA2 mAb 12G3H11 (described in Dall'Acqua et al., supra, which is hereby incorporated by reference in its entirety). In another embodiment, the antigen to which the variant binds is not IL-9. In another embodiment, the variant is not derived from Fa-hG1 or Fa-hG4 antibody described in WO2007005612, hereby incorporated by reference in its entirety, or any variant thereof. In one embodiment, the antigen to which the variant binds is not HIV-1 gp120. In another embodiment, the variant is not derived from b12 human IgG1κ antibody directed against gp120.
In a particular embodiment, the variant derives from a bispecific parent antibody. The bispecific antibody can be of any isotype, such as, e.g., IgG1, IgG2, IgG3, or IgG4, and may be a full-length antibody or an Fc-containing fragment thereof. An exemplary method for preparing a bispecific antibody is described in WO 2008/119353 (Genmab).
Dosages
It is to be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody are also to be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region.
Efficient dosages and the dosage regimens for the antibody depend on the disease or condition to be treated and may be determined by the persons skilled in the art. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is about 0.1 to 100 mg/kg, such as about 0.1 to 50 mg/kg, for example about 0.1 to 20 mg/kg, such as about 0.1 to 10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3, about 5, or about 8 mg/kg.
Antibody variants of the present invention may also be administered in combination with one or more complement factors or related components to enhance the therapeutic efficacy of the variant and/or to compensate for complement consumption. Such complement factors and related components include, but are not limited to, C1q, C4, C2, C3, C5, C6, C7, C8, C9, MBL, and factor B. The combined administration may be simultaneous, separate or sequential. In a particular embodiment, the invention provides for a kit comprising a pharmaceutical composition comprising a variant of the invention, and at least one complement factor or related component in the same or different pharmaceutical composition, together with instructions for use.
Antibody variants of the present invention may also be administered in combination therapy, i.e., combined with other therapeutic agents relevant for the disease or condition to be treated. Accordingly, in one embodiment, the antibody-containing medicament is for combination with one or more further therapeutic agents, such as a cytotoxic, chemotherapeutic or anti-angiogenic agents. Such combined administration may be simultaneous, separate or sequential.
In a further embodiment, the present invention provides a method for treating or preventing disease, such as cancer, which method comprises administration to a subject in need thereof of a therapeutically effective amount of an variant or pharmaceutical composition of the present invention, in combination with radiotherapy and/or surgery.
Method of Preparation
It is to be understood that all embodiments described herein with reference to a parent antibody, first parent antibody or second parent antibody are also to be understood as embodiments relating to a parent, first parent or second parent polypeptide comprising an Fc-domain of an immunoglobulin and a binding region.
The invention also provides isolated nucleic acids and vectors encoding a variant according to any one of the aspects described above, as well as vectors and expression systems encoding the variants. Suitable nucleic acid constructs, vectors and expression systems for antibodies and variants thereof are known in the art, and described in the Examples. In embodiments where the variant comprises not only a heavy chain (or Fc-containing fragment thereof) but also a light chain, the nucleotide sequences encoding the heavy and light chain portions may be present on the same or different nucleic acids or vectors.
The invention also provides a method for producing, in a host cell, an antibody variant according to any one of the aspects described above, wherein said variant comprises at least the Fc region of a heavy chain, said method comprising the following steps:
a) providing a nucleotide construct encoding said Fc region of said variant,
b) expressing said nucleotide construct in a host cell,
and
c) recovering said antibody variant from a cell culture of said host cell.
In some embodiments, the antibody is a heavy-chain antibody. In most embodiments, however, the antibody will also contain a light chain and thus said host cell further expresses a light-chain-encoding construct, either on the same or a different vector.
Host cells suitable for the recombinant expression of antibodies are well-known in the art, and include CHO, HEK-293, PER-C6, NS/0 and Sp2/0 cells. In one embodiment, said host cell is a cell which is capable of Asn-linked glycosylation of proteins, e.g. a eukaryotic cell, such as a mammalian cell, e.g. a human cell. In a further embodiment, said host cell is a non-human cell which is genetically engineered to produce glycoproteins having human-like or human glycosylation. Examples of such cells are genetically-modified Pichia pastoris (Hamilton et al., Science 301 (2003) 1244-1246; Potgieter et al., J. Biotechnology 139 (2009) 318-325) and genetically-modified Lemna minor (Cox et al., Nature Biotechnology 12 (2006) 1591-1597).
In one embodiment, said host cell is a host cell which is not capable of efficiently removing C-terminal lysine K447 residues from antibody heavy chains. For example, Table 2 in Liu et al. (2008) J Pharm Sci 97: 2426 (incorporated herein by reference) lists a number of such antibody production systems, e.g. Sp2/0, NS/0 or transgenic mammary gland (goat), wherein only partial removal of C-terminal lysines is obtained. In one embodiment, the host cell is a host cell with altered glycosylation machinery. Such cells have been described in the art and can be used as host cells in which to express variants of the invention to thereby produce an antibody with altered glycosylation. See, for example, Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, as well as EP1176195; WO03/035835; and WO99/54342. Additional methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473), U.S. Pat. No. 6,602,684, WO00/61739A1; WO01/292246A1; WO02/311140A1; WO 02/30954A1; Potelligent™ technology (Biowa, Inc. Princeton, N.J.); GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); US 20030115614; Okazaki et al., 2004, JMB, 336: 1239-49.
The invention also relates to an antibody obtained or obtainable by the method of the invention described above.
In a further aspect, the invention relates to a host cell capable of producing an antibody variant of the invention. In one embodiment, the host cell has been transformed or transfected with a nucleotide construct of the invention.
The present invention is further illustrated by the following examples which should not be construed as further limiting.
EXAMPLES
Example 1
Design and Generation of 7D8 Mutants
The human monoclonal antibody HuMab-7D8 (described in WO 2004/035607) was used as a model antibody. It belongs to a group of human anti-CD20 IgG1 antibodies, including ofatumumab (HuMax-CD20, 2F2). These antibodies target a unique membrane-proximal epitope on the CD20 molecule and show strong CDC.
To test the functional relevance of oligomeric Fc-Fc interactions in complement activation and CDC, amino acids in the hydrophobic patch at the Fc:Fc interface were mutated to potentially disrupt the Fc-Fc side-on interaction and CDC efficacy of 7D8. In a first set of mutants (Table 3), mutations were introduced to change the charge at positions that were chosen based on the 1HZH crystal structure and described to be exposed in hydrophobic patches in the CH2-CH3 domain (Burton Mol Immunol 1985 March; 22(3):161-206)).
From the first set of mutations, I253D and H433A were found to induce the strongest effect on loss of CDC by 7D8 (e.g., Example 5). The 1HZH crystal structure shows that I253 and H433 bind two different pockets on the opposing Fc positions of the partnering antibody. Based on these data, a second set of mutations was synthesized, around the I253 and H433 positions in the crystal structure to further study the importance of residues at the Fc:Fc side-on interface for CDC. The second set of mutations around the I253 and H433 positions that potentially destabilize the Fc:Fc interface and consequently CDC are listed in Table 4.
To exclude the possibility that disruption of direct binding sites for C1q were the cause of the observed effects on CDC, a double mutant was generated based on two single mutants that showed loss of CDC, to test its ability to restore the loss of CDC by the single mutants. This principle is schematically represented in FIG. 1D The double mutant is listed in Table 5 and a structural representation is shown in FIG. 4 and FIG. 5.
Mutants were prepared using the Quikchange site-directed mutagenesis kit (Stratagene, US). Briefly, a forward and a reverse primer encoding the desired mutation were used to replicate full length plasmid DNA template encoding the 7D8 heavy chain with IgG1m(f) allotype. The resulting DNA mixture was digested using DpnI to remove source plasmid DNA and used to transform E. coli. Mutant plasmid DNA isolated from resulting colonies was checked by DNA sequencing (Agowa, Germany). Plasmid DNA mixtures encoding both heavy and light chain of antibodies were transiently transfected to Freestyle HEK293F cells (Invitrogen, US) using 293fectin (Invitrogen, US) essentially as described by the manufacturer.
TABLE 3
Set 1 mutations introduced in the CH2—CH3 domain of 7D8.
Charge
Charge mutant
Mutation
WT aa
aa
I253D
=
−
I253Y
=
=
I253A
=
=
Q311A
=
=
H433A
δ+
=
N434A
=
=
H435A
Δ+
=
H435R
δ+
+
(=) no charge
(−) negative charge
(+) positive charge
(δ+) partial positive charge
TABLE 4
Set 2 mutations introduced in the CH2—CH3 domain of 7D8.
Charge
Charge mutant
Mutation(s)
WT aa
aa
I253K
=
+
I253R
=
+
I253D/H433A
=/δ+
−/=
H310E
δ+
−
H310R
δ+
+
H310K
δ+
+
Q311K
=
+
K322A
+
=
E345R
−
+
E382R
−
+
G385D
=
−
H433D
δ+
−
H433R
δ+
+
Y436C
=
=
Y436D
=
−
Q438D
=
−
K439E
+
−
S440K
=
+
(=) no charge
(−) negative charge
(+) positive charge
(δ+) partial positive charge
TABLE 5
Double mutations introduced in the CH2—CH3 domain of 7D8
to combine two single mutations that each show loss of CDC.
Charge
Charge mutant
Mutations
WT aa
aa
K439E/S440K
+/=
−/+
(=) no charge
(−) negative charge
(+) positive charge
Example 2
CD20 Binding on Cells by 7D8 Mutants
Binding of purified antibody samples to CD20-positive cells was analyzed by FACS analysis. The 1st set of mutations (Table 3) was tested on Daudi cells and the second set of mutations (Table 4) was tested on Raji cells. 105 cells were incubated in 50 μL in polystyrene 96-well round-bottom plates (Greiner bio-one 650101) with serial dilutions of antibody preparations (range 0.04 to 10 μg/mL in 3-fold dilutions for 1st set on Daudi and range 0.003 to 10 μg/mL in 3-fold dilutions for 2nd set on Raji) in RPMI1640/0.1% BSA at 4° C. for 30 min. After washing twice in RPMI1640/0.1% BSA, cells were incubated in 100 μL with secondary antibody at 4° C. for 30 min. As a secondary antibody, fluorescein isothiocyanate (FITC)-conjugated rabbit-anti-human IgG (F0056, Dako, Glostrup, Denmark; 1/100) was used for all experiments on Daudi cells and for experiments with 7D8 antibodies on Raji cells. For the experiments with purified 7D8 antibodies on Raji cells, R-phycoerythrin (R-PE)-conjugated goat F(ab′)2 anti-human kappa light chain (2062-09, SouthernBiotech; 1/500) was used as a secondary antibody. Next, cells were washed twice in PBS/0.1% BSA/0.02% azide, resuspended in 100 μL PBS/0.1% BSA/0.02% azide and analyzed on a FACS Cantoll (BD Biosciences). Binding curves were analyzed using non-linear regression (sigmoidal dose-response with variable slope) using GraphPad Prism V5.01 software (GraphPad Software, San Diego, Calif., USA).
Binding of 7D8 antibody to Daudi cells was not affected by the introduction of the point mutations in the CH2-CH3 domain and was identical for all tested mutants and wild type 7D8. Further, binding of 7D8 antibody to Raji cells was not significantly affected by the introduction of the point mutations in the CH2-CH3 domain compared to wild type 7D8, except for E345R. Diminished binding of IgG1-7D8-E345R was detected on CD20-positive Raji cells at test concentrations above 0.3 μg/mL. Also for H433D and H433R diminished binding was detected at the highest antibody concentration tested (10 μg/mL). The diminished binding by IgG1-7D8-E345R, H433D and H433R could be explained by shielding of the epitope of the secondary antibody since direct labeling of E345R and H433R resulted in similar or even increased binding to Daudi cells. The increased avidity can be explained by the increased Fc-Fc side-on binding by E345R and H433R in comparison to wild-type IgG1-7D8.
Combining the K439E and S440K mutations did not affect binding of the 7D8 antibody to Raji cells and was identical to that of the single mutants and wild type 7D8.
Example 3
C1q Binding ELISA by 7D8 Mutants
C1q binding by the 7D8 mutants was tested in an ELISA, in which the purified antibodies were coated on the plastic surface, bringing about random antibody multimerization. Pooled human serum was used as a source of C1q.
96-well Microlon ELISA plates (Greiner, Germany) were coated overnight at 4° C. with a dilution series of the antibodies in PBS (range 0.58-10.0 μg/mL in 1.5-fold dilutions). Plates were washed and blocked with 200 μL/well 0.5×PBS supplemented with 0.025% Tween 20 and 0.1% gelatine. With washings in between incubations, plates were sequentially incubated with 3% pooled human serum (Sanquin, product # M0008) for 1 h at 37° C., with 100 μL/well rabbit anti-human C1q (DAKO, product # A0136, 1/4.000) for 1 h at RT, and with 100 μL/well swine anti-rabbit IgG-HRP (DAKO, P0399, 1:10.000) as detecting antibody for 1 h at RT. Development was performed for circa 30 min with 1 mg/mL 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany). The reaction was stopped by the addition of 100 μL 2% oxalic acid. Absorbance was measured at 405 nm in a microplate reader (Biotek, Winooski, Vt.). Log transformed data were analyzed by fitting sigmoidal dose-response curves with variable slope using GraphPad Prism software. EC50 values of the mutants were normalized per plate against wild type IgG1-7D8 and multiplied by the average of all wild type IgG1-7D8 data.
As shown in FIG. 6 and Table 6, the tested point mutations had minimal effect on C1q binding as measured by ELISA. For the IgG1-7D8-I253D mutant, a slightly less efficient C1q binding was measured in the ELISA (higher EC50 value). Coating efficacy was tested for all antibodies and was found to be similar for all antibodies.
TABLE 6
EC50 for C1q binding in ELISA
Mean EC50
Antibody
(μg/mL)1
SD1
Significance2
IgG1-7D8-WT
2.048
0
Na
IgG1-7D8-I253D
3.838
1.341
*
IgG1-7D8-I253Y
2.209
0.385
Ns
IgG1-7D8-I253A
2.556
0.187
Ns
IgG1-7D8-Q311A
2.182
0.062
ns
IgG1-7D8-H433A
3.327
1.719
ns
IgG1-7D8-N434A
2.120
0.492
ns
IgG1-7D8-H435A
2.267
0.317
ns
IgG1-7D8-H435R
1.242
0.492
ns
1Mean and SD were calculated from at least 3 experiments.
2Statistics: 1 way ANOVA on log transformed data using Dunnett's Multiple Comparison Test (GraphPad Prism 5.01). Significance was calculated in comparison to wild type IgG1-7D8: (na) not applicable (ns) not significant (*) p = 0.01 to 0.05 (**) p = 0.001 to 0.01 (***) p < 0.001.
Example 4
C1q Binding on Cells by 7D8 Mutants
Coating of antibodies on a plastic surface results in an artificial static system of antibody binding and Fc-tail presentation. Therefore, complement binding was also tested in a cell-based assay, in which C1q binding to antibody-opsonized CD20-positive B cells was measured by FACS analysis. In experiments with set 1 mutants, Daudi or Raji cells were suspended on ice in 90 μL RPMI 1640 media with 10% FBS (2×106 cells/mL). 10 μL of a concentration series of C1q (Complement Technologies, Tyler, Tex.) was added (final concentration range varies between 0-60 μg/mL and 0-140 μg/mL depending on the maximal binding). Then, 10 μL of purified antibody (10 μg/mL final concentration, i.e. saturating conditions) was added and the reaction mixtures were immediately transferred to a 37° C. water bath and incubated for one hour. In experiments with set 2 mutants, test mAb was added to Daudi cells in bulk, then varying concentrations of C1q were added to aliquots and the mixtures incubated as above. Cells were washed three times with PBS/1% BSA and incubated for 30 minutes at room temperature with rabbit FITC-labeled anti-C1q antibody (DakoCytomation, 10 ug/mL). Cells were washed with PBS/1% BSA and resuspended in PBS or fixed in 2% formaldehyde in PBS. Flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences) and mean fluorescence intensities were converted to molecules of equivalent soluble fluorescence (MESF) using calibrated beads (Spherotech). The dissociation constants (KD values) for binding of C1q to CD20-positive cells opsonised with the indicated 7D8 antibodies were calculated using SigmaPlot® software (Systat Software Inc., Washington). Average KD values were calculated from repeated binding experiments (4 times on Daudi cells, 3 times on Raji cells) and compared to the KD value for C1q binding on cells opsonized with wild type 7D8 (Table 7 and Table 8).
Set 1 mutants were tested on both Daudi and Raji cells and gave the same results. In contrast to the C1q ELISA results, most tested mutants showed decreased C1q binding avidity (increased KD) on both antibody-opsonized Daudi (Table 7A) and Raji cells (Table 8). Compared to wild type 7D8, IgG1-7D8-Q311A and H435A showed little to no decrease, I253A, I253Y and N434A a more pronounced decrease, and I253D and H433A a very drastic decrease in C1q binding avidity on opsonized Daudi or Raji cells. IgG1-7D8-H435R showed a slightly higher avidity (lower KD) for C1q binding than wild type 7D8 on both cell types, which, however, was not significant.
Set 2 mutants were tested on Daudi cells. Compared to wild type 7D8, IgG1-7D8-E345R, E382R and H433R showed increased binding avidity on opsonized Daudi cells, reflected by the lower KD values (Table 7B). All other Set 2 mutants showed decreased binding avidity compared to wild type 7D8, with G385D, Y436D, Q438D, K439E and S440K showing drastically increased KD values (Table 7B) and H433D and Y436C showing such a drastically reduced binding that no reliable KD value could be measured.
The double mutant IgG1-7D8-K439E/S440K showed restored C1q binding on antibody-opsonized Daudi cells, while both single mutants showed decreased C1q binding compared to wild type 7D8. The binding avidity of the K439E/S440K double mutant was even slightly increased compared to wild type 7D8 (Table 7C). Mixtures of single mutants IgG1-7D8-K439E and IgG1-7D8-K440E were able to completely restore C1q binding which was comparable to C1q binding of wild type 7D8 (Table 7C).
The discrepancy between the unchanged C1q binding in the ELISA (Example 3) and the affected C1q binding in the cell-based assay by the IgG1-7D8 mutants, shows that the tested CH3 positions that are involved in the Fc:Fc interaction between antibody molecules, do not influence C1q binding directly, but are important determinants that affect the dynamic positioning of antibody Fc-tails when bound on cells, and thereby also the strength of the C1q binding.
TABLE 7A
KD values for C1q binding to antibody-opsonized Daudi cells (mutants set 1)
KD (nM)
KD (nM)
KD (nM)
KD (nM)
KD (nM)
KD (nM)
Average
P-
mAb
Exp. 1
Exp. 2
Exp. 3
Exp. 4
Exp. 10
Exp. 11
KD (nM)
sd
value*
7D8
7.7
9.3
4.2
4.3
11.8
13.3
8.4
3.7
na**
7D8-I253A
33.0
20.4
16.7
15.7
21.5
8.0
0.007
7D8-I253Y
58.5
37.0
21.1
48.7
41.3
16.1
0.001
7D8-I253D
146.5
176.1
101.7
205.2
157.4
44.2
<0.001
7D8-Q311A
14.3
13.0
9.6
5.9
10.7
3.8
0.379
7D8-H433A
168.0
76.1
45.2
180.7
117.5
67.0
0.003
7D8-N434A
36.7
47.8
28.3
48.7
42.6
9.7
<0.001
7D8-H435A
7.8
10.9
5.0
10.9
8.6
2.8
0.925
7D8-H435R
5.2
8.7
2.6
3.0
4.9
2.8
0.147
*Compared to wild type 7D8 (t-test)
**(na) not applicable
TABLE 7B
KD values for C1q binding to antibody-opsonized Daudi cells (mutants set 2)
KD (nM)
KD (nM)
KD (nM)
KD (nM)
KD (nM)
KD (nM)
KD (nM)
Average
P-
mAb
Exp. 5
Exp. 6
Exp. 7
Exp. 8
Exp. 9
Exp. 10
Exp. 11
KD (nM)
sd
value*
Ofatumumab
6
5.4
4
2.7
12.47
12.8
7.2
4.3
0.6192
7D8
11.8
13.3
8.4***
3.7
na**
7D8-H310K
32.4
216
124
130
0.0371
7D8-E345R
3.5
0.17
0.35
2.7
1.7
1.7
0.0106
7D8-E382R
3.5
1.18
1.13
3.3
2.3
1.3
0.0150
7D8-G385D
77
71
74
4
<0.0001
7D8-H433D****
(1227)
(2694)
(1961)
1037
0.0013
7D8-H433R
5.2
0.72
1.78
5.69
1.6
3
2.3
0.0205
7D8-Y436C****
(2420)
(128)
(1274)
1621
0.0576
7D8-Y436D
431
504
468
52
<0.0001
7D8-Q438D
767
667
717
70
<0.0001
7D8-K439E
418
304
361
81
<0.0001
7D8-S440K
170
48
109
87
0.0131
7D8-
103161
246
5291
7106
0.0681
I253D/H433A
*Compared to wild type 7D8 (t-test)
**(na) not applicable
***Average KD of 7D8 was calculated from experiments 1, 2, 3, 4, 10 and 11.
****No reliable fitting curve and KD value could be measured due to too weak binding of these mutants.
TABLE 7C
KD values for C1q binding to antibody-opsonized Daudi cells (double mutant)
KD (nM)
KD (nM)
KD (nM)
KD (nM)
KD (nM)
KD (nM)
KD (nM)
Average
mAb
Exp. 5
Exp. 6
Exp. 7
Exp. 8
Exp. 9
Exp. 10
Exp. 11
KD (nM)
sd
P-value*
7D8
11.8
13.3
8.4***
3.7
na**
7D8-K439E
418
304
361
81
<0.0001
7D8-S440K
170
48
109
87
0.0131
7D8-
4.6
1.63
1.01
2.9
2.6
1.6
0.0196
K439E/S440K
7D8-K439E +
3.6
3.05
3.1
3.3
0.3
0.0555
7D8-S440K mix
*Compared to wild type 7D8 (t-test)
**(na) not applicable
***Average KD of 7D8 was calculated from experiments 1, 2, 3, 4, 10 and 11.
TABLE 8
KD values for C1q binding to antibody-opsonized Raji cells
(mutants set 1)
KD
KD
(nM)
(nM)
KD (nM)
Average
P-
mAb
Exp. 1
Exp. 2
Exp. 3
KD (nM)
sd
value*
7D8
4.8
7.0
10.9
6.5
3.1
na**
7D8-I253A
10.0
25.7
20.1
18.6
7.9
0.020
7D8-I253Y
24.3
45.6
46.2
38.7
12.4
0.001
7D8-I253D
70.0
172.0
85.2
109.1
55.0
0.005
7D8-Q311A
4.1
10.1
12.2
9.1
3.5
0.280
7D8-H433A
124.8
85.0
84.0
97.9
23.3
<0.001
7D8-N434A
35.9
46.7
35.2
44.9
12.5
<0.001
7D8-H435A
5.4
9.9
6.6
7.3
2.3
0.721
7D8-H435R
3.5
6.2
4.5
4.7
1.4
0.721
*Compared to wild type 7D8 (t-test)
**(na) not applicable
Example 5
C1q Efficacy by 7D8 Mutants in a CDC Assay on CD20-Positive Raji Cells
C1q efficacy using cells opsonized with IgG1-7D8 mutants was tested in a CDC assay to investigate the impact of the observed changes in C1q binding avidity on CDC activity. Therefore, a CDC assay was performed using C1q-depleted normal human serum that was supplemented with a defined concentration series of C1q. 0.1×106 Raji cells were pre-incubated in round-bottom 96-well plates (Nunc, Rochester, N.Y.) with 10 μg/mL purified antibody and a concentration series human C1q (0.005, 0.025, 0.1, 0.3, 1.0, 5.0, 30.0 μg/mL) at RT for 15 min in a total volume of 100 μL RPMI1640 medium, supplemented with 0.1% BSA. Next, 25 μL C1q-depleted serum (Quidel, San Diego, Calif.) was added and incubated at 37° C. in a water bath for 30 min or in an incubator for 45 min. After incubation, the reaction was stopped by placing the samples on ice. Cell lysis was determined on FACS by using propidium iodide (PI, Sigma Aldrich, Zwijndrecht, the Netherlands) viable cell exclusion assay. % lysis was determined as follows: % lysis=(number of PI pos cells/total number of cells)×100%.
The lysis by wild type 7D8 in the presence of 30 μg/mL C1q minus the lysis when no C1q was added, was set to 100%. CH50 values (the C1q concentration resulting in 50% lysis) were calculated from fitting sigmoidal dose-response curves on log-transformed data using GraphPad Prism software. CH50 values of the mutants were normalized to wild type 7D8 (Table 9).
The data in Table 9 show that, in accordance with the C1q binding avidity measurements, IgG1-7D8-Q311A, E382R and H435A showed no decrease in C1q efficacy; I253A, I253Y, G385D, N434A and Y436C a significant decrease in C1q-efficacy; and I253D, H310K, K322A, H433A, H433D, Y436D, Q438D, K439E and S440K almost completely lost the capacity to induce CDC with all C1q concentrations tested.
IgG1-7D8-H435R and H433R used C1q slightly more efficient which resulted in more efficient CDC than wild type 7D8. IgG1-7D8-E345R showed a drastic increase in C1q efficacy, which resulted in significantly higher CDC lysis compared to wild type 7D8 (Table 9).
FIG. 7 shows that combining the K439E and S440K mutation, which both result in loss of CDC as a single mutant, restored CDC in the C1q efficacy assay when both mutations were combined in one molecule (K439E/S440K double mutant) or when both single mutants were combined (K439E+S440K mix).
TABLE 9
CH50 for C1q efficacy in a CDC assay on Raji cells
Mean CH50
Antibody
n(1)
(μg/mL)(2)
SD(2)
Significance(3)
IgG1-7D8-WT
8
0.49
0.26
na
IgG1-7D8-I253A
3
11.16
16.31
***
IgG1-7D8-I253D
3
>30(4)
0.00
nd
IgG1-7D8-I253Y
3
16.07
12.50
***
IgG1-7D8-H310K
3
>30
0.00
nd
IgG1-7D8-Q311A
3
0.63
0.58
ns
IgG1-7D8-K322A
6
>30
0.00
nd
IgG1-7D8-E345R
3
0.03
0.01
***
IgG1-7D8-E382R
3
0.77
0.476
ns
IgG1-7D8-G385D
3
22.51
12.97
***
IgG1-7D8-H433A
3
>30
0.00
nd
IgG1-7D8-H433D
3
>30
0.00
nd
IgG1-7D8-H433R
3
0.16
0.09
ns
IgG1-7D8-N434A
3
21.16
15.32
***
IgG1-7D8-H435A
3
0.96
0.20
ns
IgG1-7D8-H435R
3
0.24
0.15
ns
IgG1-7D8-Y436C
3
23.03
12.07
***
IgG1-7D8-Y436D
3
>30
0.00
nd
IgG1-7D8-Q438D
3
>30
0.00
nd
IgG1-7D8-K439E
3
>30
0.00
nd
IgG1-7D8-S440K
3
>30
0.00
nd
IgG1-7D8-
3
>30
0.00
nd
I253D/H433A
IgG1-7D8-
3
0.09
0.71
ns
K439E/S440K
IgG1-7D8-K439E +
3
1.33
1.48
ns
IgG1-7D8-S440K mix
(1)(n) Number of experiments
(2)Mean and SD were calculated from all performed experiments.
(3)Statistics: 1 way ANOVA on log transformed data using Dunnett's Multiple Comparison Test (GraphPad Prism 5.01). Significance was calculated in comparison to wild type IgG1-7D8: (na) not applicable (nd) not determined (ns) not significant (*) p = 0.01 to 0.05 (**) p = 0.001 to 0.01 (***) p < 0.001.
(4)When lysis did not reach 50%, the CH50 was set to >30 μg/mL.
(5)No P-value could be determined for mutants that did not reach 50% lysis. However, these are assumed to be significantly different from IgG1-7D8-WT.
Example 6
CDC by 7D8 Mutants in a CDC Assay on CD20-Positive Cells
0.1×106 cells were pre-incubated in round-bottom 96-well plates (Nunc, Rochester, N.Y.) with antibody concentration series (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg/mL) in a total volume of 80 μL for 15 min on a shaker at RT. Next, 20 μL normal human serum was added as a source of C1q (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by adding 30 μL ice cold RPMI medium, supplemented with 0.1% BSA. Cell lysis was determined on FACS by using propidium iodide.
For the CDC assays on Daudi cells, EC50 values (the antibody concentration resulting in 50% lysis) were calculated from fitting sigmoidal dose-response curves on log-transformed data using GraphPad Prism software. EC50 values of the mutants were normalized to wild type 7D8 (Table 10 and Table 11).
Table 10 shows that on Daudi cells, IgG1-7D8-I253A, Q311A, E382R, H433R and H435A showed no difference in CDC compared to wild type 7D8; a significant worse CDC (higher EC50) than wild type 7D8 was found for IgG1-7D8-I253D, I253Y, H310K, G385D, H433A, H433D, N434A, Y436C, Y436D, Q438D, K439E, S440K and I253D/H433A, which only induced CDC at higher antibody concentrations; The C1q binding deficient mutant IgG1-7D8-K322A, which was included as control, almost completely lost the capacity to induce CDC and did not reach EC50 at the tested concentrations; IgG1-7D8-H435R showed more efficient CDC than wild type 7D8 on Daudi cells. Importantly, in accordance with the C1q efficacy CDC assay, E345R showed drastically better CDC than wild type 7D8 with a 10-fold lower EC50 value on Daudi cells (Table 10). FIG. 8 shows that combining the K439E and S440K mutation, which both result in loss of CDC as a single mutant, restored CDC when both mutations were combined in one molecule (K439E/S440K double mutant) or when both single mutants were combined (K439E+S440K mix).
Table 11 shows that similar data were found for the IgG1-7D8 mutants on Raji cells.
TABLE 10
EC50 calculated from the CDC assay on Daudi cells
Mean EC50
Antibody
n(1)
(μg/mL)(2)
SD(2)
Significance(3)
IgG1-7D8
12
0.48
0.11
na
IgG1-7D8-I253A
4
0.79
0.15
ns
IgG1-7D8-I253D
5
3.33
1.05
***
IgG1-7D8-I253Y
4
1.77
0.43
***
IgG1-7D8-H310K
3
3.03
0.30
***
IgG1-7D8-Q311A
4
0.42
0.12
ns
IgG1-7D8-K322A
>30(4)
Nd
***(5)
IgG1-7D8-E345R
4
0.04
0.01
***
IgG1-7D8-E382R
4
0.76
0.25
ns
IgG1-7D8-G385D
3
2.12
0.45
***
IgG1-7D8-H433A
5
3.44
1.17
***
IgG1-7D8-H433D
4
4.73
2.57
***
IgG1-7D8-H433R
4
0.33
0.14
ns
IgG1-7D8-N434A
4
1.77
0.46
***
IgG1-7D8-H435A
4
0.81
0.27
ns
IgG1-7D8-H435R
5
0.28
0.06
**
IgG1-7D8-Y436C
4
1.90
1.21
***
IgG1-7D8-Y436D
3
1.88
0.45
***
IgG1-7D8-Q438D
3
2.61
0.38
***
IgG1-7D8-K439E
4
2.34
0.38
***
IgG1-7D8-S440K
4
1.78
0.46
***
IgG1-7D8-I253D/H433A
4
4.77
1.36
***
IgG1-7D8-K439E/S440K
4
0.33
0.08
ns
IgG1-7D8-K439E +
4
0.48
0.17
ns
IgG1S440K
(1)(n) Number of experiments
(2)Mean and SD were calculated from all performed experiments.
(3)Statistics: 1 way ANOVA on log transformed data using Dunnett's Multiple Comparison Test (GraphPad Prism 5.01). Significance was calculated in comparison to wild type 7D8: (na) not applicable (nd) not determined (ns) not significant (*) p = 0.01 to 0.05 (**) p = 0.001 to 0.01 (***) p < 0.001.
(4)When lysis did not reach 50%, the EC50 was set to >30 μg/mL.
(5)No P-value could be determined for mutants that did not reach EC50. However, these are assumed to be significantly different from wild 7D8-WT.
TABLE 11
EC50 calculated from the CDC assay on Raji cells
Mean EC50
Antibody
n(1)
(μg/mL)(2)
SD(2)
Significance(3)
IgG1-7D8
13
1.54
0.77
Na
IgG1-7D8-I253A
4
5.55
3.19
*
IgG1-7D8-I253D
6
>30(4)
0.00
***(5)
IgG1-7D8-I253Y
4
28.95
2.09
***
IgG1-7D8-H310K
2
19.29
15.15
***
IgG1-7D8-Q311A
4
1.72
0.42
Ns
IgG1-7D8-K322A
>30
***
IgG1-7D8-E345R
4
0.16
0.09
***
IgG1-7D8-E382R
4
2.96
1.27
Ns
IgG1-7D8-G385D
2
17.40
17.82
***
IgG1-7D8-H433A
6
22.60
9.30
***
IgG1-7D8-H433D
4
>30
0.00
***
IgG1-7D8-H433R
4
1.42
0.67
Ns
IgG1-7D8-N434A
4
23.02
6.16
***
IgG1-7D8-H435A
4
2.22
1.47
Ns
IgG1-7D8-H435R
6
0.61
0.21
**
IgG1-7D8-Y436C
2
11.93
10.13
**
IgG1-7D8-Y436D
2
16.58
3.93
***
IgG1-7D8-Q438D
2
19.49
14.87
***
IgG1-7D8-K439E
4
21.51
9.96
***
IgG1-7D8-S440K
4
19.53
12.71
***
IgG1-7D8-I253D/H433A
4
>30
0.00
***
IgG1-7D8-K439E/S440K
4
1.34
0.45
Ns
IgG1-7D8-K439E +
4
1.58
0.64
Ns
IgG1S440K
(1)(n) Number of experiments
(2)Mean and SD were calculated from all performed experiments.
(3)Statistics: 1 way ANOVA on log transformed data using Dunnett's Multiple Comparison Test (GraphPad Prism 5.01). Significance was calculated in comparison to wild type 7D8: (na) not applicable (nd) not determined (ns) not significant (*) p = 0.01 to 0.05 (**) p = 0.001 to 0.01 (***) p < 0.001.
(4)When lysis did not reach CH50, the CH50 was set to >30 μg/mL.
(5)No P-value could be determined for mutants that did not reach EC50. However, these are assumed to be significantly different from wild 7D8-WT.
Example 7
Ranking of 7D8 Mutants According to their Capacity to Induce CDC
For the tested 7D8 mutants, a correlation was found between C1q binding on Daudi cells (described in Example 4) and C1q efficacy assays on Raji cells (described in Example 5), and between C1q binding on Daudi cells and CDC assays on Daudi and Raji cells (described in Example 6) (correlation data Table 13). Therefore, the KD values of the C1q binding assays on Daudi cells were used to rank all tested 7D8 mutants according to their capacity to induce CDC, as shown in Table 12.
TABLE 12
Ranking of all tested 7D8 mutants according to descending
KD values for C1q binding on Daudi cells, which serve
as a representative for their capacity to induce CDC.
C1q binding on Daudi cells
Antibody
n(1)
KD (nM)(2)
SD
IgG1-7D8-E345R
4
1.7
1.7
IgG1-7D8-E382R
4
2.3
1.3
IgG1-7D8-K439E/S440K
4
2.6
1.6
IgG1-7D8-H433R
5
3.0
2.3
IgG1-7D8-K439E + IgG1S440K
3
3.3
0.3
IgG1-7D8-H435R
3
4.9
2.8
IgG1-7D8-H435A
3
8.6
2.8
IgG1-7D8
7
8.7
3.5
IgG1-7D8-Q311A
3
10.7
3.8
IgG1-7D8-I253A*
3
21.5
8.0
IgG1-7D8-I253Y*
3
41.3
16.1
IgG1-7D8-N434A*
3
42.6
9.7
IgG1-7D8-G385D*
2
74.0
4.0
IgG1-7D8-S440K*
2
109.0
87.0
IgG1-7D8-H433A*
3
117.5
16.1
IgG1-7D8-H310K*
2
124.0
130.0
IgG1-7D8-I253D*
3
157.4
44.2
IgG1-7D8-K439E*
2
361.0
81.0
IgG1-7D8-Y436D*
2
468.0
52.0
IgG1-7D8-Q438D*
2
717.0
70.0
IgG1-7D8-Y436C*
2
(1274.0)
1621.0
IgG1-7D8-H433D*
2
(1961.0)
1037.0
IgG1-7D8-I253D/H433A*
2
(5291.0)
7106.0
*No reliable fitting curve.
Italicized KD values could not be measured due to too weak binding of these mutants.
TABLE 13
correlation between C1q binding on Daudi cells (Example 4)
and C1q efficacy assays on Raji cells (Example 5), and
between C1q binding on Daudi cells and CDC assays
on Daudi and Raji cells (Example 06). Data were log
transformed before the correlation was analyzed.
Parameter
C1q efficacy Raji
CDC Raji
CDC Daudi
Number of XY
21
21
21
Pairs
Pearson r
0.8600
0.8668
0.8959
95%
0.6812 to 0.9420
0.6952 to 0.9449
0.7569 to 0.9573
confidence
interval
P value
<0.0001
<0.0001
<0.0001
(two-tailed)
P value
***
***
***
summary
Is the
Yes
Yes
Yes
correlation
significant?
(alpha = 0.05)
R squared
0.7396
0.7513
0.8026
Example 8
Design and Generation of CD38 Antibody 005 Mutants
The human monoclonal antibody HuMab 005 is a fully human IgG1,κ antibody described in WO/2006/099875. Here, it was used as a model antibody for validation of the identified Fc mutations to enhance CDC activity. The tested mutations are listed in Table 14.
DNA constructs for the different mutants were prepared and transiently transfected as described in Example 1, using the heavy chain of HuMab 005 with IgG1m(f) allotype as a template for mutagenesis reactions.
TABLE 14
set of mutations that were introduced in the CH2—CH3
domain of 005 (HuMax-CD38).
Charge
Charge
Mutation
WT aa
mutant aa
I253D
=
−
E345R
−
+
H433A
δ+
=
K439E
+
−
S440K
=
+
(=) no charge
(−) negative charge
(+) positive charge
(δ+) partial positive charge
Example 9
CD38 Binding on Cells by HuMab-005 Mutants
Binding of unpurified antibody samples to CD38-positive Daudi and Raji cells was analyzed by FACS analysis. 105 cells were incubated in 100 μL in polystyrene 96-well round-bottom plates with serial dilutions of antibody preparations (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg/mL) in RPMI1640/0.1% BSA at 4° C. for 30 min. After washing twice in RPMI1640/0.1% BSA, cells were incubated in 50 μL with FITC-conjugated rabbit F(ab′)2 anti-human IgG (cat. no. F0056; DAKO; 1:150) at 4° C. for 30 min. Next, cells were washed twice in PBS/0.1% BSA/0.02% azide, resuspended in 100 μL PBS/0.1% BSA/0.02% azide and analyzed on a FACS Cantoll (BD Biosciences). Binding curves were analyzed using GraphPad Prism V5.01 software. As a negative control, supernatant of mock-transfected cells was used.
Binding of HuMab 005 to Daudi cells was not much affected by the introduction of point mutations in the CH2-CH3 domain. All tested antibodies bound Daudi cells in a dose-dependent manner. Binding was similar to wild type HuMab-005 for all tested mutants, with the exception of 005-E345R, which showed slightly decreased binding. However, without being bound by any theory, the lower binding might be a result of decreased binding by the secondary antibody, analogous to IgG1-7D8-E345 in Example 2. The actual binding avidity by 005-E345R might be similar or even increased compared 005-WT, however we could not confirm this because of lack of directly labeled antibodies.
Binding of HuMab-005 to Raji cells was also not much affected by the introduction of point mutations in the CH2-CH3 domain. All tested antibodies bound Raji cells in a dose-dependent manner. Maximal binding was similar to that of wild type 005 for the 005-I253D and H433A mutants and lower for the 005-E435R, K439E, S440K mutants and the combination of 005-K439E+005-S440K. However, without being bound by any theory, the lower binding might be a result of decreased binding by the secondary antibody, analogous to IgG1-7D8-E345R in example 2 (shielding of the epitope).
Example 10
CDC Assay on CD38-Positive Cells by Mutants of the CD38 Antibody 005
0.1×106 Daudi or Raji cells were pre-incubated in round-bottom 96-well plates with a concentration series of unpurified antibodies (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg/mL) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of C1q (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
The CDC enhancing capacity of the E435R mutation, which was shown to enhance CDC activity of both 7D8 and 005 antibodies on Daudi and Raji cells, was further analyzed on Wien133 cells with different concentration normal human serum (NHS). 0.1×106 Wien133 cells were pre-incubated for 15 min on a shaker at RT in round-bottom 96-well plates with a concentration series of unpurified antibodies (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg/mL) in a total volume of 50 μL. Next, NHS was added as a source of C1q to reach a final concentration of either 20% or 50% NHS in a total volume of 100 μL. The reaction mixture was incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
Identified mutations in the CH2-CH3 region that resulted in either loss or increased CDC activity for the CD20 antibody 7D8, were found to have the same effect on the 005 antibody recognizing CD38. FIG. 9 shows that 005-I253D, H443A, K439E and S440K showed complete loss of CDC activity on both Daudi (FIG. 9A) and Raji (FIG. 9B) cells, whereas the 005-E345R mutant showed strongly enhanced CDC activity on both cell lines. Comparable to 7D8 data, a combination of 005-K439E+005-S440K, which both result in loss of CDC as a single mutant, resulted in restored CDC. Surprisingly, 005-E435R even strongly induced CDC on Wien133 cells, for which wild type 005 is not capable to induce killing by CDC (FIG. 9C). CDC killing by 005-E345R on Wien133 cells was observed with both 20% and 50% serum concentrations (FIG. 9C). Also on Raji cells, both 7D8-E345R and 005-E345R showed enhanced CDC in vitro in 50% serum, with similar efficacy as in 20% serum (FIG. 9D).
As the E345R mutation in the CH2-CH3 region resulted in enhanced CDC activity in both the tested CD20 antibody 7D8 and CD38 antibody 005, the E345R mutation is considered to be a general antibody modification that can be applied to induce or enhance CDC.
Example 11
IgG1 Antibodies Containing the CDC-Enhancing Mutation E345R are Less Sensitive to Inhibition of CDC by Fc Binding Peptide DCAWHLGELVWCT than Wild Type Antibodies
By mutating amino acid positions in the hydrophobic patch at the Fc:Fc interface of IgG, CDC efficacy was found to be either disturbed or enhanced. The involvement of the interactions at the Fc-Fc interface, and thus possibly the formation of an oligomeric (e.g., hexameric ring) structure as observed in the b12 crystal structure, in CDC efficacy was further explored. Therefore, a 13-residue peptide (DCAWHLGELVWCT (SEQ ID NO:7)) was used that targets a consensus binding site in the hydrophobic patch region on the surface of wild type IgG Fc (Delano et al., Science 2000 Feb. 18; 287(5456):1279-83). Indeed, the identification of the consensus binding site on the surface of IgG Fc as an adaptive region that is primed for interaction with a variety of distinct molecules (Delano et al., Science 2000 Feb. 18; 287(5456):1279-83), is consistent with the identification of the core amino acids in the hydrophobic patch that are involved in the Fc-Fc interaction in the IgG1 b12 crystal structure (Saphire et al., Science 2001 Aug. 10; 293(5532):1155-9). Interactions that are present in all of the binding interfaces are mediated by a shared set of six amino acids (Met-252, Ile-253, Ser-254, Asn-434, His-435, and Tyr-436), as well as shared backbone contacts (Delano et al., Science 2000 Feb. 18; 287(5456):1279-83). Accordingly, the Fc binding peptide is expected to affect the Fc-Fc interaction and consequently CDC efficacy.
0.1×106 Daudi cells were pre-incubated in 75 μL with 1.0 μg/mL unpurified antibody in round-bottom 96-well plates for 10 min at room temperature on a shaker. 25 μL of a concentration series (range 0.06-60 μg/mL final concentration) of the Fc binding peptide DCAWHLGELVWCT was added to the opsonized cells and incubated for 10 min on a shaker at RT. Next, 25 μL NHS was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by adding 25 μL ice cold RPMI medium, supplemented with 0.1% BSA. 15 μL propidium iodide was added and cell lysis was determined by FACS analysis.
CDC mediated by wild type 005 (FIG. 10A) or 7D8 (FIG. 10B) was found to be inhibited by the Fc-binding peptide DCAWHLGELVWCT in a dose-dependent manner. These competition data suggest again the involvement of the Fc-Fc interactions at the hydrophobic patch of IgG in CDC efficacy. The CDC-enhanced IgG1-005-E345R and IgG1-7D8-E345R mutants were both less sensitive for competition by the Fc-binding peptide compared to their corresponding wild type antibodies, suggesting that the E345R mutation results in increased stability of the Fc-Fc interaction, and consequently increased CDC.
Example 12
Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) of CD38 Expressing Cells by Variants of CD38 Antibody HuMAb 005
Daudi cells were harvested (5×106 cells/ml), washed (twice in PBS, 1200 rpm, 5 min) and collected in 1 mL RPMI 1640 medium supplemented with 10% cosmic calf serum (CCS) (HyClone, Logan, Utah, USA), to which 200 μCi 51Cr (Chromium-51; Amersham Biosciences Europe GmbH, Roosendaal, The Netherlands) was added. The mixture was incubated in a shaking water bath for 1 hour at 37° C. After washing of the cells (twice in PBS, 1200 rpm, 5 min), the cells were resuspended in RPMI 1640 medium supplemented with 10% CCS, counted by trypan blue exclusion and diluted to a concentration of 1×105 cells/mL.
Meanwhile, peripheral blood mononuclear cells (PBMCs) were isolated from fresh buffy coats (Sanquin, Amsterdam, The Netherlands) using standard Ficoll density centrifugation according to the manufacturer's instructions (lymphocyte separation medium; Lonza, Verviers, France). After resuspension of cells in RPMI 1640 medium supplemented with 10% CCS, cells were counted by trypan blue exclusion and concentrated to 1×107 cells/m L.
For the ADCC experiment, 50 μL 51Cr-labeled Daudi cells (5.000 cells) were pre-incubated with 15 μg/mL CD38 antibody IgG1-005 or mutant IgG1-005-E345R in a total volume of 100 μL RPMI medium supplemented with 10% CCS in a 96-well microtiter plate. After 10 min at RT, 50 μL PBMCs (500.000 cells) were added, resulting in an effector to target ratio of 100:1. The maximum amount of cell lysis was determined by incubating 50 μL 51Cr-labeled Daudi cells (5,000 cells) with 100 μL 5% Triton-X100. The amount of spontaneous lysis was determined by incubating 5,000 51Cr-labeled Daudi cells in 150 μL medium, without any antibody or effector cells. The level of antibody-independent cell lysis was determined by incubating 5,000 Daudi cells with 500,000 PBMCs without antibody. Subsequently, the cells were incubated 4 hr at 37° C., 5% CO2. To determine the amount of cell lysis, the cells were centrifuged (1200 rpm, 3 min) and 75 μL of supernatant was transferred to micronic tubes, after which the released 51Cr was counted using a gamma counter. The measured counts per minute (cpm) were used to calculate the percentage of antibody-mediated lysis as follows:
(cpm sample−cpm Ab-independent lysis)/(cpm max. lysis−cpm spontaneous lysis)×100%
Table 15 shows the calculated EC50 values of IgG1-005-wt and IgG1-005-E345R in the performed ADCC assay. Four samples were tested. IgG1-005-E345R shows a significant lower EC50 value than IgG1-005-wt of all four tested samples.
TABLE 15
Calculated EC50 values of the four performed experiments.
IgG1-005-wt
IgG1-005-E345R
ADCC
EC50
EC50
A
5.7
1.2
B
8.3
4.0
C
14.1
4.1
D
5.0
0.6
average
8.3
2.5
ng/ml
SEM
4.1
1.9
TTEST
2-tail
P =
0.04
Factor enhanced
3.3
times
FIG. 11 shows that compared to wild type antibody HuMab-005, mutant IgG1-005-E345R demonstrated enhanced efficacy of ADCC capacity, being able to induce ADCC at lower concentrations.
Example 13
FcRn Binding and Pharmacokinetic Analysis of 7D8 Mutants Compared to Wild Type 7D8
The neonatal Fc receptor (FcRn) is responsible for the long plasma half-life of IgG by protecting IgG from degradation. After internalization of the antibody, FcRn binds to antibody Fc regions in endosomes, where the interaction is stable in the mildly acidic environment (pH 6.0). Upon recycling to the plasma membrane, where the environment is neutral (pH7.4), the interaction is lost and the antibody is released back into the circulation. This influences the plasma half-life of IgG.
The capability of the 7D8 mutant IgG1-7D8-E354R to interact with FcRn from mouse, cynomolgus monkey and human was tested in an ELISA. All incubations were done at room temperature. 96 well plates were coated with 5 μg/mL (100 μL/well) recombinantly produced biotinylated extracellular domain of FcRn (mouse, human or cynomolgus) (FcRnECDHis-B2M-BIO), diluted in PBST plus 0.2% BSA; 1 hour. Plates were washed 3 times with PBST, and 3-fold serially diluted (in PBST/0.2% BSA, pH 6.0) wild type IgG1-7D8 or IgG1-7D8-E354R was added, and plates were incubated for 1 hour. Plates were washed with PBST/0.2% BSA, pH 6.0. Goat-anti-human IgG(Fab′2)—HRP (Jackson Immuno Research, cat no:109-035-097) diluted in PBST/0.2% BSA, pH 6.0 was added, and plates were incubated for 1 hour. After washing, ABTS was added as substrate and plates were incubated in the dark for 30 minutes. Absorbance was read at 405, using an EL808 ELISA reader.
The mice in this study were housed in a barrier unit of the Central Laboratory Animal Facility (Utrecht, The Netherlands) and kept in filter-top cages with water and food provided ad libitum. All experiments were approved by the Utrecht University animal ethics committee.
To analyse pharmacokinetics of the 7D8 mutants in vivo, SCID mice (C.B-17/IcrCrl-scid-BR, Charles-River) were injected intravenously with 100 μg (5 mg/kg) wild type 7D8, IgG1-7D8-E354R, —S440K or K322A; 3 mice per group.
50 μL blood samples were collected from the saphenous vein at 10 minutes, 4 hours, 24 hours, 2 days, 7 days, 14 days and 21 days after antibody administration. Blood was collected into heparin containing vials and centrifuged for 5 minutes at 10,000 g. Plasma was stored at −20° C. until determination of mAb concentrations.
Human IgG concentrations were determined using a sandwich ELISA. Mouse mAb anti-human IgG-kappa clone MH16 (# M1268, CLB Sanquin, The Netherlands), coated to 96-well Microlon ELISA plates (Greiner, Germany) at a concentration of 2 μg/mL was used as capturing antibody. After blocking plates with PBS supplemented with 2% chicken serum, samples were added, serially diluted in ELISA buffer (PBS supplemented with 0.05% Tween 20 and 2% chicken serum), and incubated on a plate shaker for 1 h at room temperature (RT). Plates were subsequently incubated with goat anti-human IgG immunoglobulin (#109-035-098, Jackson, West Grace, Pa.) and developed with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany). Absorbance was measured in a microplate reader (Biotek, Winooski, Vt.) at 405 nm.
SCID mice were chosen because they have low plasma IgG concentrations and therefore relatively slow clearance of IgG. This provides a PK model that is very sensitive for detecting changes in clearance due to diminished binding of the Fcγ-part to the neonatal Fc receptor (FcRn).
Statistical testing was performed using GraphPad PRISM version 4 (Graphpad Software).
FIG. 12 shows that both wild HuMab-7D8 and IgG1-7D8-E345R bound well to mouse, human and cynomolgus FcRn. Binding of IgG1-7D8-E345R was slightly better than that of wild type 7D8.
FIG. 13 shows the plasma concentrations in time. There was no difference in the change of plasma concentrations (clearance) over time of wild type HuMab-7D8 versus either one of IgG1-7D8-E345R, —S440K or K322A.
Example 14
Use of the Fc-Fc Stabilizing Mutation E345R for Increased Bactericidal Activity of IgG Antibodies Against Bacteria that Express Fc-Binding Surface Proteins
The complement cascade system is an important host defense mechanism against pathogens and can be divided in three different activation routes to recognize pathogens: i) the antibody-mediated classical pathway, which is activated upon C1q binding to the pathogen-bound antibody, ii) the lectin and iii) the alternative pathway, in which the complement system directly recognizes and is triggered by the pathogen in the absence of antibody. The three pathways converge at the step of C3 cleavage and C3b deposition. Microorganisms have developed multiple mechanisms of complement evasion, one of which is mediated by Protein A (Joiner Ann. Rev. Microbiol. (1988) 42:201-30; Foster Nat Rev Microbiol (2005) December; 3(12):948-58). Protein A was first identified in the cell wall of Staphylococcus aureus and is well known for its binding to the Fc region of IgG (Deisenhofer et al., Biochem (1981) 20, 2361-70; Uhlen et al., J. Biol. Chem. (1984) 259, 1695-1702). So far, the antiphagocytotic effect of Protein A and its role in the pathogenesis of S. aureus was explained by the interaction between Protein A and IgG, which results in an incorrect antibody orientation to be recognized by the neutrophil Fc receptor (Foster Nat Rev Microbiol (2005) December; 3(12):948-58).
Example 11 shows that CDC mediated by B cell-specific IgG1 antibodies was inhibited by the competing Fc-binding peptide DCAWHLGELVWCT. The peptide targets the consensus binding site on IgG Fc that coincides with the binding site for Protein A, Protein G and rheumatoid factor (Delano et al., Science 2000 Feb. 18; 287(5456):1279-83). Based on these data, it is believed that the Protein A-mediated bacterial complement evasion mechanism could work by competing for Fc binding, resulting in destabilization of the Fc-Fc interaction of a microbe-specific antibody, and consequently inhibition of antibody-mediated complement activation. Moreover, Example 11 also shows that B cell-specific IgG1 antibodies containing the CDC-enhancing E345R mutation were less sensitive to inhibition of CDC by the competing Fc-binding peptide DCAWHLGELVWCT than the parent wild type antibodies. By extrapolating these results to Fc binding proteins expressed on microbes, increased stabilization of the IgG1 Fc-Fc interactions by the E345R mutation would make microbe-specific antibodies less prone to complement inhibition by an escape strategy of the pathogen via Fc binding competition by microbial surface proteins, such as Protein A. Consequently, introduction of the E345R mutation in IgG antibodies directed against a bacterium would result in increased C3b deposition on bacteria and increased bactericidal activity compared to the parent wild type antibodies.
As an in vitro measure for complement-mediated bacterial killing, both phagocytosis by neutrophils and the generation of C3a in the plasma, which coincides with C3b deposition on the bacteria, can be determined as described below. Indeed, it has been described that C3b deposition on S. aureus results in enhanced phagocytosis and correlates with bacterial killing (Rooijakkers et. al., Nature Immunology 2005: 6, 920-927).
S. aureus will be labelled with FITC by incubating an exponentially growing bacterial culture with 100 μg/mL FITC for 1 h at 37° C. in 0.1 M carbonate buffer (pH 9.6). Human polymorph nuclear cells (PMN) will be isolated using a Ficoll gradient. FITC-labelled bacteria will be opsonized with a concentration series of specific antibodies with or without the mutation E345R. Phagocytosis will be performed in vitro by incubating 1×108 opsonized FITC-labelled bacteria with human PMN in the presence of 25% IgG-depleted serum as complement source for 25 min at 37° C. in a total volume of 200 μL under vigorous shaking. The cells will be fixed and erythrocytes lyzed by incubation with BD FACS lysing solution for 15 min at room temperature. After washing, phagocytosis will be measured by FACS. The neutrophil population will be selected through forward and side scatter gating and phagocytosis will be expressed as the mean fluorescence in the neutrophil population. Alternatively, C3a generation will be measured in the samples by ELISA as a measure for complement activation and C3b deposition.
It is expected that the S. aureus-specific antibodies containing the E345R mutation will induce more complement activation and phagocytosis by neutrophils than the parent wild type antibodies. An example of an antibody that could be used in such experiments is the chimeric monoclonal IgG1 pagibaximab (BSYX-A110; Biosynexus), targeting Lipoteichoic acid (LTA) that is embedded in the cell wall of staphylococci (Baker, Nat Biotechnol. 2006 December; 24(12):1491-3; Weisman et al., Int Immunopharmacol. 2009 May; 9(5):639-44).
Example 15
Use of CDC-Inhibiting Mutations that Restrict CDC Activation to Target Cells Simultaneously Bound by a Mixture of Two Different Therapeutic Monoclonal Antibodies
As described in Example 6, CD20 antibody 7D8 mutations K439E and S440K decreased the CDC efficacy as monoclonal antibodies. Mixing 7D8 antibodies containing these mutations restored CDC. Efficient CDC was thus restricted to cells bound by both mutant antibodies simultaneously. As described in Example 10, CD38 antibody 005 mutations K439E and S440K decreased the CDC efficacy as monoclonal antibodies. Mixing 005 antibodies containing these mutations restored CDC. Efficient CDC was thus restricted to cells bound by both mutant antibodies simultaneously.
It can be advantageous to restrict the induction of efficient CDC to target cells that express two specific antigens simultaneously, exploiting their combined expression to improve selectivity of CDC induction. To restrict CDC induction to cells bound by both CD20 and CD38 antibodies simultaneously, the pair of 7D8-K439E and 005-S440K or the pair of 7D8-S440K and 005-K439E will be added separately or mixed 1:1 in CDC experiments as follows. 0.1×106 Daudi or Raji cells will be pre-incubated in round-bottom 96-well plates with a concentration series of unpurified antibodies or antibody mixture (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg/mL) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum will be added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction will be stopped by putting the plates on ice. 10 μL propidium iodide will be added and cell lysis will be determined by FACS. It is expected, that 7D8-K439E, 005-S440K, 7D8-S440K and 005-K439E will display limited CDC efficacy. It is expected, that the simultaneous addition of 7D8-K439E and 005-S440K will restore efficient CDC specifically on cells expressing both CD20 and CD38. Likewise, it is expected that the mixture of 7D8-S440K and 005-K439E will restore efficient CDC specifically on cells expressing both CD20 and CD38.
Example 16
Increased Specificity of Enhanced CDC by Combining E345R with Complementary Inhibiting Mutations K439E and S440K in a Mixture of Two Different Monoclonal Antibodies
As described in Example 6, CD20 antibody 7D8 mutations K439E and S440K decreased the CDC efficacy as monoclonal antibodies. Mixing 7D8 antibodies containing these mutations restored CDC. Efficient CDC was thus restricted to cells bound by both mutant antibodies simultaneously. As described in Example 10, CD38 antibody 005 mutations K439E and S440K decreased the CDC efficacy as monoclonal antibodies. Mixing 005 antibodies containing these mutations restored CDC. Efficient CDC was thus restricted to cells bound by both mutant antibodies simultaneously.
It can be advantageous to restrict the enhancement of CDC induction to target cells that express two specific antigens simultaneously, exploiting their combined expression to improve selectivity of enhanced CDC induction. It can also be advantageous to restrict the enhancement of CDC induction to target cells that are bound by mixtures of at least two different antibodies simultaneously, said antibodies binding an identical cell surface antigen at two different epitopes simultaneously, or at two cross-competing, similar, or identical epitopes.
Therefore, to restrict enhanced CDC induction to cells bound by both CD20 and CD38 antibodies simultaneously, the CDC enhancing mutation E345R was combined with CDC inhibiting mutations in the antibodies 7D8-E345R/K439E, 7D8-E345R/S440K, 005-E345R/S440K and 005-E345R/K439E. These antibodies were added separately or mixed 1:1 in CDC experiments as follows. 0.1×106 Wien133 cells (other cell types such as Daudi or Raji cells may also be used) were pre-incubated in round-bottom 96-well plates with a concentration series of unpurified antibodies (final concentration 0.056-10,000 ng/mL in 3-fold dilutions for 7D8-E345R/K439E, 7D8-E345R/S440K, 005-E345R/S440K or 005-E345R/K439E) or antibody mixtures (final concentrations 0.01 μg/mL CD20 antibody mixed with 0-333 ng/mL in 3-fold dilutions CD38 antibody; or 3.3 μg/mL CD38 antibody mixed with 0.0056-1,000 ng/mL in 3-fold dilutions CD20 antibody) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
A concentration series of 005-E345R/K439E or 005-E345R/S440K antibody was mixed with a fixed concentration of 0.01 μg/mL 7D8 double mutant antibody (maximal concentration with minimal CDC on Wien133 cells as a single agent as determined from FIG. 14A) to make the complementary combinations 005-E345R/K439E+7D8-E345R/S440K or 005-E345R/S440K+7D8-E345R/K439E. FIG. 14C shows that the 005 double mutant CD38 antibodies induced CDC dose-dependently in the presence of fixed concentration of the complementary 7D8-E345R/K439E or 7D8-E345R/S440K CD20 antibody, respectively. The CDC efficacy by these complementary combinations (FIG. 14C) was comparable to the 005-E345R single mutant (enhancer) antibody as a single agent (FIG. 14B). In contrast, in the presence of irrelevant antibody b12, both 005-E345R/K439E and 005-E345R/S440K showed hardly any CDC in the concentration series tested (comparable to 005-E345R/K439E or 005-E345R/S440K as single agents shown in FIG. 14B).
A concentration series of 7D8-E345R/K439E or 7D8-E345R/S440K antibody was mixed with a fixed concentration of 3.3 μg/mL 005 double mutant antibody (showing a little but limited CDC on Wien133 cells as a single agent as determined from FIG. 14B) to make the complementary combinations 7D8-E345R/K439E+005-E345R/S440K or 7D8-E345R/S440K+005-E345R/K439E. FIG. 14D shows that the 7D8 double mutant CD20 antibodies induced CDC very efficiently in the presence of the complementary 005-E345R/K439E or 005-E345R/S440K CD38 antibody respectively, even at the lowest concentrations tested, resembling not more than a few 7D8 double mutant antibody molecules per cell. To eliminate the contribution of increased Fc-tail density on the cell membrane to the observed enhanced CDC by the mixture of 7D8 and 005 antibodies with complementary K439E and S440K mutations, also antibody combinations with non-complementary mutations were tested. FIG. 14D shows that non-complementary combinations showed much lower CDC efficacy than complementary combinations, as a result of less efficient Fc-Fc interaction than the complementary combinations.
These data suggest that the induction of (enhanced) CDC by therapeutic antibodies can be limited to cells that bind simultaneous a mixture of two complementary antibodies, in this case with different antigen specificities, thereby increasing target cell specificity by requiring co-expression of both antigens.
As can be seen in FIGS. 14A and 14B, 7D8-E345R/K439E, 005-E345R/S440K, 7D8-E345R/S440K and 005-E345R/K439E displayed limited CDC efficiency in comparison to 7D8-E345R alone. It is further seen, that the mixture of 7D8-E345R/K439E and 7D8-E345R/S440K enabled CDC with enhanced efficiency compared to wildtype 7D8 antibody as single agent. Likewise, it was observed that the mixture of 005-E345R/K439E and 005-E345R/S440K enabled CDC with enhanced efficiency compared to wildtype 005 antibody as single agent (data not shown).
Example 17
Use of CDC-Inhibiting Mutations that Restrict Efficient CDC Activation to Antibody Complexes Exclusively Consisting of Therapeutically Administered Antibodies
As described in Example 6, the CD20 antibody 7D8 double mutant K439E/S440K restored the CDC efficiency diminished by K439E or S440K single point mutants. As described in Example 10, the CD38 antibody 005 double mutant K439E/S440K restored the CDC efficiency inhibited by K439E or S440K single point mutants. As observed, the single point mutations disrupt the Fc:Fc interaction with the unmutated amino acid on the facing side of the Fc:Fc interface. Introduction of the compensatory mutation on the facing side of the Fc:Fc interface restored CDC efficiency. Efficient CDC was thus apparently restricted to antibody complexes exclusively consisting of antibodies containing both mutations.
In another example, the induction of CDC is restricted to antibody complexes exclusively consisting of therapeutically administered antibodies. To restrict CDC induction to cells bound by therapeutically CD20 or by CD38 antibodies exclusively, the CDC inhibiting mutations K439E and S440K will be combined in the antibodies 7D8-K439E/S440K or 005-K439E/S440K. These antibodies will be added separately in CDC experiments in the absence or presence of non-target specific IgG as follows. 0.1×106 Daudi or Raji cells will be pre-incubated in round-bottom 96-well plates with a concentration series of unpurified antibodies or antibody mixture (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg/mL) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum will be added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction will be stopped by putting the plates on ice. 10 μl propidium iodide will be added and cell lysis will be determined by FACS.
It is expected, that 7D8-K439E/S440K will induce CDC with efficiency similar to wildtype 7D8 antibody. Addition of non-specific IgG to 7D8-K439E/S440K is expected not to affect the efficiency of CDC induction for this antibody. Likewise, it is expected that 005-K439E/S440K will enable CDC with efficiency similar to wildtype HuMAb 005. Addition of non-specific IgG to 005-K439E/S440K is expected not to affect the efficiency of CDC induction for this antibody.
Example 18
Use of CDC-Inhibiting Mutations that Restrict Enhanced CDC Activation to Antibody Complexes Exclusively Consisting of Therapeutically Administered Antibodies
As described in Example 6, the CD20 antibody 7D8 double mutant K439E/S440K restored the CDC efficiency diminished by K439E or S440K single point mutants. As described in Example 10, the CD38 antibody HuMAb 005 double mutant K439E/S440K restored the CDC efficiency inhibited by K439E or S440K single point mutants. As observed, the single point mutations disrupt the Fc:Fc interaction with the unmutated amino acid on the facing side of the Fc:Fc interface. Introduction of the compensatory mutation on the facing side of the Fc:Fc interface restored CDC efficiency. Efficient CDC was thus apparently restricted to antibody complexes exclusively consisting of antibodies containing both mutations.
In another example, the enhancement of CDC induction is restricted to antibody complexes exclusively consisting of therapeutically administered antibodies. By screening and selection of mutations that stimulate the Fc:Fc interaction exploited for CDC stimulation, one could identify mutations that can form CDC-inducing antibody complexes with serum antibodies not specific for the antigen target of interest. To restrict enhanced CDC induction to cells bound by complexes of CD20 or by CD38 antibodies exclusively, the CDC enhancing mutation E345R will be combined with CDC inhibiting mutations in the antibodies 7D8-E345R/K439E/S440K or 005-E345R/K439E/S440K. These antibodies will be added separately in CDC experiments in the absence or presence of non-target specific IgG as follows. 0.1×106 Daudi or Raji cells will be pre-incubated in round-bottom 96-well plates with a concentration series of unpurified antibodies or antibody mixture (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg/mL) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum will be added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction will be stopped by putting the plates on ice. 10 μl propidium iodide will be added and cell lysis will be determined by FACS.
It is expected that 7D8-E345R/K439E/S440K will induce CDC with enhanced efficiency compared to wildtype HuMAb 7D8. Addition of non-specific IgG to 7D8-E345R/K439E/S440K is expected not to affect the efficiency of CDC induction compared to wildtype 7D8 antibody. Likewise, it is expected that the 005-E345R/K439E/S440K will enable CDC with enhanced efficiency compared to wildtype 005 antibody. Addition of non-specific IgG to 005-E345R/K439E/S440K is expected not to affect the efficiency of CDC induction relative to wildtype 005 antibody.
Example 19
Use of a Mutant Screening Approach to Identify Mutations Stimulating Fc:Fc Interaction Mediated Antibody Oligomerization Detected by a CDC Assay
As described in Examples 6 and 10, amino acid mutations were identified that stimulated CDC for antibodies recognizing two different target antigens, CD20 and CD38, on multiple cell lines expressing variable levels of said antigens. Surprisingly, the single point mutation E345R proved sufficient to endow CDC-dependent cell lysis of Wien133 cells to the anti-CD38 antibody 005, which failed to lyse these cells by CDC in wild type IgG1 format.
Other mutations on or at the periphery of the Fc:Fc interface could stimulate oligomerization and CDC in an analogous fashion. Alternatively, mutations could indirectly stimulate oligomerization, for example by allosterically inducing Fc:Fc interactions.
To determine if other amino acid mutations could stimulate Fc-mediated antibody oligomerization, a library of anti-CD38 IgG1-005 mutants was screened using CDC assays, both individually and mixed in a pairwise fashion to select for example amino acid pairs interacting across the Fc:Fc interface. However, the same strategy can be applied to other antibodies, such as another IgG1 or an IgG3 antibody.
A focused library of mutations at the positions indicated in Table 15 was generated. Mutations were introduced into the IgG1-005 Fc region using the Quikchange site-directed mutagenesis kit (Stratagene, US). Briefly, for each desired mutation position, a forward and a reverse primer encoding a degenerate codon at the desired location were used to replicate full length plasmid DNA template of the 005 heavy chain with IgG1m(f) allotype. The resulting DNA mixtures were digested using DpnI to remove source plasmid DNA and used to transform E. coli. Resulting colonies were pooled and cultured and plasmid DNA was isolated from these pools and retransformed into E. coli to obtain clonal colonies. Mutant plasmid DNA isolated from resulting colonies was checked by DNA sequencing (LGC genomics, Berlin, Germany). Expression cassettes were amplified from plasmid DNA by PCR and DNA mixes containing both a mutant heavy and a wildtype light chain of IgG1-005 were transiently transfected to Freestyle HEK293F cells (Invitrogen, US) using 293fectin (Invitrogen, US) essentially as described by the manufacturer. Supernatants of transfected cells containing antibody mutants were collected. Mutant antibody supernatants were screened in CDC assays both individually and in pairwise mixtures as follows.
0.1×106 Daudi or Wien-133 cells (other cells types such as Raji cells may be used) were pre-incubated in round-bottom 96-well plates with 1.0 ug/ml of unpurified antibodies in a total volume of 100 μL for 15 min on a shaker at RT. Next, 30 μL normal human serum was added as a source of complement (30% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μl propidium iodide was added and cell lysis was determined by FACS.
Mutations described in Table 16, Table 17 and Table 18 were selected for their ability to enhance oligomerization as detected by CDC efficiency, either as a single mutant or when mixed with other mutants for example facing the mutation across the Fc:Fc interface. Mutations can optionally be further screened for their ability to not compromise FcRn, Protein-A or Protein-G binding, ADCC, ADCP or other effector functions mediated by the Fc domain. Combining such stimulating point mutations into one Fc domain can stimulate oligomerization and CDC efficiency even further.
Mutations in the CH2-CH3 region incorporated in the CD38 antibody 005 were tested for their ability to inhibit oligomerization as determined by CDC on Daudi cells. Lysis of the mutant antibody was compared to wild type 005, for which lysis was set to 100%. The cut-off for inhibition was set to 66% lysis. Measured in this way, most of the tested mutations inhibited CDC (see Table 16).
Mutations in the CH2-CH3 region incorporated in the CD38 antibody 005 were tested for their ability to enhance oligomerization as determined by CDC on Wien133 cells (Table 17). Wild type CD38 antibody 005 is not able to induce CDC on Wien133 cells. Mutants displaying ≥39% cell lysis were scored as enhancing. Completely unexpectedly, virtually all obtained substitutions of amino acids E345 and E430 stimulated cell lysis by CDC. To verify this result, amino acids E345, E430 and S440 were substituted with each possible mutation by site directed mutagenesis and tested for their ability to enhance oligomerization as determined by CDC of Wien133 cells using a new human serum batch, yielding slightly more efficient lysis (Table 18). Again, all substitutions of E345 and E430 induced efficient CDC of Wien133 cells.
The following preferred mutations caused ≥39% cell lysis of Wien133 cells: P247G, I253V, S254L, Q311L, Q311W, E345A, E345C, E345D, E345F, E345G, E345H, E345I, E345K, E345L, E345M, E345N, E345P, E345Q, E345R, E345S, E345T, E345V, E345W, E345Y, D/E356G, D/E356R, T359R, E382L, E382V, Q386K, E430A, E430C, E430D, E430F, E430G, E430H, E430I, E430L, E430M, E430N, E430P, E430Q, E430R, E430S, E430T, E430V, E430W, E430Y, Y436I, S440Y and S440W.
TABLE 16
Percentage lysis of daudi cells in the presence of 1.0 μg/ml IgG1-005 antibody point mutations.
IgG1-005 wildtype lysed 66% of cells under these conditions. For each of the individual positions
which have been substituted by another amino acid are given in the outer left column.
The substituted amino acid for each particular position is given followed by the measured
percentage lysis indicated in paranteses ( ) in the horizontal rows of the individual positions.
Position
P247
A (42)
C (67)
D (91)
F (93)
G (95)
H (80)
I (89)
K (96)
L (13)
I253
A (17)
D (12)
K (13)
M (6)
N (5)
R (7)
S (6)
V (94)
S254
E (14)
F (75)
G (100)
H (46)
I (93)
K (86)
L (99)
P (4)
T (8)
H310
K (6)
W (87)
Q311
A (53)
C (72)
E (5)
F (90)
G (68)
H (72)
I (92)
K (93)
L (96)
E345
A (85)
C (91)
F (95)
G (86)
H (83)
I (96)
K (94)
L (98)
M (94)
D/E356
G (88)
I (95)
L (94)
R (97)
T (97)
V (98)
T359
G (88)
N (93)
P (87)
R (96)
E382
F (3)
K (3)
L (99)
M (90)
P (3)
V (96)
W (3)
G385
D (28)
H (9)
Q (24)
R (27)
S (14)
T (10)
Q386
A (56)
C (18)
D (6)
E (9)
F (11)
G (10)
H (26)
I (42)
K (98)
E430
A (97)
F (97)
G (99)
H (98)
L (95)
P (95)
Q (90)
R (96)
S (94)
N434
D (5)
E (5)
K (5)
R (5)
S (6)
W (98)
Y436
I (98)
K (7)
L (10)
R (35)
S (8)
T (7)
W (6)
Q438
E (5)
K (6)
S (5)
T (8)
W (10)
Y (31)
K439
A (6)
D (5)
H (5)
L (5)
P (8)
T (4)
Y (7)
S440
A (61)
C (10)
D (95)
E (24)
F (13)
G (40)
I (8)
N (33)
R (11)
K447
E (20)
*del (90)
Position
P247
M (83)
N (78)
R (93)
S (93)
T (10)
V (9)
W (82)
I253
S254
W (7)
H310
Q311
N (53)
P (97)
R (87)
S (66)
T (54)
W (93)
Y (85)
E345
N (97)
P (74)
R (98)
S (93)
T (82)
V (92)
W (95)
Y (95)
D/E356
T359
E382
G385
Q386
L (15)
N (25)
P (6)
R (10)
S (43)
T (12)
V (53)
W (13)
Y (42)
E430
V (98)
N434
Y436
Q438
K439
S440
T (28)
Y (98)
K447
*where “del” means that there was a deletion of the amino acid residue at the indicated position.
TABLE 17
Percentage lysis of Wien-133 cells in the presence on 1.0 μg/ml IgG1-005 antibody point
mutants. IgG1-005 wildtype lysed 3% of cells under these conditions. For each of the
individual positions which have been substituted by another amino acid are given in the
outer left column. The substituted amino acid for each particular position is given
followed by the measured percentage lysis indicated in paranteses ( ) in the horizontal
rows of the individual positions.
Position
P247
A (5)
C (5)
D (12)
F (16)
G (50)
H (11)
I (10)
K (14)
L (4)
I253
A (11)
D (9)
K (3)
M (3)
N (3)
R (4)
S (3)
V (51)
S254
E (14)
F (10)
G (32)
H (2)
I (15)
K (12)
L (65)
P (2)
T (9)
H310
K (3)
W (13)
Q311
A (9)
C (4)
E (3)
F (19)
G (4)
H (6)
I (28)
K (16)
L (55)
E345
A (57)
C (22)
F (48)
G (47)
H (49)
I (59)
K (42)
L (72)
M (67)
D/E356
G (39)
I (31)
L (30)
R (64)
T (32)
V (13)
T359
G (2)
N (3)
P (4)
R (40)
E382
F (2)
K (2)
L (44)
M (21)
P (3)
V (53)
W (2)
G385
D (5)
H (4)
N (18)
Q (4)
R (14)
S (4)
T (4)
Q386
A (3)
C (4)
D (4)
E (4)
F (3)
G (3)
H (3)
I (4)
K (60)
E430
A (54)
F (68)
G (55)
H (57)
L (58)
P (56)
Q (31)
R (39)
S (20)
N434
D (2)
E (2)
K (2)
R(2)
S (3)
W (18)
Y436
I (49)
K (3)
L (4)
R (3)
S (3)
T (2)
W (3)
Q438
E (3)
K (3)
S (2)
T (2)
W (2)
Y (2)
K439
A (3)
D (2)
H (2)
L (2)
P (2)
T (2)
Y (4)
S440
A (3)
C (3)
D (6)
E (2)
F (2)
G (3)
I (2)
N (2)
R (2)
Position
P247
M (13)
N (7)
R (10)
S (7)
T (4)
V (3)
W (9)
I253
S254
W (9)
H310
Q311
N (6)
P (12)
R (18)
S (9)
T (3)
W (41)
Y (12)
E345
P (51)
R (64)
S (60)
T (53)
V (67)
W (52)
Y (70)
D/E356
T359
E382
G385
Q386
L (3)
N (4)
P (2)
R (4)
S (3)
T (3)
V (3)
W (3)
Y (4)
E430
V (53)
N434
Y436
Q438
K439
S440
T (3)
Y (64)
TABLE 18
Percentage lysis of Wien-133 cells in the presence on 1.0 μg/ml IgG1-005
antibody point mutants. IgG1-005 wildtype lysed 12% of cells under these conditions.
Each of the individual positions which have been substituted by another amino acid
are given in the outer left column. The substituted amino acid for each particular
position is given followed by the measured percentage lysis indicated in
paranteses ( ) in the horizontal rows of the individual positions.
Position
E345
A (94)
C (87)
D (76)
F (95)
G (95)
H (94)
I (93)
K (97)
L (94)
M (96)
E430
A (95)
C (79)
D (91)
F (96)
G (96)
H (95)
I (96)
K (83)
L (94)
M (75)
S440
A (12)
C (8)
D (41)
E (9)
F (7)
G (8)
H (26)
I (7)
K (6)
L (7)
Position
E345
N (93)
P (97)
Q (98)
R (94)
S (93)
T (92)
V (96)
W (93)
Y (94)
E430
N (95)
P (97)
Q (86)
R (92)
S (96)
T (97)
V (96)
W (98)
Y (97)
S440
M (8)
N (12)
P (10)
Q (21)
R (9)
T (10)
V (7)
W (86)
Y (90)
Example 20
In Vivo Efficacy of IgG1-7D8-E345R in a Subcutaneous B Cell Lymphoma Xenograft Model
The in vivo anti-tumor efficacy of the IgG1-7D8-E345R antibody was evaluated in a subcutaneous model with Raji-luc #2D1 cells. These cells show ˜300,000 CD20 molecules per cell (determined by QIFIKIT analysis, data not shown) and high complement defense receptor expression. Cells were cultured in RPMI with 10% cosmic calf serum (HyClone, Logan, Utah), penicillin and streptomycin, 1% (v/v) sodium Pyruvate and 1 μg/mL puromycin (P-8833, Sigma, Zwijndrecht). Cells were harvested in log-phase (approximately 70% confluency). Six to eleven weeks old female SCID mice (C.B-17/IcrPrkdc-scid/CRL) were used (Charles-River). At day 0, 5×106 Raji-luc #2D1 cells in 200 μL PBS were subcutaneously injected in the right flank of each mouse. The tumor development was monitored by caliper measurement. When average tumor volume was 100 mm3 (around day 7), the mice were sorted into groups (n=9) and treated by intraperitoneal (i.p.) injection of a single dose of 50 μg antibody per mouse (2.5 mg/kg). All antibody samples were supplemented with irrelevant antibody b12 to obtain a total antibody concentration of 0.5 mg/mL. Treatment groups are shown in Table 18. Seven days after treatment, blood samples were obtained to determine human IgG serum levels to check correct antibody administration. Tumors were measured at least twice per week using caliper (PLEXX) until an endpoint tumor volume of 1500 mm3, tumors showed ulcerations or until serious clinical signs were observed.
TABLE 18
Treatment groups and dosing.
Group
Antibody
Dose
1. wild type
IgG1-7D8-WT
50 μg (=2.5 mg/kg)
2. CDC-enhancing mutant
IgG1-7D8-E345R
50 μg (=2.5 mg/kg)
3. Irrelevant Ab control
IgG1-b12
50 μg (=2.5 mg/kg)
FIG. 15A shows mean tumor growth on day 22, when all groups were still complete. Wild type antibody IgG1-7D8 slightly inhibited tumor growth compared to negative control antibody IgG1-b12, although this was not statistically significant. Only IgG1-7D8-E345R inhibited tumor growth significantly compared to the negative control antibody IgG1-b12 (one-way ANOVA analysis p<0.01).
FIG. 15B shows a Kaplan-Meier plot of the percentage mice with tumor sizes smaller then 700 mm3. Compared to mice treated with negative control antibody IgG1-b12, tumor formation was significantly delayed in mice treated with IgG1-7D8-E345R antibody (Mantel-Cox analysis p<0.01), but not in mice treated with wild type IgG1-7D8.
These data show that the E345R mutation enhanced the in vivo anti-tumor efficacy of the CD20 antibody 7D8.
Example 21
In Vivo Efficacy of IgG1-005-E345R in a Subcutaneous B Cell Lymphoma Xenograft Model
The in vivo anti-tumor efficacy of the IgG1-005-E345R antibody was evaluated in a subcutaneous model with Raji-luc #2D1 cells. These cells show ˜150,000 CD38 molecules per cell (determined by QIFIKIT analysis, data not shown) and high complement defense receptor expression. The protocol for tumor inoculation and measurement is basically the same as described in Example 20. At day 0, 5×106 Raji-luc #2D1 cells in 200 μL PBS were s.c. injected in the right flank of SCID mice. When average tumor volume was 100 mm3 (around day 7), the mice were sorted into groups (n=7) and treated by i.p. injection of a single dose of 500 μg antibody per mouse (25 mg/kg). Treatment groups are shown in Table 19. Tumors were measured until an endpoint tumor volume of 1500 mm3 or until tumors showed ulcerations or serious clinical signs were observed to avoid major discomfort.
FIG. 16A shows mean tumor growth on day 21, when all groups were still complete. Wild type antibody IgG1-005 slightly inhibited tumor growth, although this was not statistically significant. Only IgG1-005-E345R significantly inhibited tumor growth compared to the irrelevant antibody control at day 21 (One-way ANOVA p<0.05).
FIG. 16B shows a Kaplan-Meier plot of the percentage mice with tumor sizes smaller then 500 mm3. Tumor formation was significantly delayed in mice treated with IgG1-005-E345R antibody compared to mice treated with negative control antibody IgG1-b12 (Mantel-Cox analysis p<0.001) or wild type IgG1-005 (p<0.05).
These data show that introduction of the E345R mutation in the CD38 antibody 005 resulted in enhanced in vivo anti-tumor activity.
TABLE 19
Treatment groups and dosing.
Group
Antibody
Dose
1. wild type
IgG1-005-WT
500 μg (=25 mg/kg)
2. CDC-enhancing mutant
IgG1-005-E345R
500 μg (=25 mg/kg)
3. Irrelevant Ab control
IgG1-b12
500 μg (=25 mg/kg)
Example 22
Monovalent Target Binding Further Enhances the CDC Efficacy of E345R Antibodies
A molecular surface of the IgG1 hexameric ring observed in the b12 crystal structure demonstrates that for each IgG in the hexameric ring, one of the two C1q binding sites is facing upwards and the other site is facing downwards of the ring structure, and also one Fab-arm of each antibody is oriented up and one is oriented down, resulting in only one Fab-arm per antibody to take part in antigen binding, suggesting monovalent binding per antibody molecule in the hexameric antibody ring. Monovalency might bring antibodies upon antigen binding in a hexamerization compatible orientation. To test this hypothesis, the CDC efficacy of a bispecific CD38/EGFR antibody with the E345R mutation was tested on CD38-positive, EGFR-negative Wien133 cells, to which this bispecific antibody can only bind monovalently via CD38, and compared to the CDC efficacy of the bivalent binding CD38 antibody, also with the E345R mutation. The human monoclonal antibody HuMax-EGFr (2F8, described in WO 2004/056847) was used as a basis for the EGFR antibodies described in this example.
Bispecific antibodies were generated in vitro according to the DuoBody™ platform, i.e. 2-MEA-induced Fab-arm exchange as described in WO 2011/147986. The basis for this method is the use of complementary CH3 domains, which promote the formation of heterodimers under specific assay conditions. To enable the production of bispecific antibodies by this method, IgG1 molecules carrying certain mutations in the CH3 domain were generated: in one of the parental IgG1 antibody the F405L mutation, in the other parental IgG1 antibody the K409R mutation. To generate bispecific antibodies, these two parental antibodies, each antibody at a final concentration of 0.5 mg/mL, were incubated with 25 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 μL TE at 37° C. for 90 min. The reduction reaction is stopped when the reducing agent 2-MEA is removed by using spin columns (Microcon centrifugal filters, 30 k, Millipore) according to the manufacturer's protocol.
For the CDC assay, 0.1×106 Wien133 cells were pre-incubated in round-bottom 96-well plates with a concentration series of antibodies (0.01 to 10.0 μg/mL) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
FIG. 17 shows that, as expected, CD38 antibodies without the E345R mutation (wild type IgG1-005 and IgG-b12-K409R×IgG1-005-F405L) did not induce killing of Wien133 cells. Also the EGFR antibody IgG1-2F8-E345R/F405L, that did not bind the EGFR-negative Wien133 cells (data not shown), did not induce CDC, as expected. The introduction of the K409R mutation did not influence the capacity of the IgG1-005-E345R antibody to induce ˜60% killing on Wien133 cells (described in Example 10). Interestingly, the bispecific CD38/EGFR antibody IgG1-005-E345R/K409R×IgG1-2F8-E345R/F405L, which can only bind monovalently to the CD38-positive, EGFR-negative Wien133 cells, showed increased maximal CDC killing (from ˜60% to ˜100% killing).
These data show that monovalent targeting can further enhance the maximal killing capacity of antibodies containing the CDC enhancing E345R mutation. Furthermore, these data show that the E345R oligomerization enhancing mutation, as measured by enhancing CDC activity, can be applied to other antibody formats, such as DuoBody.
Example 23
The Oligomerization Enhancing E345R Mutation can be Applied to Other Antibody Formats Such as DuoBody™
The effect of the E345R mutation was tested in a bispecific antibody of the DuoBody format. CDC assays were performed with CD20/CD38 bispecific antibodies on CD20-positive, CD38-positive Wien133 and Raji cells.
Bispecific antibodies were generated as described in Example 22. For the CDC assay, 0.1×106 Wien133 or Raji cells were pre-incubated in round-bottom 96-well plates with a concentration series of antibodies (0.01 to 30.0 μg/mL) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
FIG. 18 shows that introduction of the E345R mutation enhanced CDC of the bispecific IgG1-005-F405L×IgG1-7D8-K409R antibody on Wien 133 (FIG. 18A) and Raji (FIG. 18B) cells. These data show that the E345R oligomerization enhancing mutation can be applied to other antibody formats to enhance CDC activity.
Example 24
E345R Rescues CDC by EGFR Antibody 2F8, which can be Further Enhanced by Monovalent Target Binding
As described in Examples 6, 10 and 26, E345R enhanced or rescued CDC for antibodies recognizing different hematological tumor targets (CD20 and CD38). To extend the analysis to a solid tumor antigen, the effect of E345R on the CDC capacity of the EGFR antibody 2F8 was tested on A431 epidermoid carcinoma cells. Furthermore, the effect of monovalent EGFR targeting on E345R-mediated CDC induction was tested using a bispecific EGFRxCD20 antibody (IgG1-2F8-E345R/F405L×IgG1-7D8-E345R/K409R) on EGFR-positive, CD20-negative A431 cells.
Bispecific antibodies were generated as described in Example 22. For the CDC assay, 5×106 A431 cells/mL were labeled with 100 μCi 51Cr for 1 h at 37° C. Cells were washed three times with PBS and resuspended in medium at a concentration of 1×105 cells/mL. 25,000 labeled cells were incubated in round-bottom 96-well plates with a concentration series of unpurified antibodies (0-30 μg/mL in 3-fold dilutions) in a total volume of 100 μL for 15 min at RT. Next, 50 μL normal human serum dilution was added as a source of complement (25% final concentration) and incubated in a 37° C. incubator for 1 h. Cells were spun down (3 min at 300×g) and 25 μL supernatant was added to 100 μL microscint in a white 96 well optiplate (PerkinElmer) for incubation on a shaker (750 rpm) for 15 min. 51Cr release was determined as counts per minute (cpm) on a scintillation counter. Maximum lysis (100%) was determined by the 51Cr level measured in the supernatant of Triton X-100-treated cells. Spontaneous lysis was determined by the 51Cr level measured in the supernatant of cells incubated without antibody. Specific cell lysis was calculated according to the formula: Specific lysis=100×(cpm sample−cpm spont)/(cpm max−cpm spont).
FIG. 19 shows that IgG1-2F8-E345R/F405L is able to lyse A431 cells by CDC, whereas wild type 2F8 is not capable of killing A431 cells. These data show that CDC activity can be rescued in the EGFR antibody 2F8 by introduction of the E345R mutation. This potentially extends the applicability of the CDC enhancing E345R mutation to antibodies targeting solid tumor antigens.
Bispecific EGFRxCD20 antibody IgG-2F8-E345R/F405L×IgG1-7D8-E345R/K409R, showed further enhancement of CDC on the EGFR-positive, CD20-negative A431 cells.
These data further support the hypothesis that monovalency facilitates the formation of Fc-Fc interactions and subsequent CDC induction as postulated for a CD38 binding antibody described in Example 22.
Example 25
E345R Enhances or Rescues CDC by CD38 Antibody 003 and CD20 Antibodies 11B8 and Rituximab
As described in Examples 6, 10 and 24, E345R enhances or induces CDC activity of several antibodies with different target specificities (CD20, CD38 and EGFR), as was tested on multiple cell lines expressing variable levels of said antigens. Therefore, introduction of the E345R mutation was considered to be a general mechanism to enhance or rescues CDC for existing antibodies. To further support this, the effect of the E345R mutation on CDC was tested for more antibodies with variable intrinsic CDC efficacy on Daudi and Wien133 cells: CD38 antibody 003, described in WO 2006/099875 and CD20 antibodies rituximab (type I) and 11B8 (type II), described in WO 2005/103081. CD20 antibodies can be divided in two subgroups (Beers et al. Seminars in Hematology 47, (2) 2010, 107-114). Type I CD20 antibodies display a remarkable ability to activate complement and elicit CDC by redistributing the CD20 molecules in the plasma membrane into lipid rafts, which cluster the antibody Fc regions and enabling improved C1q binding. Type II CD20 antibodies do not appreciably change CD20 distribution and without concomitant clustering, they are relatively ineffective in CDC.
0.1×106 Daudi or Raji cells were pre-incubated in round-bottom 96-well plates with a concentration series of unpurified antibodies (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0 μg/mL) in a total volume of 70 μL for 15 min on a shaker at RT. Next, 30 μL normal human serum was added as a source of C1q (30% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
FIG. 20 shows that the E345R mutation enhanced CDC for all tested antibodies on both (A) Daudi and (B) Wien133 cells. Interestingly, at the used concentrations all antibodies that did not induce CDC in the wild type format, induced CDC efficiently after introduction of the E345R mutation: CD38 mAb 003 and CD20 type II mAb 11B8 on Daudi cells, and CD38 mAbs 005 and 003 and CD20 type II mAb 11B8 on Wien133 cells. These data suggest that enhancement of antibody oligomerization, more specifically by introduction of an E345R mutation, is a general mechanism to enhance or rescue CDC by existing antibodies.
Example 26
E345R Enhances Internalization of Tissue Factor Antibodies
To test if enhanced oligomerization can induce increased antibody internalization, colocalization studies of wild type and E345R mutated Tissue Factor (TF) antibodies with the lysosomal marker LAMP1 were performed by confocal microscopy.
SK-OV-3 cells were grown on glass coverslips (thickness 1.5 micron, Thermo Fisher Scientific, Braunschweig, Germany) in standard tissue culture medium at 37° C. for 1 day. Cells were pre-incubated for 1 hour with 50 μg/mL leupeptin (Sigma) to block lysosomal activity, after which 10 μg/mL Tissue Factor (TF) antibody (WO 2010/066803) was added. The cells were incubated for an additional 1, 3 or 16 hours at 37° C. Hereafter, cells were washed with PBS and incubated for 30 minutes at room temperature (RT) with 4% formaldehyde (Klinipath). Slides were washed with blocking buffer (PBS supplemented with 0.1% saponin [Roche] and 2% BSA [Roche]) and incubated for 20 minutes with blocking buffer containing 20 mM NH4Cl to quench formaldehyde. Slides were washed again with blocking buffer and incubated for 45 minutes at RT with a cocktail of mouse-anti-human CD107a-APC (BD Pharmingen) to identify lysosomal LAMP1 and goat-anti-human IgG-FITC (Jackson) to identify TF antibodies. Slides were washed again with blocking buffer and mounted overnight on microscope slides using 20 μL mounting medium (6 gram Glycerol [Sigma] and 2.4 gram Mowiol 4-88 [0 mnilabo] was dissolved in 6 mL distilled water to which 12 mL 0.2M Tris [Sigma] pH8.5 was added followed by incubation for 10 min at 50-60° C.; mounting medium was aliquoted and stored at −20° C.). Slides were imaged with a Leica SPE-II confocal microscope (Leica Microsystems) equipped with a 63×1.32-0.6 oil immersion objective lens and LAS-AF software.
12-bit grayscale TIFF images were analyzed for colocalization using MetaMorph® software (version Meta Series 6.1, Molecular Devices Inc, Sunnyvale Calif., USA). Images were imported as stacks and background was subtracted. Identical thresholds settings were used (manually set) for all FITC images and all APC images. Colocalization was depicted as the pixel intensity of FITC in the region of interest (ROI), were the ROI is composed of all APC positive regions. To compare different slides stained with different TF antibodies, the images were normalized using the pixel intensity of APC. Mouse-anti-human CD107a-APC was used to stain the lysosomal marker LAMP1 (CD107a). The pixel intensity of LAMP1 should not differ between various TF antibodies imaged.
Normalized values for colocalization of FITC and APC are expressed as arbitrary units according to the formula [(TPI FITC×percentage colocalization)/100]×[1/TPI APC]
Percentage colocalization=TPI FITC that colocalizes with an APC pixel/TPI APC
TPI, total pixel Intensity
FIG. 21 depicts the amount of FITC pixel intensity of wild type and E345R mutated TF antibodies that overlap with APC-labeled lysosomal marker. For each antibody or condition tested, three different images were analyzed from one slide containing ˜1, 3 or >5 cells. Variation was observed between the different images within each slide. Still, it was evident that the E345R mutation for antibodies 011 and 098 resulted in increased lysosomal colocalization after 1 hour incubation, when compared with wild type 011 and 098. These results indicate that mutation E345R induces more rapid internalization and lysosomal colocalization and could therefore potentiate antibody drug conjugates.
Example 27
Enhanced CDC by E345R Mutation in Rituximab in Different B Cell Lines with Similar CD20 Expression but Different Levels of Membrane-Bound Complement Regulatory Proteins
Examples 25 and 28 show that the CDC efficacy of wild type rituximab on Daudi and Wien133 cells was enhanced by introducing the E345R mutation. This enhanced CDC efficacy results from the E345R-mediated stabilization of Fc-Fc interactions. The concomitantly formed hexameric antibody ring structure on the target cell membrane can then promote efficient generation of the membrane attack complex by facilitating the capture and concentration of activated complement components close to the cell membrane. As a result of this efficient complement activation, the inhibiting effects of membrane-bound complement regulatory proteins (mCRP) could be partly overcome. Overexpression of mCRPs, such as CD55, CD46 and CD59, is considered as a barrier for successful immunotherapy with monoclonal anti-tumor antibodies (Jurianz et al., Mol Immunol 1999 36:929-39; Fishelson et al. Mol Immunol 2003 40:109-23, Gorter et al., Immunol Today 1999 20:576-82, Zell et al., Clin Exp Immunol. 2007 December 150(3):576-84). Therefore, the efficacy of rituximab-E345R was compared to that of wild type rituximab on a series of B cell lines with different levels of the mCRPs CD46, CD55 and CD59, but comparable levels of the CD20 target expression.
The B cell lines Daudi, WIL2-S, WSU-NHL, MEC-2 and ARH-77 express comparable amounts of CD20 molecules (˜250.000 specific antibody-binding capacity—sABC) as determined by QIFIKIT analysis (data not shown). To compare the expression levels of complement regulatory proteins between these cell lines, QIFIKIT analysis was performed to determine the levels of CD46 (mouse anti-human CD46, CBL488, clone J4.48 Chemicon), CD55 (mouse anti-human CD55, CBL511, Clone BRIC216, Chemicon), and CD59 (mouse anti-human CD59, MCA1054x, clone MEM-43, Serotec).
For the CDC assay, 0.1×106 of cells were pre-incubated in round-bottom 96-well plates with a saturating antibody concentration series (0.002-40.0 μg/mL in 4-fold dilutions) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS. The maximal CDC-mediated killing was calculated from two independent experiments using the top of best-fit values of a non-linear fit in GraphPad PRISM 5.
FIG. 22A-D shows that introduction of E345R in wild type rituximab resulted in enhanced CDC efficacy as observed by an increased maximal lysis and decreased EC50 for all tested B cell lines.
FIG. 22E shows that the maximal CDC-mediated killing induced by the rituximab-E345R mutant was always higher than by wild type rituximab, independent of the expression levels of the membrane-bound complement regulatory proteins. These data indicate that introduction of E345R enhances the therapeutic potential of monoclonal antibodies as the tumor cells are less effective in evading antibody-mediated complement attack by the E345R containing antibodies.
Example 28
Comparison of CDC Kinetics for Wild Type and E345R Antibodies
Introduction of the Fc:Fc interaction stabilizing E345R mutation has been shown to enhance or rescue CDC as observed by decreased EC50 values and increased maximal lysis for different antibodies on different cell lines described in Example 6 (CD20 antibody 7D8 on Daudi and Raji), Example 10 (CD38 antibody 005 on Daudi, Raji and Wien133) and Example 25 (CD38 antibody 003 and CD20 antibodies rituximab and 11B8 on Daudi and Wien133). Next, the kinetics of the CDC reactions were analyzed to further unravel the difference in CDC efficacy between wild type and E345R antibodies.
0.1×106 Raji cells were pre-incubated in round-bottom 96-well plates with antibody at a saturating concentration (10.0 μg/mL) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for different periods of time, varying between 0 and 60 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
FIG. 23A shows that wild type CD20 antibody IgG1-7D8 showed a maximal CDC-mediated killing of 80% of the Raji cells, which was already reached after 5 min under the tested conditions. However, for IgG-7D8-E345R, 80% killing of Raji cells was observed even faster, after 3 min. Maximal lysis by IgG-7D8-E345R (95%) was also reached after 5 minutes.
FIG. 23B shows that also for wild type CD20 antibody rituximab, which is less potent than 7D8 to induce CDC on the used Raji cells, introduction of the E345R mutation resulted in faster killing of the target cells. Wild type rituximab showed a maximal CDC-mediated killing of 32%, which was reached after 20 minutes. Rituximab-E345R reached 32% killing already after approximately 3 minutes and remarkably, maximal lysis by rituximab-E345R (85%) was also reached after 20 minutes.
FIG. 23C+D shows that the used Raji cells, which are resistant for CDC-mediated killing by wild type CD38 antibodies IgG1-003 and IgG1-005, could be killed fast by introducing the E345R mutation. IgG1-003-E345R and IgG1-005-E345R showed maximal CDC (50% and 60%, respectively) already after 5 min.
In summary, E345R antibodies are more potent than their wild type counterparts, which results from a combination of higher efficacy (lower EC50), increased maximal lysis and a faster kinetics of the CDC reaction.
Example 29
Comparison of CDC Kinetics for Bispecific Antibodies with or without the E345R Mutation
In example 23 it is described that the E345R mutation can be applied to the CD38xCD20 bispecific antibody IgG1-005-F405L×IgG1-7D8-K409R that was generated by the DuoBody platform, resulting in an enhanced killing capacity as observed by a decreased EC50 in CDC assays on Raji and Wien133 cells. Next, the kinetics of the CDC reaction was analyzed to further unravel the difference in CDC efficacy between the CD38xCD20 bispecific antibodies with and without E345R.
0.1×106 Raji cells were pre-incubated in round-bottom 96-well plates with antibody at a saturating concentration (10.0 μg/mL) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for different periods of time, varying between 0 and 60 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
FIG. 24 shows that the bispecific antibody IgG1-005-F405L×IgG1-7D8-K409R induced a maximal CDC-mediated killing of 83%, which was reached after 10 minutes. Introduction of E345R resulted in an increased maximal killing by IgG1-005-E345R—F405L×IgG1-7D8-E345R-K409R (98%), which was already reached after 2 minutes. These data indicate that introducing the Fc-Fc stabilizing E345R mutation in the bispecific antibody results in an accelerated CDC-mediated killing of the target cells.
Example 30
Comparison of CDC Kinetics for Monovalent Binding Antibodies with and without E345R
Example 22 shows that monovalent target binding further enhanced the CDC efficacy of E345R antibodies as observed by increased maximal lysis with a CD38xEGFR bispecific antibody on the CD38-positive, EGFR-negative Wien133 cells. Next, the kinetics of the CDC reaction was analyzed to further unravel the difference in CDC-mediated killing capacity between monovalently binding antibodies with and without E345R.
Bispecific CD38xEGFR and CD20xEGFR antibodies, with or without the E345R mutation, were generated in vitro according to the DuoBody platform as described in Example 22. CDC efficacy of the CD38xEGFR bispecific antibodies was tested on the CD38-positive, EGFR-negative Raji cells, to which the bispecific antibodies can only bind monovalently via CD38. 0.1×106 Raji cells were pre-incubated in round-bottom 96-well plates with antibody at a saturating concentration (10.0 μg/mL) in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for different periods of time, varying between 0 and 60 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
Bispecific antibody CD38xEGFR (IgG1-005-K409R×IgG1-2F8-F405L) was also tested and induced a maximal CDC-mediated killing of 55%, which was reached after approximately 10 minutes. Introduction of E345R resulted in an increased maximal killing (96%), which was already reached within 5 minutes (data not shown).
Bispecific antibody CD20xEGFR (IgG1-7D8-K409R×IgG1-2F8-F405L) was also tested and induced a maximal CDC-mediated killing of 85%, which was reached after approximately 5 minutes. However, with the CD20xEGFR antibody with introduced E345R, 85% lysis was observed faster, after 2 minutes. Maximal lysis by the E345R CD20xEGFR antibody (97%) was also reached after 5 minutes (data not shown).
In summary, introduction of the E345R mutation in these monovalent binding antibodies resulted in more potent antibodies, which results from a combination of increased maximal lysis and a faster kinetics of the CDC reaction.
Example 31
CDC by a Combination of Therapeutic and E345R/Q386K Antibodies
As described in Example 19, mutant CD38 antibodies derived from IgG1-005 could induce efficient CDC on Wien133 cells when the E345 position of the wild type antibody was substituted to any amino acid other than Glutamate (E). This suggests that oligomerization, as a prerequisite of CDC, is hindered by the presence of the Glutamate side chain at position 345 of the antibody. Since E345 on one Fc is in close proximity to Q386 on the facing second Fc moiety in the hexameric antibody ring structure, the E345-mediated hindrance of oligomerization in a first antibody could possibly be removed by substitutions at the Q386 position of a second antibody. This would then enable E345 in the first antibody to interact better with the mutated 386 position in the second antibody in case both antibodies are combined. To test this hypothesis, CDC assays were performed on Wien133, in which wild type antibodies (IgG1-003, IgG1-005 or IgG1-11B8) were mixed with IgG1-005-E345R/Q386K or IgG1-005-E345R/Q386K/E430G as an example.
0.1×106 Wien133 cells were pre-incubated in round-bottom 96-well plates with a concentration series of unpurified IgG1-005-E345R/Q386K, IgG1-005-E345R/Q386K/E430G or control antibody (0.0001-20.0 μg/mL in 3.33-fold dilutions) in the presence or absence of 1.0 or 10.0 μg/mL wild type IgG1-003, IgG1-005 or IgG1-11B8 antibody in a total volume of 100 μL for 15 min on a shaker at RT. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
FIG. 26A/B/C shows that CD38 antibody IgG1-005-E345R/Q386K induced CDC-mediated lysis of Wien133 cells in a dose-dependent fashion (dashed line). Combining IgG1-005-E345R/Q386K with 1 or 10 μg/mL wild type CD38 antibody IgG1-003 (FIG. 26A) or wild type CD20 antibody IgG1-11B8 (FIG. 26B) resulted in an increased maximal cell lysis. Combining IgG1-005-E345R/Q386K with wild type IgG1-005 inhibited CDC in a dose-dependent fashion, possibly by competing for the binding site (FIG. 26C).
FIG. 26D/E/F shows similar results for CD38 antibody IgG1-005-E345R/Q386K/E430G.
These data indicate that wild type antibodies IgG1-003 and IgG1-11B8 participated in antibody oligomerization and CDC activation when combined with IgG1-005-E345R/Q386K or IgG1-005-E345R/Q386K/E430G. In such combinations, the hindrance of oligomerization by the E345-position that is present in the wild type antibody could be, at least partly, removed by the Q386K substitution in the mutant antibody. This application is in particular interesting to improve therapies with antibodies that are wild type in the E345 position, such as rituximab, ofatumumab, daratumumab or trastuzumab. Also, such oligomerization-inducing antibodies might promote formation of cell-bound complexes with patient-own antibodies directed against target cells like tumor cells or bacteria.
Example 19 describes multiple amino acids in addition to E345 that enhance CDC upon mutation, for example E430 and S440, of which specific mutations induced efficient CDC on Wien133 cells when incorporated in CD38 antibody IgG1-005. With the exception of I253 and Y436 mutants, the identified oligomerization-enhancing mutations contact unmutated amino acids on the facing second Fc moiety in the hexameric ring structure. Therefore, the identified oligomerization-enhancing mutations, both alone or combined, can be expected to also promote oligomerization with unmutated antibodies, and further optimization of such mutants could be achieved by a selection strategy similar to that applied in example 19.
Example 32
E345R Induced CDC in IgG2, IgG3 and IgG4 Antibody Isotypes
To test if the introduction of oligomerization-promoting mutations can stimulate the CDC activity of non-IgG1 antibody isotypes, isotypic variants of the CD38 antibody IgG1-005 were generated with constant domains of human IgG2, IgG3 or IgG4 yielding IgG2-005, IgG3-005 and IgG4-005 by methods known in the art. Furthermore, the oligomerization enhancing E345R mutation was introduced in all these antibodies, yielding IgG2-005-E345R, IgG3-005-E345R and IgG4-005-E345R. In a similar way, also IgG2-003 and IgG2-003-E345R were generated from CD38 antibody IgG1-003. CDC efficacy of the different isotypes was compared in an in vitro CDC assay.
0.1×106 Wien133 cells were pre-incubated in round-bottom 96-well plates with 10 μg/mL unpurified antibodies in a total volume of 100 μL for 15 min on a shaker at RT. IgG1-005-E345R was added at 3.0 μg/mL. Next, 25 μL normal human serum was added as a source of complement (20% final concentration) and incubated in a 37° C. incubator for 45 min. The reaction was stopped by putting the plates on ice. 10 μL propidium iodide was added and cell lysis was determined by FACS.
FIG. 27 shows that IgG2-005, IgG2-003, IgG3-005 and IgG4-005 were unable to lyse either (A) Daudi or (B) Wien133 cells efficiently under the tested conditions (the observed ˜20% lysis was considered as background). Introduction of the E345R mutation enabled potent CDC on Daudi cells by all IgG isotypes tested. These results were confirmed using CDC on Wien133 cells, albeit that IgG3-005-E345R displayed limited CDC activity relative to the other isotypic variants. These data indicate that besides IgG1, an oligomerization enhancing mutation such as E345R can also be applied to promote CDC activity of IgG2, IgG3 and IgG4 antibodies.
Example 33
CDC by IgG1-005 and IgG1-005-E345R in an Ex Vivo CDC Assay on Patient-Derived CD38-Positive B Cell Chronic Lymphocytic Leukemia (CLL) Cells
Cryopreserved primary cells from CLL patient samples were obtained from the hematopathology biobank from CDB-IDIBAPS-Hospital Clinic (Dr. Elias Campo, Hematopathology Unit, Department of Pathology, Hospital Clinic, Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain), or from clinical studies by the National Heart, Lung, and Blood Institute (NHLBI) (Dr. Adrian Wiestner, NHLBI, Hematology Branch of the National Institutes of Health (NIH), Bethesda). Informed consent was obtained from all patients in accordance with the Institutional Ethics Committee of the Hospital Clinic (Barcelona, Spain) or the Institutional Review Board of the NIH and the Declaration of Helsinki. All samples were genetically and immunophenotypically characterized.
The CLL samples were categorized into two groups according to their CD38 expression as determined by FACS: five samples were included in the CD38 high group (between 50% and 98% of the CD38 expression on Daudi cells) and four samples were included in the CD38 low group (between 0.5% and 3% of the CD38 expression on Daudi cells).
Fluorescently labeled CLL cells (labeling with 5 μM Calcein AM) were incubated with a concentration series of antibody (0.01-10 μg/mL in 10-fold dilutions). Next, normal human serum was added to the antibody-opsonized cells (100,000 cells/well) as a source of complement (10% final concentration) and incubated for 45 min at 37° C. Supernatans were recovered and fluorescence was read in a Synergy™ HT fluorometer as a measure for cell lysis. Cell killing was calculated as follows: Specific lysis=100×(sample-spontaneous lysis)/(max lysis−spontaneous lysis) where max lysis is determined by a sample of cells treated with 1% Triton, and spontaneous lysis is determined from a sample where cells were incubated in the presence of 10% NHS without antibody.
FIG. 28 shows that IgG1-005-E345R strongly enhanced CDC efficacy compared to wild type IgG1-005 on both CLL primary cells with high CD38 expression and CLL primary cells with low CD38 expression.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. Any and all combination of embodiments disclosed in dependent claims is also contemplated to be within the scope of the invention.
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14130543
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genmab b.v.
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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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Apr 1st, 2022 06:04PM
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Apr 1st, 2022 06:04PM
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Genmab A/S
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Health Care
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Pharmaceuticals & Biotechnology
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cph:gen
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Genmab A/S
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Jun 30th, 2015 12:00AM
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Mar 10th, 2011 12:00AM
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https://www.uspto.gov?id=US09068011-20150630
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Monoclonal antibodies against c-Met
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Isolated monoclonal antibodies which bind to human c-Met, the hepatocyte growth factor receptor, and related antibody-based compositions and molecules, are disclosed. Pharmaceutical compositions comprising the antibodies and therapeutic and diagnostic methods for using the antibodies are also disclosed.
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9068011
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1. An isolated human monoclonal antibody which binds human c-Met and comprises:
(i) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 34, 185 and 36 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 38, 39 and 206,
(ii) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 191, 192 and 193 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 78, 79 and 208,
(iii) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 194, 195 and 196 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 209, 210 and 104;
(iv) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 197, 198 and 116 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 118, 119 and 211, or
(v) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 199, 200 and 201 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 126, 212 and 128.
2. An isolated human monoclonal antibody which binds human c-Met and comprises a heavy and light chain variable region, wherein:
(a) the VH region comprises the sequence of SEQ ID NO: 33 and the VL region comprises the sequence of SEQ ID NO: 37,
(b) the VH region comprises the sequence of SEQ ID NO: 65 and a VL region comprising the sequence of SEQ ID NO: 69,
(c) the VH region comprises the sequence of SEQ ID NO: 73 and the VL region comprises the sequence of SEQ ID NO: 77,
(d) the VH region comprises the sequence of SEQ ID NO:81 and the VL region comprises the sequence of SEQ ID NO: 85,
(e) the VH region comprises the sequence of SEQ ID NO:89 and the VL region comprises the sequence of SEQ ID NO: 93,
(f) the VH region comprises the sequence of SEQ ID NO:97 and the VL region comprises the sequence of SEQ ID NO: 101,
(g) the VH region comprises the sequence of SEQ ID NO: 113 and the VL region comprises the sequence of SEQ ID NO: 117,
(h) the VH region comprises the sequence of SEQ ID NO: 121 and the VL region comprises the sequence of SEQ ID NO: 125,
(i) the VH region comprises the sequence of SEQ ID NO: 129 and the VL region comprises the sequence of SEQ ID NO: 133,
(j) the VH region comprises the sequence of SEQ ID NO: 159 and the VL region comprises the sequence of SEQ ID NO: 160,
(k) the VH region comprises the sequence of SEQ ID NO: 161 and the VL region comprises the sequence of SEQ ID NO: 162,
(l) the VH region comprises the sequence of SEQ ID NO: 163 and the VL region comprises the sequence of SEQ ID NO: 164,
(m) the VH region comprises the sequence of SEQ ID NO: 165 and the VL region comprises the sequence of SEQ ID NO: 166,
(n) the VH region comprises the sequence of SEQ ID NO: 137 and the VL region comprises the sequence of SEQ ID NO: 138,
(o) the VH region comprises the sequence of SEQ ID NO: 139 and the VL region comprises the sequence of SEQ ID NO: 140,
(p) the VH region comprises the sequence of SEQ ID NO: 141 and the VL region comprises the sequence of SEQ ID NO: 142, or
(q) the VH region comprises the sequence of SEQ ID NO: 143 and the VL region comprises the sequence of SEQ ID NO: 144.
3. The antibody of claim 1, wherein the antibody binds to the SEMA domain of c-Met, wherein the antibody inhibits binding of HGF to the SEMA domain with an IC50 less than 10 μg/mL as determined by TR-FRET.
4. The antibody of claim 1, wherein the antibody binds to A431 cells with an EC50 of 10 nM or less, as determined by FACS.
5. The antibody of claim 4, wherein the antibody is a bivalent antibody.
6. The antibody of claim 1, wherein the antibody binds to c-Met with an affinity constant (KD) of 20 nM or less, as determined by bio-layer interferometry.
7. The antibody of claim 1, wherein the antibody binds to Rhesus monkey c-Met, wherein the signal of antibody binding to Rhesus monkey c-Met is at least 5 times that of a negative control antibody, as determined by FACS.
8. The antibody of claim 1, wherein the antibody inhibits binding of HGF to the extracellular domain of c-Met, wherein the antibody inhibits binding more than 40%, as determined by ELISA.
9. The antibody of claim 1, wherein the antibody is capable to inhibit the viability of KP4 cells, wherein the antibody inhibits the viability of KP4 cells by more than 10% after applying 66.7 nM of the antibody to 10,000 KP4 cells for 3 days.
10. The antibody of claim 1, wherein the antibody is a full-length antibody.
11. The antibody of claim 1, wherein the antibody is conjugated to another moiety.
12. The antibody of claim 1, wherein the antibody is an effector-function-deficient antibody.
13. The antibody of claim 1, wherein the antibody is a monovalent antibody.
14. The antibody of claim 13, wherein the monovalent antibody comprises:
(i) a variable region of an antibody of claim 1 or an antigen binding part of the said region, and
(ii) a CH region of an immunoglobulin or a fragment thereof comprising the CH2 and CH3 regions, wherein the CH region or fragment thereof has been modified such that the region corresponding to the hinge region and, if the immunoglobulin is not an IgG4 subtype, other regions of the CH region do not comprise any amino acid residues, which are capable of forming disulfide bonds with an identical CH region or other covalent or stable non-covalent inter-heavy chain bonds with an identical CH region in the presence of polyclonal human IgG.
15. The antibody of claim 14, wherein the immunoglobulin referred to in step (ii) is of the IgG4 subtype.
16. The antibody of claim 14, wherein the heavy chain has been modified such that the entire hinge has been deleted.
17. The antibody of claim 1, wherein the antibody has been modified to make it less flexible in the hinge region, wherein the hinge region has been modified by:
(i) deleting the hinge region of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 214) and substituting it with the IgG2 hinge region of the sequence: ERKCCVECPPCP (SEQ ID NO: 215);
(ii) deleting position 220 modified hinge region has the sequence of EPKSDKTHTCPPCP (SEQ ID NO: 216);
(iii) substituting cysteine at position 220 with any other natural amino acid (X) so the modified hinge region has the sequence of EPKSXDKTHTCPPCP (SEQ ID NO: 217);
(iv) deleting the hinge region of sequence EPKSCDKTHTCPPCP (SEQ ID NO: 214) ;
(v) deleting the hinge region of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 214) and substituting it with the IgG3 hinge region of the sequence ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP (SEQ ID NO: 218); or
(vi) substituting threonine at position 223 with cysteine, and deleting lysine at position 222 and threonine at position 225, so the modified hinge region has the sequence of EPKSCDCHCPPCP (SEQ ID NO: 219),
wherein the amino acid positions are based on the EU index as described in Kabat.
18. The antibody of claim 17, wherein the hinge region has been modified by substituting cysteine at position 220 with serine so the modified hinge region has the sequence of EPKSSDKTHTCPPCP (SEQ ID NO: 220).
19. The antibody of claim 17, wherein the antibody is of the IgG2 subtype.
20. The antibody of claim 1, wherein the antibody has been modified to reduce core-fucosylation below 10%, as determined by high performance anion-exchange chromatography coupled with pulsed amperometric detection.
21. The antibody of claim 1, wherein the antibody is a bispecific antibody, comprising a c-Met binding site and a second antigen-binding site having a different binding specificity.
22. A pharmaceutical composition comprising an antibody as defined in claim 1 and a pharmaceutically acceptable carrier.
23. A kit for detecting the presence of c-Met in a sample comprising
an antibody of claim 1; and
instructions for use of the kit.
24. The antibody of claim 1, wherein the antibody comprises:
a) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 34, 35 and 36 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 38, 39 and 40,
b) (i) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 66, 67 and 68 and a VL region comprising the CDR1 , 2 and 3 sequences of SEQ ID NO: 70, 71 and 72,
(ii) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 74, 75 and 76 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 78, 79 and 80,
(iii) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 82, 83 and 84 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 86, 87 and 88, or
(iv) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 90, 91 and 92 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 94, 95 and 96,
c) (i) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 98, 99 and 100 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 102, 103 and 104, or
(ii) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 130, 131 and 132 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 134, 135 and 136,
d) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 114, 115 and 116 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 118, 119 and 120, or
e) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 122, 123 and 124 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 126, 127 and 128.
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RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national stage filing of International Application No. PCT/EP2011/053646 filed Mar. 10, 2011, which claims priority to Denmark Patent Application No. PA 2010 00862 filed on Sep. 24, 2010, to Denmark Patent Application No. PA 2010 00191 filed on Mar. 10, 2010, and US Provisional Application No. 61/312,622 filed on Mar. 10, 2010. The contents of the aforementioned applications are hereby incorporated by reference.
FIELD OF THE INVENTION
The present invention relates to monoclonal antibodies directed to human c-Met, the hepatocyte growth factor receptor, and to uses of such antibodies, in particular their use in the treatment of cancer.
BACKGROUND OF THE INVENTION
c-Met is a membrane-spanning receptor tyrosine kinase protein. The primarily single chain precursor is post-translationally cleaved to produce the mature form of the c-Met heterodimer that consists of an extracellular α-chain (50 kDa) and a longer transmembrane β-chain (145 kDa), which are disulfide-linked (Birchmeier et al. 2003. Nat Rev Mol Cell Biol 4:915). The extracellular part of c-Met is composed of three domain types. The N-terminal SEMA domain is formed by the whole α-subunit and part of the β-subunit, and encompasses homology to semaphorin proteins. The SEMA domain is followed by a cysteine-rich domain and further by four immunoglobulin-(Ig)-like domains. The cytoplasmic part contains a juxtamembrane kinase domain and a carboxy-terminal tail that is essential for downstream signaling. The only known high affinity ligand for c-Met, hepatocyte growth factor (HGF), is mainly expressed by fibroblasts under normal conditions (Li and Tseng 1995. J Cell Physiol 163:61) and by tumor cells (Ferracini et al. 1995. Oncogene 10:739). HGF (also called scatter factor: SF) is synthesized as a precursor that is converted proteolytically into an active α/β heterodimer. Based on the crystal structure of the receptor-binding fragment, HGF is thought to bind c-Met as a dimer (Chirgadze et al. 1999. Nat Struct Biol 6:72). The HGF-α chain binds with high affinity to the Ig-like domain in c-Met, whereas the HGF-β chain binds with low affinity to the c-Met SEMA domain (Basilico et al. 2008. J Biol Chem 283:21267). The latter interaction is responsible for c-Met dimerization and receptor tyrosine kinase activation upon binding of the active HGF heterodimer. Receptor autophosphorylation results in a unique docking site for recruitment of effectors, of which Gab1 (growth factor receptor-bound protein 2 [Grb2]-associated binder 1) binding is essential for the major c-Met downstream signaling pathways (Comoglio et al. 2008. Nat Rev Drug Discov 7:504):
Ras-ERK1/2 pathway: proliferation.
Ras-Rac pathway: invasion, motility, epithelial-to-mesenchymal transition.
PI3K-Akt pathway: survival.
c-Met is expressed on the surface of epithelial and endothelial cells of many organs during embryogenesis and in adulthood, including the liver, pancreas, prostate, kidney, muscle, and bone marrow. c-Met activation plays an essential role in the so-called “invasive growth” programme that consists of a series of processes, including proliferation, motility, angiogenesis and protection from apoptosis (Boccaccio and Comoglio 2006. Nat Rev Cancer 6:637). These c-Met-regulated processes occur under normal physiological conditions during embryonic development, hepatic and cardiac injury repair, and pathologically during oncogenesis (Eder et al. 2009. Clin Cancer Res 15:2207).
Inappropriate c-Met signaling occurs in virtually all types of solid tumors, such as bladder, breast, cervical, colorectal, gastric, head and neck, liver, lung, ovarian, pancreatic, prostate, renal, and thyroid cancers, as well as in various sarcomas, hematopoietic malignancies, and melanoma (Birchmeier et al. 2003. Nat Rev Mol Cell Biol 4:915; Comoglio et al. 2008. Nat Rev Drug Discov 7:504; Peruzzi and Bottaro 2006. Clin Cancer Res 12:3657). The underlying mechanisms for tumorigenicity of c-Met are typically achieved in three different ways:
autocrine HGF/c-Met loops,
c-Met or HGF overexpression,
kinase-activating mutations in the c-Met receptor coding sequence.
Most notably, activating c-Met mutations have been identified in patients with hereditary papillary renal cancer (Schmidt et al. 1997. Nat Genet 16:68). Constitutive activation of c-Met contributes to one or a combination of proliferative, invasive, survival, or angiogenic cancer phenotypes. Gene silencing of endogenously expressed c-Met in tumor cells has been shown to result in lack of proliferation and tumor growth and regression of established metastasis, as well as decreased generation of new metastases (Corso et al. 2008. Oncogene 27:684).
As c-Met contributes to multiple stages of cancer development, from initiation through progression to metastasis, c-Met and its ligand HGF have become leading candidates for targeted cancer therapies (Comoglio et al. 2008. Nat Rev Drug Discov 7:504; Knudsen and Vande Woude 2008. Curr Opin Genet Dev 18:87). Several strategies are being explored to reach this goal:
Decoy receptors: subregions of HGF or c-Met or molecular analogs can act antagonistic as stoichiometric competitors by blocking ligand binding or receptor dimerization. One example of such an antagonistic subregion of HGF is NK4 (Kringle Pharma).
Small molecule tyrosine kinase inhibitors (TKIs): Three c-Met-specific TKIs in different stages of clinical evaluation are ARQ197 (ArQule), JNJ 38877605 (Johnson & Johnson) and PF-04217903 (Pfizer).
Anti-HGF monoclonal antibodies, such as AMG102, rilotumumab (Amgen), HuL2G7 (Takeda), and AV-299 (Schering).
Anti-c-Met monoclonal antibodies have been described in WO2005016382, WO2006015371, WO2007090807, WO2007126799 WO2009007427, WO2009142738 and van der Horst et al. (van der Horst et al. 2009. Neoplasoa 11:355). MetMAb (Genentech) is a humanized monovalent (one-armed) OA-5D5 antibody that binds to the extracellular domain of c-Met, thereby preventing HGF binding and subsequent receptor activation (Jin et al. 2008. Cancer Res 68:4360). In mouse xenograft models, treatment with MetMAb was found to inhibit tumor growth of HGF-driven orthotopic glioblastoma and subcutanous pancreatic tumors (Jin et al. 2008. Cancer Res 68:4360; Martens et al. 2006. Clin Cancer Res 12:6144). h224G11 (Pierre Fabre) (Corvaia and Boute 2009. Abstract 835 AACR 100th Annual Meeting) is a humanized bivalent anti-c-Met IgG1 antibody. Anti-tumor effects of this antibody have been observed in mice (Goetsch et al. 2009. Abstract 2792 AACR 100th Annual Meeting). CE-355621 (Pfizer) is a human IgG2 that blocks ligand binding by binding to the extracellular domain of c-Met and inhibits HGF-dependent growth in tumor xenograft models (Tseng et al. 2008.3 Nucl Med 49:129).
In conclusion, several anti-c-Met products are being investigated, but so far no anti-c-Met product has yet been approved for therapeutic use. There remains a need for effective and safe products for treating serious c-Met-related diseases, such as cancer.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide novel highly specific and effective monoclonal anti-c-Met antibodies for medical use. The antibodies of the invention exhibit c-Met binding characteristics that differ from the antibodies described in the art. In preferred embodiments, the antibodies of the invention have a high affinity towards human c-Met, are antagonistic and have a favorable pharmacokinetic profile for use in human patients.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Alignment of HuMabs heavy chain variable region sequences. On the basis of these sequences, consensus sequence can be defined for some of the CDR sequences. These consensus sequences are given in Table 4.
VH Comparison 1: IgHV1-18-1 (SEQ ID NO: 221), VH1016-181(SEQ ID NO: 129), VH1016-066, (SEQ ID NO: 136), VH1016-065, (SEQ ID NO: 139), VH1016-069, (SEQ ID NO: 97), VH1016-082, (SEQ ID NO: 141), VH1016-089, (SEQ ID NO: 143), Consensus (SEQ ID NO: 222).
VH Comparison 2: IgHV1-69-4, (SEQ ID NO: 223), VH1016-005, (SEQ ID NO: 1), VH1016-031, (SEQ ID NO: 145), VH1016-006, SEQ ID NO: 9), VH1016-007, SEQ ID NO: 147), VH1016-011, SEQ ID NO: 149), Consensus (SEQ ID NO: 224).
VH Comparison 3: IgHV3-30-3-1, (SEQ ID NO: 225), VH1016-017, (SEQ ID NO: 151), VH1016-025, (SEQ ID NO: 153), VH1016-022, SEQ ID NO: 25), Consensus (SEQ ID NO: 25).
VH Comparison 4: IgHV3-23-1, (SEQ ID NO: 226), VH1016-040, (SEQ ID NO: 155), VH1016-045, (SEQ ID NO: 49), VH1016-039, (SEQ ID NO: 157), Consensus (SEQ ID NO: 227).
VH Comparison 5: IgHV4-30-2-1, (SEQ ID NO: 228), VH1016-068 ID NO: 89), VH1016-078,(SEQ ID NO: 159)VH1016-084(SEQ ID NO: 161), VH1016-061, (SEQ ID NO: 65), VH1016-062, (SEQ ID NO: 73), VH1016-063, (SEQ ID NO: 163), VH1016-064, (SEQ ID NO: 81), VH1016-087, (SEQ ID NO: 165), Consensus (SEQ ID NO: 165).
VH Comparison 6: IgHV5-51-1, (SEQ ID NO: 229), VH1016-016, (SEQ ID NO: 167), VH1016-028, (SEQ ID NO: 169), VH1016-008, (SEQ ID NO: 17), VH1016-012, (SEQ ID NO: 171), VH1016-035, (SEQ ID NO: 41), VH1016-095, (SEQ ID NO: 173), VH1016-093, (SEQ ID NO: 175), VH1016-096, (SEQ ID NO: 105), VH016-104, (SEQ ID NO: 177), Consensus (SEQ ID NO: 105).
FIG. 2: Alignment of HuMabs light chain variable region sequences. On the basis of these sequences, consensus sequence can be defined for some of the CDR sequences. These consensus sequences are given in Table 4.
IGKV1-12*01, (SEQ ID NO: 230), VL1016-065, (SEQ ID NO: 140), VL1016-066, (SEQ ID NO: 138), VL1016-069, (SEQ ID NO: 101), VL1016-089, (SEQ ID NO: 144), VL1016-082(SEQ ID NO: 142), VL1016-181, (SEQ ID NO: 133), Consensus (SEQ ID NO: 133).
IGKV1D-16*01, (SEQ ID NO: 231), VL1016-005, (SEQ ID NO: 5), VL1016-031, (SEQ ID NO: 146), VL1016-006, (SEQ ID NO: 13), VL1016-007, (SEQ ID NO: 148), VL1016-011(SEQ ID NO: 150), Consensus (SEQ ID NO: 150).
IGKV1-12*01, (SEQ ID NO: 230), VL1016-017 (SEQ ID NO: 152), VL1016-022, (SEQ ID NO: 29), VL1016-025, (SEQ ID NO: 154), Consensus (SEQ ID NO: 152).
IGKV3-11*01, (SEQ ID NO: 232), VL1016-039, (SEQ ID NO: 158), VL1016-040, (SEQ ID NO: 156), VL1016-045, (SEQ ID NO: 53), Consensus (SEQ ID NO: 53).
IGKV1-2*01, (SEQ ID NO: 230), VL016-061, (SEQ ID NO: 69), VL1016-062, (SEQ ID NO: 77), VL1016-063, (SEQ ID NO: 164), VL1016-064, (SEQ ID NO: 85), VL1016-068(SEQ ID NO: 93), VL1016-084, (SEQ ID NO: 162), Consensus (SEQ ID NO: 85).
IGKV1-13*02, (SEQ ID NO: 233), VL1016-008, (SEQ ID NO: 21), VL1016-012, (SEQ ID NO: 172), VL1016-035, (SEQ ID NO: 45), VL1016-104, (SEQ ID NO: 178), VL1016-093 (SEQ ID NO: 176), VL1016-096, (SEQ ID NO: 109), VL1016-016, (SEQ ID NO: 168), VL1016-028, (SEQ ID NO: 170), VL1016-095, (SEQ ID NO: 174), Consensus (SEQ ID NO: 174).
FIG. 3: Binding curves of monovalent and bivalent forms of anti-c-Met antibodies to c-Met expressing A431 cells. Data shown are MFI of one representative experiment. Because IgG1-024 and Uni-068 did not show saturated binding to A431 cells it was not possible to calculate an accurate EC50 value.
FIG. 4: Binding of antibodies to c-Met expressed on Rhesus monkey epithelial cells. Data shown are MFI of one experiment.
FIG. 5: Anti-c-Met antibody-induced inhibition of HGF binding to the extracellular domain of the c-Met receptor. Data shown is one representative experiment.
FIG. 6: HGF binding inhibition curves of the various anti-c-Met antibodies for binding to cMetSEMA—567His8 tested with TR-FRET. Data shown are mean MFI±standard deviation of three independent experiments.
FIG. 7: Percentage inhibition of viable KP4 cells after anti-c-Met antibody treatment compared to untreated cells (0%). Data shown are percentages inhibition of viable cells of two independent experiments±the standard deviation. IgG1-1016-022 was only positive in one experiment.
FIG. 8: Efficacy of anti-c-Met antibodies to inhibit tumor growth in a KP4 xenograft model in SCID mice. Mice were treated with 400 μg antibody at day 9 followed weekly with a maintenance dose of 200 μg. Median tumor sizes per treatment group are shown.
FIG. 9: Efficacy of anti-c-Met antibodies to inhibit tumor growth in a KP4 xenograft model in SCID mice. Mice were treated with 400 μg antibody at day 9 followed weekly with a maintenance dose of 200 μg. Effect of treatment on tumor incidence in time. Shown is the percentage tumor free mice (tumor sizes <500 mm3). Tumor formation is delayed in mice treated with antagonistic antibodies compared to control antibodies.
FIG. 10: Efficacy of anti-c-Met antibodies to inhibit tumor growth in an MKN45 xenograft model in SCID mice. Mice were treated with 40 mg/kg antibody on day 7 and 20 mg/kg antibody on days 14, 21 and 28. Median tumor sizes until 50% of the mice reached the 700 mm3 endpoint, per treatment group are shown.
FIG. 11: Efficacy of anti-c-Met antibodies to inhibit tumor growth in an MKN45 xenograft model in SCID mice. Mice were treated with 40 mg/kg antibody on day 7 and 20 mg/kg antibody on days 14, 21 and 28. The percentage mice with tumor sizes smaller then 700 mm3 is shown in a Kaplan Meier plot. Tumor formation is delayed in mice treated with anti-c-Met antibodies compared to isotype control antibody.
FIG. 12: KP4 viability assay to determine the effect of antibody flexibility on agonistic activity. The IgA2m(1) format did not induce proliferation, in contrast to IgA1 and IgG1 formats of the same antibody. Variants of the 5D5 anti-c-Met antibody (see U.S. Pat. No. 6,468,529 and Example 2) were used in this experiment.
FIG. 13: Non-reduced SDS-PAGE analysis of the flexibility mutants of (069). No aberrant multimers or degradation products were observed whereas the light chain paring was visible as a 50 kD band ((LC)2) in the C220S, ΔC220 and IgG1-hinge IgG3 mutants.
FIG. 14: Antigen binding ELISA to measure c-Met binding of hinge mutants of c-Met antibodies. All mutants bind with comparable affinity to c-Met as shown in ELISA.
FIG. 15: c-Met phosphorylation as readout for agonistic activity of antibodies against c-Met. FIG. 15 shows Western blot results of A549 lysates; membranes stained with antibodies against phosphorylated c-met, total c-Met or β-actin.
FIG. 16: Proliferation assay with NCI-H441 cells. Cell mass was determined after 7 days incubation in the presence of antibody or controls and plotted as percentage of non-treated samples (set as 100%).
FIG. 17: KP4 viability assay. The effect of antibodies against c-Met on the overall viability of KP4 cells was tested. The ability of IgG1-1016-069 to reduce the viability of KP4 was retained and/or improved by introducing mutations that decrease the flexibility of the antibodies.
FIG. 18: Down-modulation as measured as total c-Met levels in A549 lysates using ELISA. All variants of antibody (069) retained the down-modulating capacity.
FIG. 19: ADCC assay to compare high and low fucose versions of antibody IgG1-1016-069.
FIG. 20: Lack of binding of c-Met antibodies to cells in whole blood in FACS binding assay. Results are shown for B cells; monocytes and granulocytes.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “c-Met”, when used herein, refers to the hepatocyte growth factor receptor (Genbank accession NM 000245) and includes any variants, isoforms and species homologs of human c-Met which are naturally expressed by cells or are expressed on cells transfected with the c-Met gene.
The term “immunoglobulin” refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. See for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain constant region. The heavy chain constant region typically is comprised of three domains, CH1, CH2, and CH3. Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL or VL) and a light chain constant region. The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Typically, the numbering of amino acid residues in this region is performed by the method described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) (phrases such as variable domain residue numbering as in Kabat or according to Kabat herein refer to this numbering system for heavy chain variable domains or light chain variable domains). Using this numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of VH CDR2 and inserted residues (for instance residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.
The term “antibody” (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions with a half life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q, the first component in the classical pathway of complement activation. An anti-c-Met antibody may also be a bispecific antibody, diabody, or similar molecule (see for instance PNAS USA 90(14), 6444-8 (1993) for a description of diabodies). Indeed, bispecific antibodies, diabodies, and the like, provided by the present invention may bind any suitable target in addition to a portion of c-Met. As indicated above, the term antibody herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that retain the ability to specifically bind to the antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antibody” include (i) a Fab′ or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or a monovalent antibody as described in WO2007059782 (Genmab); (ii) F(ab′)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CH1 domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends Biotechnol. 2003 November; 21(11):484-90); (vi) camelid or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 January; 5(1):111-24) and (vii) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present invention, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present invention are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. An antibody as generated can possess any isotype.
As used herein, “isotype” refers to the immunoglobulin class (for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encoded by heavy chain constant region genes.
The term “monovalent antibody” means in the context of the present invention that an antibody molecule is capable of binding a single molecule of the antigen, and thus is not able of antigen crosslinking.
An “antibody deficient in effector function” or an “effector-function-deficient antibody” refers to an antibody which has a significantly reduced or no ability to activate one or more effector mechanisms, such as complement activation or Fc receptor binding. Thus, effector-function deficient antibodies have significantly reduced or no ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC). An example of such an antibody is IgG4.
An “anti-c-Met antibody” is an antibody as described above, which binds specifically to the antigen c-Met.
The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
As used herein, a human antibody is “derived from” a particular germline sequence if the antibody is obtained from a system using human immunoglobulin sequences, for instance by immunizing a transgenic mouse carrying human immunoglobulin genes or by screening a human immunoglobulin gene library, and wherein the selected human antibody is at least 90%, such as at least 95%, for instance at least 96%, such as at least 97%, for instance at least 98%, or such as at least 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, outside the heavy chain CDR3, a human antibody derived from a particular human germline sequence will display no more than 20 amino acid differences, e.g. no more than 10 amino acid differences, such as no more than 9, 8, 7, 6 or 5, for instance no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.
In a preferred embodiment, the antibody of the invention is isolated. An “isolated antibody,” as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (for instance an isolated antibody that specifically binds to c-Met is substantially free of antibodies that specifically bind antigens other than c-Met). An isolated antibody that specifically binds to an epitope, isoform or variant of human c-Met may, however, have cross-reactivity to other related antigens, for instance from other species (such as c-Met species homologs). Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment of the present invention, two or more “isolated” monoclonal antibodies having different antigen-binding specificities are combined in a well-defined composition.
When used herein in the context of two or more antibodies, the term “competes with” or “cross-competes with” indicates that the two or more antibodies compete for binding to c-Met, e.g. compete for c-Met binding in the assay described in the Examples herein. For some pairs of antibodies, competition in the assay of Examples is only observed when one antibody is coated on the plate and the other is used to compete, and not vice versa. The term “competes with” when used herein is also intended to cover such combinations antibodies.
The term “epitope” means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen binding peptide (in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide).
The terms “monoclonal antibody” as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term “human monoclonal antibody” refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The human monoclonal antibodies may be generated by a hybridoma which includes a B cell obtained from a transgenic or transchromosomal nonhuman animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, fused to an immortalized cell.
As used herein, the term “binding” in the context of the binding of an antibody to a predetermined antigen typically is a binding with an affinity corresponding to a KD of about 10−7 M or less, such as about 10−8 M or less, such as about 10−9 M or less, about 10−10 M or less, or about 10−11 M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1,000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The amount with which the affinity is lower is dependent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is highly specific), then the amount with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000 fold.
The term “kd” (sec−1), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the koff value.
The term “ka” (M−1×sec−1), as used herein, refers to the association rate constant of a particular antibody-antigen interaction.
The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.
The term “KA” (M−1), as used herein, refers to the association equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing the Ka by the kd.
As used herein, the term “inhibits growth” (e.g. referring to cells, such as tumor cells) is intended to include any measurable decrease in the cell growth when contacted with an anti-c-Met antibody as compared to the growth of the same cells not in contact with an anti-c-Met antibody, e.g., the inhibition of growth of a cell culture by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. Such a decrease in cell growth can occur by a variety of mechanisms, e.g. effector cell phagocytosis, ADCC, CDC, and/or apoptosis.
The present invention also provides antibodies comprising functional variants of the VL region, VH region, or one or more CDRs of the antibodies of the examples. A functional variant of a VL, VH, or CDR used in the context of an anti-c-Met antibody still allows the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity/avidity and/or the specificity/selectivity of the parent antibody and in some cases such an anti-c-Met antibody may be associated with greater affinity, selectivity and/or specificity than the parent antibody.
Such functional variants typically retain significant sequence identity to the parent antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences may e.g. be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970) algorithm.
The sequence of CDR variants may differ from the sequence of the CDR of the parent antibody sequences through mostly conservative substitutions; for instance at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements.
In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected in the following table:
Amino acid residue classes for conservative substitutions
Acidic Residues
Asp (D) and Glu (E)
Basic Residues
Lys (K), Arg (R), and His (H)
Hydrophilic Uncharged Residues
Ser (S), Thr (T), Asn (N), and
Gln (Q)
Aliphatic Uncharged Residues
Gly (G), Ala (A), Val (V), Leu (L),
and Ile (I)
Non-polar Uncharged Residues
Cys (C), Met (M), and Pro (P)
Aromatic Residues
Phe (F), Tyr (Y), and Trp (W)
The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which an expression vector has been introduced, e.g. an expression vector encoding an antibody of the invention. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK293 cells, NS/0 cells, and lymphocytic cells.
The term “transgenic non-human animal” refers to a non-human animal having a genome comprising one or more human heavy and/or light chain transgenes or transchromosomes (either integrated or non-integrated into the animal's natural genomic DNA) and which is capable of expressing fully human antibodies. For example, a transgenic mouse can have a human light chain transgene and either a human heavy chain transgene or human heavy chain transchromosome, such that the mouse produces human anti-c-Met antibodies when immunized with c-Met antigen and/or cells expressing c-Met. The human heavy chain transgene may be integrated into the chromosomal DNA of the mouse, as is the case for transgenic mice, for instance HuMAb mice, such as HCo7 or HCo12 mice, or the human heavy chain transgene may be maintained extrachromosomally, as is the case for transchromosomal KM mice as described in WO02/43478. Similar mice, having a larger human Ab gene repertoire, include HCo7 and HCo20 (see e.g. WO2009097006). Such transgenic and transchromosomal mice (collectively referred to herein as “transgenic mice”) are capable of producing multiple isotypes of human monoclonal antibodies to a given antigen (such as IgG, IgA, IgM, IgD and/or IgE) by undergoing V-D-J recombination and isotype switching. Transgenic, nonhuman animal can also be used for production of antibodies against a specific antigen by introducing genes encoding such specific antibody, for example by operatively linking the genes to a gene which is expressed in the milk of the animal.
“Treatment” refers to the administration of an effective amount of a therapeutically active compound of the present invention with the purpose of easing, ameliorating, arresting or eradicating (curing) symptoms or disease states.
An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of an anti-c-Met antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the anti-c-Met antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.
An “anti-idiotypic” antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
Further Aspects and Embodiments of the Invention
As described above, in a first aspect, the invention relates to a monoclonal antibody which binds human c-Met.
Monoclonal antibodies of the present invention may e.g. be produced by the hybridoma method first described by Kohler et al., Nature 256, 495 (1975), or may be produced by recombinant DNA methods. Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies may be obtained from any suitable source. Thus, for example, monoclonal antibodies may be obtained from hybridomas prepared from murine splenic B cells obtained from mice immunized with an antigen of interest, for instance in form of cells expressing the antigen on the surface, or a nucleic acid encoding an antigen of interest. Monoclonal antibodies may also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or non-human mammals such as rats, dogs, primates, etc.
In one embodiment, the antibody of the invention is a human antibody. Human monoclonal antibodies directed against c-Met may be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. Such transgenic and transchromosomic mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as “transgenic mice”.
The HuMAb mouse contains a human immunoglobulin gene miniloci that encodes unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous p and K chain loci (Lonberg, N. et al., Nature 368, 856-859 (1994)). Accordingly, the mice exhibit reduced expression of mouse IgM or κ and in response to immunization, the introduced human heavy and light chain transgenes, undergo class switching and somatic mutation to generate high affinity human IgG,κ monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook of Experimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65-93 (1995) and Harding, F. and Lonberg, N. Ann. N.Y. Acad. Sci 764 536-546 (1995)). The preparation of HuMAb mice is described in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994), Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845-851 (1996). See also U.S. Pat. Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, 5,770,429, 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.
The HCo7 mice have a JKD disruption in their endogenous light chain (kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)), a CMD disruption in their endogenous heavy chain genes (as described in Example 1 of WO 01/14424), a KCo5 human kappa light chain transgene (as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)), and a HCo7 human heavy chain transgene (as described in U.S. Pat. No. 5,770,429).
The HCo12 mice have a JKD disruption in their endogenous light chain (kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)), a CMD disruption in their endogenous heavy chain genes (as described in Example 1 of WO 01/14424), a KCo5 human kappa light chain transgene (as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)), and a HCo12 human heavy chain transgene (as described in Example 2 of WO 01/14424).
In the KM mouse strain, the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al., EMBO J. 12, 811-820 (1993) and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of WO 01/09187. This mouse strain carries a human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also carries a human heavy chain transchromosome composed of chromosome 14 fragment hCF (SC20) as described in WO 02/43478.
Splenocytes from these transgenic mice may be used to generate hybridomas that secrete human monoclonal antibodies according to well known techniques.
Further, human antibodies of the present invention or antibodies of the present invention from other species may be identified through display-type technologies, including, without limitation, phage display, retroviral display, ribosomal display, and other techniques, using techniques well known in the art and the resulting molecules may be subjected to additional maturation, such as affinity maturation, as such techniques are well known in the art (see for instance Hoogenboom et al., J. Mol. Biol. 227, 381 (1991) (phage display), Vaughan et al., Nature Biotech 14, 309 (1996) (phage display), Hanes and Plucthau, PNAS USA 94, 4937-4942 (1997) (ribosomal display), Parmley and Smith, Gene 73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245 (1992), Cwirla et al., PNAS USA 87, 6378-6382 (1990), Russel et al., Nucl. Acids Research 21, 1081-1085 (1993), Hogenboom et al., Immunol. Reviews 130, 43-68 (1992), Chiswell and McCafferty TIBTECH 10, 80-84 (1992), and U.S. Pat. No. 5,733,743). If display technologies are utilized to produce antibodies that are not human, such antibodies may be humanized.
In one embodiment, the antibody of the invention is of isotype IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM.
In a first main embodiment of the antibody of the invention, the antibody competes for binding to soluble cMetECDHis with an immobilized antibody, wherein said immobilized antibody comprises a VH region comprising the sequence of SEQ ID NO:33 and a VL region comprising the sequence of SEQ ID NO:37 (024), preferably wherein the antibody competes for more than 50%, such as more than 75% with said immobilized antibody, when determined as described in Example 17.
In a further embodiment, the antibody does not compete for binding to soluble cMetECDHis with an antibody selected from the group consisting of:
a) an immobilized antibody comprising a VH region comprising the sequence of SEQ ID NO:1 and a VL region comprising the sequence of SEQ ID NO:5 (005)
b) an immobilized antibody comprising a VH region comprising the sequence of SEQ ID NO:17 and a VL region comprising the sequence of SEQ ID NO:21 (008)
c) an immobilized antibody comprising the VH region and the VL region of antibody 5D5, and
d) an immobilized antibody comprising a VH region comprising the sequence of SEQ ID NO:49 and a VL region comprising the sequence of SEQ ID NO:53 (045),
preferably wherein the antibody competes for less than 25%, such as less than 20% with said immobilized antibody, when determined as described in Example 17.
In a further embodiment, the antibody binds to the same epitope as an antibody selected from the group consisting of:
a) an antibody comprising a VH region comprising the sequence of SEQ ID NO:33 and a VL region comprising the sequence of SEQ ID NO:37 (024)
b) an antibody comprising a VH region comprising the sequence of SEQ ID NO:65 and a VL region comprising the sequence of SEQ ID NO:69 (061)
c) an antibody comprising a VH region comprising the sequence of SEQ ID NO:73 and a VL region comprising the sequence of SEQ ID NO:77 (062)
d) an antibody comprising a VH region comprising the sequence of SEQ ID NO:81 and a VL region comprising the sequence of SEQ ID NO:85 (064)
e) an antibody comprising a VH region comprising the sequence of SEQ ID NO:89 and a VL region comprising the sequence of SEQ ID NO:93 (068)
f) an antibody comprising a VH region comprising the sequence of SEQ ID NO:97 and a VL region comprising the sequence of SEQ ID NO:101 (069)
g) an antibody comprising a VH region comprising the sequence of SEQ ID NO:113 and a VL region comprising the sequence of SEQ ID NO:117 (098)
h) an antibody comprising a VH region comprising the sequence of SEQ ID NO:121 and a VL region comprising the sequence of SEQ ID NO:125 (101), and
i) an antibody comprising a VH region comprising the sequence of SEQ ID NO:129 and a VL region comprising the sequence of SEQ ID NO:133 (181).
In a further embodiment, the antibody comprises a VH CDR3 region having the sequence as set forth in
a) SEQ ID NO:36 (024)
b) SEQ ID NO:193, such as a VH CDR3 region as set forth in SEQ ID NO:68, 76, 84 or 92 (061, 062, 064, 068)
c) SEQ ID NO:196, such as a VH CDR3 region as set forth in SEQ ID NO:100 or 132 (069, 181)
d) SEQ ID NO:116 (098), or
e) SEQ ID NO:201, such as a VH CDR3 region as set forth in SEQ ID NO:124 (101).
In a further embodiment, the antibody comprises:
a) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:34, 185 and 36 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:38, 39 and 206, such as an antibody comprising a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:34, 35 and 36 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:38, 39 and 40 (024)
b) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:191, 192 and 193 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:78, 79 and 208, such as an antibody comprising
a. a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:66, 67 and 68 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:70, 71 and 72 (061)
b. a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:74, 75 and 76 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:78, 79 and 80, (062)
c. a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:82, 83 and 84 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:86, 87 and 88, (064), or
d. a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:90, 91 and 92 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:94, 95 and 96, (068)
c) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:194, 195 and 196 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:209, 210 and 104, such as an antibody comprising
a. a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:98, 99 and 100 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:102, 103 and 104, (069), or
b. a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:130, 131 and 132 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:134, 135 and 136, (181)
d) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:197, 198 and 116 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:118, 119 and 211, such as an antibody comprising a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:114, 115 and 116 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:118, 119 and 120 (098), or
e) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 199, 200 and 201 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 126, 212 and 128, such as an antibody comprising a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:122, 123 and 124 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:126, 127 and 128 (101).
In a further embodiment, the antibody comprises:
a) a VH region comprising the sequence of SEQ ID NO:33 and, preferably, a VL region comprising the sequence of SEQ ID NO:37 (024)
b) a VH region comprising the sequence of SEQ ID NO:61 and, preferably, a VL region comprising the sequence of SEQ ID NO:69 (061)
c) a VH region comprising the sequence of SEQ ID NO:73 and, preferably, a VL region comprising the sequence of SEQ ID NO:77 (062)
d) a VH region comprising the sequence of SEQ ID NO:81 and, preferably, a VL region comprising the sequence of SEQ ID NO:85 (064)
e) a VH region comprising the sequence of SEQ ID NO:89 and, preferably, a VL region comprising the sequence of SEQ ID NO:93 (068)
f) a VH region comprising the sequence of SEQ ID NO:97 and, preferably, a VL region comprising the sequence of SEQ ID NO:101 (069)
g) a VH region comprising the sequence of SEQ ID NO:113 and, preferably, a VL region comprising the sequence of SEQ ID NO:117 (098)
h) a VH region comprising the sequence of SEQ ID NO:121 and, preferably, a VL region comprising the sequence of SEQ ID NO:125 (101)
i) a VH region comprising the sequence of SEQ ID NO:129 and, preferably, a VL region comprising the sequence of SEQ ID NO:133 (181)
j) a VH region comprising the sequence of SEQ ID NO:159 and, preferably, a VL region comprising the sequence of SEQ ID NO:160 (078)
k) a VH region comprising the sequence of SEQ ID NO:161 and, preferably, a VL region comprising the sequence of SEQ ID NO:162 (084)
l) a VH region comprising the sequence of SEQ ID NO:163 and, preferably, a VL region comprising the sequence of SEQ ID NO:164 (063)
m) a VH region comprising the sequence of SEQ ID NO:165 and, preferably, a VL region comprising the sequence of SEQ ID NO:166 (087)
n) a VH region comprising the sequence of SEQ ID NO:137 and, preferably, a VL region comprising the sequence of SEQ ID NO:138 (066)
o) a VH region comprising the sequence of SEQ ID NO:139 and, preferably, a VL region comprising the sequence of SEQ ID NO:140 (065)
p) a VH region comprising the sequence of SEQ ID NO:141 and, preferably, a VL region comprising the sequence of SEQ ID NO:142 (082)
q) a VH region comprising the sequence of SEQ ID NO:143 and, preferably, a VL region comprising the sequence of SEQ ID NO:144 (089), or
r) a variant of any of said antibodies, wherein said variant preferably has at most 1, 2 or 3 amino-acid modifications, more preferably amino-acid substitutions, such as conservative amino-acid substitutions in said sequences.
In one embodiment, the antibody comprises a VH region comprising the CDR3 sequence of SEQ ID NO:100 and a VL region comprising the CDR3 sequence of SEQ ID NO: 104, (069).
In one embodiment, the antibody comprises a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:98, 99 and 100 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:102, 103 and 104 (069).
In one embodiment, the antibody comprises a VH region comprising the sequence of SEQ ID NO:97 and a VL region comprising the sequence of SEQ ID NO:101 (069).
In another main embodiment of the antibody of the invention:
the antibody competes for binding to soluble cMetECDHis with an immobilized antibody, wherein said immobilized antibody comprises a VH region comprising the sequence of SEQ ID NO:9 and a VL region comprising the sequence of SEQ ID NO:13 (006), preferably wherein the antibody competes for more than 50%, such as more than 75% with said immobilized antibody, when determined as described in Example 17, and
the antibody does not compete binding to soluble cMetECDHis with an immobilized antibody comprising a VH region comprising the sequence of SEQ ID NO:49 and a VL region comprising the sequence of SEQ ID NO:53 (045), preferably wherein the antibody competes less than 50%, e.g. less than 25%, such as less than 20% with said immobilized antibody, when determined as described in Example 17 and
the antibody binds to the SEMA domain of c-Met, preferably wherein the antibody is able to inhibit binding of HGF to the SEMA domain with an IC50 of less than 10 μg/mL, such as less than 2 μg/mL as described in Example 9.
In a further embodiment, the antibody does not compete for binding to soluble cMetECDHis with an immobilized antibody comprising a VH region comprising the sequence of SEQ ID NO:33 and a VL region comprising the sequence of SEQ ID NO:37 (024), preferably wherein the antibody competes for less than 25%, such as less than 20% with said immobilized antibody, when determined as described in Example 17.
In a further embodiment, the antibody binds to the same epitope as an antibody selected from the group consisting of:
a) an antibody comprising a VH region comprising the sequence of SEQ ID NO:1 and a VL region comprising the sequence of SEQ ID NO:5 (005)
b) an antibody comprising a VH region comprising the sequence of SEQ ID NO:9 and a VL region comprising the sequence of SEQ ID NO:13 (006)
c) an antibody comprising a VH region comprising the sequence of SEQ ID NO:25 and a VL region comprising the sequence of SEQ ID NO:29 (022), and
d) an antibody comprising a VH region comprising the sequence of SEQ ID NO:57 and a VL region comprising the sequence of SEQ ID NO:61 (058).
In a further embodiment, the antibody comprises a VH CDR3 region having the sequence as set forth in
a) SEQ ID NO:181, such as a VH CDR3 region as set forth in SEQ ID NO:4 or 12 (005, 006)
b) SEQ ID NO:28 (022), or
c) SEQ ID NO:60 (058).
In a further embodiment, the antibody comprises:
a) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:179, 180 and 181 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:6, 7 and 202, such as an antibody comprising
a. a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:2, 3 and 4 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:6, 7 and 8, (005), or
b. a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:10, 11 and 12 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:14, 15 and 16, (006)
b) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:26, 184 and 28 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:30, 31 and 205, such as an antibody comprising a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:26, 27 and 28 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:30, 31 and 32 (022), or
c) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 189, 190 and 60 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 62, 63 and 207, such as an antibody comprising a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:58, 59 and 60 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:62, 63 and 64 (058)
In an even further embodiment, the antibody comprises:
a) a VH region comprising the sequence of SEQ ID NO:1 and, preferably, a VL region comprising the sequence of SEQ ID NO:5 (005)
b) a VH region comprising the sequence of SEQ ID NO:9 and, preferably, a VL region comprising the sequence of SEQ ID NO:13 (006)
c) a VH region comprising the sequence of SEQ ID NO:25 and, preferably, a VL region comprising the sequence of SEQ ID NO:29 (022)
d) a VH region comprising the sequence of SEQ ID NO:57 and, preferably, a VL region comprising the sequence of SEQ ID NO:61 (058)
e) a VH region comprising the sequence of SEQ ID NO:145 and, preferably, a VL region comprising the sequence of SEQ ID NO:146 (031)
f) a VH region comprising the sequence of SEQ ID NO:147 and, preferably, a VL region comprising the sequence of SEQ ID NO:148 (007)
g) a VH region comprising the sequence of SEQ ID NO:149 and, preferably, a VL region comprising the sequence of SEQ ID NO:150 (011)
h) a VH region comprising the sequence of SEQ ID NO:151 and, preferably, a VL region comprising the sequence of SEQ ID NO:152 (017)
i) a VH region comprising the sequence of SEQ ID NO:153 and, preferably, a VL region comprising the sequence of SEQ ID NO:154 (025), or
j) a variant of any of said antibodies, wherein said variant preferably has at most 1, 2 or 3 amino-acid modifications, more preferably amino-acid substitutions, such as conservative amino-acid substitutions in said sequences.
In another main embodiment of the antibody of the invention:
the antibody competes for binding to soluble cMetECDHis with an immobilized antibody, wherein said immobilized antibody comprises a VH region comprising the sequence of SEQ ID NO:49 and a VL region comprising the sequence of SEQ ID NO:53 (045), preferably wherein the antibody competes for more than 50%, such as more than 75% with said immobilized antibody, when determined as described in Example 17, and
the antibody does not compete binding to soluble cMetECDHis with an immobilized antibody, wherein said immobilized comprises a VH region comprising the sequence of SEQ ID NO:9 and a VL region comprising the sequence of SEQ ID NO:13 (006), preferably wherein the antibody competes for less than 25%, such as less than 20% with said immobilized antibody, when determined as described in Example 17.
In a further embodiment, the antibody does not compete for binding to soluble cMetECDHis with an antibody selected from the group consisting of:
a) an immobilized antibody comprising a VH region comprising the sequence of SEQ ID NO:17 and a VL region comprising the sequence of SEQ ID NO:21 (008), and
b) an immobilized antibody comprising a VH region comprising the sequence of SEQ ID NO:33 and a VL region comprising the sequence of SEQ ID NO:37 (024),
preferably wherein the antibody competes for less than 25%, such as less than 20% with said immobilized antibody, when determined as described in Example 17.
In a further embodiment, the antibody binds to the same epitope as an antibody comprising a VH region comprising the sequence of SEQ ID NO:49 and a VL region comprising the sequence of SEQ ID NO:53 (045).
In a further embodiment, the antibody comprises a VH CDR3 region having the sequence as set forth in SEQ ID NO:188, such as a VH CDR3 region as set forth in SEQ ID NO:52 (045).
In a further embodiment, the antibody comprises a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 186, 187 and 188 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 54, 55 and 56, such as an antibody comprising a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:50, 51 and 52 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:54, 55 and 56 (045).
In a further embodiment, the antibody comprises:
a) a VH region comprising the sequence of SEQ ID NO:49 and, preferably, a VL region comprising the sequence of SEQ ID NO:53 (045)
b) a VH region comprising the sequence of SEQ ID NO:155 and, preferably, a VL region comprising the sequence of SEQ ID NO:156 (040)
c) a VH region comprising the sequence of SEQ ID NO:157 and, preferably, a VL region comprising the sequence of SEQ ID NO:158 (039), or
d) a variant of any of said antibodies, wherein said variant preferably has at most 1, 2 or 3 amino-acid modifications, more preferably amino-acid substitutions, such as conservative amino-acid substitutions in said sequences.
In a further embodiment, the antibody binds to the SEMA domain of c-Met, preferably wherein the antibody is able to inhibit binding of HGF to the SEMA domain with an IC50 of less than 10 μg/mL, such as less than 2 μg/mL as described in Example 9.
In another main embodiment of the antibody of the invention, the antibody binds to the same epitope as an antibody comprising a VH region comprising the sequence of SEQ ID NO:17 and a VL region comprising the sequence of SEQ ID NO:21 (008) or binds to the same epitope as an antibody comprising a VH region comprising the sequence of SEQ ID NO:41 and a VL region comprising the sequence of SEQ ID NO:45 (035) or binds to the same epitope as an antibody comprising a VH region comprising the sequence of SEQ ID NO:105 and a VL region comprising the sequence of SEQ ID NO:109 (096).
In a further embodiment, the antibody comprises a VH CDR3 region having the sequence as set forth in SEQ ID NO:183, such as a VH CDR3 region as set forth in SEQ ID NO:20, 44 or 108 (008, 035, 096).
In a further embodiment, the antibody comprises a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:18, 182 and 183 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:22, 203 and 204, such as an antibody comprising
a) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:18, 19 and 20 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:22, 23 and 24, (008), or
b) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:42, 43 and 44 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:46, 47 and 48, (035), or
c) a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:106, 107 and 108 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:110, 111 and 112 (096).
In a further embodiment, the antibody comprises:
a) a VH region comprising the sequence of SEQ ID NO:17 and, preferably, a VL region comprising the sequence of SEQ ID NO:21 (008)
b) a VH region comprising the sequence of SEQ ID NO:41 and, preferably, a VL region comprising the sequence of SEQ ID NO:45 (035)
c) a VH region comprising the sequence of SEQ ID NO:105 and, preferably, a VL region comprising the sequence of SEQ ID NO:109 (096) or
d) a variant of any of said antibodies, wherein said variant preferably has at most 1, 2 or 3 amino-acid modifications, more preferably amino-acid substitutions, such as conservative amino-acid substitutions in said sequences.
In a further embodiment, the antibody binds to A431 cells with an EC50 of 10 nM or less, such as an EC50 of 2 nM or less, preferably as determined according to Example 13.
In an even further embodiment, the antibody binds to c-Met with an affinity constant (KD) of 20 nM or less, such as an affinity of 5 nM or less, preferably as determined according to Example 14.
In an even further embodiment, the antibody binds to Rhesus c-Met, preferably wherein the signal of antibody binding to Rhesus c-Met is at least 5 times that of a negative control antibody, as determined according to Example 15.
In an even further embodiment, the antibody inhibits binding of HGF to the extracellular domain of c-Met, preferably wherein the antibody inhibits binding more than 40%, such as more than 50%, e.g. more than 60%, e.g. more than 70%, e.g. more than 80%, e.g. more than 90%, as determined according to Example 16.
In a yet even further embodiment, the antibody is capable of inhibit the viability of KP4 cells, preferably wherein the antibody is capable of inhibit the viability of more than 10%, such as more than 25%, e.g. more than 40%, preferably as described in Example 19.
Antibody Formats
The present invention provides antagonistic and non-antagonistic anti-c-Met antibodies. Whereas some antibodies act antagonistically on target cells regardless of whether they are monovalent or bivalent, for other antibodies, the functional effect depends on the valency. As shown in Example 19 herein, antibodies 024, 062, 064, 068, 069, 098, 101, 181, for instance, (which are all in the same cross-blocking group see Example 17) have antagonistic properties in a KP4 viability assay regardless of the format. Antibodies 022 and 058, on the other hand, behave antagonistically in this assay in a monovalent format, but agonistically (or at least non-antagonistically) in a bivalent format. Thus, depending on the desired functional properties for a particular use, particular antibodies can be selected from the set of antibodies provided in the present invention and/or their format can be adapted to change the valency.
Furthermore, the antibody of the invention can be of any isotype. The choice of isotype typically will be guided by the desired effector functions, such as ADCC induction. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either of the human light chain constant regions, kappa or lambda, may be used. If desired, the class of an anti-c-Met antibody of the present invention may be switched by known methods. For example, an antibody of the present invention that was originally IgM may be class switched to an IgG antibody of the present invention. Further, class switching techniques may be used to convert one IgG subclass to another, for instance from IgG1 to IgG2. Thus, the effector function of the antibodies of the present invention may be changed by isotype switching to, e.g., an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses. In one embodiment an antibody of the present invention is an IgG1 antibody, for instance an IgG1,κ.
Down-modulation of c-Met induced by antagonistic antibodies represents a mechanism of action of therapeutic c-Met antibodies. Accordingly, in one aspect of the invention antibodies with reduced agonistic properties, but with retained ability to induce down-modulation of c-Met are desirable.
It has been discovered that by reducing the conformational flexibility of the antibodies the potential residual agonistic activity of the bivalent IgG1 antibodies are minimized.
Accordingly, in a further embodiment, the antibody of the invention has been modified to make it less flexible, such as by hinge region mutations.
The largest conformational changes are the result of the flexibility of the hinge, which allows a wide range of Fab-Fc angles (Ollmann Saphire, E., R. L. Stanfield, M. D. M. Crispin, P. W. H. I. Parren, P. M. Rudd, R. A. Dwek, D. R. Burton and I. A. Wilson. 2002. Contrasting IgG structures reveal extreme asymmetry and flexibility. J. Mol. Biol. 319: 9-18). One way to reduce Fab-arm flexibility in immunoglobulins is to prevent the formation of disulphide bonds between the light and the heavy chain by means of genetic modification. In a natural IgG1 antibody the light chain is connected covalently with the heavy chain via a disulphide bond, connecting the C-terminal cysteine of the light chain to the cysteine at position 220 (C220 EU numbering) in the hinge of the Fc of the heavy chain. By either mutating amino acid C220 to serine or any other natural amino acid, by removing C220 by removing the complete hinge, or by replacing the IgG1 hinge with an IgG3 hinge, a molecule is formed in which the light chains are connected via their C-terminal cysteines, analogous to the situation found in the human isotype IgA2m(1). This results in a reduced flexibility of the Fabs relative to the Fc and consequently reduced cross-linking capacity, as shown in the Examples.
Another strategy to reduce the flexibility of an IgG1 molecule is to replace the IgG1 hinge with the IgG2 hinge or IgG2-like hinge. (Dangl et al. EMBO J. 1988; 7:1989-94). This hinge region has two properties distinct from that of IgG1, which are considered to render the molecules less flexible. First, compared to IgG1 hinge the IgG2 hinge is 3 amino acids shorter. Second, the IgG2 hinge contains an additional cysteine, thus three instead of two inter-heavy chain disulphide bridges will be formed. Alternatively, a variant of the IgG1 hinge that resembles the IgG2 hinge can be introduced. This mutant (TH746-9) (WO2010063746) contains mutation T223C and two deletions (K222 and T225) in order to create a shorter hinge with an additional cysteine.
In a further embodiment, the antibody of the invention is of the IgG1 subtype, wherein the hinge region has been modified by:
(i) deleting the hinge region of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 214) and substituting it with the IgG2 hinge region of the sequence: ERKCCVECPPCP (SEQ ID NO: 215) (IgG1 Hinge-IgG2);
(ii) deleting position 220 so the modified hinge region has the sequence of EPKSDKTHTCPPCP (SEQ ID NO: 216) (IgG1 ΔC220);
(iii) substituting cysteine at position 220 with any other natural amino acid (X) so the modified hinge region has the sequence of EPKSXDKTHTCPPCP (SEQ ID NO: 217) (IgG1 C220X);
(iv) deleting the hinge region of sequence EPKSCDKTHTCPPCP (SEQ ID NO: 214) (UniBody IgG1);
(v) deleting the hinge region of the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 214) and substituting it with the IgG3 hinge region of the sequence ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP (SEQ ID NO: 218) (IgG1 Hinge-IgG3); or
(vi) substituting threonine at position 223 with cysteine, and deleting lysine at position 222 and threonine at position 225, so the modified hinge region has the sequence of EPKSCDCHCPPCP (SEQ ID NO: 219) (IgG1 TH7Δ6-9).
In one embodiment of the invention, the antibody of the invention is of the IgG1 subtype, wherein the hinge region has been modified by deleting position 220 so the modified hinge region has the sequence of EPKSDKTHTCPPCP (SEQ ID NO: 216) (IgG1 ΔC220) or by substituting cysteine at position 220 with any other natural amino acid (X) so the modified hinge region has the sequence of EPKSXDKTHTCPPCP (SEQ ID NO: 217) (IgG1 C220X);
In a further embodiment, the antibody of the invention is of the IgG1 subtype, wherein the hinge region has been modified by substituting cysteine at position 220 with serine so the modified hinge region has the sequence of EPKSSDKTHTCPPCP (SEQ ID NO: 220) (IgG1 C220S).
In a further embodiment, the antibody of the invention is of IgG2 subtype.
In a further embodiment, the antibody of the invention is glyco-engineered to reduce fucose and thus enhance ADCC, e.g. by addition of compounds to the culture media duringantibody production as described in US2009317869 or as described in van Berkel et al.(2010) Biotechnol. Bioeng. 105:350 or by using FUT8 knockout cells, e.g. as described inYamane-Ohnuki et al (2004) Biotechnol. Bioeng 87:614. ADCC may alternatively beoptimized using the method described by Umalia et al. (1999) Nature Biotech 17:176.
In one embodiment, the antibody comprises a VH region comprising the CDR3 sequence of SEQ ID NO:100 and a VL region comprising the CDR3 sequence of SEQ ID NO: 104 (069) of the IgG1 subtype, wherein the hinge region has been modified by substituting cysteine at position 220 with serine so the modified hinge region has the sequence of EPKSSDKTHTCPPCP (SEQ ID NO: 220) (IgG1C220S).
In one embodiment, the antibody comprises a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:98, 99 and 100 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:102, 103 and 104 (069) of the IgG1 subtype, wherein the hinge region has been modified by substituting cysteine at position 220 with serine so the modified hinge region has the sequence of EPKSSDKTHTCPPCP (SEQ ID NO: 220) (IgG1 C220S).
In one embodiment, the antibody comprises a VH region comprising the sequence of SEQ ID NO:97 and a VL region comprising the sequence of SEQ ID NO:101 (069) of the IgG1 subtype, wherein the hinge region has been modified by substituting cysteine at position 220 with serine so the modified hinge region has the sequence of EPKSSDKTHTCPPCP (SEQ ID NO: 220) (IgG1 C220S).
Various publications have demonstrated the correlation between reduced core-fucosylation and enhanced ADCC activity in vitro (Shields R L. 2002 JBC; 277:26733-26740, Shinkawa T. 2003 JBC; 278(5):3466-3473, Umaña P. Nat Biotechnol. 1999 February; 17(2):176-80).
In a further embodiment, the antibody of the invention has been modified to reduce core-fucosylation below 10%, such as below 5% as determined with high performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). This may be achieved by methods well known in the prior art, e.g. kifunensine treatment or production in FUT8 negative cells.
In a further embodiment, the antibody of the invention has been engineered to enhance complement activation, e.g. as described in Natsume et al. (2009) Cancer Sci. 100:2411.
In one embodiment, the antibody of the invention is a full-length antibody, preferably an IgG1 antibody, in particular an IgG1,κ antibody. In another embodiment, the antibody of the invention is an antibody fragment or a single-chain antibody.
Antibodies fragments may e.g. be obtained by fragmentation using conventional techniques, and the fragments screened for utility in the same manner as described herein for whole antibodies. For example, F(ab′)2 fragments may be generated by treating antibody with pepsin. The resulting F(ab′)2 fragment may be treated to reduce disulfide bridges to produce Fab′ fragments. Fab fragments may be obtained by treating an IgG antibody with papain; Fab′ fragments may be obtained with pepsin digestion of IgG antibody. An F(ab′) fragment may also be produced by binding Fab′ described below via a thioether bond or a disulfide bond. A Fab′ fragment is an antibody fragment obtained by cutting a disulfide bond of the hinge region of the F(ab′)2. A Fab′ fragment may be obtained by treating an F(ab′)2 fragment with a reducing agent, such as dithiothreitol. Antibody fragment may also be generated by expression of nucleic acids encoding such fragments in recombinant cells (see for instance Evans et al., J. Immunol. Meth. 184, 123-38 (1995)). For example, a chimeric gene encoding a portion of an F(ab′)2 fragment could include DNA sequences encoding the CH1 domain and hinge region of the H chain, followed by a translational stop codon to yield such a truncated antibody fragment molecule.
As explained above, in one embodiment, the anti-c-Met antibody of the invention is a bivalent antibody.
In another embodiment, the anti-c-Met antibody of the invention is a monovalent antibody.
In one embodiment, the antibody of the invention is a Fab fragment or a one-armed antibody, such as described in US20080063641 (Genentech) or other monovalent antibody, e.g. such as described in WO2007048037 (Amgen).
In a preferred embodiment, a monovalent antibody has a structure as described in WO2007059782 (Genmab) (incorporated herein by reference) having a deletion of the hinge region. Accordingly, in one embodiment, the antibody is a monovalent antibody, wherein said anti-c-Met antibody is constructed by a method comprising:
i) providing a nucleic acid construct encoding the light chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the VL region of a selected antigen specific anti-c-Met antibody and a nucleotide sequence encoding the constant CL region of an Ig, wherein said nucleotide sequence encoding the VL region of a selected antigen specific antibody and said nucleotide sequence encoding the CL region of an Ig are operably linked together, and wherein, in case of an IgG1 subtype, the nucleotide sequence encoding the CL region has been modified such that the CL region does not contain any amino acids capable of forming disulfide bonds or covalent bonds with other peptides comprising an identical amino acid sequence of the CL region in the presence of polyclonal human IgG or when administered to an animal or human being;
ii) providing a nucleic acid construct encoding the heavy chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the VH region of a selected antigen specific antibody and a nucleotide sequence encoding a constant CH region of a human Ig, wherein the nucleotide sequence encoding the CH region has been modified such that the region corresponding to the hinge region and, as required by the Ig subtype, other regions of the CH region, such as the CH3 region, does not comprise any amino acid residues which participate in the formation of disulphide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising an identical amino acid sequence of the CH region of the human Ig in the presence of polyclonal human IgG or when administered to an animal human being, wherein said nucleotide sequence encoding the VH region of a selected antigen specific antibody and said nucleotide sequence encoding the CH region of said Ig are operably linked together;
iii) providing a cell expression system for producing said monovalent antibody;
iv) producing said monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii).
Similarly, in one embodiment, the anti-c-Met antibody is a monovalent antibody, which comprises
(i) a variable region of an antibody of the invention as described herein or an antigen binding part of the said region, and
(ii) a CH region of an immunoglobulin or a fragment thereof comprising the CH2 and CH3 regions, wherein the CH region or fragment thereof has been modified such that the region corresponding to the hinge region and, if the immunoglobulin is not an IgG4 subtype, other regions of the CH region, such as the CH3 region, do not comprise any amino acid residues, which are capable of forming disulfide bonds with an identical CH region or other covalent or stable non-covalent inter-heavy chain bonds with an identical CH region in the presence of polyclonal human IgG.
In a further embodiment hereof, the heavy chain of the monovalent anti-c-Met antibody has been modified such that the entire hinge has been deleted.
In another further embodiment, said monovalent antibody is of the IgG4 subtype, but the CH3 region has been modified so that one or more of the following amino acid substitutions have been made:
Numbering of CH3 mutations
EU index
KABAT*
G4*
Mutations
E378
E357
E357A or E357T or E357V or E357I
S387
S364
S364R or S364K
T389
T366
T366A or T366R or T366K or T366N
L391
L368
L368A or L368V or L368E or
L368G or L368S or L368T
D427
D399
D399A or D399T or D399S
F436
F405
F405A or F405L or F405T
or F405D or F405R or F405Q
or F405K or F405Y
Y438
Y407
Y407A or Y407E or Y407Q or Y407K or Y407F
F436
F405
(F405T and Y407E) or (F405D and Y407E)
and Y438
and Y407
D427
D399
(D399S and Y407Q) or (D399S and Y407K)
and Y438
and Y407
or (D399S and Y407E)
*KABAT indicates amino acid numbering according to Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). EU index indicates amino acid numbering according to EU index as outlined in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).
In another further embodiment, the sequence of said monovalent antibody has been modified so that it does not comprise any acceptor sites for N-linked glycosylation.
Anti-c-Met antibodies of the invention also include single chain antibodies. Single chain antibodies are peptides in which the heavy and light chain Fv regions are connected. In one embodiment, the present invention provides a single-chain Fv (scFv) wherein the heavy and light chains in the Fv of an anti-c-Met antibody of the present invention are joined with a flexible peptide linker (typically of about 10, 12, 15 or more amino acid residues) in a single peptide chain. Methods of producing such antibodies are described in for instance U.S. Pat. No. 4,946,778, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994), Bird et al., Science 242, 423-426 (1988), Huston et al., PNAS USA 85, 5879-5883 (1988) and McCafferty et al., Nature 348, 552-554 (1990). The single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used.
In one embodiment, the anti-c-Met antibody of the invention is an effector-function-deficient antibody. In one embodiment, the effector-function-deficient anti-c-Met antibody is a stabilized IgG4 antibody, which has been modified to prevent Fab-arm exchange (van der Neut Kolfschoten et al. (2007) Science 317(5844):1554-7). Examples of suitable stabilized IgG4 antibodies are antibodies, wherein arginine at position 409 in a heavy chain constant region of human IgG4, which is indicated in the EU index as in Kabat et al., is substituted with lysine, threonine, methionine, or leucine, preferably lysine (described in WO2006033386 (Kirin)) and/or wherein the hinge region has been modified to comprise a Cys-Pro-Pro-Cys (SEQ ID NO: 213) sequence.
In a further embodiment, the stabilized IgG4 anti-c-Met antibody is an IgG4 antibody comprising a heavy chain and a light chain, wherein said heavy chain comprises a human IgG4 constant region having a residue selected from the group consisting of: Lys, Ala, Thr, Met and Leu at the position corresponding to 409 and/or a residue selected from the group consisting of: Ala, Val, Gly, Ile and Leu at the position corresponding to 405, and wherein said antibody optionally comprises one or more further substitutions, deletions and/or insertions, but does not comprise a Cys-Pro-Pro-Cys (SEQ ID NO: 213) sequence in the hinge region. Preferably, said antibody comprises a Lys or Ala residue at the position corresponding to 409 or the CH3 region of the antibody has been replaced by the CH3 region of human IgG1, of human IgG2 or of human IgG3. See also and WO2008145142 (Genmab).
In an even further embodiment, the stabilized IgG4 anti-c-Met antibody is an IgG4 antibody comprising a heavy chain and a light chain, wherein said heavy chain comprises a human IgG4 constant region having a residue selected from the group consisting of: Lys, Ala, Thr, Met and Leu at the position corresponding to 409 and/or a residue selected from the group consisting of: Ala, Val, Gly, Ile and Leu at the position corresponding to 405, and wherein said antibody optionally comprises one or more further substitutions, deletions and/or insertions and wherein said antibody comprises a Cys-Pro-Pro-Cys (SEQ ID NO: 213) sequence in the hinge region. Preferably, said antibody comprises a Lys or Ala residue at the position corresponding to 409 or the CH3 region of the antibody has been replaced by the CH3 region of human IgG1, of human IgG2 or of human IgG3.
In a further embodiment, the effector-function-deficient anti-c-Met antibody is an antibody of a non-IgG4 type, e.g. IgG1, IgG2 or IgG3 which has been mutated such that the ability to mediate effector functions, such as ADCC, has been reduced or even eliminated. Such mutations have e.g. been described in Dall'Acqua W F et al., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J Virol.; 75(24):12161-12168 (2001).
Conjugates
In a further embodiment, the present invention provides an anti-c-Met antibody conjugated to a therapeutic moiety, such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant, or a radioisotope. Such conjugates are referred to herein as “immunoconjugates”. Immunoconjugates which include one or more cytotoxins are referred to as “immunotoxins”.
A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Suitable therapeutic agents for forming immunoconjugates of the present invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydro-testosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin), antibiotics (such as dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), diphtheria toxin and related molecules (such as diphtheria A chain and active fragments thereof and hybrid molecules), ricin toxin (such as ricin A or a deglycosylated ricin A chain toxin), cholera toxin, a Shiga-like toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins. Other suitable conjugated molecules include ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, diphtherin toxin, and Pseudomonas endotoxin. See, for example, Pastan et al., Cell 47, 641 (1986) and Goldenberg, Calif. A Cancer Journal for Clinicians 44, 43 (1994). Therapeutic agents, which may be administered in combination with a an anti-c-Met antibody of the present invention as described elsewhere herein, may also be candidates for therapeutic moieties useful for conjugation to an antibody of the present invention.
In another embodiment, an anti-c-Met antibody of the invention comprises a conjugated nucleic acid or nucleic acid-associated molecule. In one such facet of the present invention, the conjugated nucleic acid is a cytotoxic ribonuclease, an antisense nucleic acid, an inhibitory RNA molecule (e.g., a siRNA molecule) or an immunostimulatory nucleic acid (e.g., an immunostimulatory CpG motif-containing DNA molecule). In another embodiment, an anti-c-Met antibody of the invention is conjugated to an aptamer or a ribozyme.
In one embodiment, anti-c-Met antibodies comprising one or more radiolabeled amino acids are provided. A radiolabeled anti-c-Met antibody may be used for both diagnostic and therapeutic purposes (conjugation to radiolabeled molecules is another possible feature). Non-limiting examples of labels for polypeptides include 3H, 14C, 15N, 35S, 90Y, 99Tc, and 125I, 131I, and 186Re.
Anti-c-Met antibodies may also be chemically modified by covalent conjugation to a polymer to for instance increase their circulating half-life. Exemplary polymers, and methods to attach them to peptides, are illustrated in for instance U.S. Pat. Nos. 4,766,106, 4,179,337, 4,495,285 and No. 4,609,546. Additional polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., a PEG with a molecular weight of between about 1,000 and about 40,000, such as between about 2,000 and about 20,000).
Any method known in the art for conjugating the anti-c-Met antibody to the conjugated molecule(s), such as those described above, may be employed, including the methods described by Hunter et al., Nature 144, 945 (1962), David et al., Biochemistry 13, 1014 (1974), Pain et al., J. Immunol. Meth. 40, 219 (1981) and Nygren, J. Histochem. and Cytochem. 30, 407 (1982). Such antibodies may be produced by chemically conjugating the other moiety to the N-terminal side or C-terminal side of the anti-c-Met antibody or fragment thereof (e.g., an anti-c-Met antibody H or L chain) (see, e.g., Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). Such conjugated antibody derivatives may also be generated by conjugation at internal residues or sugars, where appropriate. The agents may be coupled either directly or indirectly to an anti-c-Met antibody of the present invention. One example of indirect coupling of a second agent is coupling by a spacer moiety. In one embodiment, the anti-c-Met antibody of the present invention is attached to a chelator linker, e.g. tiuxetan, which allows for the antibody to be conjugated to a radioisotope.
Bispecific Antibodies
In a further aspect, the invention relates to a bispecific molecule comprising a first antigen binding site from an anti-c-Met antibody of the invention as described herein above and a second antigen binding site with a different binding specificity, such as a binding specificity for a human effector cell, a human Fc receptor, a T cell receptor, a B cell receptor or a binding specificity for a non-overlapping epitope of c-Met, i.e. a bispecific antibody wherein the first and second antigen binding sites do not compete for binding to c-Met, e.g. when tested as described in Example 17.
Exemplary bispecific antibody molecules of the invention comprise (i) two antibodies one with a specificity to c-Met and another to a second target that are conjugated together, (ii) a single antibody that has one chain or arm specific to c-Met and a second chain or arm specific to a second molecule, and (iii) a single chain antibody that has specificity to c-Met and a second molecule. In one embodiment, the second molecule is a cancer antigen/tumor-associated antigen such as carcinoembryonic antigen (CEA), prostate specific antigen (PSA), RAGE (renal antigen), α-fetoprotein, CAMEL (CTL-recognized antigen on melanoma), CT antigens (such as MAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin antigens (e.g., MUC1, mucin-CA125, etc.), ganglioside antigens, tyrosinase, gp75, C-myc, Marti, MelanA, MUM-1, MUM-2, MUM-3, HLA-B7, Ep-CAM or a cancer-associated integrin, such as α5β3 integrin. In another embodiment, the second molecule is an angiogenic factor or other cancer-associated growth factor, such as a vascular endothelial growth factor, a fibroblast growth factor, epidermal growth factor, angiogenin or a receptor of any of these, particularly receptors associated with cancer progression (for instance one of the HER1-HER4 receptors). In one embodiment, a bispecific antibody of the present invention is a diabody.
Nucleic Acid Sequences, Vectors and Host Cells
In a further aspect, the invention relates to nucleic acid sequences, such as DNA sequences, encoding heavy and light chains of an antibody of the invention.
In one embodiment, the nucleic acid sequence encodes an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177 and 178.
In another particular embodiment, the nucleic acid sequence encodes a VH amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175 and 177.
In another particular embodiment, the nucleic acid sequence encodes a VL amino acid sequence selected from the group consisting: SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176 and 178.
In an even further aspect, the invention relates to an expression vector, or a set of expression vectors, encoding an antibody of the invention. The heavy and light chain of the antibody may be encoded by the same vector or by different vector.
Such expression vectors may be used for recombinant production of antibodies of the invention.
In one embodiment, the expression vector of the invention comprises a nucleotide sequence encoding one or more of the amino acid sequences selected from the group consisting of: SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177 and 178.
In another particular embodiment, the expression vector of the invention comprises a nucleotide sequence encoding one or more of the VH amino acid sequences selected from the group consisting of: SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175 and 177.
In another particular embodiment, the expression vector of the invention comprises a nucleotide sequence encoding one or more of the VL amino acid sequences selected from the group consisting of: SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176 and 178.
In a further embodiment, the expression vector further comprises a nucleotide sequence encoding the constant region of a light chain, a heavy chain or both light and heavy chains of an antibody, e.g. a human antibody.
An expression vector in the context of the present invention may be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (a nucleic acid sequence comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, an anti-c-Met antibody-encoding nucleic acid is comprised in a naked DNA or RNA vector, including, for example, a linear expression element (as described in for instance Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a compacted nucleic acid vector (as described in for instance U.S. Pat. No. 6,077,835 and/or WO 00/70087), a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sized nucleic acid vector (as described in for instance Schakowski et al., Mol Ther 3, 793-800 (2001)), or as a precipitated nucleic acid vector construct, such as a CaP04-precipitated construct (as described in for instance WO 00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such nucleic acid vectors and the usage thereof are well known in the art (see for instance U.S. Pat. Nos. 5,589,466 and No. 5,973,972).
In one embodiment, the vector is suitable for expression of the anti-c-Met antibody in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989), pET vectors (Novagen, Madison Wis.) and the like).
An expression vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be employed. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), and Grant et al., Methods in Enzymol 153, 516-544 (1987)).
An expression vector may also or alternatively be a vector suitable for expression in mammalian cells, e.g. a vector comprising glutamine synthetase as a selectable markers, such as the vectors described in (Bebbington (1992) Biotechnology (NY) 10:169-175).
A nucleic acid and/or vector may also comprises a nucleic acid sequence encoding a secretion/localization sequence, which can target a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or into cell culture media. Such sequences are known in the art, and include secretion leader or signal peptides.
In an expression vector of the invention, anti-c-Met antibody-encoding nucleic acids may comprise or be associated with any suitable promoter, enhancer, and other expression-facilitating elements. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter/enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly (A) termination sequences, an origin of replication for plasmid product in E. coli, an antibiotic resistance gene as selectable marker, and/or a convenient cloning site (e.g., a polylinker). Nucleic acids may also comprise an inducible promoter as opposed to a constitutive promoter such as CMV IE.
In one embodiment, the anti-c-Met-antibody-encoding expression vector may be positioned in and/or delivered to the host cell or host animal via a viral vector.
In an even further aspect, the invention relates to a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, which produces an antibody of the invention as defined herein. Examples of host cells include yeast, bacterial, and mammalian cells, such as CHO or HEK cells. For example, in one embodiment, the present invention provides a cell comprising a nucleic acid stably integrated into the cellular genome that comprises a sequence coding for expression of an anti-c-Met antibody of the present invention. In another embodiment, the present invention provides a cell comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a sequence coding for expression of an anti-c-Met antibody of the invention.
In a further aspect, the invention relates to a hybridoma which produces an antibody of the invention as defined herein. In an even further aspect, the invention relates to a transgenic non-human animal or plant comprising nucleic acids encoding a human heavy chain and a human light chain, wherein the animal or plant produces an antibody of the invention of the invention.
In a further aspect, the invention relates to a method for producing an anti-c-Met antibody of the invention, said method comprising the steps of
a) culturing a hybridoma or a host cell of the invention as described herein above, and
b) purifying the antibody of the invention from the culture media.
Compositions
In a further main aspect, the invention relates to a pharmaceutical composition comprising:
an anti-c-Met antibody as defined herein, and
a pharmaceutically-acceptable carrier.
The pharmaceutical composition of the present invention may contain one antibody of the present invention or a combination of different antibodies of the present invention.
The pharmaceutical compositions may be formulated in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995. A pharmaceutical composition of the present invention may e.g. include diluents, fillers, salts, buffers, detergents (e.g., a nonionic detergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and/or other materials suitable for inclusion in a pharmaceutical composition.
Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible with a compound of the present invention. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, carboxymethyl cellulose colloidal solutions, tragacanth gum and injectable organic esters, such as ethyl oleate, and/or various buffers. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
Pharmaceutical compositions of the present invention may also comprise pharmaceutically acceptable antioxidants for instance (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Pharmaceutical compositions of the present invention may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the compositions.
The pharmaceutical compositions of the present invention may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the pharmaceutical composition. The compounds of the present invention may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid alone or with a wax, or other materials well known in the art. Methods for the preparation of such formulations are generally known to those skilled in the art.
Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients e.g. as enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients e.g. from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
The pharmaceutical composition may be administered by any suitable route and mode. In one embodiment, a pharmaceutical composition of the present invention is administered parenterally. “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion.
In one embodiment that pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.
Uses
In a further main aspect, the invention relates to an anti-c-Met antibody of the invention for use as a medicament.
The anti-c-Met antibodies of the invention may be used for a number of purposes. In particular, the antibodies of the invention may be used for the treatment of various forms of cancer, including metastatic cancer and refractory cancer. Such cancer may be HGF-dependent or HGF-independent.
In one embodiment, the anti-c-Met antibodies of the invention are used for the treatment of a form of cancer selected from the group consisting of: bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, endometrial cancer, esophogeal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer (such as non-small cell lung cancer (NSCLC)), nasopharyngeal cancer, ovarian cancer, pancreatic cancer, gall bladder cancer, prostate cancer and thyroid cancer.
In another embodiment, the anti-c-Met antibodies of the invention are used for the treatment of a form of cancer selected from the group consisting of: osteosarcoma, rhabdomyosarcoma and synovial sarcoma.
In another embodiment, the anti-c-Met antibodies of the invention are used for the treatment of a form of cancer selected from the group consisting of: Kaposi's sarcoma, leiomyosarcoma, malignant fibrous histiocytoma and fibrosarcoma.
In another embodiment, the anti-c-Met antibodies of the invention are used for the treatment of hematopoietic malignancies, such as a malignancy selected from the group consisting of: acute myelogenous leukemia, adult T cell leukemia, chronic myeloid leukemia, lymphoma and multiple myeloma.
In a further embodiment, the anti-c-Met antibodies of the invention are used for the treatment of a neoplasm selected from the group consisting of: glioblastoma, astrocytoma, melanoma, mesothelioma and Wilm's tumor.
In a further embodiment, the anti-c-Met antibodies of the invention are used for the treatment of MiT tumors, including clear cell sarcoma (CCS), alveolar soft part sarcoma (ASPS) and translocation-associated renal cell carcinoma.
In another embodiment, agonistic anti-c-Met antibodies of the invention are used for the regulation of cytokine production and the induction of endothelial progenitor cell mobilization, e.g. in patients with coronary heart disease (Yang et al. (2009) Clin Exp Pharmacol Physiol. 36:790).
In another embodiment, agonistic anti-c-Met antibodies of the invention are used for inhibiting or improving chronic renal failure (Mizuno et al. (2008) Front Biosci. 13:7072).
Similarly, the invention relates to a method for inhibiting growth and/or proliferation of a tumor cell expressing c-Met, comprising administration, to an individual in need thereof, of an effective amount of an antibody of the invention.
In one embodiment, said tumor cell is involved in a form of cancer selected from the group consisting of: bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, gall bladder cancer, prostate cancer, thyroid cancer, osteosarcoma, rhabdomyosarcoma, synovial sarcoma, Kaposi's sarcoma, leiomyosarcoma, malignant fibrous histiocytoma, fibrosarcoma, acute myelogenous leukemia, adult T cell leukemia, chronic myeloid leukemia, lymphoma, multiple myeloma, glioblastoma, astrocytoma, melanoma, mesothelioma and Wilm's tumor.
Also, the invention relates to the use of a monoclonal antibody that binds to human c-Met for the preparation of a medicament for the treatment of cancer, such as one of the specific cancer indications mentioned above.
In an embodiment, selection of patients to be treated with an anti-c-Met antibody is based on the level of (over)expression of c-Met and/or HGF on the relevant tumor cells of said patients.
In a further embodiment of the methods of treatment of the present invention, the efficacy of the treatment is being monitored during the therapy, e.g. at predefined points in time, by determining c-Met expression levels on the relevant tumor cells.
Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.
The efficient dosages and the dosage regimens for the anti-c-Met antibodies depend on the disease or condition to be treated and may be determined by the persons skilled in the art. An exemplary, non-limiting range for a therapeutically effective amount of a compound of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3, about 5, or about 8 mg/kg.
A physician or veterinarian having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the anti-c-Met antibody employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Administration may e.g. be parenteral, such as intravenous, intramuscular or subcutaneous. In one embodiment, the anti-c-Met antibodies may be administered by infusion in a weekly dosage of from 10 to 500 mg/m2, such as of from 200 to 400 mg/m2. Such administration may be repeated, e.g., 1 to 8 times, such as 3 to 5 times. The administration may be performed by continuous infusion over a period of from 2 to 24 hours, such as of from 2 to 12 hours. In one embodiment, the anti-c-Met antibodies may be administered by slow continuous infusion over a long period, such as more than 24 hours, in order to reduce toxic side effects.
In one embodiment the anti-c-Met antibodies may be administered in a weekly dosage of from 250 mg to 2000 mg, such as for example 300 mg, 500 mg, 700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times, such as from 4 to 6 times. Such regimen may be repeated one or more times as necessary, for example, after 6 months or 12 months. The dosage may be determined or adjusted by measuring the amount of compound of the present invention in the blood upon administration by for instance taking out a biological sample and using anti-idiotypic antibodies which target the antigen binding region of the anti-c-Met antibodies of the present invention.
In one embodiment, the anti-c-Met antibodies may be administered by maintenance therapy, such as, e.g., once a week for a period of 6 months or more.
An anti-c-Met antibody may also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and/or reduce the risk of recurrence when a cancer is in remission.
Anti-c-Met antibodies may also be administered in combination therapy, i.e., combined with other therapeutic agents relevant for the disease or condition to be treated. Accordingly, in one embodiment, the antibody-containing medicament is for combination with one or more further therapeutic agent, such as a cytotoxic, chemotherapeutic or anti-angiogenic agent.
Such combined administration may be simultaneous, separate or sequential. For simultaneous administration the agents may be administered as one composition or as separate compositions, as appropriate. The present invention thus also provides methods for treating a disorder involving cells expressing c-Met as described above, which methods comprise administration of an anti-c-Met antibody of the present invention combined with one or more additional therapeutic agents as described below.
In one embodiment, the present invention provides a method for treating a disorder involving cells expressing c-Met in a subject, which method comprises administration of a therapeutically effective amount of an anti-c-Met antibody of the present invention and at least one additional therapeutic agent to a subject in need thereof.
In one embodiment, the present invention provides a method for treating or preventing cancer, which method comprises administration of a therapeutically effective amount of an anti-c-Met antibody of the present invention and at least one additional therapeutic agent to a subject in need thereof.
In one embodiment, such an additional therapeutic agent may be selected from an antimetabolite, such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine or cladribine.
In another embodiment, such an additional therapeutic agent may be selected from an alkylating agent, such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin.
In another embodiment, such an additional therapeutic agent may be selected from an anti-mitotic agent, such as taxanes, for instance docetaxel, and paclitaxel, and vinca alkaloids, for instance vindesine, vincristine, vinblastine, and vinorelbine.
In another embodiment, such an additional therapeutic agent may be selected from a topoisomerase inhibitor, such as topotecan or irinotecan, or a cytostatic drug, such as etoposide and teniposide.
In another embodiment, such an additional therapeutic agent may be selected from a growth factor inhibitor, such as an inhibitor of ErbB1 (EGFR) (such as an anti-EGFR antibody, e.g. zalutumumab, cetuximab, panitumumab or nimotuzumab or other EGFR inhibitors, such as gefitinib or erlotinib), an inhibitor of ErbB2 (Her2/neu) (such as an anti-HER2 antibody, e.g. trastuzumab, trastuzumab-DM1 or pertuzumab) or an inhibitor of both EGFR and HER2, such as lapatinib).
In another embodiment, such an additional therapeutic agent may be selected from a tyrosine kinase inhibitor, such as imatinib (Glivec, Gleevec STI571) or lapatinib, PTK787/ZK222584.
In another embodiment, the present invention provides a method for treating a disorder involving cells expressing c-Met in a subject, which method comprises administration of a therapeutically effective amount of an anti-c-Met antibody of the present invention and at least one inhibitor of angiogenesis, neovascularization, and/or other vascularization to a subject in need thereof.
Examples of such angiogenesis inhibitors are urokinase inhibitors, matrix metalloprotease inhibitors (such as marimastat, neovastat, BAY 12-9566, AG 3340, BMS-275291 and similar agents), inhibitors of endothelial cell migration and proliferation (such as TNP-470, squalamine, 2-methoxyestradiol, combretastatins, endostatin, angiostatin, penicillamine, SCH66336 (Schering-Plough Corp, Madison, N.J.), R115777 (Janssen Pharmaceutica, Inc, Titusville, N.J.) and similar agents), antagonists of angiogenic growth factors (such as such as ZD6474, SU6668, antibodies against angiogenic agents and/or their receptors (such as VEGF (e.g. bevacizumab), bFGF, and angiopoietin-1), thalidomide, thalidomide analogs (such as CC-5013), Sugen 5416, SU5402, antiangiogenic ribozyme (such as angiozyme), interferon α (such as interferon α2a), suramin and similar agents), VEGF-R kinase inhibitors and other anti-angiogenic tyrosine kinase inhibitors (such as SU011248), inhibitors of endothelial-specific integrin/survival signaling (such as vitaxin and similar agents), copper antagonists/chelators (such as tetrathiomolybdate, captopril and similar agents), carboxyamido-triazole (CAI), ABT-627, CM101, interleukin-12 (IL-12), IM862, PNU145156E as well as nucleotide molecules inhibiting angiogenesis (such as antisense-VEGF-cDNA, cDNA coding for angiostatin, cDNA coding for p53 and cDNA coding for deficient VEGF receptor-2).
Other examples of such inhibitors of angiogenesis, neovascularization, and/or other vascularization are anti-angiogenic heparin derivatives (e.g., heperinase III), temozolomide, NK4, macrophage migration inhibitory factor, cyclooxygenase-2 inhibitors, inhibitors of hypoxia-inducible factor 1, anti-angiogenic soy isoflavones, oltipraz, fumagillin and analogs thereof, somatostatin analogues, pentosan polysulfate, tecogalan sodium, dalteparin, tumstatin, thrombospondin, NM-3, combrestatin, canstatin, avastatin, antibodies against other targets, such as anti-alpha-v/beta-3 integrin and anti-kininostatin antibodies.
In one embodiment, a therapeutic agent for use in combination with an anti-c-Met antibody for treating the disorders as described above may be an anti-cancer immunogen, such as a cancer antigen/tumor-associated antigen (e.g., epithelial cell adhesion molecule (EpCAM/TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen (CEA), tumor-associated glycoprotein 72 (TAG-72), gp100, Melan-A, MART-1, KDR, RCAS1, MDA7, cancer-associated viral vaccines (e.g., human papillomavirus vaccines) or tumor-derived heat shock proteins.
In one embodiment, a therapeutic agent for use in combination with an anti-c-Met antibody for treating the disorders as described above may be an anti-cancer cytokine, chemokine, or combination thereof. Examples of suitable cytokines and growth factors include IFNγ, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα (e.g., INFα2b), IFNβ, GM-CSF, CD40L, Flt3, ligand, stem cell factor, ancestim, and TNFα. Suitable chemokines may include Glu-Leu-Arg (ELR)-negative chemokines such as IP-10, MCP-3, MIG, and SDF-1α from the human CXC and C-C chemokine families. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins.
In one embodiment, a therapeutic agent for use in combination with an anti-c-Met antibody for treating the disorders as described above may be a cell cycle control/apoptosis regulator (or “regulating agent”). A cell cycle control/apoptosis regulator may include molecules that target and modulate cell cycle control/apoptosis regulators such as (i) cdc-25 (such as NSC 663284), (ii) cyclin-dependent kinases that overstimulate the cell cycle (such as flavopiridol (L868275, HMR1275), 7-hydroxystaurosporine (UCN-01, KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase modulators (such as BIBR1532, SOT-095, GRN163 and compositions described in for instance U.S. Pat. Nos. 6,440,735 and No. 6,713,055). Non-limiting examples of molecules that interfere with apoptotic pathways include TNF-related apoptosis-inducing ligand (TRAIL)/apoptosis-2 ligand (Apo-2L), antibodies that activate TRAIL receptors, IFNs, and anti-sense Bcl-2.
In one embodiment, a therapeutic agent for use in combination with an anti-c-Met antibody for treating the disorders as described above may be a hormonal regulating agent, such as agents useful for anti-androgen and anti-estrogen therapy. Examples of such hormonal regulating agents are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol/estinyl, an antiandrogene (such as flutaminde/eulexin), a progestin (such as such as hydroxyprogesterone caproate, medroxy-progesterone/provera, megestrol acepate/megace), an adrenocorticosteroid (such as hydrocortisone, prednisone), luteinizing hormone-releasing hormone (and analogs thereof and other LHRH agonists such as buserelin and goserelin), an aromatase inhibitor (such as anastrazole/arimidex, aminoglutethimide/cytraden, exemestane) or a hormone inhibitor (such as octreotide/sandostatin).
In one embodiment, a therapeutic agent for use in combination with an anti-c-Met antibody for treating the disorders as described above may be an anti-anergic agent, such ascompounds are molecules that block the activity of CTLA-4, e.g. ipilimumab.
In one embodiment, a therapeutic agent for use in combination with an anti-c-Met antibody for treating the disorders as described above may be an anti-cancer nucleic acid or an anti-cancer inhibitory RNA molecule.
Examples of other anti-cancer agents, which may be relevant as therapeutic agents for use in combination with an anti-c-Met antibody for treating the disorders as described above are differentiation inducing agents, retinoic acid analogues (such as all trans retinoic acid, 13-cis retinoic acid and similar agents), vitamin D analogues (such as seocalcitol and similar agents), inhibitors of ErbB3, ErbB4, IGF-IR, insulin receptor, PDGFRa, PDGFRbeta, Flk2, Flt4, FGFR1, FGFR2, FGFR3, FGFR4, TRKA, TRKC, RON (such as an anti-RON antibody), Sea, Tie, Tie2, Eph, Ret, Ros, Alk, LTK, PTK7 and similar agents.
Examples of other anti-cancer agents, which may be relevant as therapeutic agents for use in combination with an anti-c-Met antibody for treating the disorders as described above are estramustine and epirubicin.
Examples of other anti-cancer agents, which may be relevant as therapeutic agents for use in combination with an anti-c-Met antibody for treating the disorders as described above are a HSP90 inhibitor like 17-allyl amino geld-anamycin, antibodies directed against a tumor antigen such as PSA, CA125, KSA, integrins, e.g. integrin β1, or inhibitors of VCAM.
Examples of other anti-cancer agents, which may be relevant as therapeutic agents for use in combination with an anti-c-Met antibody for treating the disorders as described above are calcineurin-inhibitors (such as valspodar, PSC 833 and other MDR-1 or p-glycoprotein inhibitors), TOR-inhibitors (such as sirolimus, everolimus and rapamcyin). and inhibitors of “lymphocyte homing” mechanisms (such as FTY720), and agents with effects on cell signaling such as adhesion molecule inhibitors (for instance anti-LFA).
In one embodiment, the anti-c-Met antibody of the invention is for use in combination with one or more other therapeutic antibodies, such as ofatumumab, zanolimumab, daratumumab, ranibizumab, Zenapax, Simulect, Remicade, Humira, Tysabri, Xolair, raptiva and/or rituximab.
Other therapeutic antibodies which may be used in combination with the antibody of the present invention are anti-c-Met antibodies that bind to other regions of c-Met, such as the antibodies described in WO2005016382, WO2006015371, WO2007090807, WO2007126799 or WO2009007427 (all incorporated herein by reference).
In another embodiment, two or more different antibodies of the invention as described herein are used in combination for the treatment of disease. Particularly interesting combinations include two or more non-competing antibodies. Such combination therapy may lead to binding of an increased number of antibody molecules per cell, which may give increase efficacy, e.g. via activation of complement-mediated lysis.
In addition to the above, other embodiments of combination therapies of the invention include the following:
For the treatment of non-small-cell lung cancer, an anti-c-Met antibody in combination with EGFR inhibitors, such as an anti-EGFR antibody, e.g. zalutumumab, cetuximab, panitumumab or nimotuzumab or other EGFR inhibitors, such as gefitinib or erlotinib), or in combination with an an inhibitor of ErbB2 (Her2/neu) (such as an anti-HER2 antibody, e.g. trastuzumab, trastuzumab-DM1 or pertuzumab) or in combination with an inhibitor of both EGFR and HER2, such as lapatinib, or in combination with a HER3 inhibitor.
For the treatment of glioma, an anti-c-Met antibody in combination with temozolomide or an angiogenesis inhibitor, such as bevacizumab.
For the treatment of colorectal cancer an anti-c-Met antibody in combination with one or more compounds selected from: gemcitabine, bevacizumab, FOLFOX, FOLFIRI , XELOX, IFL, oxaliplatin, irinotecan, 5-FU/LV, Capecitabine, UFT, EGFR targeting agents, such as cetuximab. panitumumab, zalutumumab; VEGF inhibitors, or tyrosine kinase inhibitors such as sunitinib.
For the treatment of prostate cancer an anti-c-Met antibody in combination with one or more compounds selected from: hormonal/antihormonal therapies; such as antiandrogens, Luteinizing hormone releasing hormone (LHRH) agonists, and chemotherapeutics such as taxanes, mitoxantrone, estramustine, 5FU, vinblastine, ixabepilone,
Radiotherapy—Surgery
In one embodiment, the present invention provides a method for treating a disorder involving cells expressing c-Met in a subject, which method comprises administration of a therapeutically effective amount of an anti-c-Met antibody, such as an anti-c-Met antibody of the present invention, and radiotherapy to a subject in need thereof.
In one embodiment, the present invention provides a method for treating or preventing cancer, which method comprises administration of a therapeutically effective amount of an anti-c-Met antibody, such as an anti-c-Met antibody of the present invention, and radiotherapy to a subject in need thereof.
In one embodiment, the present invention provides the use of an anti-c-Met antibody, such as an anti-c-Met antibody of the present invention, for the preparation of a pharmaceutical composition for treating cancer to be administered in combination with radiotherapy.
Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals to a patient is provided. The source of radiation may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used in practicing such methods include, e.g., radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-111.
In a further embodiment, the present invention provides a method for treating or preventing cancer, which method comprises administration to a subject in need thereof of a therapeutically effective amount of an anti-c-Met antibody, such as an anti-c-Met antibody of the present invention, in combination with surgery.
Diagnostic Uses
The anti-c-Met antibodies of the invention may also be used for diagnostic purposes. Thus, in a further aspect, the invention relates to a diagnostic composition comprising an anti-c-Met antibody as defined herein.
In one embodiment, the anti-c-Met antibodies of the present invention may be used in vivo or in vitro for diagnosing diseases wherein activated cells expressing c-Met play an active role in the pathogenesis, by detecting levels of c-Met, or levels of cells which contain c-Met on their membrane surface. This may be achieved, for example, by contacting a sample to be tested, optionally along with a control sample, with the anti-c-Met antibody under conditions that allow for formation of a complex between the antibody and c-Met.
Thus, in a further aspect, the invention relates to a method for detecting the presence of c-Met antigen, or a cell expressing c-Met, in a sample comprising:
contacting the sample with an anti-c-Met antibody of the invention under conditions that allow for formation of a complex between the antibody and c-Met; and
analyzing whether a complex has been formed.
In one embodiment, the method is performed in vitro.
More specifically, the present invention provides methods for the identification of, and diagnosis of invasive cells and tissues, and other cells targeted by anti-c-Met antibodies of the present invention, and for the monitoring of the progress of therapeutic treatments, status after treatment, risk of developing cancer, cancer progression, and the like.
Suitable labels for the anti-c-Met antibody and/or secondary antibodies used in such techniques are well-known in the art.
In a further aspect, the invention relates to a kit for detecting the presence of c-Met antigen, or a cell expressing c-Met, in a sample comprising
an anti-c-Met antibody of the invention or a bispecific molecule of the invention; and
instructions for use of the kit.
In one embodiment, the present invention provides a kit for diagnosis of cancer comprising a container comprising an anti-c-Met antibody, and one or more reagents for detecting binding of the anti-c-Met antibody to c-Met. Reagents may include, for example, fluorescent tags, enzymatic tags, or other detectable tags. The reagents may also include secondary or tertiary antibodies or reagents for enzymatic reactions, wherein the enzymatic reactions produce a product that may be visualized.
Anti-Idiotypic Antibodies
In a further aspect, the invention relates to an anti-idiotypic antibody which binds to an anti-c-Met antibody of the invention as described herein.
An anti-idiotypic (Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. An Id antibody may be prepared by immunizing an animal of the same species and genetic type as the source of an anti-c-Met mAb with the mAb to which an anti-Id is being prepared. The immunized animal typically can recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody).
An anti-Id antibody may also be used as an “immunogen” to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. An anti-anti-Id may be epitopically identical to the original mAb, which induced the anti-Id. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to identify other clones expressing antibodies of identical specificity.
The present invention is further illustrated by the following examples which should not be construed as further limiting.
EXAMPLES
Example 1
Expression Constructs for c-Met
Codon-optimized constructs for expression of c-Met, the extracellular domain (ECD) (aa 1-932 and a C-terminal His6 tag or the SEMA domain of c-Met (aa 1-567 and a C-terminal His9 tag), in HEK or CHO cells, were generated. The proteins encoded by these constructs are identical to Genbank accession NM 000245 for c-Met. The constructs contained suitable restriction sites for cloning and an optimal Kozak sequence (Kozak et al. (1999) Gene 234: 187-208). The constructs were cloned in the mammalian expression vector pEE13.4 (Lonza Biologics) (Bebbington (1992) Biotechnology (NY) 10:169-175), obtaining pEE13.4cMet, pEE13.4cMetECDHis and pEE13.4cMetSEMA-567His8.
Example 2
Expression Constructs for 5D5v1, 5D5 and G11-HZ
Codon-optimized constructs for expression of the heavy chain (HC) and the light chain (LC) of the IgG1 antibodies 5D5v1, 5D5 and G11-HZ in HEK cells, were generated. The proteins encoded by these constructs are identical to those described in U.S. Pat. No. 6,468,529 (sequence numbers 3 and 4) for 5D5v1 heavy chain and light chain, WO 2006/015371 A2 (FIG. 13) for 5D5 heavy chain and light chain and WO 2009/007427 A2 (sequence was extracted from multiple figures) for 224G11 heavy and light chain. 224G11 is also termed G11-HZ herein.
Example 3
Transient Expression in HEK-293F Cells
Freestyle™ 293-F (a HEK-293 subclone adapted to suspension growth and chemically defined Freestyle medium, (HEK-293F)) cells were obtained from Invitrogen and transfected with the appropriate plasmid DNA, using 293fectin (Invitrogen) according to the manufacturer's instructions. Expression of c-Met was tested by means of FACS analysis as described below. In the case of antibody expression, the appropriate heavy chain and light chain expression vectors were co-expressed.
Example 4
Transient Expression in CHO Cells
pEE13.4cMet was transiently transfected in Freestyle™ CHO-S (Invitrogen) cell line using Freestyle MAX transfection reagent (Invitrogen). Expression of c-Met was tested by means of FACS analysis as described below.
Example 5
Cloning and Expression of Monovalent Antibodies (UniBody® Molecules)
For the expression of monovalent antibodies in mammalian cells the HC constant region of IgG4, missing the hinge region (Ch) (amino acids E99-P110) and containing the 2 mutations F405T and Y407E in the CH3 region, was synthesized as a codon optimized construct in mammalian expression vector pcDNA3.3 (Invitrogen) and named pUniTE. A separate vector was constructed by inserting the codon optimized constant region of the human kappa light chain region in pcDNA3.3 and named pKappa.
Relevant VH and VL regions were inserted respectively in pUniTE and pKappa resulting in vectors for the expression of the heavy and light chains of the specific antibodies. Cotransfection of the heavy and light chain vectors of a specific antibody in HEK-293F (Invitrogen) cells, resulted in the transient production of monovalent antibodies with the desired specificities. Purification was performed using Protein A affinity column chromatography (as described in Example 11).
Example 6
Purification of His-Tagged c-Met
cMetECDHis and cMetSEMAHis were expressed in HEK-293F cells. The His-tag in cMetECDHis and cMetSEMAHis enables purification with immobilized metal affinity chromatography. In this process, a chelator fixed onto the chromatographic resin is charged with Co2+ cations. cMetECDHis and cMetSEMAHis containing supernatants were incubated with the resin in batch mode (i.e. solution). The His-tagged protein binds strongly to the resin beads, while other proteins present in the culture supernatant do not bind strongly. After incubation the beads are retrieved from the supernatant and packed into a column. The column is washed in order to remove weakly bound proteins. The strongly bound cMetECDHis and cMetSEMAHis proteins are then eluted with a buffer containing imidazole, which competes with the binding of His to Co2+. The eluent is removed from the protein by buffer exchange on a desalting column.
Example 7
Immunization Procedure of Transgenic Mice
Antibodies 005, 006, 007, 008, 011, 012, 016, 017, 022, 024, 025, 028, 031, 035, 039, 040, 045, 093, 095, 096, 101 and 104 were derived from the following immunizations: one HCo20 mouse (1 female, strain GG2713), one HCo17 mouse (female, strain GG2714) and two HCo12-Balb/C mice (2 females, strain GG2811) (Medarex, San Jose, Calif., USA; for references see paragraph on HuMab mouse above, WO2009097006 and US2005191293) were immunized every fortnight alternating with 5×106 NCI-H441 tumor cells intraperitoneal (IP) and 20 μg of cMetECDHis protein coupled to the hapten Keyhole Limpet Hemocyanin (KLH) subcutaneous (SC).
Antibodies 058, 061, 062, 063, 064, 065, 066, 068, 069, 078, 082, 084, 087, 089, 098 and 181 were derived from the following immunizations: two HCo20 mice (1 male and 1 female, strain GG2713) and one HCo12-Balb/C mouse (1 male, strain GG2811) (Medarex, San José, Calif., USA; for references see paragraph on HuMab mouse above) were immunized every fortnight alternating with 5×106 CHO-K1SV cells transient transfected with cMetECD intraperitoneal (IP) and 20 μg of cMetECDHis protein coupled to the hapten Keyhole Limpet Hemocyanin (KLH) subcutaneous (SC).
A maximum of eight immunizations was performed per mouse, four IP and four SC immunizations at the tail base. The first immunization with cells was done in complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, Mich., USA). For all other immunizations, cells were injected IP in PBS and KLH-coupled cMetECD was injected SC using incomplete Freund's adjuvant (IFA; Difco Laboratories, Detroit, Mich., USA). Mice with at least two sequential c-Met specific antibody titers of 200 (serum dilutions of 1/200) or higher, detected in the antigen specific screening FMAT assay as described in Example 8, were fused.
Example 8
Homogeneous Antigen Specific Screening Assay
The presence of anti-c-Met antibodies in sera of immunized mice or HuMab (human monoclonal antibody) hybridoma or transfectoma culture supernatant was determined by homogeneous antigen specific screening assays (four quadrant) using Fluorometric Micro volume Assay Technology (FMAT; Applied Biosystems, Foster City, Calif., USA). For this, a combination of 3 cell based assays and one bead based assay was used. In the cell based assays, binding to TH1016-cMet (HEK-293F cells transiently expressing the extracellular domain of the c-Met receptor; produced as described above) and HT29 (which express c-Met at the cell surface) as well as HEK293 wild-type cells (negative control which does not express c-Met) was determined. For the bead based assay, binding to SB1016-cMet (cMetECDHis obtained from transient transfected HEK-293F cells as described above, biotinylated and coupled to streptavidin-coated beads) was determined. Samples were added to the cells/beads to allow binding to c-Met. Subsequently, binding of HuMab was detected using a fluorescent conjugate (Goat anti-Human IgG-Cy5; Jackson ImmunoResearch). The chimeric c-Met specific antibody 5D5v1 (produced in HEK-293F cells) was used as a positive control and HuMab-mouse pooled serum and HuMab-KLH were used as negative controls. The samples were scanned using an Applied Biosystems 8200 Cellular Detection System (8200 CDS) and ‘counts x fluorescence’ was used as read-out. Samples were stated positive when counts were higher than 50 and counts x fluorescence were at least three times higher than the negative control HuMab-KLH.
Example 9
HuMab Hybridoma Generation
HuMab mice with sufficient antigen-specific titer development (defined as above) were sacrificed and the spleen and lymph nodes flanking the abdominal aorta and vena cava were collected. Fusion of splenocytes and lymph node cells to a mouse myeloma cell line was done by electrofusion using a CEEF 50 Electrofusion System (Cyto Pulse Sciences, Glen Burnie, Md., USA), essentially according to the manufacturer's instructions. Fusion plates were screened with the antigen specific binding assay as described above and positives from this assay were tested in an ERK-phosphorylation Alphascreen® SureFire® assay and affinity ranking Octet assay as described below. Antibodies 031, 035, 087 and 089 were expanded and cultured based upon standard protocols (e.g. as described in Coligan J. E., Bierer, B. E., Margulies, D. H., Shevach, E. M. and Strober, W., eds. Current Protocols in Immunology, John Wiley & Sons, Inc., 2006).
In parallel antibodies 005, 006, 007, 008, 011, 012, 016, 017, 022, 024, 025, 028, 035, 039, 040, 045, 058, 061, 062, 063, 064, 065, 066, 068, 069, 078, 082, 084, 093, 095, 096, 098, 101, 104 and 181 were cloned using the ClonePix system (Genetix, Hampshire, UK). Specific primary well hybridomas were seeded in semisolid medium made from 40% CloneMedia (Genetix, Hampshire, UK) and 60% HyQ 2× complete media (Hyclone, Waltham, USA) and approximately 100 sub clones of each primary well were picked. The sub clones were retested in the antigen specific binding assay as described previously and IgG levels were measured using Octet in order to select the best specific and producing clone per primary well for further expansion. Further expansion and culturing of the resulting HuMab hybridomas was done based upon standard protocols (e.g. as described in Coligan J. E., Bierer, B. E., Margulies, D. H., Shevach, E. M. and Strober, W., eds. Current Protocols in Immunology, John Wiley & Sons, Inc., 2006).
Example 10
Mass Spectrometry of Purified Antibodies
Small 0.8 ml aliquots of antibody containing hybridoma supernatant from 6-well or Hyperflask stage were purified using PhyTip columns containing Protein G resin (PhyNexus Inc., San Jose, USA) on a Sciclone ALH 3000 workstation (Caliper Lifesciences, Hopkinton, USA). The PhyTip columns were used according to manufacturers instructions, but buffers were replaced by: Binding Buffer PBS (B. Braun, Medical B.V., Oss, Netherlands) and Elution Buffer 0.1M Glycine-HCl pH 2.7 (Fluka Riedel-de Haen, Buchs, Germany). After purification, samples were neutralized with 2M Tris-HCl pH 9.0 (Sigma-Aldrich, Zwijndrecht, Netherlands). Alternatively, in some cases larger volumes of culture supernatant were purified using Protein A affinity column chromatography.
After purification, the samples were placed in a 384-well plate (Waters, 100 ul square well plate, part #186002631). Samples were deglycosylated overnight at 37° C. with N-glycosidase F. DTT (15 mg/mL) was added (1 μl/well) and incubated for 1 h at 37° C. Samples (5 or 6 ul) were desalted on an Acquity UPLC™ (Waters, Milford, USA) with a BEH300 C18, 1.7 μm, 2.1×50 mm column at 60° C. MQ water and LC-MS grade acetonitrile (Biosolve, cat no 01204101, Valkenswaard, The Netherlands) with both 0.1% formic acid (Fluka, cat no 56302, Buchs, Germany), were used as Eluens A and B, respectively. Time-of-flight electrospray ionization mass spectra were recorded on-line on a micrOTOF™ mass spectrometer (Bruker, Bremen, Germany) operating in the positive ion mode. Prior to analysis, a 900-3000 m/z scale was calibrated with ES tuning mix (Agilent Technologies, Santa Clara, USA). Mass spectra were deconvoluted with DataAnalysis™ software v. 3.4 (Bruker) using the Maximal Entropy algorithm searching for molecular weights between 5 and 80 kDa.
After deconvolution the resulting heavy and light chain masses for all samples were compared in order to find duplicate antibodies. In the comparison of the heavy chains the possible presence of C-terminal lysine variants was taken into account. This resulted in a list of unique antibodies, where unique is defined as a unique combination of heavy and light chains. In case duplicate antibodies were found, the results from other tests were used to decide which antibody was the best material to continue experiments with.
Example 11
Sequence Analysis of the Anti-c-Met Antibody Variable Domains and Cloning in Expression Vectors
Total RNA of the anti-c-Met HuMabs was prepared from 5×106 hybridoma cells and 5′-RACE-Complementary DNA (cDNA) was prepared from 100 ng total RNA, using the SMART RACE cDNA Amplification kit (Clontech), according to the manufacturer's instructions. VH (variable region of heavy chain) and VL (variable region of light chain) coding regions were amplified by PCR and in frame cloned into the constant region vectors pG1f (Containing the codon optimized, fully synthetic, constant region of the heavy chain of human IgG1 (allotype f) in the mammalian expression vector pEE6.4 (Lonza Biologics, Slough, UK (Bebbington et al. (1992) Biotechnology 10:169-175)) and pKappa (Containing the codon optimized, fully synthetic, constant region of the human kappa light chain (allotype Km3) in the mammalian expression vector pEE12.4 (Lonza Biologics, Slough, UK (Bebbington et al. (1992) Biotechnology 10:169-175)) using a ligation independent cloning strategy (Aslanidis et al. 1990 Nucleic Acids Res. 18:6069-6074). For each HuMab, 12 VL clones and 8 VH clones were sequenced and their theoretical masses were calculated and compared to the available antibody mass spectrometry data. The sequences are given in the Sequence Listing and in Table 1 here below. CDR sequences are defined according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Table 2 and Table 3 give an overview of the antibody sequence information and most homologous germline sequences.
TABLE 1
Heavy chain variable region (VH), light chain variable region (VL)
and CDR sequences of HuMabs
SEQ ID No: 1
VH 005
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGFG
WVRQAPGQGLEWMGRISPILGIANYAQMFQGRVTIT
ADKSTSTAYMELSSLRSEDTAVYYCARDVGYDWPDT
FDIWGQGTMVIVSS
SEQ ID No: 2
VH 005, CDR1
SYGFG
SEQ ID No: 3
VH 005, CDR2
RISPILGIANYAQMFQG
SEQ ID No: 4
VH 005, CDR3
DVGYDWPDTFDI
SEQ ID No: 5
VL 005
DIQMTQSPSSLSASVGDVTITCRASQGISSWLAWY
QQKPEKAPKSLIYAASSLQSGVPSRFSGGGSGTDFTL
TISSLQPEDFATYYCQQYNSFPPTFGQGTKVEIK
SEQ ID No: 6
VL 005, CDR1
RASQGISSWLA
SEQ ID No: 7
VL 005, CDR2
AASSLQS
SEQ ID No: 8
VL 005, CDR3
QQYNSFPPT
SEQ ID No: 9
VH 006
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSFGIG
WVRQAPGQGLEWMGRIFPILGTANYAQMFQGRVTIT
ADKSTSTAYMELTSLRSEDTAVYYCARDVGYDSADAF
DIWGQGTMVTVSS
SEQ ID No: 10
VH 006, CDR1
SFGIG
SEQ ID No: 11
VH 006, CDR2
RIFPILGTANYAQMFQG
SEQ ID No: 12
VH 006, CDR3
DVGYDSADAFDI
SEQ ID No: 13
VL 006
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWY
QQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQYNSYPPTFGQGTKVEIK
SEQ ID No: 14
VL 006, CDR1
RASQGISSWLA
SEQ ID No: 15
VL 006, CDR2
AASSLQS
SEQ ID No: 16
VL 006, CDR3
QQYNSYPPT
SEQ ID No: 17
VH 008
EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGW
VRQMPGKGLEWMGIIPGDSETRYSPSFQGQVTISA
DKSISTAYLQWSSLKASDTAMYYCARQEITGEFDYW
GQGTLVTVSS
SEQ ID No: 18
VH 008, CDR1
SYWIG
SEQ ID No: 19
VH 008, CDR2
IIYPGDSETRYSPSFQG
SEQ ID No: 20
VH 008, CDR3
QEITGEFDY
SEQ ID No: 21
VL 008
AIQLTQSPSSLSASVGDRVTITCRASQGISSAGAWYQ
QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTI
SSLQPEDFATYYCQQFNSYPRTFGQGTKVEIK
SEQ ID No: 22
VL 008, CDR1
RASQGISSALA
SEQ ID No: 23
VL 008, CDR2
DASSLES
SEQ ID No: 24
VL 008, CDR3
QQFNSYPRT
SEQ ID No: 25
VH 022
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMH
WVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTIS
RDNSKNTLYLQMNSLRAEDTAVYYCARELLWFGELW
GYFDLWGRGTLVTVSS
SEQ ID No: 26
VH 022, CDR1
SYAMH
SEQ ID No: 27
VH 022, CDR2
VISYDGSNKYYADSVKG
SEQ ID No: 28
VH 022, CDR3
ELLWFGELWGYFDL
SEQ ID No: 29
VL 022
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWY
QHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQEASSFTWTFGQGTKVEIK
SEQ ID No: 30
VL 022, CDR1
RASQGISSWLA
SEQ ID No: 31
VL 022, CDR2
AASSLQS
SEQ ID No: 32
VL 022, CDR3
QEASSFTWT
SEQ ID No: 33
VH 024
EVQLLESGGGLVQPGGSLRLSCVASGFTFSSYAMSW
VRQAPGKGLEWVSAISGSSGGSTYYVDSVKGRFTIS
RANSKNTLYLQMNSLRAEDTAVYYCAKDLDRGWMG
YFGYWGQGTLVTVSS
SEQ ID No: 34
VH 024, CDR1
SYAMS
SEQ ID No: 35
VH 024, CDR2
AISGSSGGSTYYVDSVKG
SEQ ID No: 36
VH 024, CDR3
DLDRGWMGYFGY
SEQ ID No: 37
VL 024
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWY
QHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQANSFPTFGQGTRLEIK
SEQ ID No: 38
VL 024, CDR1
RASQGISSWLA
SEQ ID No: 39
VL 024, CDR2
AASSLQS
SEQ ID No: 40
VL 024, CDR3
QQANSFPT
SEQ ID No: 41
VH 035
EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGW
VRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISA
DKSISTAYLQWNSLKASDTAMYYCARQEITGEFDYW
GQGTLVTVSS
SEQ ID No: 42
VH 035, CDR1
SYWIG
SEQ ID No: 43
VH 035, CDR2
IIYPGDSDTRYSPSFQG
SEQ ID No: 44
VH 035, CDR3
QEITGEFDY
SEQ ID No: 45
VL 035
AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ
QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTI
SSLQPEDFATYYCQQFNSYPMYTFGQGTKLEIK
SEQ ID No: 46
VL 035, CDR1
RASQGISSALA
SEQ ID No: 47
VL 035, CDR2
DASSLES
SEQ ID No: 48
VL 035, CDR3
QQFNSYPMYT
SEQ ID No: 49
VH 045
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSW
VRQAPGKGLEWVSVISGSGGITYYADSVKGRFTISR
DNSKNTLYLQMNSLRAEDTAVYYCARDRGWGSDYW
GQGTLVTVSS
SEQ ID No: 50
VH 045, CDR1
SYAMS
SEQ ID No: 51
VH 045, CDR2
VISGSGGITYYADSVKG
SEQ ID No: 52
VH 045, CDR3
DRGWGSDY
SEQ ID No: 53
VL 045
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQ
QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTI
SSLEPEDFAVYYCQQRSNWPFTFGPGTKVDIK
SEQ ID No: 54
VL 045, CDR1
RASQSVSSYLA
SEQ ID No: 55
VL 045, CDR2
DASNRAT
SEQ ID No: 56
VL 045, CDR3
QQRSNWPFT
SEQ ID No: 57
VH 058
EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYYMYW
VRQTPEKRLEWVATISDDGSYTYYPDSVKGRFTISRD
NAKNNLYLQMSSLKSEDTAMYYCAREGLYYYGSGSY
YNQDYWGQGTLVTVSS
SEQ ID No: 58
VH 058, CDR1
DYYMY
SEQ ID No: 59
VH 058, CDR2
TISDDGSYTYYPDSVKG
SEQ ID No: 60
VH 058, CDR3
EGLYYYGSGSYYNQDY
SEQ ID No: 61
VL 058
AIQLTQSPSSLSASVGDRVTITCRASQGLSSALAWYR
QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTI
SSLQPEDFATYYCQQFTSYPQITFGQGTRLEIK
SEQ ID No: 62
VL 058, CDR1
RASQGLSSALA
SEQ ID No: 63
VL 058, CDR2
DASSLES
SEQ ID No: 64
VL 058, CDR3
QQFTSYPQIT
SEQ ID No: 65
VH 061
QLQLQESGSGLVKPSQTLSLTCAVSGGSISSGGHSW
SWIRQPPGKGLEWIGX1IYHSGNTYDNPSLKSRVTIA
VDRSKNQLSLKLSFLTAADTAVYYCARSSYDFLTDWG
QGTLVTVSS, wherein X1 is any amino acid,
preferably C, S, Y or A
SEQ ID No: 66
VH 061, CDR1
SGGHSWS
SEQ ID No: 67
VH 061, CDR2
X1IYHSGNTYDNPSLKS, wherein X1 is any amino
acid, preferably C, S, Y or A
SEQ ID No: 68
VH 061, CDR3
SSYDFLTD
SEQ ID No: 69
VL 061
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWY
QHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQANGFPITFGQGTRLEIK
SEQ ID No: 70
VL 061, CDR1
RASQGISSWLA
SEQ ID No: 71
VL 061, CDR2
AASSLQS
SEQ ID No: 72
VL 061, CDR3
QQANGFPIT
SEQ ID No: 73
VH 062
QLQLQESGSGLVKPSQTLSLTCAVSGGSISSGGHSW
SWIRQPPGKGLEWIGX1IYHSGNTYDNPSLKSRVTIA
VDRSKNQLSLKLSFVTAADTAVYYCARSSYDILTDWG
QGTLVTVSS, wherein X1 is any amino acid,
preferably C, S, Y or A
SEQ ID No: 74
VH 062, CDR1
SGGHSWS
SEQ ID No: 75
VH 062, CDR2
X1IYHSGNTYDNPSLKS, wherein X1 is any amino
acid, preferably C, S, Y or A
SEQ ID No: 76
VH 062, CDR3
SSYDILTD
SEQ ID No: 77
VL 062
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWY
QHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQANGFPITFGQGTRLEIK
SEQ ID No: 78
VL 062, CDR1
RASQGISSWLA
SEQ ID No: 79
VL 062, CDR2
AASSLQS
SEQ ID No: 80
VL 062, CDR3
QQANGFPIT
SEQ ID No: 81
VH 064
QLQLQESGSGLVKPSQTLSLTCAVSGGSISSGGHSW
SWIRQPPGKGLEWIGX1IYHSGNTYDNPSLKSRVTIS
VDRSKNQVSLKLSSVTAADTAVYYCARSSYDILTDW
GQGTLVTVSS, wherein X1 is any amino acid,
preferably C, S, Y or A
SEQ ID No: 82
VH 064, CDR1
SGGHSWS
SEQ ID No: 83
VH 064, CDR2
X1IYHSGNTYDNPSLKS, wherein X1 is any amino
acid, preferably C, S, Y or A
SEQ ID No: 84
VH 064, CDR3
SSYDILTD
SEQ ID No: 85
VL 064
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWY
QHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQANGFPITFGQGTRLEIK
SEQ ID No: 86
VL 064, CDR1
RASQGISSWLA
SEQ ID No: 87
VL 064, CDR2
AASSLQS
SEQ ID No: 88
VL 064, CDR3
QQANGFPIT
SEQ ID No: 89
VH 068
QLQLQESGSGLVKPSQTLSLTCAVSGGSISSGGYSW
SWIRQPPGKGLEWIGX1IYHSGSTYYNPSLKSRVTIS
VDRSKNQFSLKLSSVTAADTAVYYCARSSYDILTDW
GQGTLVTVSS, wherein X1 is any amino acid,
preferably C, S, Y or A
SEQ ID No: 90
VH 068, CDR1
SGGYSWS
SEQ ID No: 91
VH 068, CDR2
X1IYHSGSTYYNPSLKS, wherein X1 is any amino
acid, preferably C, S, Y or A
SEQ ID No: 92
VH 068, CDR3
SSYDILTD
SEQ ID No: 93
VL 068
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWY
QHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQANSFPITFGQGTRLEIK
SEQ ID No: 94
VL 068, CDR1
RASQGISSWLA
SEQ ID No: 95
VL 068, CDR2
AASSLQS
SEQ ID No: 96
VL 068, CDR3
QQANSFPIT
SEQ ID No: 97
VH 069
QVQLVQSGAEVKKPGASVKVSCETSGYTFTSYGISW
VRQAPGHGLEWMGWISAYNGYTNYAQKLQGRVTMT
TDTSTSTAYMELRSLRSDDTAVYYCARDLRGTNYFDY
WGQGTLVTVSS
SEQ ID No: 98
VH 069, CDR1
SYGIS
SEQ ID No: 99
VH 069, CDR2
WISAYNGYTNYAQKLQG
SEQ ID No: 100
VH 069, CDR3
DLRGTNYFDY
SEQ ID No: 101
VL 069
DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWF
QHKPGKAPKLLIYAASSLLSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQANSFPITFGQGTRLEIK
SEQ ID No: 102
VL 069, CDR1
RASQGISNWLA
SEQ ID No: 103
VL 069, CDR2
AASSLLS
SEQ ID No: 104
VL 069, CDR3
QQANSFPIT
SEQ ID No: 105
VH 096
EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGW
VRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISA
DKSISTAYLQWSSLKASDTAMYYCARQEITGDFDYW
GQGTLVTVSS
SEQ ID No: 106
VH 096, CDR1
SYWIG
SEQ ID No: 107
VH 096, CDR2
IIYPGDSDTRYSPSFQG
SEQ ID No: 108
VH 096, CDR3
QEITGDFDY
SEQ ID No: 109
VL 096
AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ
QKPGKAPNLLIYAASSLESGVPSRFSGSGSGTDFTLTI
SSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK
SEQ ID No: 110
VL 096, CDR1
RASQGISSALA
SEQ ID No: 111
VL 096, CDR2
AASSLES
SEQ ID No: 112
VL 096, CDR3
QQFNSYPLT
SEQ ID No: 113
VH 098
QVQLVQSGAEVKKPGASVKVSCKASGYTFTNFGISW
VRQAPGQGLEWMGWISAFNGHTDYSQKVQGRVTM
TTDTSTSTAYMELRSLRSDDTAVFYCARSHYYGSGSP
FDYWGQGTLVTVSS
SEQ ID No: 114
VH 098, CDR1
NFGIS
SEQ ID No: 115
VH 098, CDR2
WISAFNGHTDYSQKVQG
SEQ ID No: 116
VH 098, CDR3
SHYYGSGSPFDY
SEQ ID No: 117
VL 098
DIQMTQSPSSLSASVGDRVTITCRASQGISNWLAWY
QQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCHQYKSYPWTFGQGTKVEIK
SEQ ID No: 118
VL 098, CDR1
RASQGISNWLA
SEQ ID No: 119
VL 098, CDR2
AASSLQS
SEQ ID No: 120
VL 098, CDR3
HQYKSYPWT
SEQ ID No: 121
VH 101
QVQLVQSGGEVKKPGASVKVSCKASGYTFTRHGITW
VRQAPGQGLEWMGWISADNGNTNYAQKFQDRVTM
TTDTSTSTAYMELRSLRSDDTAVYFCARVFRYFDWLL
PYFDYWGQGTLVTVST
SEQ ID No: 122
VH 101, CDR1
RHGIT
SEQ ID No: 123
VH 101, CDR2
WISADNGNTNYAQKFQD
SEQ ID No: 124
VH 101, CDR3
VFRYFDWLLPYFDY
SEQ ID No: 125
VL 101
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY
QQKPGQAPRLLIYGVFSRATGIPDRFSGSGSGTDFTL
TISRLEPEDFAVYYCQQYGSSPYTFGQGTKLEIK
SEQ ID No: 126
VL 101, CDR1
RASQSVSSSYLA
SEQ ID No: 127
VL 101, CDR2
GVFSRAT
SEQ ID No: 128
VL 101, CDR3
QQYGSSPYT
SEQ ID No: 129
VH 181
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISW
VRQAPGQGLEWMGWISTYNGYTNYAQKLQGRVTMT
TDTSTSTAYMELRSLRSDDTAVYYCARDLRGTAYFDY
WGQGTLVTVSS
SEQ ID No: 130
VH 181, CDR1
SYGIS
SEQ ID No: 131
VH 181, CDR2
WISTYNGYTNYAQKLQG
SEQ ID No: 132
VH 181, CDR3
DLRGTAYFDY
SEQ ID No: 133
VL 181
DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWY
QHKPGKAPKLLIYAASSLLSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQANSFPITFGQGTRLEIK
SEQ ID No: 134
VL 181, CDR1
RASQGISNWLA
SEQ ID No: 135
VL 181, CDR2
AASSLLS
SEQ ID No: 136
VL 181, CDR3
QQANSFPIT
SEQ ID No: 137
VH 066
QVQLVQSGAEVKKPGASVKVSCEASGYTFTSYGISW
VRQAPGHGLEWMGWISAYNGYTNYAQKLQGRVTMT
ADTSTSTAYMELRSLRSDDTAVYYCARDLRGTNYFDY
WGQGTLVTVSS
SEQ ID No: 138
VL 066
DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWY
QHKPGKAPKLLIYAASSLLSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQANSFPITFGQGTRLEIK
SEQ ID No: 139
VH 065
QVQLVQSGAEVKKPGASVKVSCEASGYTFTNYGISW
VRQAPGHGLEWMGWISAYNGYTNYAQKLQGRVTMT
TDTSTTTAYMELRSLRSDDTAVYYCARDLRGTNYFDY
WGQGTLVTVSS
SEQ ID No: 140
VL 065
DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWY
QHKPGKAPKLLIYAASSLLSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQANSFPITFGQGTRLEIK
SEQ ID No: 141
VH 082
QVQLVQSGAEVKKPGASVKVSCETSGYTFTSYGISW
VRQAPGHGLEWMGWISAYNGYTNYAQKLQGRVTMT
TDTSTSTAYMELRSLRSDDTAVYYCARDLRGTNYFDY
WGQGTLVTVSS
SEQ ID No: 142
VL 082
DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWY
QHKPGKAPKLLIYAASSLLSGVPSRFSGSGSGTDGTL
TISSLQPEDFATYYCQQANSFPITFGQGTRLEIK
SEQ ID No: 143
VH 089
QVQLVQSGAEVKKPGASVKVSCETSGYTFTSYGISW
VRQAPGHGLEWMGWISAYNGTNYAQKLQGRVTMT
TDTSTSTAYMELRSLRSDDTAVYYCARDLRGTNYFDY
WGQGTLVTVSS
SEQ ID No: 144
VL 089
DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWF
QHKPGKAPKLLIYAASSLLSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQANSGPITFGQGTRLEIK
SEQ ID No: 145
VH 031
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGFG
WVRQAPGQGLEWMGRISPILGITNYAQMFQGRVTIT
ADKSTSTAYMELSSLRSEDTAVYYCARDVGYDQPDT
FDIWGQGTMVIVSS
SEQ ID No: 146
VL 031
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWY
QQKPEKAPKSLIYAASSLQSGVPSRFSGGGSGTDFTL
TISSLQPEDFATYYCQQYNSFPPPTFGQGTKVEIK
SEQ ID No: 147
VH 007
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGIG
WVRQAPGQGLEWMGRIFPILGTANYAQMFQGRVTIT
ADKSTSTAYIELTSLRSEDTAVYYCARDVGYDSADAF
DIWGQGTMVTVSS
SEQ ID No: 148
VL 007
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWY
QQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQYNSYPPTFGQGTKVEIK
SEQ ID No: 149
VH 011
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGIG
WVRQAPGQGLEWMGRVFPILGTANYAQMFQGRVTI
TADKSTSTAYMELTSLRSEDTAVYYCARDVGYDSAD
AFDIQGQGTMVTVSS
SEQ ID No: 150
VL 011
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWY
QQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQYNSYPPTFGQGTKVEIK
SEQ ID No: 151
VH 017
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMH
WVRQAPGKGLEWVAFISYDGSNKYFADSVKGRFTIS
RDNSKNTLYLQMNSLRAEDTAVYYCARELLWFGELW
GYFDLWGRGTLVTVSS
SEQ ID No: 152
VL 017
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWY
QHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQEANSFTWTFGQGTKVEIK
SEQ ID No: 153
VH 025
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMH
WVRQAPGKGLEWVAFISYDGSSKDYADSVKGRFTIF
RDNSKNTLYLQMSSLRAADTAVYYCARELLWFGELW
GYFDLWGRGTLVTVSS
SEQ ID No: 154
VL 025
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWY
QHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQTNSFTWTFGQGTKVEIK
SEQ ID No: 155
VH 040
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMTW
VRQAPGKGLEWVSVISGSGGITYYADSVKGRFTISR
DNSKNTLYLQMNSLRAEDTAVYYCARDRGWGSDYW
GQGTLVTVSS
SEQ ID No: 156
VL 040
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQ
QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTI
SSLEPEDFAVYYCQQRSNWPFTFGPGTKVDIK
SEQ ID No: 157
VH 039
EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYAMSW
VRQAPGKGLEWVSAISGSGGITYYADSEKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCAKDRGWGSDCWG
QGTLVTVSS
SEQ ID No: 158
VL 039
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQ
QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTI
SSLEPEDFAVYYCQQRSNWPFTFGPGTKVDIK
SEQ ID No: 159
VH 078
QLQLQESGSGLVKPSQTLSLTCAVSGGSISSGGHSW
SWIRQPPGKGLEWIGCLYHSGNTYYNPSLKSRVTISV
DRSKNQFSLKLSSVTAADTAVYYCARSSYDILTDWG
QGILVTVSS
SEQ ID No: 160
VL 078
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWY
QHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQANSFPITFGQGTRLEIK
SEQ ID No: 161
VH 084
QLQLQESGSGLVKPSQTLSLTCGVSGGSISSGGHSW
SWIRQPPGKGLEWIGCLYHSGNTYYNPSLKSRVTISV
DRSKNQFSLKLSSVTAADTAVYYCARSSYDILTDWG
QGTLVTVSS
SEQ ID No: 162
VL 084
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWY
QHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQANSFPITFGQGTRLEIK
SEQ ID No: 163
VH 063
QLQLQESGSGLVKPSQTLSLTCAVSGGSISSGGHSW
SWIRQPPGKGLEWIGCIYHSGNTYDNPSLKSRVTIAV
DRSKNQLSLKLSFVTAADTAVYYCARSSYDILTDWG
QGTLVTVSS
SEQ ID No: 164
VL 063
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWY
QHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQANGFPITFGQGTRLEIK
SEQ ID No: 165
VH 087
QLQLQESGSGLVKPSQTLSLTCAVSGGSISSGGHSW
SWIRQPPGKGLEWIGCIYHSGNTYDNPSLKSRVTISV
DRSKNQFSLKLSSVTAADTAVYYCARSSYDILTDWG
QGTLVTVSS
SEQ ID No: 166
VL 087
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWY
QHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTL
TISSLQPEDFATYYCQQANGFPITFGQGTRLEIK
SEQ ID No: 167
VH 016
EVQLVQSGAEVKKPGESLKISCKGSGYIFTSYWIGW
VRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISA
DKSISTAYLQWSSLKASDTAMYYCARQEVTGDFDYW
GQGTLVTVSS
SEQ ID No: 168
VL 016
AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ
QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTI
SSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK
SEQ ID No: 169
VH 028
EVQLVQSGGEVKKPGESLKISCKGSGYSFTSYWIGW
VRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISA
DKSISTAYLQWSSLKASDTAMYYCARQEVTGDFDYW
GQGTLVTVSS
SEQ ID No: 170
VL 028
AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ
QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTI
SSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK
SEQ ID No: 171
VH 012
EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGW
VRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISA
DKSISTAYLQWSSLKASDTAMYYCARQEITGEFDYW
GQGTLVTVSS
SEQ ID No: 172
VL 012
AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ
QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTI
SSLQPEDFATYYCQQFNSYPRTFGQGTKVEIK
SEQ ID No: 173
VH 095
EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGW
VRQMPGKGLEWMGIIYPGDSNTRYSPSFQGQVTISA
DKSISTAYLQWSSLKASDTAMYYCARQEITGDFDYW
GQGTLVTVSS
SEQ ID No: 174
VL 095
AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ
QKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTI
SSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK
SEQ ID No: 175
VH 093
EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGW
VRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISA
DKSISTAYLQWSSLKASDTAMYYCARQEITGDFDYW
GQGTLVTVSS
SEQ ID No: 176
VL 093
AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ
QKPGKAPNLLIYAASSLESGVPSRFSGSGSGTDFTLTI
SSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK
SEQ ID No: 177
VH 104
EVQLVQSGAEVKKPGESLKISCKGSGYSFISYWIGW
VRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISA
DKSISTAYLQWSSLKASDTAMYYCARQEITGDFDYW
GQGTLVTVSS
SEQ ID No: 178
VL 104
AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQ
QKPGKAPKLLIYVASSLESGVPSRFSGSGSGTDFTLTI
TSLQPEDFATYYCQQFNSYPITFGQGTRLEIK
TABLE 2
Mouse origin and heavy chain sequence homologies
Antibody:
mouse number:
mouse strain:
germline VH:
TH1016-005
339732
HCo12B, C1
IgHV1-69-4
TH1016-006
339732
HCo12B, C1
IgHV1-69-4
TH1016-008
339732
HCo12B, C1
IgHV5-51-1
TH1016-022
339733
HCo12B, C1
IgHV3-30-3*1
TH1016-024
339733
HCo12B, C1
IgHV3-23-1
TH1016-035-D09
339732
HCo12B, C1
IgHV5-51-1
TH1016-045
339282
HCo17, C1
IgHV3-23-1
TH1016-058
343191
HCo12B, C2
IgHV3-11-3
TH1016-061
348072
HCo20, C2
IgHV4-30-2*1
TH1016-062
348072
HCo20, C2
IgHV4-30-2*1
TH1016-064
348072
HCo20, C2
IgHV4-30-2*1
TH1016-068
348072
HCo20, C2
IgHV4-30-2*1
TH1016-069
348072
HCo20, C2
IgHV1-18-1
TH1016-096
339732
HCo12B, C1
IgHV5-51-1
TH1016-098
347330
HCo20, C2
IgHV1-18-1
TH1016-101
340659
HCo20, C1
IgHV1-18-1
TH1016-181
348072
HCo20, C2
IgHV1-18-1
TABLE 3
Mouse origin and light chain sequence homologies
Antibody:
mouse number:
mouse strain:
germline:
PC1016-005
339732
HCo12B, C1
IGKV1D-16*01
PC1016-006
339732
HCo12B, C1
IGKV1D-16*01
PC1016-008
339732
HCo12B, C1
IGKV1-13*02
PC1016-022
339733
HCo12B, C1
IGKV1-12*01
PC1016-024
339733
HCo12B, C1
IGKV1-12*01
P1016-035
339732
HCo12B, C1
IGKV1-13*02
PC1016-045
339282
HCo17, C1
IGKV3-11*01
PC1016-058
343191
HCo12B, C2
IGKV1-13*02
PC1016-061
348072
HCo20, C2
IGKV1-12*01
PC1016-062
348072
HCo20, C2
IGKV1-12*01
PC1016-064
348072
HCo20, C2
IGKV1-12*01
PC1016-068
348072
HCo20, C2
IGKV1-12*01
PC1016-069
348072
HCo20, C2
IGKV1-12*01
PC1016-096
339732
HCo12B, C1
IGKV1-13*02
PC1016-098
347330
HCo20, C2
IGKV1D-16*01
PC1016-101
340659
HCo20, C1
IGKV3-20*01
PC1016-181
348072
HCo20, C2
IGKV1-12*01
FIGS. 1 and 2 give an alignment of HuMabs sequences. On the basis of these sequences, consensus sequence can be defined for some of the CDR sequences. These consensus sequences are given in Table 4.
TABLE 4
Consensus sequences
SEQ ID No: 179
IgHV1-69-4
CDR1
SX1X2X3X4
wherein X1 = Y or F, X2 = A or G,
005-006
X3 = F or I, X4 = S or G.
Preferably, wherein X1 = Y or F,
X2 = G, X3 = F or I and X4 = G.
SEQ ID No: 180
IgHV1-69-4
CDR2
RX1X2PILGX3X4NYAQX5FQG
wherein X1 = I or V, X2 = I, S or F,
005-006
X3 = I or T, X4 = A or T, X5 = K or M.
Preferably, wherein X1 = I or V,
X2 = S or F, X3 = I or T, X4 = A or T
and X5 = M.
SEQ ID No: 181
IgHV1-69-4
CDR3
DVGYDX1X2DX3FDI
wherein X1 = W or S, X2 = P or A,
005-006
X3 = T or A
SEQ ID No: 182
IgHV5-51-1
CDR2
IIYPGDSX1TRYSPSFQG
wherein X1 = D, E or N
008-035
SEQ ID No: 183
IgHV5-51-1
CDR3
QEX1TGX2FDY
wherein X1 = V or I, X2 = E or D
008-035-096
SEQ ID No: 184
IgHV3-30-3*1
CDR2
X1ISYDGSX2KX3X4ADSVKG
wherein X1 = V or F, X2 = N or S,
022
X3 = D or Y, X4 = Y or F
SEQ ID No: 185
IgHV3-23-1
CDR2
AISGSX1GGSTYYX2DSVKG
wherein X1 = S or no aa, X2 = V or A
024
SEQ ID No: 186
IgHV3-23-1
CDR1
X1YAMX2
wherein X1 = S or N, X2 = S or T
045
SEQ ID No: 187
IgHV3-23-1
CDR2
X1ISGSGGX2TYYADSX3KG
wherein X1 = A or V, X2 = S or I,
045
X3 = V or E. Preferably, wherein
X1 = A or V, X2 = I and X3 = V or E.
SEQ ID No: 188
IgHV3-23-1
CDR3
DRGWGSDX1
wherein X1 = Y or C
045
SEQ ID No: 189
IgHV3-11-3
CDR1
DYYMX1
wherein X1 = Y or S
058
SEQ ID No: 190
IgHV3-11-3
CDR2
X1ISX2X3X4SYTX5YX6DSVKG
wherein X1 = T or Y, X2 = D or S,
058
X3 = D or S, X4 = G or S, X5 = Y or
N, X6 = P or A
SEQ ID No: 191
IgHV4-30-2*1
CDR1
SGGX1SWS
wherein X1 = Y or H
062-064-068
SEQ ID No: 192
IgHV4-30-2*1
CDR2
X1X2YHSGX3TYX4NPSLKS
wherein X1 = any amino acid,
062-064-068
preferably C, Y, S or A, X2 = I or L,
X3 = S or N, X4 = Y or D
SEQ ID No: 193
IgHV4-30-2*1
CDR3
SSYDX1LTD
wherein X1 = F or I
062-064-068
SEQ ID No: 194
IgHV1-18-1
CDR1
X1YGIS
wherein X1 = S or N
069-181
SEQ ID No: 195
IgHV1-18-1
CDR2
WISX1YNGX2TNYAQKLQG
wherein X1 = A or T, X2 = N or Y.
069-181
Preferably wherein X1 = A or T and
X2 = Y
SEQ ID No: 196
IgHV1-18-1
CDR3
DLRGTX1YFDY
wherein X1 = A or N
069-181
SEQ ID No: 197
IgHV1-18-1
CDR1
X1X2GIS
wherein X1 = N or S, X2 = F or Y
098
SEQ ID No: 198
IgHV1-18-1
CDR2
WISAX1NGX2TX3YX4QKX5QG
wherein X1 = F or Y, X2 = H or N,
098
X3 = D or N, X4 = S or A, X5 = V or L
SEQ ID No: 199
IgHV1-18-1
CDR1
X1X2GIX3
wherein X1 = R or S, X2 = H or Y,
101
X3 = T or S
SEQ ID No: 200
IgHV1-18-1
CDR2
WISAX1NGNTNYAQKX2QX3
wherein X1 = D or Y, X2 = F or L,
101
X3 = D or G
SEQ ID No: 201
IgHV1-18-1
CDR3
VX1RYFDWLLX2YFDY
wherein X1 = F or L, X2 = P or no aa
101
SEQ ID No: 202
IGKV1D-16*01
CDR3
QQYNSX1PX2T
wherein X1 = Y or F, X2 = P or W.
005-006
Preferably, wherein X1 = Y or F and
X2 = P
SEQ ID No: 203
IGKV1-13*02
CDR2
X1ASSLES
wherein X1 = D, V or A
008-035
SEQ ID No: 204
IGKV1-13*02
CDR3
QQFNSYPLX1T
wherein X1 = R, I, L, W or MY
008-035
SEQ ID No: 205
IGKV1-12*01
CDR3
QX1X2X3SFX4WT
wherein X1 = Q or E, X2 = A or T,
022
X3 = N or S; X4 = P or T
SEQ ID No: 206
IGKV1-12*01
CDR3
QQANSFPX1T
wherein X1 = I or no aa
024
SEQ ID No: 207
IGKV1-13*02
CDR3
QQFX1SYPX2IT
wherein X1 = T or N, X2 = Q or no aa
058
SEQ ID No: 208
IGKV1-12*01
CDR3
QQANX1FPIT
wherein X1 = G or S
062-064-068
SEQ ID No: 209
IGKV1-12*01
CDR1
RASQGISX1WLA
wherein X1 = S or N
069-181
SEQ ID No: 210
IGKV1-12*01
CDR2
AASSLX1S
wherein X1 = Q or L
069-181
SEQ ID No: 211
IGKV1D-16*01
CDR3
X1QYX2SYPWT
wherein X1 = H or Q, X2 = K or N
098
SEQ ID No: 212
IGKV3-20*01
CDR2
GX1X2SRAT
wherein X1 = V or A, X2 = F or S
101
Example 12
Purification of Antibodies
Culture supernatant was filtered over 0.2 μm dead-end filters and loaded on 5 ml MabSelect SuRe columns (GE Health Care) and eluted with 0.1 M sodium citrate-NaOH, pH 3. The eluate was immediately neutralized with 2M Tris-HCl, pH 9 and dialyzed overnight to 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B.Braun). Alternatively, subsequent to purification the eluate was loaded on a HiPrep Desalting column and the antibody was exchanged into 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B.Braun) buffer. After dialysis or bufferexchange samples were sterile filtered over 0.2 μm dead-end filters. Purity was determined by SDS-PAGE and concentration was measured by nephelometry and absorbance at 280 nm. Purified antibodies were stored at 4° C. Mass spectrometry was performed to identify the molecular mass of the antibody heavy and light chains expressed by the hybridomas as described in Example 10.
Example 13
Binding of Anti-c-Met Clones to Tumor Cells Expressing Membrane-Bound c-Met Measured by Means of FACS Analysis
The binding of anti-c-Met antibodies and monovalent forms thereof (also termed “UniBody molecules” herein, see Example 5) to A431 cells expressing membrane-bound c-Met (purchased at ATCC, CRL-1555) was tested using flow cytometry (FACS Canto II, BD Biosciences). Qifi analysis (Dako, Glostrup, Denmark) revealed that A431 cells express on average 30,000 copies of c-Met protein per cell. Binding of anti-c-Met antibodies and UniBody molecules was detected using a Phycoerythrin-conjugated goat-anti-human IgG antibody (Jackson). IgG1-5D5 was used as positive control antibody, and HuMab-KLH was used as isotype control antibody. EC50 values were determined by means of non-linear regression (sigmoidal dose-response with variable slope) using GraphPad Prism V4.03 software (GraphPad Software, San Diego, Calif., USA).
FIG. 3 shows that all tested anti-c-Met antibodies and UniBody molecules bound to c-Met expressed on A431 cells in a dose-dependent manner. The EC50 values for binding varied between 0.28-1.92 nM for IgG and 0.52-13.89 nM for UniBody molecules. Interestingly, antibody IgG1-024 demonstrated high unsaturated binding levels to A431 cells, which was not observed when binding to HT-29 cells (purchased at ATCC, HTB-38™) was tested (data not shown). For antibodies 022, 024, 062, 064, 069, 098, 101 and 181, no or less than 2-fold decreased EC50 values were observed between IgG1's and UniBody molecules of identical clones. Also maximum binding levels were unchanged between IgG1's and UniBody molecules. For antibodies 005, 006, 008, 035, 045 and 058, on the other hand, a more than 2-fold decrease in EC50 value as well as a decrease in maximum binding level was observed when comparing IgG1 with their UniBody counterpart. This was most likely due to the lower off-rates (Kd) of these antibodies (see Example 14).
Example 14
Affinity Ranking Octet Assay
Antibody binding to cMetECDHis was analyzed by means of Bio-Layer Interferometry (BLI) technology on the Octet System (Fortebio, Menlo Park, USA). Anti-human IgG coated biosensors (Fc-specific) were used to capture anti-c-Met antibodies according to the procedure recommended by the manufacturer. cMetECDHis derived from HEK293 cells was loaded on top of the immobilized anti-c-Met antibodies by placing the loaded biosensor into a well containing 10 μg/mL cMetECDHis diluted in 10 times diluted kinetics buffer (Fortebio). The difference in reflection of light (Δλ, nm) of the biosensor surface due to the binding of cMetECDHis was measured in real time during approximately 10 minutes and was used by the Octet software (V4.0, Fortebio) to calculate the association constant (ka [1/M×s]). Next, the loaded biosensor was placed into a well containing only kinetics buffer (10 times diluted in PBS) to determine the dissociation constant (kd [1/s]). Kinetics analysis was performed to determine the affinity (KD [M]) using model 1:1 (langmuir). As a positive control, 0.2 μg/mL 5D5 IgG1 produced in HEK293 cells, was used.
Table 5 shows that all anti-c-Met antibodies bound to cMetECDHis with nanomolar affinities in the range of 0.6-13.9 nM.
TABLE 5
Kinetic constants (ka, kd and KD) of antibodies for binding to cMetECDHis
Clone
ka [1/Ms]
kd [1/s]
KD [M]
5D5
2.14E+05
1.25E−03
5.86E−09
005
3.18E+05
2.52E−03
7.92E−09
006
4.25E+05
4.20E−03
9.89E−09
008
3.08E+05
1.57E−03
5.12E−09
022
2.36E+05
2.51E−04
1.06E−09
024
1.45E+05
2.28E−04
1.57E−09
035
2.64E+05
3.68E−03
1.39E−08
045
7.21E+05
2.07E−03
2.87E−09
058
4.64E+05
1.25E−03
2.70E−09
061
2.56E+05
1.53E−04
5.96E−10
062
2.73E+05
3.19E−04
1.17E−09
064
2.84E+05
3.24E−04
1.14E−09
068
3.21E+05
1.35E−03
4.21E−09
069
2.12E+05
2.67E−04
1.26E−09
096
1.96E+05
5.00E−04
2.55E−09
098
1.64E+05
2.97E−04
1.82E−09
101
1.69E+05
2.14E−04
1.27E−09
181
2.37E+05
5.31E−04
2.23E−09
Except for 5D5, each sample was measured once
Example 15
Binding of Anti-c-Met Antibodies to Membrane-Bound c-Met Expressed on Rhesus Monkey Epithelial Cells Measured by Means of FACS Analysis
To determine cross-reactivity with Rhesus monkey c-Met, the binding of anti-c-Met antibodies to c-Met positive Rhesus monkey epithelial cells (4MBr-5 purchased at ATCC) was tested using flow cytometry (FACS Canto II, BD Biosciences). A Phycoerythrin-conjugated goat-anti-human IgG antibody (Jackson) was used as a secondary conjugate. HuMab-KLH was used as isotype control antibody.
FIG. 4 demonstrates that all tested anti-c-Met antibodies are cross-reactive with Rhesus c-Met. At both tested concentrations (0.5 μg/mL and 10 μg/mL) the anti-c-Met antibodies were able to bind specifically to Rhesus monkey c-Met. For all antibodies, the signal was at least 5 times higher than for isotype control antibody HuMab-KLH. Interestingly, P1016-035 demonstrated much higher top-fluorescence levels (MFI of ˜200,000) compared to other c-Met specific antibodies. This difference was not observed on cell lines expressing human c-Met receptor.
Example 16
Blocking of HGF Binding to the Extracellular Domain of c-Met Determined with Enzyme-Linked Immuno Sorbent Assay (ELISA)
An ELISA was performed to analyze if anti-c-Met antibodies could block binding of hepatocyte growth factor (HGF) to the c-Met receptor. Therefore, coated extracellular domain of c-Met was incubated with an unlabeled anti-c-Met antibody and fluorescently labeled HGF. Non-blocking antibodies do not compete with the labeled HGF for c-Met binding, resulting in maximal fluorescent signal. Blocking antibodies compete with labeled HGF for c-Met binding, resulting in a decreased fluorescent signal.
HGF (ProSpec Tany, Rehovot, Israel) was fluorescently labeled by conjugation with Europium3+ (PerkinElmer, Turku, Finland). ELISA wells were coated overnight at 4° C. with 0.5 μg/mL recombinant human c-Met extracellular domain (R&D systems, Minneapolis, USA) diluted in PBS. Next, the ELISA wells were washed with PBST (PBS supplemented with 0.05% Tween-20 [Sigma-Aldrich, Zwijndrecht, The Netherlands]) and blocked for one hour at room temperature (RT) with PBST supplemented with 2% (v/v) chicken serum (Gibco, Paisley, Scotland). After washing with PBST, the ELISA wells were incubated for one hour at RT protected from light with a mixture of 50 μL serially diluted anti-c-Met antibody (0.128-10,000 ng/mL in 5-fold dilutions) and 50 μL of 0.44 μg/mL Europium3+-conjugated HGF in PBST, Next, unbound Europium3+-conjugated HGF was washed away with PBST and bound Europium3+-conjugated HGF was incubated for 30 minutes at RT in the dark with Delfia Enhancement Solution (PerkinElmer) to increase the fluorescent signal. Mean fluorescence intensity at 615 nm was measured using the EnVision 2101 Multilabel reader (PerkinElmer) applying the following settings: Lance/Delfia dual mirror, emission filter 615, excitation filter 340 nm, delay time 400 μs, window 400 μs, 100 flashes, 2000 μs per cycle and bidirectional row-by-row reading. To determine IC50 values, the binding curves were analyzed with non-linear regression (sigmoidal dose-response with variable slope, top-values constrained to a shared value for all data-sets) using GraphPad Prism V4.03 software (GraphPad Software, San Diego, Calif., USA).
FIG. 5 depicts representative examples of HGF binding inhibition curves of anti-c-Met antibodies for binding to the extracellular domain of recombinant human c-Met. 5D5 was used as positive control antibody. All anti-c-Met antibodies in the experiment shown were able to compete with Europium3+-conjugated HGF for binding to recombinant c-Met. IC50 values varied between 0.0011-0.0794 μg/mL. Without adding Europium3+-conjugated HGF, roughly ˜600 relative fluorescent units (RFU) were detected, indicating the signal when maximal inhibition is accomplished. When binding of Europium3+-conjugated HGF was not inhibited, approximately 66,000 RFU were detected. Antibodies 005, 006, 058, 101 and the positive control antibody 5D5 were able to inhibit 84.5-92.1% of HGF binding to the c-Met receptor. All other antibodies were able to inhibit at least 55% of HGF binding to c-Met. Since HGF can bind the c-Met receptor at both the SEMA domain and the Ig region, some antibodies may inhibit only one of these interactions. To determine which interaction was inhibited, a cMetSEMAHis-based inhibition of time-resolved fluorescence resonance energy transfer (TR-FRET) assay was performed.
Example 17
Competition of Anti-c-Met Antibodies for Binding to Soluble cMetECDHis Measured with Sandwich-ELISA
First, the optimal coating concentrations of the tested anti-c-Met antibodies and the optimal cMetECDHis concentration were determined. Therefore, ELISA wells were coated overnight at 4° C. with anti-c-Met HuMabs serially diluted in PBS (8 μg/mL in 2-fold dilutions). Next, the ELISA wells were washed with PBST (PBS supplemented with 0.05% Tween-20 [Sigma-Aldrich, Zwijndrecht, The Netherlands]) and blocked for one hour at room temperature (RT) with PBSTC (PBST supplemented 2% [v/v] chicken serum [Gibco, Paisley, Scotland]). Subsequently, the ELISA wells were washed with PBST and incubated for one hour at RT with biotinylated cMetECDHis serially diluted in PBSTC (1 μg/mL in 2-fold dilutions). Unbound biotinylated cMetECDHis was washed away with PBST, and bound biotinylated cMetECDHis was incubated for one hour at RT with 0.1 μg/mL Streptavidin-poly-HRP (Sanquin, Amsterdam, The Netherlands) diluted in PBST. After washing, the reaction was visualized through a 15 minutes incubation with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS: dilute one ABTS tablet in 50 mL ABTS buffer [Roche Diagnostics, Almere, The Netherlands]) at RT protected from light. The colorization was stopped by adding an equal volume of oxalic acid (Sigma-Aldrich, Zwijndrecht, The Netherlands). Fluorescence at 405 nm was measured on a microtiter plate reader (Biotek Instruments, Winooski, USA). The conditions that resulted in sub-optimal (approx. 80%) binding of each antibody were determined and used for following cross-block experiments.
ELISA wells were coated with anti-c-Met antibody at a sub-optimal dose as described above. After blocking of the ELISA wells, they were incubated with the predetermined concentration of biotinylated cMetECDHis in the presence of an excess of anti-c-Met antibody. The reaction was developed as described above. Residual binding was expressed as a percentage relative to the binding observed in the absence of competitor antibody.
Table 6: When added as competitor, all anti-c-Met antibodies were able to compete for binding with their immobilized counterparts. 022, 058 and 5D5, when added as competitor antibodies, competed with antibodies 005 and 006. However, the reverse reaction revealed only partial competition by antibodies 005 and 006. These differences can be explained by the lower affinities of antibodies 005 and 006 for biotinylated cMetECDHis. Antibody 5D5, when added as competitor antibody, also demonstrated partial competition with antibodies 008 and 045, whereas no or minimal competition was observed in the reverse reaction. In addition, antibodies 024, 062, 064, 068 and 181 when added as competitor antibodies, demonstrated partial competition with antibody 101, whereas the reverse reaction demonstrated complete inhibition of cMetECDHis binding. Values higher than 100% can be explained by avidity effects and the formation of antibody-cMetECDHis complexes containing two non-competing antibodies.
Antibodies 024, 062, 064, 068, 069, 098, 101 and 181 compete with each other for binding to cMetECDHis. Antibodies 005, 006, 022 and 058 were considered to belong to one cross-block group, a group that is characterized by complete competition with 005, 006, 022, 058 and 5D5. However, antibody 5D5 was the only antibody that was also able to compete for binding with antibody 045. Another group of antibodies that compete for binding to cMetECDHis is formed by 008, 035 and G11-HZ.
TABLE 6
Competition of anti-c-Met antibodies for binding to biotinylated cMetECDHis
Immo-
bilized
anti-
Competing antibody
body
005
006
008
022
024
035
045
058
005
7.7 ± 1.1
18.2 ± 3.6
81.9 ± 3.1
4.9 ± 1.3
113.5 ± 5.0
84.9 ± 0.2
116.9 ± 7.0
3.6 ± 0.1
006
11.3 ± 0.9
14.6 ± 0.7
58.8 ±2.2
4.6 ± 0.3
113.3 ± 1.0
67.5 ± 4.2
114.5 ± 3.5
3.6 ± 0.3
008
63.9 ± 3.1
47.3 ± 1.2
5.4 ± 0.3
82.1 ± 3.0
103.2 ± 0.4
32.9 ± 1.0
100.4 ± 3.8
40.8 ± 0.8
022
37.9 ± 3.9
60.5 ± 4.0
94.1 ± 3.5
3.8 ± 1.2
99.4 ± 4.8
92.4 ± 0.4
95.7 ± 3.5
5.8 ± 0.0
024
98.4 ± 10.4
101.4* ± 16.7
104.2* ± 12.7
100.2* ± 9.0
5.4 ± 0.5
108.1* ± 5.8
98.1* ± 11.9
102.8* ± 12.8
035
36.7 ± 1.0
33.0 ± 17.6
7.2 ± 1.7
54.6 ± 6.5
121.4 ± 27.8
10.6 ± 0.3
125.0 ± 16.8
18.5 ± 2.5
045
111.4 ± 1.5
110.6 ± 3.5
98.5 ± 3.1
105.3 ± 2.5
102.4 ± 5.6
105.4 ± 5.5
21.3 ± 0.1
115.3* ± 6.5
058
31.4 ± 3.6
43.6 ± 2.1
90.2 ± 2.5
6.8 ± 0.3
109.0 ± 4.1
90.1 ± 5.4
111.7 ± 4.9
4.0 ± 0.2
062
95.8 ± 5.1
95.2 ± 6.8
97.4 ± 5.3
94.6 ± 4.0
7.3 ± 2.9
90.6 ± 11.5
97.0 ± 3.0
94.4 ± 4.3
064
90.4 ± 1.9
90.1* ± 1.4
94.6* ± 0.5
94.2 ± 3.6
7.5 ± 2.5
83.5 ± 12.2
95.0 ± 4.9
95.5 ± 0.6
068
101.1 ± 7.6
98.5 ± 6.7
101.7 ± 5.5
99.6 ± 4.0
4.7 ± 2.3
88.6 ± 12.7
100.4 ± 9.0
101.5 ± 5.1
069
102.3 ± 11.2
100.3 ± 12.3
102.1 ± 12.8
97.8 ± 12.5
6.6 ± 4.1
91.7 ± 27.3
99.8 ± 14.4
100.6 ± 14.1
098
99.6 ± 6.3
97.9 ± 6.7
99.8 ± 4.2
95.8 ± 5.4
12.9 ± 4.2
89.4 ± 20.6
96.7 ± 3.7
98.6 ± 2.9
101
91.5 ± 7.2
89.7 ± 7.9
94.0 ± 6.3
90.7 ± 5.3
40.5 ± 5.4
96.7 ± 1.9
94.7 ± 5.1
93.1 ± 5.2
181
95.9 ± 7.8
93.7 ± 8.4
98.7 ± 5.8
92.5 ± 7.4
4.3 ± 1.9
96.0 ± 9.6
96.8 ± 6.7
98.9 ± 9.8
5D5
42.3 ± 14.7
58.8 ± 19.4
90.2 ± 9.9
12.4 ± 4.7
94.2 ± 9.7
98.1
83.9 ± 13.4
6.6 ± 3.2
G11-HZ
50.5 ± 7.6
47.7 ± 2.9
33.3 ± 0.2
54.3 ± 3.7
98.8 ± 5.6
32.8 ± 4.0
72.0 ± 9.9
27.6 ± 4.3
Immo-
bilized
anti-
Competing antibody
body
062
064
068
069
098
101
181
5D5
G11-HZ
005
117.7 ± 10.7
118.2 ± 7.8
128.7 ± 9.5
124.0 ± 8.0
110.4 ± 7.6
103.2 ± 5.0
131.0 ± 7.7
2.9 ± 0.1
76.8 ± 4.4
006
118.8 ± 8.4
122.2 ± 5.3
128.6 ± 6.5
124.5 ± 1.0
110.6 ± 2.3
105.9 ± 4.1
123.5 ± 6.1
3.1 ± 0.0
54.0 ± 35.1
008
100.5 ± 2.5
107.1 ± 6.2
112.2 ± 5.1
104.1 ± 4.4
106.6 ± 2.6
101.0 ± 2.5
111.3 ± 1.3
32.4 ± 0.8
2.7 ± 0.2
022
99.4 ± 2.0
101.9 ± 3.2
104.1 ± 3.3
99.6 ± 6.0
104.8 ± 4.0
103.6 ± 5.1
107.1 ± 5.2
4.2 ± 2.1
85.9 ± 8.3
024
2.3 ± 0.6
2.3 ± 0.6
12.0 ± 5.5
2.9 ± 0.5
10.4 ± 4.2
4.8 ± 1.0
7.1 ± 2.8
95.5* ± 1.1
98.2* ± 1.3
035
119.6 ± 11.2
131.7 ± 20.0
175.1 ± 30.2
150.9 ± 24.9
126.2 ± 19.9
113.0 ± 4.6
159.1 ± 12.9
25.5 ± 9.9
7.8 ± 3.2
045
103.1 ± 3.5
103.7 ± 5.7
113.1 ± 1.4
97.0 ± 5.2
76.4 ± 11.7
101.5 ± 5.1
99.4 ± 3.8
27.8 ± 3.9
99.3 ± 5.3
058
109.1 ± 4.6
108.8 ± 4.4
118.8 ± 4.2
112.6 ± 4.0
111.8 ± 6.2
104.4 ± 0.8
121.3 ± 3.1
2.8 ± 0.4
81.5 ± 8.6
062
2.4 ± 0.5
2.2 ± 0.2
14.2 ± 1.8
2.9 ± 0.1
13.2 ± 0.9
7.8 ± 1.1
9.4 ± 1.6
97.7 ± 8.5
101.3 ± 0.9
064
2.2 ± 0.6
2.0 ± 0.2
13.0 ± 0.9
2.7 ± 0.2
14.7 ± 1.2
7.6 ± 0.8
10.1 ± 3.0
94.9* ± 4.6
102.0 ± 10.5
068
2.0 ± 0.3
2.0 ± 0.3
6.6 ± 0.7
2.4 ± 0.4
8.2 ± 1.3
4.8 ± 0.7
5.2 ± 0.6
94.8 ± 2.7
110.3 ± 6.6
069
2.2 ± 0.4
2.3 ± 0.5
10.1 ± 2.6
2.4 ± 0.7
12.5 ± 3.1
3.9 ± 0.5
6.3 ± 1.0
99.4 ± 16.2
110.4 ± 13.2
098
8.8 ± 0.6
9.3 ± 1.3
18.0 ± 2.5
3.4 ± 0.6
2.6 ± 0.4
4.0 ± 0.6
12.0 ± 2.1
94.9 ± 1.2
99.6 ± 1.2
101
36.9 ± 3.3
37.4 ± 3.7
45.9 ± 4.3
9.5 ± 1.2
9.7 ± 1.5
3.7 ± 2.4
41.9 ± 0.8
97.2 ± 4.6
98.3 ± 2.1
181
2.0 ± 0.2
2.1 ± 0.3
6.5 ± 1.1
2.2 ± 0.3
5.1 ± 1.1
2.4 ± 0.2
3.6 ± 0.2
94.2 ± 4.5
98.7 ± 6.7
5D5
97.6 ± 8.1
97.1 ± 12.7
97.8 ± 6.6
99.6 ± 3.9
97.6 ± 4.9
97.9 ± 10.6
103.4 ± 4.3
4.1 ± 1.5
97.3
G11-HZ
95.3 ± 3.1
99.2 ± 0.6
102.6 ± 1.3
95.0 ± 8.4
96.2 ± 11.8
90.1 ± 6.8
101.1 ± 5.2
29.1 ± 9.2
2.6 ± 0.4
75->100% competition
25-74% competition
0-24% competition
Data shown are percentages inhibition of binding±the stdev. of 3 independent experiments. For antibodies 035, 5D5 and G11-HZ the cross-block ELISA was performed only twice. In addition, a number of competition reactions (*) resulted in Optimal Density values higher than 5.0, which is above the detection limit of the ELISA reader. These results were discarded from the analysis resulting in duplicate measurements.
Example 18
Blocking of HGF Binding to cMetSEMA-567His8 Determined by Means of Time Resolved-Fluorescent Resonance Energy Transfer (TR-FRET)
HGF can bind the c-Met receptor at both the SEMA domain and the IgG-region. However, only HGF bound to the SEMA domain was found to be crucial for receptor activation. Therefore, the interaction of anti-c-Met antibodies with the SEMA domain of the c-Met receptor was studied using TR-FRET technology. In order to perform this homogenous proximity-based assay, hepatocyte growth factor (HGF, ProSpec Tany, Rehovot, Israel) was conjugated with a fluorescent acceptor dye; AlexaFluor-647 (Invitrogen, Breda, The Netherlands). cMetSEMA-567His8 was labeled with a fluorescent donor molecule directed against the histidine tag (Anti-6×his Europium3+, PerkinElmer, Turku, Finland). Binding of the AlexaFluor-647-conjugated HGF to the Europium3+-labeled cMetSEMA-567His8 enables an energy transfer of the donor molecule (excitation 340 nm) to the acceptor molecule (emission 665 nm). The mean fluorescent intensity at 665 nm was measured on the EnVision 2101 Multilabel reader (PerkinElmer). Competition of unlabeled anti-c-Met antibodies with AlexaFluor-647-conjugated HGF was measured by a decrease in TR-FRET signal at 665 nm, because in the unbound state, the distance between the donor and acceptor fluorophores is too large for energy transfer to occur.
All dilutions were made in 0.5×Lance detection buffer (PerkinElmer) supplemented with 2.67% Stabilizer solution (PerkinElmer) and 0.03% (v/v) Tween-20 (Riedel de Haen, Seelze, Germany). 25 μL of cMetSEMA-567His8 was added to 25 μL AlexaFluor-647 conjugated HGF, 25 μL of anti-6×his Europium3+ and 25 μL of unlabeled anti-c-Met antibody to a 96-well opti-white plate (PerkinElmer). A final concentration of 2.93 μg/mL cMetSEMA-567His8, 0.96 μg/mL AlexaFluor-647-conjugated HGF and 0.4 μg/mL anti-6×his Europium3+ was obtained. A 4-fold serial dilution of unlabeled anti-c-Met antibody ranging from 0.49-8000 ng/mL was tested. After overnight incubation at 4° C. in the dark, mean fluorescence intensity at 665 nm was measured using the EnVision 2101 Multilabel reader applying the following settings: Lance/Delfia dual mirror, emission filter 615-665 nm, excitation filter 320 nm, delay time 60 μs, window 100 μs, 100 flashes, 2000 μs per cycle and bidirectional row-by-row reading. To determine IC50 values, the binding curves were analyzed with non-linear regression (sigmoidal dose-response with variable slope) using GraphPad Prism V4.03 software (GraphPad Software, San Diego, Calif., USA).
FIG. 6 shows HGF binding inhibition curves of the various anti-c-Met antibodies for binding to cMetSEMA—567His8 tested with TR-FRET. Except for antibodies 008, 035 and 096, all antibodies were able to compete with AlexaFluor-647-conjugated HGF for binding to cMetSEMA-567His8. Antibody 022 was able to inhibit ˜80% binding of HGF, whereas antibodies 005, 006, 024, 045, 058, 061, 062, 064, 068, 069, 098, 101, 181 and the positive control antibody 5D5 were able to inhibit >90% of HGF binding to cMetSEMA-567His8. IC50 values ranging from 0.082-0.623 μg/mL were determined.
TABLE 7
IC50 values (μg/mL) and percentage of ligand
inhibition of anti-c-Met antibodies for binding to
cMetSEMA-567His8 determined with TR-FRET
mAb
IC50
% inhibition
005
0.16
92
006
0.16
92
008
ND
4
022
0.37
77
024
0.39
95
035
ND
19
045
0.17
92
058
0.15
99
061
0.49
96
062
0.58
97
064
0.07
97
068
0.26
96
069
0.54
97
096
ND
16
098
0.55
98
101
0.53
96
181
0.34
93
5D5
0.2
95
Data shown are mean MFI of three independent experiments.
Example 19
KP4 Viability Assay
C-Met antibodies were tested for their ability to inhibit viability of KP4 cells (Riken BioResource Center Cell Bank, RCB1005). KP4 cells, which express high levels of both c-Met and HGF in an autocrine manner, were seeded in a 96-wells tissue culture plate (Greiner bio-one, Frickenhausen, Germany) (10,000 cells/well) in serum-free medium (1 part HAM's F12K [Cambrex, East Rutherford, N.J.] and 1 part DMEM [Cambrex]). 66.7 nM anti-c-Met antibody dilution was prepared in serum-free medium and added to the cells. After 3 days incubation, the amount of viable cells was quantified with Alamarblue (BioSource International, San Francisco, US) according to the manufacturer's instruction. Fluorescence was monitored using the EnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland) with standard Alamarblue settings. The Alamarblue signal of antibody-treated cells was plotted as a percentage signal compared to untreated cells.
FIG. 7 depicts the percentage inhibition of viable KP4 cells after anti-c-Met antibody treatment compared to untreated cells (0%). The boxed clones are antibodies that cross compete with each other as described in Example 17. Interestingly, antibodies 024, 062, 064, 068, 069, 098, 101 and 181, which belong to the same cross-block group, were all able to inhibit KP4 viability (18-46%), both as IgG1 and as UniBody molecule. Also IgG1 molecules of antibodies 008, 061 and 096 were able to inhibit KP4 viability. In contrast, antibody 045 did not inhibit KP4 viability as IgG1 nor as UniBody molecule. For Uni-1016-045-TE this may be due to its low apparent affinity for membrane bound c-Met, as measured by FACS analysis (Example 13). The IgG1 antibodies of clones 005, 006, 022 and 058 did not inhibit KP4 viability significant, while Uni-1016-022-TE, Uni-1016-058-TE and IgG1-1016-058-wtFab did inhibit 57, 38 and 44% of KP4 viability, respectively. Uni-1016-005 and Uni-1016-006 also cross compete with clones 022 and 058 but did not inhibit KP4 viability significant. This may be due to their low apparent affinities as measured by FACS analysis (Example 13). Interestingly also IgG4-1016-058 demonstrated some inhibition of KP4 viability. This was not observed with IgG4-5D5).
Overall the data indicates that for some cross-blocking groups, monovalent binding is required to inhibit KP4 viability, whereas for other cross-blocking groups both monovalent and bivalent binding antibodies can inhibit KP4 viability.
Example 20
KP4 Xenograft Tumor Model in SCID Mice
A KP4 xenograft tumor model in SCID mice was performed to determine the efficacy of anti-c-Met HuMabs to inhibit tumor growth in vivo. Seven to eleven week-old female SCID-mice, strain C.B-17/IcrPrkdc-scid/CRL, were purchased from Charles River Laboratories Nederland (Maastricht, the Netherlands) and kept under sterile conditions in filter-top cages with food and water provided ad libitum. Microchips (PLEXX BV, Elst, The Netherlands) were placed for mouse identification. All experiments were approved by the Utrecht University animal ethics committee.
At day 0, 10×106 KP4 cells were inoculated subcutaneously in 200 μl PBS on the right flank. Mice were examined at least twice per week for clinical signs of illness. Tumor size was determined at least once a week. Volumes (mm3) are calculated from caliper (PLEXX) measurements as 0.52×(length)×(width)2, starting on day 16. On day 9, average tumor sizes were measured and mice were divided in 8 groups of 7 mice each. Anti-c-Met antibodies (008, 058, 069 and 098) were injected intraperitoneally. Antibody G11-HZ was used as a positive control antibody, whereas 5D5 and isotype-control antibodies were used as negative control antibodies. Mice received a loading dose of 400 μg/mouse followed weekly with a maintenance dose of 200 μg/mouse, for the duration of 7 weeks.
Additionally, plasma samples, collected before administration of 1st, 3rd and 5th maintenance dose and when mice were terminated, the presence of human IgG was verified using latex beads on the BNII nephelometer (Dade Behring, Atterbury, UK).
FIGS. 8 and 9 show that tumor growth of KP4 cells was inhibited by HuMabs 008, 069, 098 and positive control G11-HZ. The inhibition was compared to treatment with isotype-control antibody. Tumor growth of KP4 cells was delayed but not completely inhibited by control antibody G11-HZ. Clones 069 and 098 showed more potent inhibition compared to clones 008 and G11-HZ. Antibodies 5D5 and 058 did not inhibit tumor growth. This was consistent with in vitro data as described in Example 19. Taken together, these data indicate that for some cross-blocking groups bivalent binding antibodies can inhibit KP4 tumor growth.
Example 21
MKN45 Xenograft Tumor Model
A human gastric adenocarcinoma MKN45 xenograft tumor model in nude mice was used to determine the efficacy of anti-c-Met HuMabs to inhibit tumor growth in vivo.
Human MKN45 gastric adenocarcinoma cells were cultured at 37° C. and 5% CO2 in RPMI-1640 medium containing 100 units/mL penicillin G sodium, 100 μg/mL streptomycin sulfate, 25 μg/mL gentamicin, 20% fetal bovine serum, and 2 mM glutamine. Seven to eight weeks old female nude mice (nu/nu, Harlan) (body weights ranging from 17.0 to 26.4 g at the beginning of the study) were used. The animals were fed ad libitum water and food. The mice were housed under conditions complying with the recommendations of the Guide for Care and Use of Laboratory Animals. The animal care and use program was accredited by AAALAC. At day 0, 1×10e7 MKN45 cells were inoculated subcutaneously in 200 μl 50% matrigel in PBS in the flank of each mouse. On day 7, the animals were sorted into five groups (n=10) with an average tumor volume of 80 to 120 mm3 and treatment was started. Anti-c-Met antibodies (008, 058, 069) were injected in the tail vein (iv). Antibody G11-HZ was used as a positive control antibody and an isotype control antibody was used as a negative control antibody. All mice received 40 mg/kg antibody on day 7 and 20 mg/kg antibody on days 14, 21, and 28.
Tumors were measured twice weekly using calipers until an endpoint tumor volume of 700 mm3 or until the end of the study (day 62). FIGS. 10 and 11 show that tumor growth of MKN45 cells was significantly delayed by antibodies 008, 058, 069 and the control antibody G11-HZ compared to treatment with isotype control antibody.
Example 22
Decreasing Residual Agonistic Activity of IgG1 c-Met Antibodies by Reducing Conformational Flexibility
The natural ligand of c-Met, HGF, is a functional dimer that induces dimerization of two c-Met molecules. The subsequent intracellular phosphorylation of the intracellular domain of c-Met results in the activation of several signaling pathways which are involved in proliferation, invasion and survival of cells. Most bivalent antibodies raised against c-Met show comparable effects as HGF on cell fate, especially when the binding epitopes of the antibody are located near or in the SEMA domain of c-Met.
To minimize the potential residual agonistic activity of the bivalent IgG1 antibodies, a strategy to reduce the conformational flexibility was employed. In an IgG1 there is a large degree of freedom for the Fab arms to move relative to the Fc domain. The largest conformational changes are the result of the flexibility of the hinge, which allows a wide range of Fab-Fc angles (Ollmann Saphire, E., R. L. Stanfield, M. D. M. Crispin, P. W. H. I. Parren, P. M. Rudd, R. A. Dwek, D. R. Burton and I. A. Wilson. 2002. Contrasting IgG structures reveal extreme asymmetry and flexibility. J. Mol. Biol. 319: 9-18). One way to reduce Fab-arm flexibility in immunoglobulins is to prevent the formation of disulphide bonds between the light and the heavy chain by means of genetic modification. In a natural IgG1 antibody the light chain is connected covalently with the heavy chain via a disulphide bond, connecting the C-terminal cysteine of the light chain to the cysteine at position 220 (C220 EU numbering) in the hinge of the Fc of the heavy chain. By either mutating amino acid C220 to serine or any other natural amino acids, by removing C220 by removing the complete hinge, or by replacing the IgG1 hinge with an IgG3 hinge, a molecule is formed in which the light chains are connected via their C-terminal cysteines, analogous to the situation found in the human isotype IgA2m(1). This results in a reduced flexibility of the Fabs relative to the Fc and consequently reduced cross-linking capacity, as shown in comparative studies with IgA2m(1) and IgG1 formats of an agonistic c-Met antibody (5D5) in a KP4 viability assay (FIG. 12).
Another strategy to reduce the flexibility of an IgG1 molecule is to replace the IgG1 hinge with the IgG2 hinge or IgG2-like hinge. (Dangl et al. EMBO J. 1988; 7:1989-94). This hinge region has two properties distinct from that of IgG1, which are considered to render the molecules less flexible. First, compared to IgG1, hinge the IgG2 hinge is 3 amino acids shorter. Second, the IgG2 hinge contains an additional cysteine, thus three instead of two inter-heavy chain disulphide bridges will be formed. Alternatively, a variant of the IgG1 hinge that resembles the IgG2 hinge can be introduced. This mutant (TH7Δ6-9) (WO2010063746) contains mutation T223C and two deletions (K222 and T225) in order to create a shorter hinge with an additional cysteine.
Example 23
Generation of IgG1 Molecules with Reduced Flexibility (Stiffened IgG1 Molecules)
Cloning and Expression
Mutant IgG1 antibodies were designed and cloned using standard molecular biological techniques. An overview of the sequences of all generated hinge region mutations is shown in Table 8 below.
TABLE 8
Amino acid sequence of the hinge of mutant IgG1 antibodies. Deletions are marked by
‘-’, and mutations are highlighted in grey.
For the expression of the resulting stiffened IgG1 antibodies in mammalian cells, the HC constant region of IgG1, containing mutations in the hinge region (see above Table 8), was synthesized as a codon optimized construct in mammalian expression vector pcDNA3.3 (Invitrogen). A separate vector was constructed by inserting the codon optimized constant region of the human kappa light chain region in pcDNA3.3. VH and VL regions of clone 069 and control antibody 5D5 were inserted in the HC constant plasmid and Kappa light chain plasmid respectively resulting in vectors for the expression of the (mutated) heavy and light chains of the specific antibodies. Co-transfection of the heavy and light chain vectors of a specific antibody in HEK-293F (Invitrogen) cells, resulted in the transient production of mutant antibodies. Purification of the antibodies was performed using Protein A affinity column chromatography (as described in Example 11).
Biochemicial Characterization
Transient Expression
All mutants were expressed at sufficient levels and did not show aberrant formation of multimers as determined by MS (>99% purity) and SDS-PAGE.
The SDS-PAGE results are shown in FIG. 13. In the C220 mutants (C220S and ΔC220) and the hinge-deleted IgG1 variants (the hinge-deleted IgG1 variants are also named Unibody-IgG1 or Uni-IgG1) light chain pairing, visible as a protein band of around 50 kD in non-reduced SDS-PAGE analysis, was observed. The variant with an IgG3 hinge also showed light chain pairing, while the variant with an IgG2 hinge and the IgG1 TH7Δ6-9 mutant showed normal light-heavy chain pairing.
Example 24
c-Met Binding Properties of the Mutants
c-Met binding properties of the mutants were tested in an ELISA. ELISA plate wells were coated overnight at 4° C. with rhHGF R/Fc Chimera (R&D Systems; Cat. 358MT/CF) in PBS (1 μg/mL). Next, the wells were washed with PBST (PBS supplemented with 0.05% Tween-20 [Sigma-Aldrich, Zwijndrecht, The Netherlands]) and blocked for one hour at room temperature (RT) with PBSTC (PBST supplemented 2% [v/v] chicken serum [Gibco, Paisley, Scotland]). Subsequently, the wells were washed with PBST and incubated for one hour at RT with the anti-cMet antibodies and variants serially diluted in PBSTC (10 μg/mL in 4-fold dilutions). Unbound antibody was washed away with PBST, and antibody bound to the coat was detected by incubating for one hour at RT with goat-anti-human IgG F(ab′)2-HRPdiluted in PBST (Jackson cat. no. 109-035-097). After washing, the reaction was visualized by a 15 min incubation with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS: dilute one ABTS tablet in 50 mL ABTS buffer [Roche Diagnostics, Almere, The Netherlands]) at RT protected from light. The colorization was stopped by adding an equal volume of oxalic acid (Sigma-Aldrich, Zwijndrecht, The Netherlands). Fluorescence at 405 nm was measured on a microtiter plate reader (Biotek Instruments, Winooski, USA). All mutants bound with comparable apparent affinity (EC50) to c-Met (FIG. 14). Table 10 shows the EC50 values of the mutants obtained in this experiment.
TABLE 9
The EC50 values as determined by ELISA
IgG1-
IgG1-
IgG1-
IgG1-
1016-069-
IgG1-
Uni-
1016-069-
Uni-
IgG1-
1016-069-
1016-069-
Hinge
IgG2-
1016-069
1016-069-
IgG1-
Hinge
IgG1-
1016-069
ΔC220
C220S
IgG2
1016-069
TH7Δ6-9
TE
1016-069
IgG3
1016-069
EC50
49.5
18.87
15.56
23.03
29.61
18.81
30.08
45.43
14.18
15.39
(ng/mL)
Example 25
Reduced Agonistic Effect of Stiffened IgG1 c-Met Antibodies
Receptor Phosphorylation
To determine the agonistic properties of the stiffened antibodies the effect of the antibodies on cMet phosphorylation was performed. Upon dimerizaton of two adjacent cMet receptors by either the natural ligand HGF or most bivalent antibodies, three tyrosine residues (position 1230, 1234 and 1235) in the intracellular domain of c-Met are cross phosphorylated, which is followed by subsequent phosphorylation of several other amino acids in the intracellular domain and activation of a number of signaling cascades. The dimerization and activation of cMet can therefore be monitored by using antibodies specific for the phosphorylated receptor at these positions, and thus used as a read out for the potential agonism of the anti-c-Met antibodies.
A549 cells, CCL-185 obtained from ATCC, were grown in serum containing DMEM medium until 70% confluency was reached. After trypsinization and washing cells they were plated in a 6 well culture plate at 1*10e6 cells/well in serum containing culture medium. After overnight incubation the cells were treated with either HGF (R&D systems; cat. 294-HG) (50 ng/mL) or the panel of antibodies (30 μg/mL) and incubated for 15 minutes at 37° C. The cells were then washed twice with ice cold PBS and lysed with lysis buffer (Cell Signaling; cat. 9803) supplemented with a protease inhibitor cocktail (Roche; cat. 11836170001) and samples were stored at −80° C. Receptor activation was determined by measuring the phosphorylation by means of Western blot using phospho c-Met specific antibodies. The proteins in the cell lysate were separated on a 4-12% SDS-PAGE gel and transferred to nitrocellulose membrane that was subsequently stained with antibody specific for phosphorylated c-Met (Y1234/1235) (Cell Signaling, cat: 3129). To control for gel loading, antibodies against total c-Met and beta-actin were used. Results of the Western blots are shown in FIG. 15.
Tissue culture medium controls and cells treated with the monovalent format UniBody of antibody 5D5 did not show phosphorylation of the receptor. In contrast, Western blot analysis of cells treated with the positive control HGF or agonist antibody IgG1-1016-058 showed a clear band at the expected heigth. Antibody IgG1-1016-069 showed low, but detectable receptor phosphorylation indicating that some cross linking of the receptor takes place. However, variants that were designed to reduce the flexibility of the antibody molecule showed minimal receptor activation, down to a level comparable to the levels detected in cells treated with the monovalent control Uni-5D5-TE. (FIG. 15).
Effect of c-Met Antibodies on NCI-H441 Proliferation In Vitro
The potential proliferative agonistic activity of cMet antibodies was tested using the lung adenocarcinoma cell line NCI-H441 (ATCC, HTB-174™), which expresses high levels of c-Met, but does not produce its ligand HGF. NCI-H441 cells were seeded in a 96-wells tissue culture plate (Greiner bio-one, Frickenhausen, Germany) (5,000 cells/well) in RPMI (Lonza) without serum. Anti c-Met antibody dilutions (66.7 nM) were prepared in RPMI without serum and added to the cells. After 7 days incubation at 37° C./5% CO2, the amount of viable cells was quantified with Alamarblue (BioSource International, San Francisco, US) according to the manufacturer's instruction. Fluorescence was monitored using the EnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland) with standard Alamarblue settings.
As appears from FIG. 17 proliferation of NCI-H441 cells was strongly induced by agonistic control mAbs IgG1-058 and IgG1-5D5. Antibody IgG1-1016-069 also showed some agonistic effect compared to cells treated with the istoype control. The agonistic activity of IgG1-1016-069 could be completely removed by introducing the C220 mutants C220S and -del, and partially by the variants with the IgG2 and TH746-9 hinge or IgG2 backbone. Control samples treated with isotype control and the monovalent version of 5D5 (Uni-5D5-TE) did not induce growth of the cells.
KP4 Viability Assay
The ability to inhibit HGF dependent cells was also determined for the anti-c-Met antibody mutants in a KP4 viability assay (see Example 19 for experimental procedures). The results are shown in FIG. 17. The efficacy of IgG1-1016-069 based mutants was completely retained or slightly better in the C220 mutants. Remarkably, mutating C220 in the agonistic antibody 5D5 resulted in a marked reduction of KP4 viability. No agonistic effect of the 058 and 5D5 antibodies in IgG1 format were observed due to the high expression of HGF by KP4 (autocrine HGF loop).
Down-Modulation
Down-modulation of c-Met induced by antagonistic antibodies represents a mechanism of action of therapeutic c-Met antibodies. Accordingly, in one embodiment antibodies with reduced agonistic properties, but with retained ability to induce down-modulation of c-Met are desirable. To determine the down-modulating potential of the antibodies, A549 cells (CCL-185 obtained from ATCC) were seeded in 6-well tissue culture plates (500,000 cells/well) in serum containing cell culture medium and cultured overnight at 37° C. The next morning, anti-c-Met antibodies were added at a final concentration of 10 μg/mL and the plate was incubated another 2 days at 37° C. After washing with PBS, cells were lysed by incubating 30 min at room temperature with 250 μL Lysis buffer (Cell signaling, Danvers, USA). Total protein levels were quantified using bicinchoninic acid (BCA) protein assay reagent (Pierce) following the manufacturer's protocol. c-Met protein levels in cell lysates were quantified using a c-Met-specific sandwich ELISA. To this end, wells of ELISA plates were coated overnight at 4° C. with goat-anti-human c-Met antibody directed against the extracellular domain of c-Met (R&D systems), diluted in PBS (1 μg/mL). Next, the wells were washed with PBST (PBS supplemented with 0.05% Tween-20 [Sigma-Aldrich, Zwijndrecht, The Netherlands]) and blocked for one hour at RT with PBSTC (PBST supplemented 2% [v/v] chicken serum [Gibco, Paisley, Scotland]). Undiluted cell lysates were added (100 μL) and incubated one hour at RT. After washing with PBST, the wells were incubated one hour at RT with a mouse-antibody directed against the intracellular Tyrosine-1234 residue of human-c-Met (Cell signaling), diluted 1:1000 in PBSC. The wells were washed again with PBST and incubated one hour at RT with a goat-anti-mouse Fc-HRP antibody (Jackson) diluted 1:5000 in PBSC. Following washing with PBST, the reaction was visualized through a 30 minutes incubation with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS: dilute one ABTS tablet in 50 mL ABTS buffer [Roche Diagnostics, Almere, The Netherlands]) at RT protected from light. The colorization was stopped by adding an equal volume of oxalic acid (Sigma-Aldrich, Zwijndrecht, The Netherlands). Fluorescence at 405 nm was measured on a microtiter plate reader (Biotek Instruments, Winooski, USA). As appears from FIG. 18 all mutants of antibody 069 were able to induce down-modulation.
Example 26
Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
MKN45 cells (purchased from RIKEN BioResource Center, Tsukuba, Japan, RCB1001) were harvested (5×106 cells), washed (twice in PBS, 1500 rpm, 5 min) and collected in 1 mL RPMI 1640 medium supplemented with 10% cosmic calf serum (CCS) (HyClone, Logan, Utah, USA), to which 200 μCi 51Cr (Chromium-51; Amersham Biosciences Europe GmbH, Roosendaal, The Netherlands) was added. The mixture was incubated in a shaking water bath for 1.5 hours at 37° C. After washing of the cells (twice in PBS, 1500 rpm, 5 min), the cells were resuspended in RPMI 1640 medium supplemented with 10% CCS, counted by trypan blue exclusion and diluted to a concentration of 1×105 cells/mL.
Meanwhile, peripheral blood mononuclear cells (PBMCs) were isolated from fresh buffy coats (Sanquin, Amsterdam, The Netherlands) using standard Ficoll density centrifugation according to the manufacturer's instructions (lymphocyte separation medium; Lonza, Verviers, France). After resuspension of cells in RPMI 1640 medium supplemented with 10% CCS, cells were counted by trypan blue exclusion and concentrated to 1×107 cells/mL.
For each ADCC experiment, 50 μL 51Cr-labeled MKN45 cells (5,000 cells) were pre-incubated with 15 μg/mL cMet antibody in a total volume of 100 μL RPMI medium supplemented with 10% CCS in a 96-well microtiter plate. After 15 min at RT, 50 μL PBMCs (500,000 cells) were added, resulting in an effector to target cell ratio of 100:1. The maximum amount of cell lysis was determined by incubating 50 μL 51Cr-labeled MKN45 cells (5,000 cells) with 100 μL 5% Triton-X100. The amount of spontaneous lysis was determined by incubating 5,000 51Cr-labeled MKN45 cells in 150 μL medium, without antibody or effector cells. The level of antibody-independent cell lysis was determined by incubating 5,000 MKN45 cells with 500,000 PBMCs without antibody. Subsequently, the cells were incubated 4 hours at 37° C., 5% CO2. The cells were centrifuged (1200 rpm, 3 min) and 75 μL of supernatant was transferred to micronic tubes, after which the released 51Cr was counted using a gamma counter. The measured counts per minute (cpm) were used to calculate the percentage of antibody-mediated lysis as follows:
(cpm sample−cpm Ab-independent lysis)/(cpm max. lysis−cpm spontaneous lysis)×100%
Various publications have demonstrated the correlation between reduced core-fucosylation and enhanced ADCC activity in vitro (Shields R L. 2002 JBC; 277:26733-26740, Shinkawa T. 2003 JBC; 278(5):3466-3473). FIG. 19 demonstrates that antibody 069 does not induce lysis of MKN45 cells through ADCC. However when core-fucosylation was reduced due to the presence of kifunensine during mAb production in HEK-cells, antibody 069 was able to induce over 30% lysis of MKN45 cells. Moreover, lysis was already observed at antibody concentrations below 0.01 ug/mL. Values depicted are the mean maximum percentages 51Cr-release±the stdev from one representative in vitro ADCC experiment with MKN45 cells. 069 low-fucose was produced in HEK 293 cells in presence of kifunensine, resulting in an ˜99.5% non-core fucosylation (i.e. absence of fucose). 069 high-fucose was produced in HEK 293 cells without kifunensin, resulting in ˜2.11% non-core fucosylation, as determined with high performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) (data not shown).
Example 27
Lack of Binding of c-Met Antibodies to Human Peripheral Blood Cells
In order to address binding of clone 069 to three types of cells (B-cells, monocytes and granulocytes) present in peripheral blood a FACS binding assay was performed. Fluorescently labeled clone 069 was used to enable direct measurement on FACS without use of secondary detection antibodies. The cell populations in the blood were identified in the assay using fluorescently commercial antibodies against specific markers on the cells of interest.
Perhipheral blood from healthy volunteers (University Medical Center Utrecht) was diluted ten times in FACS buffer (PBS+0.4% BSA+0.02% NaN3) and incubated with Alexa488-conjugated c-Met antibodies and FITC-conjugated anti-CD19, -CD16 and -CD14 antibodies (final concentration 10 μg/mL) and phycoerythrin (PE)-labeled anti-CD19, -CD16 and -CD14 antibodies (BD Biosciences, San Jose Calif.) to identify cell populations (resp. B cells, granulocytes and monocytes) in a final volume of 100 μl. After 30 minutes at 4° C., samples were centrifuged (300 g, 3 min), supernatant was removed, erythrocytes were lysed by incubation (10 min, 4° C.) with 200 μl Ery-lysis solution (155 mM NH4Cl, 10 mM KHCO3, 0.1 Mm EDTA [pH 7.4]), and samples were washed twice in FACS buffer. Samples were resuspended in 100 μL FACS buffer and analyzed using a FACS Canto II (BD Biosciences).
FIG. 20 is a representative FACS plot which demonstrates that Alexa488-conjugated-069 did not bind the B cell population (CD19-PE+ cells within the lymphocyte gate). Binding of Alexa488-conjugated-rituximab was used as positive control. Binding to other cell populations was analyzed similarly and representative results for 1 of 3 donors are also plotted in FIG. 21. Antibody 069-Alexa488 did not bind to B cells, monocytes or granulocytes, whereas the positive control antibodies did demonstrate specific binding.
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13583743
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genmab a+s
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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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Apr 1st, 2022 06:04PM
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Apr 1st, 2022 06:04PM
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Genmab A/S
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Health Care
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Pharmaceuticals & Biotechnology
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cph:gen
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Genmab A/S
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Jun 15th, 2021 12:00AM
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Oct 8th, 2020 12:00AM
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https://www.uspto.gov?id=US11034772-20210615
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Bispecific anti-CD37 antibodies, monoclonal anti-CD37 antibodies and methods of use thereof
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CD37-specific bispecific antibody molecules binding to different epitopes of the human CD37 antigen which bispecific antibody molecules have enhanced Fc-Fc interactions upon binding to CD37 on the cell surface. The invention also relates to the monoclonal parental antibodies from which the first or the second binding region of the bispecific antibody molecules is obtained. The invention also relates to pharmaceutical compositions containing these molecules and the treatment of cancer and other diseases using these compositions.
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11034772
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1. An antibody which binds to human CD37 comprising:
(a) a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively, and a variable light chain (VL) region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 27, the sequence YAS, and SEQ ID NO: 31, respectively, or
(b) a VH region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 20, the sequence KAS, and SEQ ID NO: 21, respectively.
2. The antibody of claim 1, which comprises the VH region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively, and the VL region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 27, the sequence YAS, and SEQ ID NO: 31, respectively.
3. The antibody of claim 1, which comprises the VH region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, and the VL region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 20, the sequence KAS, and SEQ ID NO: 21, respectively.
4. The antibody of claim 2, which comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 22 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 29.
5. The antibody of claim 3, which comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 15 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 19.
6. The antibody of claim 2, which comprises a human IgG1 heavy chain constant region.
7. The antibody of claim 3, which comprises a human IgG1 heavy chain constant region.
8. The antibody of claim 6, wherein the human IgG1 heavy chain constant region comprises a substitution of glutamic acid at position 430 for glycine, and wherein the amino acid residue is numbered according to the EU Index.
9. The antibody of claim 7, wherein the human IgG1 heavy chain constant region comprises a substitution of glutamic acid at position 430 for glycine, and wherein the amino acid residue is numbered according to the EU Index.
10. The antibody of claim 2, which comprises a kappa light chain constant region.
11. The antibody of claim 3, which comprises a kappa light chain constant region.
12. A bispecific antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises a first antigen-binding region and the second binding arm comprises a second antigen-binding region, wherein both the first antigen-binding region and second antigen-binding region bind to human CD37, and wherein
(a) the first antigen-binding region comprises a variable heavy chain (VH) region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively, and a variable light chain (VL) region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 27, the sequence YAS, and SEQ ID NO: 31, respectively, and
(b) the second antigen-binding region comprises a VH region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, and a VL region comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 20, the sequence KAS, and SEQ ID NO: 21.
13. The bispecific antibody of claim 12, wherein:
(a) the first antigen-binding region comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 22 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 29, and
(b) the second antigen-binding region comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 15 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 19.
14. The bispecific antibody of claim 13, wherein the first binding arm comprises a first human IgG1 heavy chain constant region and the second binding arm comprises a second human IgG1 heavy chain constant region.
15. The bispecific antibody of claim 14, wherein both the first and second human IgG1 heavy chain constant regions comprise a substitution of glutamic acid at position 430 for glycine, and wherein the amino acid residue is numbered according to the EU Index.
16. The bispecific antibody of claim 14, wherein the first human IgG1 heavy chain constant region comprises a substitution of phenylalanine at position 405 for leucine, wherein the second human IgG1 heavy chain constant region comprises a substitution of lysine at position 409 for arginine, and wherein the amino acid residues are numbered according to the EU Index.
17. The bispecific antibody of claim 14, wherein the first human IgG1 heavy chain constant region comprises a substitution of lysine at position 409 for arginine, wherein the second human IgG1 heavy chain constant region comprises a substitution of phenylalanine at position 405 for leucine, and wherein the amino acid residues are numbered according to the EU Index.
18. The bispecific antibody of claim 13, wherein both the first binding arm and second binding arm comprise a human kappa light chain constant region.
19. A bispecific antibody comprising a first binding arm comprising a first heavy chain and a first light chain and a second binding arm comprising a second heavy chain and a second light chain, wherein both the first binding arm and second binding arm bind to human CD37, and wherein:
(a) the first heavy chain comprises a variable heavy chain (VH) region comprising the amino acid sequence set forth in SEQ ID NO: 22, and the first light chain comprises a variable light chain (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 29, and
(b) the second heavy chain comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 15, and the second light chain comprises a VL region comprising the amino acid sequence set forth in SEQ ID NO: 19,
wherein the first heavy chain and second heavy chain comprise heavy chain constant regions comprising the amino acid sequences set forth in SEQ ID NOs: 59 and 60, respectively, and
wherein both the first light chain and second light chain comprise a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 61.
20. A bispecific antibody comprising a first binding arm comprising a first heavy chain and a first light chain and a second binding arm comprising a second heavy chain and a second light chain, wherein both the first binding arm and second binding arm bind to human CD37, and wherein:
(a) the first heavy chain comprises a variable heavy chain (VH) region comprising the amino acid sequence set forth in SEQ ID NO: 22, and the first light chain comprises a variable light chain (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 29, and
(b) the second heavy chain comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 15, and the second light chain comprises a VL region comprising the amino acid sequence set forth in SEQ ID NO: 19,
wherein the first heavy chain and second heavy chain comprise heavy chain constant regions comprising the amino acid sequences set forth in SEQ ID NOs: 60 and 59, respectively, and
wherein both the first light chain and second light chain comprise a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 61.
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RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 16/872,140, filed May 11, 2020, which is a continuation of U.S. patent application Ser. No. 16/498,104, filed Sep. 26, 2019, which is a 35 U.S.C. 371 national stage filing of International Application No. PCT/EP2018/058479, filed Apr. 3, 2018, which claims priority to International Application No. PCT/EP2018/057836, filed Mar. 27, 2018, and U.S. Provisional Application No. 62/479,712, filed Mar. 31, 2017. The contents of the aforementioned applications are hereby incorporated by reference.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 8, 2020, is named GMI_162USCNDV_Sequence_Listing.txt and is 109,337 bytes in size.
FIELD OF THE INVENTION
The present invention relates to bispecific antibodies that specifically bind the human CD37 antigen. The invention relates in particular to CD37-specific bispecific antibody molecules binding to different epitopes of the human CD37 antigen where the bispecific antibody molecules have enhanced Fc-Fc interactions upon binding to CD37 on the cell surface and thus have enhanced effector functions. The invention also relates to new monoclonal parental antibodies from which the first or the second antigen binding region of the bispecific antibody molecules is obtained. The invention also relates to pharmaceutical compositions containing these molecules and the treatment of cancer and other diseases using these compositions.
BACKGROUND OF THE INVENTION
Leukocyte antigen CD37 (“CD37”), also known as GP52-40, tetraspanin-26, or TSPAN26, is a transmembrane protein of the tetraspanin superfamily (Maecker et al., FASEB J. 1997; 11:428-442). In normal physiology, CD37 is expressed on B cells during the pre-B to peripheral mature B-cell stages but is reportedly absent on plasma cells (Link et al., J Pathol. 1987; 152:12-21). The CD37 antigen is only weakly expressed on T-cells and myeloid cells such as monocytes, macrophages, dendritic cells and granulocytes (Schwartz-Albiez et al., J. Immunol 1988; 140(3):905-914). CD37 is broadly expressed on malignant cells in a variety of B-cell leukemias and lymphomas, including non-Hodgkin's lymphoma (NHL) and chronic lymphoid leukemia (CLL) (Moore et al. J Immunol. 1986; 137(9):3013).
Several antibody-based CD37-targeting agents are being evaluated as potential therapeutics for B-cell malignancies and other malignancies. These include, for example, radio-immuno-conjugates such as Betalutin®, antibody-drug conjugates such as IMGN529 and AGS-67E, and reformatted or Fc-engineered antibodies such as otlertuzumab and BI 836826 (Robak and Robak, Expert Opin Biol Ther 2014; 14(5):651-61). Anti-CD37 antibodies have been proposed for use as therapeutic agents in the formats described above and other formats (see, e.g., WO 2012/135740, WO 2012/007576, WO 2011/112978, WO 2009/126944, WO 2011/112978 and EP 2 241 577).
Betalutin is a mouse anti-CD37 antibody, lilotomab (formerly HH1/tetulomab), conjugated to 177-lutetium. Betalutin internalizes rapidly, inhibits B cell growth in vitro and prolongs survival in an i.v. Daudi-SCID model (Dahle et al 2013, Anticancer Res 33: 85-96).
IMGN529 is an ADC consisting of the K7153A antibody conjugated to the maytansinoid DM1 via an SMCC linker. The K7153 antibody is reported to induce apoptosis on CD37 expressing Ramos cells in the absence of cross-linking. It also induced CDC and ADCC in Burkitt's lymphoma cell lines, though the ability to induce CDC was much less compared to rituximab (Deckert et al, Blood 2013; 122(20):3500-10). These Fc-mediated effector functions of K7153A are retained in the DM-1 conjugated antibody.
Agensys is developing AGS-67E, a human anti-CD37 IgG2 mAb conjugated to monomethyl auristatin E. AGS67E induces potent cytotoxicity and apoptosis (Pereira et al, Mol Cancer Ther 2015; 14(7): 1650-1660).
Otlertuzumab (originally known as TRU-016) is a SMIP (small modular immuno pharmaceutical; SMIPS are disulfide-linked dimers of single-chain proteins comprised of one antigen binding VH/VL, a connecting hinge region, and an Fc (fragment, crystallizable) region (CH2-CH3)). Its mechanisms of action are induction of apoptosis and ADCC, but not CDC (Zhao et al 2007, Blood 110 (7), 2569-2577).
mAb37.1/BI 836826 is a chimeric antibody that is engineered for high-affinity binding to FcγRIIIa (CD16a)(Heider et al 2011, Blood 118: 4159-4168). It has pro-apoptotic activity independent of IgG Fc crosslinking, although the pro-apoptotic activity is increased by cross-linking. It shows potent ADCC of CD37+ B cell lines and primary CLL cells.
Despite these and other advances in the art, however, there is still a need for improved anti-CD37 antibodies for the treatment of cancer and other diseases.
Accordingly, it is an object of the present invention to provide anti-CD37 antibodies which may be useful in the treatment of cancer and/or other diseases. It is an object of the present invention to provide anti-CD37 antibodies which are improved with respect to CDC of human cells by human complement compared to the prior art antibodies. It is a further object to provide a bispecific antibody having binding arms obtained from two parental antibodies which bind to different epitopes on CD37 and which bispecific antibody has increased CDC and/or ADCC compared to a combination of the two parental monoclonal antibodies binding said different epitopes, and/or to either parental monoclonal antibody by itself. It is a further object to provide new monoclonal antibodies binding different epitopes on CD37 in particular it is an object to provide anti-CD37 antibodies binding new epitopes of CD37. It is a further object of the present invention to provide new monoclonal antibodies binding different epitopes on CD37 which monoclonal antibodies may serve as parental antibodies for the bispecific antibodies of the invention. It is a further object to provide bispecific antibodies which bind to two different epitopes on CD37 and which bispecific antibodies have enhanced Fc-Fc interaction upon binding to CD37 on the plasma membrane compared to a bispecific antibody of the same isotype and having identical binding arms as the bispecific antibody of the invention.
SUMMARY OF THE INVENTION
The inventors of the present invention surprisingly found that a bispecific antibody having binding specificity against two different epitopes on CD37 and having a mutation increasing the Fc-Fc interaction upon binding to CD37 on the plasma membrane was more potent in inducing CDC than a combination of two anti-CD37 antibodies each having a binding specificity towards one of the two different epitopes on CD37 and having the same mutation enhancing the Fc-Fc interaction, or either antibody having the same mutation enhancing the Fc-Fc interaction by itself. In addition, a bispecific antibody having binding specificity against two different epitopes on CD37 and having a mutation increasing the Fc-Fc interactions was more potent in inducing ADCC than a combination of two anti-CD37 antibodies each having a binding specificity towards one of the two different epitopes on CD37 and having the same mutation enhancing the Fc-Fc interaction.
Accordingly, the invention relates to novel bispecific antibodies binding to human CD37 which have advantageous properties in terms of their antigen-binding characteristics, their ability to induce CDC and ADCC, their Fc-Fc interaction upon binding to membrane-bound targets, their cytotoxic effect on CD37-expressing cells and other properties, as described herein.
Accordingly, in a first aspect the present invention relates to a bispecific antibody comprising a first and second antigen binding region binding to human CD37 having the sequence of SEQ ID NO: 62, and a first and second Fc region of a human immunoglobulin, wherein the first and second antigen binding regions bind different epitopes on CD37 and wherein the first and second Fc regions comprises one or more amino acid mutations which mutation(s) enhances the Fc-Fc interaction between the bispecific antibodies upon binding to membrane-bound CD37 compared to the Fc-Fc interaction between bispecific antibodies not having said mutation(s).
Thus, in one aspect a bispecific antibody comprising a first and second antigen binding region binding to human CD37 having the sequence of SEQ ID NO: 62, and a first and second Fc region of a human immunoglobulin, wherein the first and second antigen binding regions bind different epitopes on CD37 and wherein the first and second Fc regions comprise one or more amino acid mutations which mutation(s) enhances the Fc-Fc interaction between bispecific antibodies upon binding to membrane-bound target compared to the Fc-Fc interaction between bispecific antibodies not having said mutation(s).
In a second aspect the invention relates to an anti-CD37 antibody binding to the same epitope on human CD37 as an anti-CD37 antibody which antibody comprises:
(i) a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 16, a CDR2 sequence set forth in SEQ ID NO: 17 and a CDR3 sequence set forth in SEQ ID NO: 18, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 20, and CDR2 sequence: KAS, and CDR3 sequence set forth in SEQ ID NO: 21[010]; or
(ii) a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 9, a CDR2 sequence set forth in SEQ ID NO:10 and a CDR3 sequence set forth in SEQ ID NO: 11, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 113, and CDR2 sequence: AAS, and CDR3 sequence set forth in SEQ ID NO: 14[005].
In a third aspect the invention relates to an anti-CD37 antibody which binds to human CD37 which antibody comprises:
(i) a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 23, a CDR2 sequence set forth in SEQ ID NO: 24 and a CDR3 sequence set forth in SEQ ID NO: 25, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 27, and CDR2 sequence: YAS, and CDR3 sequence set forth in SEQ ID NO: 28; [016] or
(ii) a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 2, a CDR2 sequence set forth in SEQ ID NO: 3 and a CDR3 sequence set forth in SEQ ID NO: 4, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 6, and CDR2 sequence: EAS, and CDR3 sequence set forth in SEQ ID NO: 7. [004]
In a fourth aspect the invention relates to a pharmaceutical composition comprising the bispecific antibody or the antibody of the invention and a pharmaceutically acceptable carrier.
In a fifth aspect the invention relates to the bispecific antibody or the antibody or the composition of the invention for use as a medicament. In specific aspects they are for use in the treatment of cancer or an autoimmune disease or inflammatory disorders and in particular for use in the treatment of B-cell malignancies.
In other aspects the invention relates to methods of treatment, to combination treatments, to nucleic acid sequences encoding the antibodies of the invention, to vectors and host cells expressing such and to methods of detecting presence or a CD37 antigen or cells expressing CD37 antigens in a sample or in a subject.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B: CDC mediated by G28.1 variants on primary CLL tumor cells. The capacity to induce CDC on primary CLL tumor cells of (FIG. 1A) IgG1-G28.1-K409R-delK, IgG1-G28.1-E345R or IgG1-b12-E345R (cells: Patient derived, Newly Diagnosed/Untreated (PB=peripheral blood derived)) and (FIG. 1B) IgG1-G28.1, IgG1-G28.1-E430G or IgG1-b12 (cells: Patient derived, Newly Diagnosed/Untreated (BM=bone marrow derived)) was determined in vitro. Data shown are % lysis determined by measurement of the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry.
FIG. 2: Quantitative determination of CD37, CD46, CD55 and CD59 expression levels on CLL tumor cells. Expression levels of CD37, CD46, CD55 and CD59 on CLL cells from one patient (Patient VM-PB0005 Newly Diagnosed/Untreated) were determined by flow cytometry. Antigen quantity is shown as molecules/cell. mIgG1 is Mouse IgG1,κ Isotype Control.
FIG. 3: Binding of humanized CD37 antibodies and variants thereof to Daudi cells. Binding of IgG1-004-H5L2, IgG1-004-H5L2-E430G, IgG1-005-H1L2, IgG1-005-H1L2-E430G, IgG1-010-H5L2, IgG1-010-H5L2-E430G, IgG1-016-H5L2 and IgG1-016-H5L2-E430G to Daudi cells was determined by flow cytometry. Data shown are mean fluorescence intensity (MFI) values, for one representative experiment.
FIG. 4: Binding of G28.1 and 37.3 and variants thereof to Daudi cells. Binding of IgG1-G28.1, IgG1-G28.1-E430G, IgG1-37.3 and IgG1-37.3-E430G to Daudi cells was determined by flow cytometry. Data shown are mean fluorescence intensity (MFI) values, for one representative experiment.
FIG. 5: Binding of variants of humanized CD37 antibody IgG1-016-H5L2 to Daudi cells. Binding of IgG1-016-H5L2, IgG1-016-H5L2-E430G, IgG1-016-H5L2-F405L-E430G and IgG1-016-H5L2-LC90S-F405L-E430G to Daudi cells was determined by flow cytometry. Data shown are mean fluorescence intensity (MFI) values, for one representative experiment.
FIG. 6: Binding of CD37 antibody variants to CHO cells expressing cynomolgus CD37. Binding of IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, IgG1-G28.1 and IgG1-G28.1-E430G was determined by flow cytometry. Data shown are mean fluorescence intensity (MFI) values, for one representative experiment.
FIGS. 7A-7G: Determination of binding competition between CD37 antibodies, and CDC mediated by humanized CD37 antibodies, variants thereof and combinations of CD37 antibodies on Raji cells. (FIG. 7A) Binding competition between IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G and IgG1-016-H5L2-E434G was determined by flow cytometry. Raji cells were incubated with unlabeled antibodies for primary binding and subsequently with Alexa Fluor 488 labeled probing antibodies. Loss of binding of the A488-labeled probing antibodies after pre-incubation with an unlabeled antibody, compared to binding of the A488-labeled antibody alone, indicates binding competition between the A488-labeled and the unlabeled antibody. Data shown are duplicate values of Molecules of Equivalent Soluble Fluorochrome (MESF), for one representative experiment. (FIGS. 7B-7G) The capacity to induce CDC on Raji cells of IgG1-004-H5L2, of IgG1-005-H1L2, IgG1-010-H5L2, IgG1-016-H5L2 and IgG1-37.3, with or without E430G mutation, and combinations of these was determined in vitro. Data shown are % lysis determined by measurement of the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry.
FIG. 8: Schematic overview of binding competition between CD37 antibodies. Binding competition between IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G and IgG1-016-H5L2-E4340G to Raji cells was determined by flow cytometry, using unlabeled antibodies for primary binding and Alexa Fluor 488 labeled probing antibodies for detecting subsequent binding of a competing antibody. Color indication: black; simultaneous binding, white; competition for binding, grey; cognate antibody.
FIG. 9: CDC mediated by humanized CD37 antibodies and variants thereof on Daudi cells. The capacity to induce CDC on Daudi cells of IgG1-004-H5L2, IgG1-004-H5L2-E430G, IgG1-005-H1L2, IgG1-005-H1L2-E430G, IgG1-010-H5L2, IgG1-010-H5L2-E430G, IgG1-016-H5L2 and IgG1-016-H5L2-E430G was determined in vitro. Data shown are % lysis determined by measurement of the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry.
FIGS. 10A-10C: CDC mediated by G28.1 and 37.3 and variants thereof, and CDC in Daudi cells mediated by humanized CD37 antibodies with different Fc-Fc interaction enhancing mutations on Daudi cells. (FIG. 10A) The capacity to induce CDC on Daudi cells of IgG1-G28.1, IgG1-G28.1-E430G, IgG1-37.3 and IgG1-37.3-E430G was determined in vitro. Data shown are % lysis determined by measurement of the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry. (FIGS. 10B and 10C) The capacity to induce CDC on Daudi cells of (FIG. 10A) IgG1-010-H5L2-K409R-E430G, IgG1-010-H5L2-E345R-K409R, IgG1-010-H5L2-E345K-K409R, IgG1-010-H5L2-K409R-E430S, IgG1-010-H5L2-RRGY and (FIG. 10B) IgG1-016-H5L2-LC90S-F405L-E430G, IgG1-016-H5L2-E345K-F405L, IgG1-016-H5L2-F405L-E430S and IgG1-016-H5L2-E345R-F405L was determined in vitro. Data shown are % lysis (maximum killing, at an antibody concentration of 10 μg/mL) determined by measurement of the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry, for one representative experiment. Error bars indicate the variation within the experiment (performed in duplicate).
FIG. 11: CDC mediated by variants of humanized antibody IgG1-016-H5L2 on Daudi cells. The capacity to induce CDC on Daudi cells of IgG1-016-H5L2, IgG1-016-H5L2-E430G, IgG1-016-H5L2-F405L-E430G and IgG1-016-H5L2-LC90S-F405L-E430G was determined in vitro. Data shown are % lysis determined by measurement of the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry.
FIGS. 12A-12E: CDC mediated by bispecific CD37 antibodies with an Fc-Fc interaction enhancing mutation, (combinations of) CD37 antibodies with an Fc-Fc interaction enhancing mutation, and monovalent CD37-binding antibodies with an Fc-Fc interaction enhancing mutation on Daudi cells; and CDC activity of CD37 antibody variants with an Fc-Fc interaction enhancing mutation, and combinations thereof, on OCI-Ly-7 cells.
(FIG. 12A) The capacity to induce CDC on Daudi cells of bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G, IgG1-005-H1L2-E430G, IgG1-016-H5L2-E430G, a combination of IgG1-005-H1L2-K409R-E430G plus IgG1-016-H5L2-F405L-E430G, bsIgG1-b12-F405L-E430Gx005-H1L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G, and (FIG. 12B) bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, a combination of IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G, bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G was determined in vitro. Data shown are % lysis determined by measurement of the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry. (FIG. 12C) The capacity to induce OCI-Ly-7 cells of the CD37 bispecific antibody bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, the CD37 monospecific bivalent (monoclonal) antibodies IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, a combination of IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G, the monovalent CD37 antibodies bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G, bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G and a combination of bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G plus bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G was determined in vitro. Data shown are % lysis determined by measurement of the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry. (FIG. 12D) EC50 values of CDC induction by bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G plus bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G, as determined in 2 independent experiments. (FIG. 12E) EC50 values of CDC induction by bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G, as determined in 3 independent experiments.
FIGS. 13A and 13B: CDC mediated by bispecific CD37 antibodies and by bispecific CD37 antibodies with an Fc-Fc interaction enhancing mutation on Daudi cells. The capacity to induce CDC on Daudi cells of (FIG. 13A) bsIgG1-016-H5L2-F405Lx005-H1L2-K409R and bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G, and (FIG. 13B) bsIgG1-016-H5L2-F405Lx010-H5L2-K409R and bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G was determined in vitro. Data shown are % lysis determined by measurement of the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry.
FIGS. 14A and 14B: CDC mediated by bispecific CD37 antibodies with an Fc-Fc interaction enhancing mutation, (combinations of) CD37 antibodies with an Fc-Fc interaction enhancing mutation, and monovalent binding CD37 antibodies with an Fc-Fc interaction enhancing mutation on primary CLL tumor cells. The capacity to induce CDC on primary CLL tumor cells (Patient: VM-BM0091 Newly Diagnosed/Untreated (BM=bone marrow derived)) of (FIG. 14A) bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G, IgG1-005-H1L2-K409R-E430G, IgG1-016-H5L2-F405L-E430G, a combination of IgG1-005-H1L2-K409R-E430G plus IgG1-016-H5L2-F405L-E430G, bsIgG1-b12-F405L-E430Gx005-H1L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G, and (FIG. 14B) bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, a combination of IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G, bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G was determined in vitro. Data shown are % lysis determined by measurement of the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry.
FIG. 15: CDC mediated by a bispecific CD37 antibody with an Fc-Fc interaction enhancing mutation on B cell lymphoma cell lines. The capacity of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, at a concentration of 10 μg/mL, to induce CDC on a range of B cell lymphoma cell lines was determined in vitro. Expression levels of CD37 were determined by quantitative flow cytometry, and are shown as molecules/cell, average±SD of 2 experiments. White bars indicate susceptible to CDC (>10% lysis, average of 2 experiments) mediated by bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, black bars indicate unsusceptible to CDC (<10% lysis, average of 2 experiments) mediated by bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G.
FIGS. 16A-16C: ADCC mediated by bispecific CD37 antibodies with an Fc-Fc interaction enhancing mutation, (combinations of) CD37 antibodies with an Fc-Fc interaction enhancing mutation, and monovalent binding CD37 antibodies with an Fc-Fc interaction enhancing mutation on Daudi and Raji cells. The capacity to induce ADCC of (FIG. 16A) bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G, IgG1-005-H1L2-K409R-E430G, IgG1-016-H5L2-F405L-E430G and a combination of IgG1-005-H1L2-K409R-E430G plus IgG1-016-H5L2-F405L-E430G on Daudi cells, and (FIG. 16B) bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G and a combination of IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G on Daudi cells, and (FIG. 16C) bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, a combination of IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G, bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G on Raji cells was determined in vitro using a chromium release assay. Data shown are % specific lysis; error bars indicate variation within the assay, with 5 replicates (FIG. 16A, FIG. 16B) or 6 replicates (FIG. 16C) per data point.
FIGS. 17A-17D: Quantitative determination of CD37, CD46, CD55 and CD59 expression levels on (FIG. 17A) CLL, (FIG. 17B) FL, (FIG. 17C) MCL or (FIG. 17D) DLBCL tumor cells. Expression levels on tumor cells were determined by flow cytometry. Antigen quantity is shown as antibody binding capacity.
FIGS. 18A-18C: CDC mediated by a bispecific CD37 antibody with an Fc-Fc interaction enhancing mutation on primary tumor cells of patients with CLL, FL, MCL, DLBCL or B-NHL (not further specified). The capacity of bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G to induce CDC on tumor cells derived from patients with (FIG. 18A) CLL, (FIG. 18B) FL and (FIG. 18C) MCL, DLBCL or B-NHL (not further specified) was determined by flow cytometry. CDC induction is presented as the percentage lysis determined by the fraction of 7-AAD positive tumor cells, using 100 μg/mL (FIGS. 18A and 18B) or 10 μg/mL (FIG. 18C) of bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G.
FIGS. 19A and 19B: Binding of a bispecific CD37 antibody with an Fc-Fc interaction enhancing mutation to B cells in human or cynomolgus monkey blood. Binding of Alexa-488 labeled bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to B cells in (FIG. 19A) human or (FIG. 19B) cynomolgus monkey blood was determined by flow cytometry. Alexa-488 labeled IgG1-b12 was used as a negative control antibody. Data are shown as geometric mean A488 fluorescence intensity values, for one representative donor/animal. Error bars show variation within the experiment (duplicate measurements).
FIGS. 20A and 20B: Cytotoxicity of a bispecific CD37 antibody with an Fc-Fc interaction enhancing mutation and an FcγR-interaction enhanced monoclonal CD37 specific antibody to B cells in human or cynomolgus monkey blood. (FIG. 20A) Cytotoxicity of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-CD37-B2-S239D-1332E to B cells in human blood and (FIG. 20B) of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to B cells in cynomolgus monkey blood was determined in a whole blood cytotoxicity assay. IgG1-b12 was used as a negative control antibody. Data are shown as % B cell depletion for one representative donor/animal. Error bars show variation within the experiment (duplicate measurements).
FIGS. 21A-21D: CDC mediated by a bispecific CD37 antibody with an Fc-Fc interaction enhancing mutation, a CD20-specific antibody or a combination thereof. (FIGS. 21A-21D) The capacity to induce CDC on tumor cells derived from 2 CLL patients of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, ofatumumab or a combination thereof, at indicated concentrations, was determined ex vivo. Data are shown as the % of viable B cells.
FIGS. 22A and 22B: Dose-effect relationship for 3 weekly doses of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G in the JVM-3 model. (FIG. 22A) Tumor growth of JVM-3 xenografts after treatment with different doses of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or isotype control antibody (IgG1-b12). Mean and SEM of each group (n=10) is shown per time point. (FIG. 22B) Tumor size per mouse at day 25. Mean and SEM are indicated per treatment group. Differences were analyzed by Mann Whitney test. Statistically significant differences were indicated as follows: **: p<0.01; ***: p<0.001.
FIGS. 23A and 23B: Dose-effect relationship for 3 weekly doses of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G in the Daudi-luc model. (FIG. 23A) Tumor growth (measured by luciferase activity, bioluminescence) of Daudi-luc xenografts after treatment with different doses of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or isotype control antibody (IgG1-b12). Mean and SEM of each group (n=9) is shown per time point. (FIG. 23B) Luciferase activity per mouse at day 36. Mean and SEM are indicated per treatment group. Differences were analyzed by One Way Anova, Uncorrected Fisher's LSD. Statistically significant differences were indicated as follows: **: p<0.01; ***: p<0.001.
FIGS. 24A-24D: Plasma concentrations of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-b12 following intravenous injection in SCID mice.
SCID mice were injected with a single i.v. dose of (FIGS. 24A and 24B) 100 μg (5 mg/kg) or (FIGS. 24C and 24D) 500 μg (25 mg/kg) of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or IgG1-b12.
FIG. 25. Analysis of binding of CD37 antibodies to CD37 variants with alanine mutations in the extracellular domains. Zscore (fold change) was defined as (normalized gMFI[aa position]−μ)/σ, where μ and σ are the mean and standard deviation (SD) of the normalized gMFI of all mutants. Residues where the where the zscore was lower than −1.5 (indicated by the dotted line) were considered ‘loss of binding mutants’. Number above the x-axis refer to amino acid positions. Note that x-axis is non-continuous: the left part (until the striped line) of the axis represents aa residues in the small extracellular loop of human CD37 which are not alanines or cysteines; the right part of the axis represents aa residues in the large extracellular loop of human CD37 which are not alanines or cysteines. The dotted line indicates a zscore (fold change) of −1.5.
FIGS. 26A-26F CDC mediated by mixtures of CD37 antibodies with an Fc-Fc interaction enhancing mutation plus clinically established CD20 antibody products on Raji cells. CDC-mediated killing of Raji cells (% lysis expressed as the PI-positive cell fraction as determined by flow cytometry) for antibody concentration dilution series of 1:0, 3:1, 1:1, 3:1 and 0:1 antibody mixtures (10 μg/mL final concentration) of CD37 antibodies with an Fc-Fc interaction enhancing mutation plus standard of care CD20 antibody products MabThera (rituximab), Arzerra (ofatumumab) and Gazyva (obinutuzumab, GA101): (FIG. 26A) mixtures with IgG1-37.3-E430G, (FIG. 26B) mixtures with IgG1-G28.1-E430G, (FIG. 26C) mixtures with IgG1-004-E430G, (FIG. 26D) mixtures with IgG1-005-E430G, (FIG. 26E) mixtures with IgG1-010-E430G and (FIG. 26F) mixtures with IgG1-016-E430G.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “CD37”, as used herein, refers to Leukocyte Antigen CD37, also known as GP52-40, tetraspanin-26, and TSPAN26, which is a heavily glycosylated transmembrane protein with four transmembrane domains (TMs) and one small and one large extracellular domain. Homo sapiens, i.e., human, CD37 protein is encoded by a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 62 (human CD37 protein: UniprotKB/Swissprot P11049). In this amino acid sequence, residues 112 to 241 correspond to the large extracellular domain, residues 39 to 59 to the small extracellular domain, while the remaining residues correspond to transmembrane and cytoplasmic domains. Macaca fascicularis, i.e., cynomolgus monkey, CD37 protein is encoded by a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 63 (cynomolgus CD37 protein: Genbank accession no. XP_005589942). Unless contradicted by context the term “CD37” means “human CD37”. The term “CD37” includes any variants, isoforms and species homologs of CD37 which are naturally expressed by cells, including tumor cells, or are expressed on cells transfected with the CD37 gene or cDNA.
The term “human CD20” or “CD20” refers to human CD20 (UniProtKB/Swiss-Prot No P11836) and includes any variants, isoforms and species homologs of CD20 which are naturally expressed by cells, including tumor cells, or are expressed on cells transfected with the CD20 gene or cDNA. Species homologs include rhesus monkey CD20 (Macaca mulatta; UniProtKB/Swiss-Prot No H9YXP1) and cynomolgus monkey CD20 (Macaca fascicularis).
The term “antibody binding CD37”, “anti-CD37 antibody”, “CD37-binding antibody”, “CD37-specific antibody”, “CD37 antibody” which may be used interchangeably herein, refers to any antibody binding an epitope on the extracellular part of CD37.
The term “antibody” (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q, the first component in the classical pathway of complement activation. As indicated above, the term antibody herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that are antigen-binding fragments, i.e., retain the ability to specifically bind to the antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term “antibody” include (i) a Fab′ or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or a monovalent antibody as described in WO2007059782 (Genmab); (ii) F(ab′)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CH1 domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends Biotechnol. 2003 November; 21(11):484-90); (vi) camelid or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 January; 5(1):111-24) and (vii) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present invention, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present invention, as well as bispecific formats of such fragments, are discussed further herein. For the bispecific antibodies of the invention such fragments are linked to an Fc domain. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. An antibody as generated can possess any isotype.
The term “bispecific antibody” refers to antibody having specificities for at least two different, typically non-overlapping, epitopes. Such epitopes may be on the same or different targets. For the present invention the epitopes are on the same target, namely CD37. Examples of different classes of bispecific antibodies comprising an Fc region include but are not limited to: asymmetric bispecific molecules, e.g., IgG-like molecules with complementary CH3 domains; and symmetric bispecific molecules, e.g., recombinant IgG-like dual targeting molecules wherein each antigen-binding region of the molecule binds at least two different epitopes.
Examples of bispecific molecules include but are not limited to Triomab® (Trion Pharma/Fresenius Biotech, WO/2002/020039), Knobs-into-Holes (Genentech, WO 1998/50431), CrossMAbs (Roche, WO 2009/080251, WO 2009/080252, WO 2009/080253), electrostatically-matched Fc-heterodimeric molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO 2010/129304), LUZ-Y (Genentech), DIG-body, PIG-body and TIG-body (Pharmabcine), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono, WO2007110205), Bispecific IgG1 and IgG2 (Pfizer/Rinat, WO 2011/143545), Azymetric scaffold (Zymeworks/Merck, WO2012058768), mAb-Fv (Xencor, WO 2011/028952), XmAb (Xencor), Bivalent bispecific antibodies (Roche, WO 2009/080254), Bispecific IgG (Eli Lilly), DuoBody® molecules (Genmab A/S, WO 2011/131746), DuetMab (Medimmune, US2014/0348839), Biclonics (Merus, WO 2013/157953), NovImmune (KABodies, WO 2012/023053), FcAAdp (Regeneron, WO 2010/151792), (DT)-Ig (GSK/Domantis), Two-in-one Antibody or Dual Action Fabs (Genentech, Adimab), mAb2 (F-Star, WO 2008/003116), Zybody™ molecules (Zyngenia), CovX-body (CovX/Pfizer), FynomAbs (Covagen/Janssen Cilag), DutaMab (Dutalys/Roche), iMab (MedImmune), Dual Variable Domain (DVD)-Ig™ (Abbott), dual domain double head antibodies (Unilever; Sanofi Aventis, WO 2010/0226923), Ts2Ab (MedImmune/AZ), BsAb (Zymogenetics), HERCULES (Biogen Idec, U.S. Pat. No. 7,951,918), scFv-fusions (Genentech/Roche, Novartis, Immunomedics, Changzhou Adam Biotech Inc, CN 102250246), TvAb (Roche, WO2012/025525, WO2012/025530), ScFv/Fc Fusions, SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS), Interceptor (Emergent), Dual Affinity Retargeting Technology (Fc-DART™) (MacroGenics, WO2008/157379, WO2010/080538), BEAT (Glenmark), Di-Diabody (Imclone/Eli Lilly) and chemically crosslinked mAbs (Karmanos Cancer Center), and covalently fused mAbs (AIMM therapeutics).
The term “full-length antibody”, as used herein, refers to an antibody (e.g., a parent or variant antibody) which contains all heavy and light chain constant and variable domains corresponding to those that are normally found in a wild-type antibody of that class or isotype.
The term “chimeric antibody” as used herein, refers to an antibody wherein the variable region is derived from a non-human species (e.g. derived from rodents) and the constant region is derived from a different species, such as human. Chimeric antibodies may be generated by antibody engineering. “Antibody engineering” is a term used generic for different kinds of modifications of antibodies, and which is a well-known process for the skilled person. In particular, a chimeric antibody may be generated by using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Thus, the chimeric antibody may be a genetically or an enzymatically engineered recombinant antibody. It is within the knowledge of the skilled person to generate a chimeric antibody, and thus, generation of the chimeric antibody according to the present invention may be performed by other methods than described herein. Chimeric monoclonal antibodies for therapeutic applications are developed to reduce antibody immunogenicity. They may typically contain non-human (e.g. murine) variable regions, which are specific for the antigen of interest, and human constant antibody heavy and light chain domains. The terms “variable region” or “variable domains” as used in the context of chimeric antibodies, refers to a region which comprises the CDRs and framework regions of both the heavy and light chains of the immunoglobulin.
The term “oligomer”, as used herein, refers to a molecule that consists of more than one but a limited number of monomer units (e.g. antibodies) in contrast to a polymer that, at least in principle, consists of an unlimited number of monomers. Exemplary oligomers are dimers, trimers, tetramers, pentamers and hexamers. Likewise, “oligomerization” such as e.g. “hexamerization”, as used herein, means that there is an increase in the distribution of antibodies and/or other dimeric proteins comprising target-binding regions according to the invention into oligomers, such as hexamers. The increased formation of oligomers such as hexamers is due to increased Fc-Fc interaction after binding to membrane-bound targets.
The term “antigen-binding region”, “antigen binding region”, “binding region” or antigen binding domain, as used herein, refers to a region of an antibody which is capable of binding to the antigen. This binding region is typically defined by the VH and VL domains of the antibody which may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). The antigen can be any molecule, such as a polypeptide, e.g. present on a cell, bacterium, or virion or in solution. The terms “antigen” and “target” may, unless contradicted by the context, be used interchangeably in the context of the present invention.
The term “target”, as used herein, refers to a molecule to which the antigen binding region of the antibody binds. The target includes any antigen towards which the raised antibody is directed. The term “antigen” and “target” may in relation to an antibody be used interchangeably and constitute the same meaning and purpose with respect to any aspect or embodiment of the present invention.
The term “humanized antibody” as used herein, refers to a genetically engineered non-human antibody, which contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the six non-human antibody complementarity-determining regions (CDRs), which together form the antigen binding site, onto a homologous human acceptor framework region (FR) (see WO92/22653 and EP0629240). In order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e. the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modeling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back-mutations, may be applied to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties.
Humanized antibodies can be generated using immunized rabbits, humanization of rabbit antibodies using germline humanization (CDR-grafting) technology, and, if necessary, by back-mutating residues which may be critical for the antibody binding properties, as identified in structural modeling, to rabbit residues. Screening for potential T cell epitopes can be applied.
The term “human antibody” as used herein, refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Human monoclonal antibodies of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed, e.g., viral or oncogenic transformation of B-lymphocytes or phage display techniques using libraries of human antibody genes.
A suitable animal system for preparing hybridomas that secrete human monoclonal antibodies is the murine system. Hybridoma production in the mouse is a very well established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.
Human monoclonal antibodies can be generated using e.g. transgenic or transchromosomal mice or rabbits carrying parts of the human immune system rather than the mouse or rabbit system.
The term “immunoglobulin” refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. See for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain constant region (abbreviated herein as CH or CH). The heavy chain constant region typically is comprised of three domains, CH1, CH2, and CH3. Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL or VL) and a light chain constant region (abbreviated herein as CL or CL). The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise stated or contradicted by context, CDR sequences herein are identified according to IMGT rules (Brochet X., Nucl Acids Res. 2008; 36:W503-508 and Lefranc M P., Nucleic Acids Research 1999; 27:209-212; see also internet http address http://www.imgt.org/). Unless otherwise stated or contradicted by context, reference to amino acid positions in the constant regions in the present invention is according to the EU-numbering (Edelman et al., Proc Natl Acad Sci USA. 1969 May; 63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242).
When used herein, unless contradicted by context, the term “Fab-arm” or “arm” refers to one heavy chain-light chain pair and is used interchangeably with “half molecules” herein. Accordingly, a “Fab-arm” comprises the variable regions of the heavy chain and light chain as well as the constant region of the light chain and the constant region of the heavy chain which comprises the CH1 region, the hinge, the CH2 region and the CH3 region of an immunoglobulin. The “CH1 region” refers e.g. to the region of a human IgG1 antibody corresponding to amino acids 118-215 according to the EU numbering. Thus, the Fab fragment comprises the binding region of an immunoglobulin.
The term “fragment crystallizable region”, “Fc region”, “Fc fragment” or “Fc domain”, which may be used interchangeably herein, refers to an antibody region comprising, arranged from amino-terminus to carboxy-terminus, at least a hinge region, a CH2 domain and a CH3 domain. An Fc region of an IgG1 antibody can, for example, be generated by digestion of an IgG1 antibody with papain. The Fc region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q, the first component in the classical pathway of complement activation. The term “hinge region”, as used herein, is intended to refer to the hinge region of an immunoglobulin heavy chain. Thus, for example the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to the EU numbering.
The term “core hinge” or “core hinge region” as used herein refers to the four amino acids corresponding to positions 226-229 of a human IgG1 antibody.
The term “CH2 region” or “CH2 domain”, as used herein, is intended to refer the CH2 region of an immunoglobulin heavy chain. Thus, for example the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the EU numbering. However, the CH2 region may also be any of the other isotypes or allotypes as described herein.
The term “CH3 region” or “CH3 domain” as used herein, is intended to refer to the CH3 region of an immunoglobulin heavy chain. Thus, for example the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering. However, the CH3 region may also be any of the other isotypes or allotypes as described herein.
As used herein, the term “isotype” refers to the immunoglobulin class (for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encoded by heavy chain constant region genes.
The term “monovalent antibody” means in the context of the present invention that an antibody molecule is capable of binding a single molecule of the antigen, and thus is not capable of antigen crosslinking.
A “CD37 antibody” or “anti-CD37 antibody” is an antibody as described above, which binds specifically to the antigen CD37.
A “CD37×CD37 antibody” or “anti-CD37×CD37 antibody” is a bispecific antibody, which comprises two different antigen-binding regions, one of which binds specifically to a first epitope on the antigen CD37 and a second which binds specifically to a different epitope on CD37.
In an embodiment, the bispecific antibody of the invention is isolated. An “isolated bispecific antibody,” as used herein, is intended to refer to a bispecific antibody which is substantially free of other antibodies having different antigenic specificities (for instance an isolated bispecific antibody that specifically binds to CD37 is substantially free of monospecific antibodies that specifically bind to CD37).
The term “epitope” means a protein determinant capable of binding to an antigen-binding region of an antibody (“paratope”). Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. Epitope mapping techniques can determine “structural epitopes” or “functional epitopes”. Structural epitopes are defined as those residues within a structure that are in direct contact with the antibody and can for example be assessed by structure based methods such as X-ray crystallography. A structural epitope may comprise amino acid residues directly involved in the binding of an antibody as well as other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked or covered by antibody (in other words, the amino acid residue is within the footprint of the antibody). Functional epitope are defined as those residues that make energetic contributions to the antigen-antibody binding interaction and can for example be assessed by site-directed mutagenesis such as alanine scanning (Cunningham, B. C., & Wells, J. A. (1993) Journal of Molecular Biology; Clackson, T., & Wells, J. (1995) Science, 267(5196), 383-386). A functional epitope may comprise amino acid residues directly involved in the binding of an antibody as well as other amino acid residues which are not directly involved in the binding, such as amino acid residues which cause conformational changes to the location of residues involved in direct interactions (Greenspan, N. S., & Di Cera, E. (1999) Nature Biotechnology, 17(10), 936-937). In case of antibody-antigen interactions, the functional epitope may be used to distinguish antibody molecules between each other. A functional epitope may be determined by use of the method of alanine scanning as described in Example 17. Thus, amino acids in the protein may be substituted with alanines thereby generating a series of mutant proteins, binding of the antigen-binding region of the antibody to the mutant protein is reduced as compared to a wild type protein; reduced binding being determined as standardized log(fold change) (expressed as z-scores) in binding of said antibody being less than −1.5 as set forth in Example 17.
The term “monoclonal antibody” as used herein refers to a preparation of antibody molecules essentially of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term “human monoclonal antibody” refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The human monoclonal antibodies may be generated by a hybridoma which includes a B cell obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, fused to an immortalized cell.
As used herein, the term “binding” in the context of the binding of an antibody to a predetermined antigen typically is a binding with an affinity corresponding to a KD of about 10−6 M or less, e.g. 10−7 M or less, such as about 10−8 M or less, such as about 10−9 M or less, about 10−10 M or less, or about 10−11 M or even less when determined by for instance BioLayer Interferometry (BLI) technology in a Octet HTX instrument using the antibody as the ligand and the antigen as the analyte, and wherein the antibody binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its KD of binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. The amount with which the KD of binding is lower is dependent on the KD of the antibody, so that when the KD of the antibody is very low, then the amount with which the KD of binding to the antigen is lower than the KD of binding to a non-specific antigen may be at least 10,000-fold (that is, the antibody is highly specific).
The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.
“Affinity”, as used herein, and “KD” are inversely related, that is, higher affinity is intended to refer to lower KD, and lower affinity is intended to refer to higher KD.
As used herein, an antibody which “competes” or “cross-competes” is used interchangeably with an antibody which “blocks” or “cross-blocks” with another antibody, i.e. a reference antibody, and means that the antibody and the reference antibody compete for binding to human CD37, e.g. as determined in the assay described in Examples 7 herein. In one embodiment the antibody binds with less than 50%, such as less than 20%, such as less than 15% of its maximum binding in the presence of the competing reference antibody.
As used herein, an antibody which “does not compete” or “does not cross-compete” or “does not block” with another antibody, i.e. a reference antibody, means that the antibody and the reference antibody do not compete for binding to human CD37, e.g. as determined in the assay described in Examples 7 herein. For some pairs of antibody and reference antibody, non-competition in the assay of Example 7 is only observed when one antibody is bound to an antigen on a cell and the other is used to compete, and not vice versa. The term “does not compete with” or “non-competition” or “non-blocking” when used herein is also intended to cover such combinations of antibodies. In one embodiment the antibody binds with at least 75%, such as least 80%, such as at least 85% of its maximum binding in the presence of the reference antibody.
The term “Fc-Fc interaction enhancing mutation”, as used herein, refers to a mutation in IgG antibodies that strengthens Fc-Fc interactions between neighboring IgG antibodies that are bound to a cell surface target. This may result in enhanced oligomer formation such as e.g. hexamerization of the target-bound antibodies, while the antibody molecules remain monomeric in solution as described in WO 2013/004842; WO 2014/108198 both which are hereby incorporated by reference.
The term “Fc effector functions” or “Fc-mediated effector functions” as used herein, is intended to refer to functions that are a consequence of binding a polypeptide or antibody to its target, such as an antigen, on a cell membrane, and subsequent interaction of the IgG Fc domain with molecules of the innate immune system (e.g. soluble molecules or membrane-bound molecules). Examples of Fc effector functions include (i) C1q-binding, (ii) complement activation, (iii) complement-dependent cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (ADCC), (v) Fc-gamma receptor-binding, (vi) antibody-dependent cellular phagocytosis (ADCP), (vii) complement-dependent cellular cytotoxicity (CDCC), (viii) complement-enhanced cytotoxicity, (ix) binding to complement receptor of an opsonized antibody mediated by the antibody, (x) opsonisation, and (xi) a combination of any of (i) to (x).
When used herein the term “heterodimeric interaction between the first and second CH3 regions” refers to the interaction between the first CH3 region of the first Fc-region and the second CH3 region of the second Fc-region in a first-CH3/second-CH3 heterodimeric protein. A bispecific antibody is an example of a heterodimeric protein.
When used herein the term “homodimeric interactions of the first and second CH3 regions” refers to the interaction between a first CH3 region and another first CH3 region in a first-CH3/first-CH3 homodimeric protein and the interaction between a second CH3 region and another second CH3 region in a second-CH3/second-CH3 homodimeric protein. A monoclonal antibody is an example of a homodimeric protein.
The term “reducing conditions” or “reducing environment” refers to a condition or an environment in which a substrate, such as e.g. a cysteine residue in the hinge region of an antibody, is more likely to become reduced than oxidized.
The present invention also provides bispecific antibodies comprising functional variants of the VL regions, VH regions, or one or more CDRs of the bispecific antibodies of the examples. A functional variant of a VL, VH, or CDR used in the context of a bispecific antibody still allows each arm of the bispecific antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity and/or the specificity/selectivity of the parent bispecific antibody and in some cases such a bispecific antibody may be associated with greater affinity, selectivity and/or specificity than the parent bispecific antibody. Such functional variants typically retain significant sequence identity to the parent bispecific antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences may e.g. be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970) algorithm.
Exemplary variants include those which differ from VH and/or VL and/or CDR regions of the parent bispecific antibody sequences mainly by conservative substitutions; for instance 10, such as 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements. Preferably, a variant contains at most 10 amino acid substitutions in the VH and/or VL region of the parent antibody, such as at most 9, 8, 7, 6, 5, 4, 3, 2 or at most 1 amino acid substitution. Preferably such substitutions are conservative substitutions especially so if the substitutions are in a CDR sequence.
In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected in the following table:
Amino Acid Residue Classes for Conservative Substitutions
Acidic Residues
Asp (D) and Glu (E)
Basic Residues
Lys (K), Arg (R), and His (H)
Hydrophilic Uncharged Residues
Ser (S), Thr (T), Asn (N), and
Gln (Q)
Aliphatic Uncharged Residues
Gly (G), Ala (A), Val (V), Leu (L),
and Ile (I)
Non-polar Uncharged Residues
Cys (C), Met (M), and Pro (P)
Aromatic Residues
Phe (F), Tyr (Y), and Trp (W)
In the context of the present invention, the following notations are, unless otherwise indicated, used to describe a mutation; i) substitution of an amino acid in a given position is written as e.g. K409R which means a substitution of a Lysine in position 409 with an Arginine; and ii) for specific variants the specific three or one letter codes are used, including the codes Xaa and X to indicate any amino acid residue. Thus, the substitution of Lysine with Arginine in position 409 is designated as: K409R, and the substitution of Lysine with any amino acid residue in position 409 is designated as K409X. In case of deletion of Lysine in position 409 it is indicated by K409*.
The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which an expression vector has been introduced, e.g. an expression vector encoding an antibody of the invention. Recombinant host cells include, for example, transfectomas, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NS0 cells, and lymphocytic cells.
The term “treatment” refers to the administration of an effective amount of a therapeutically active bispecific antibody of the present invention with the purpose of easing, ameliorating, arresting or eradicating (curing) symptoms or disease states.
The term “effective amount” or “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of a bispecific antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the bispecific antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.
The term “anti-idiotypic antibody” refers to an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
EMBODIMENTS OF THE INVENTION
In a first main embodiment the invention provides a bispecific antibody comprising a first and second antigen binding region binding to human CD37 having the sequence of SEQ ID NO: 62, and a first and second Fc region of a human immunoglobulin, wherein the first and second antigen binding regions bind different epitopes on CD37 and wherein the first and second Fc regions comprises one or more amino acid mutations which mutation(s) enhances the Fc-Fc interaction between the bispecific antibodies upon binding to membrane-bound targets compared to the Fc-Fc interaction between bispecific antibodies not having said mutation(s). Hereby a bispecific anti-CD37 antibody is provided which binds two different epitopes on CD37. Preferably the two epitopes are such that both binding arms can bind the same protein and thus such that each binding arm does not block binding of the other arm and/or does not compete for binding with the other binding arm of the bispecific molecule. Also, the bispecific antibody comprises a mutation that enhances the Fc-Fc interaction between two or more of the bispecific antibodies of the invention. This has the effect that the bispecific molecules form oligomers upon binding to CD37 expressed on the plasma membrane of the target cell. The Fc-Fc interaction is enhanced compared to a molecule that is identical except for the mutation. Preferably the mutation is in the Fc region of the bispecific molecule. In one embodiment it is a single amino acid substitution in the Fc region of the bispecific molecule. It is preferably a symmetric substitution meaning that both half molecules (parental antibodies) have the mutation. It is a further advantage of the present bispecific antibody that it has enhanced CDC and/or ADCC effector functions compared to an identical bispecific molecule not having the Fc-Fc interaction enhancing mutation. Surprisingly the bispecific molecule also has improved CDC and/or ADCC compared to a combination of the two parental monoclonal anti-CD37 antibodies which are mutated to have enhanced Fc-Fc interactions, and improved CDC and/or ADCC compared to either parental monoclonal anti-CD37 antibody which is mutated to have enhanced Fc-Fc interactions by itself. Thus, the bispecific antibody of the invention is more potent in inducing CDC and/or ADCC than a combination of an antibody having the first antigen binding region and a second antibody having the second antigen binding region and where both antibodies comprise the Fc-Fc interaction enhancing mutation, or compared to the single monoclonal anti-CD37 antibodies having the first or the second antigen binding regions and which comprise the Fc-Fc interaction enhancing mutation.
In an embodiment of the invention the first antigen binding region of the bispecific antibody is obtained from an antibody which competes for binding to human CD37 with a CD37 antibody comprising the CDR sequences:
VH CDR1 sequence set forth in SEQ ID NO: 16,
VH CDR2 sequence set forth in SEQ ID NO: 17,
VH CDR3 sequence set forth in SEQ ID NO: 18,
VL CDR1 sequence set forth in SEQ ID NO: 20,
VL CDR2 sequence KAS, and
VL CDR3 sequence set forth in SEQ ID NO: 21. [010]
Preferably competition for binding is determined according to example 7.
In another embodiment the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as a CD37 antibody comprising the CDR sequences:
VH CDR1 sequence set forth in SEQ ID NO: 16,
VH CDR2 sequence set forth in SEQ ID NO: 17,
VH CDR3 sequence set forth in SEQ ID NO: 18,
VL CDR1 sequence set forth in SEQ ID NO: 20,
VL CDR2 sequence KAS, and
VL CDR3 sequence set forth in SEQ ID NO: 21. [010]
In a further embodiment of the invention the first antigen binding region of the bispecific antibody comprises the CDR sequences:
VH CDR1 sequence set forth in SEQ ID NO: 16,
VH CDR2 sequence set forth in SEQ ID NO: 17,
VH CDR3 sequence set forth in SEQ ID NO: 18,
VL CDR1 sequence set forth in SEQ ID NO: 20,
VL CDR2 sequence KAS, and
VL CDR3 sequence set forth in SEQ ID NO: 21. [010]
In a further embodiment of the invention the first antigen binding region of the bispecific antibody of the invention comprise the VH and VL sequences:
(i) VH sequence set forth in SEQ ID NO: 15 and VL sequence set forth in SEQ ID NO: 19 or
(ii) VH sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity and a VL sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity with the VH sequence and VL sequences of SEQ ID Nos 15 and 19.
In a further embodiment of the invention the first antigen binding region of the bispecific antibody is obtained from an antibody which competes for binding to human CD37 with a CD37 antibody comprising the CDR sequences:
VH CDR1 sequence set forth in SEQ ID NO: 9,
VH CDR2 sequence set forth in SEQ ID NO: 10,
VH CDR3 sequence set forth in SEQ ID NO: 11,
VL CDR1 sequence set forth in SEQ ID NO: 13,
VL CDR2 sequence: AAS, and
VL CDR3 sequence set forth in SEQ ID NO: 14. [005]
In a further embodiment of the invention the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as a CD37 antibody comprising the CDR sequences:
VH CDR1 sequence set forth in SEQ ID NO: 9,
VH CDR2 sequence set forth in SEQ ID NO: 10,
VH CDR3 sequence set forth in SEQ ID NO: 11,
VL CDR1 sequence set forth in SEQ ID NO: 13,
VL CDR2 sequence: AAS, and
VL CDR3 sequence set forth in SEQ ID NO: 14. [005]
In one embodiment of the invention the first antigen binding region of the bispecific antibody has a functional epitope comprising one or more of the amino acids Y182, D189, T191, I192, D194, K195, V196, I197 and P199 of SEQ ID No: 62 (CD37).
In one embodiment of the invention said first antigen binding region binds to a functional epitope comprising one or more of the amino acids selected from the group consisting of: Y182, D189, T191, I192, D194, K195, V196, I197 and P199 of SEQ ID No: 62 (CD37).
In one embodiment of the invention the first antigen binding region of the bispecific antibody binds to a functional epitope on CD37, wherein binding to a mutant CD37 in which any one or more of the amino acid residues at positions corresponding to positions Y182, D189, T191, I192, D194, K195, V196, I197 and P199 of SEQ ID no 62 (CD37). has/have been substituted with alanines, is reduced as compared to wild type CD37 having the amino acid sequence set forth in SEQ ID NO: 62; reduced binding being determined as zscore (fold change) in binding of said antibody being lowed that −1.5, wherein zscore (fold change) in binding is calculated as set forth in Example 17.
In a further embodiment of the invention the first antigen binding region of the bispecific antibody comprises the CDR sequences:
VH CDR1 sequence set forth in SEQ ID NO: 9,
VH CDR2 sequence set forth in SEQ ID NO: 10,
VH CDR3 sequence set forth in SEQ ID NO: 11,
VL CDR1 sequence set forth in SEQ ID NO: 13,
VL CDR2 sequence: AAS, and
VL CDR3 sequence set forth in SEQ ID NO: 14. [005]
In a further embodiment of the invention the first antigen binding region of the bispecific antibody comprise the VH and VL sequences:
(i) VH sequence set forth in SEQ ID NO: 8 and VL sequence set forth in SEQ ID NO: 12 or
(ii) VH sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity and a VL sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity with the VH sequence and VL sequences of SEQ ID Nos 8 and 12.
In a further embodiment of the invention the second antigen binding region of the bispecific antibody is obtained from an antibody which competes for binding to human CD37 with a CD37 antibody comprising the CDR sequences selected from the group comprising:
(i) VH CDR1 sequence set forth in SEQ ID NO: 23,
VH CDR2 sequence set forth in SEQ ID NO: 24,
VH CDR3 sequence set forth in SEQ ID NO: 25,
VL CDR1 sequence set forth in SEQ ID NO: 27,
VL CDR2 sequence: YAS, and
VL CDR3 sequence set forth in SEQ ID NO: 28; [016]
(ii) VH CDR1 sequence set forth in SEQ ID NO: 2,
VH CDR2 sequence set forth in SEQ ID NO: 3,
VH CDR3 sequence set forth in SEQ ID NO: 4,
VL CDR1 sequence set forth in SEQ ID NO: 6,
VL CDR2 sequence: EAS, and
VL CDR3 sequence set forth in SEQ ID NO: 7; [004]
(iii) VH CDR1 sequence set forth in SEQ ID NO: 40,
VH CDR2 sequence set forth in SEQ ID NO: 41,
VH CDR3 sequence set forth in SEQ ID NO: 42,
VL CDR1 sequence set forth in SEQ ID NO: 44,
VL CDR2 sequence: FAK, and
VL CDR3 sequence set forth in SEQ ID NO: 45; [G28.1] and
(iv) VH CDR1 sequence set forth in SEQ ID NO: 47,
VH CDR2 sequence set forth in SEQ ID NO: 48,
VH CDR3 sequence set forth in SEQ ID NO: 49,
VL CDR1 sequence set forth in SEQ ID NO: 51,
VL CDR2 sequence: VAT and
VL CDR3 sequence set forth in SEQ ID NO: 52. [37.3]
In a further embodiment of the invention the second antigen binding region of the bispecific antibody is obtained from an antibody which competes for binding to human CD37 with a CD37 antibody comprising the CDR sequences selected from the group consisting of:
(i) VH CDR1 sequence set forth in SEQ ID NO: 23,
VH CDR2 sequence set forth in SEQ ID NO: 24,
VH CDR3 sequence set forth in SEQ ID NO: 25,
VL CDR1 sequence set forth in SEQ ID NO: 27,
VL CDR2 sequence: YAS, and
VL CDR3 sequence set forth in SEQ ID NO: 28; [016]
(ii) VH CDR1 sequence set forth in SEQ ID NO: 2,
VH CDR2 sequence set forth in SEQ ID NO: 3,
VH CDR3 sequence set forth in SEQ ID NO: 4,
VL CDR1 sequence set forth in SEQ ID NO: 6,
VL CDR2 sequence: EAS, and
VL CDR3 sequence set forth in SEQ ID NO: 7; [004]
(iii) VH CDR1 sequence set forth in SEQ ID NO: 40,
VH CDR2 sequence set forth in SEQ ID NO: 41,
VH CDR3 sequence set forth in SEQ ID NO: 42,
VL CDR1 sequence set forth in SEQ ID NO: 44,
VL CDR2 sequence: FAK, and
VL CDR3 sequence set forth in SEQ ID NO: 45; [G28.1] and
(iv) VH CDR1 sequence set forth in SEQ ID NO: 47,
VH CDR2 sequence set forth in SEQ ID NO: 48,
VH CDR3 sequence set forth in SEQ ID NO: 49,
VL CDR1 sequence set forth in SEQ ID NO: 51,
VL CDR2 sequence: VAT and
VL CDR3 sequence set forth in SEQ ID NO: 52. [37.3]
Hereby bispecific antibodies are provided wherein the first and second antigen binding regions bind different epitopes on human CD37. The inventors of the present invention have found that the antibodies having the CDR sequences of antibody 005 (SEQ ID NOs 9, 10, 11 and 13, 13a, 14) and antibody 010 (SEQ ID NOs 16, 17, 18 and 20, 20a, 21) compete for binding to human CD37 and do not compete for binding to CD37 with any of the antibodies having the CDR sequences of antibodies 016 (SEQ ID NOs 23, 24, 25 and 27, 27a, 28), 004 (SEQ ID NOs 2, 3, 4 and 6, 6a, 7), G28.1 (SEQ ID NOs 40, 41, 42 and 44, 44a, 45) and 37.3 (SEQ ID NOs 47, 48, 49 and 51, 51a, 52). The 016, 004, G28.1 and 37.3 antibodies have however been found to compete with each other for binding to human CD37. Thus, a bispecific antibody comprising a first binding arm which is obtained from an antibody which competes for binding with either of or both of the 005 or 010 antibodies and a second binding arm which is obtained from an antibody which competes for binding with any of 016, 004, G28.1 and 37.3 or with all of these is a bispecific antibody which has specificity for two different epitopes on CD37. The inventors have surprisingly found that such bispecific antibodies have favorable CDC potency on CD37 expressing cells compared to treating such cells with a combination of the two monoclonal antibodies which do not compete for binding to CD37. Additionally, the inventors have surprisingly found that such bispecific antibodies have favorable ADCC potency on CD37 expressing cells compared to treating such cells with a combination of the two monoclonal antibodies which do not compete for binding to CD37.
In one embodiment of the invention the bispecific antibody comprises a first antigen binding region which is obtained from an antibody which competes for binding to human CD37 with an antibody having the CDR sequences of antibody 010 and a second binding region which is obtained from an antibody which competes for binding to human CD37 with the antibody having the CDR sequences of 016.
In another embodiment of the invention the bispecific antibody comprises a first antigen binding region which is obtained from an antibody which competes for binding to human CD37 with an antibody having the CDR sequences of antibody 010 and a second binding region which is obtained from an antibody which competes for binding to human CD37 with the antibody having the CDR sequences of 004.
In another embodiment of the invention the bispecific antibody comprises a first antigen binding region which is obtained from an antibody which competes for binding to human CD37 with an antibody having the CDR sequences of antibody 010 and a second binding region which is obtained from an antibody which competes for binding to human CD37 with the antibody having the CDR sequences of G28.1.
In another embodiment of the invention the bispecific antibody comprises a first antigen binding region which is obtained from an antibody which competes for binding to human CD37 with an antibody having the CDR sequences of antibody 010 and a second binding region which is obtained from an antibody which competes for binding to human CD37 with the antibody having the CDR sequences of 37.3.
In one embodiment of the invention the bispecific antibody comprises a first antigen binding region which is obtained from an antibody which competes for binding to human CD37 with an antibody having the CDR sequences of antibody 005 and a second binding region which is obtained from an antibody which competes for binding to human CD37 with the antibody having the CDR sequences of 016.
In another embodiment of the invention the bispecific antibody comprises a first antigen binding region which is obtained from an antibody which competes for binding to human CD37 with an antibody having the CDR sequences of antibody 005 and a second binding region which is obtained from an antibody which competes for binding to human CD37 with the antibody having the CDR sequences of 004.
In another embodiment of the invention the bispecific antibody comprises a first antigen binding region which is obtained from an antibody which competes for binding to human CD37 with an antibody having the CDR sequences of antibody 005 and a second binding region which is obtained from an antibody which competes for binding to human CD37 with the antibody having the CDR sequences of G28.1.
In another embodiment of the invention the bispecific antibody comprises a first antigen binding region which is obtained from an antibody which competes for binding to human CD37 with an antibody having the CDR sequences of antibody 005 and a second binding region which is obtained from an antibody which competes for binding to human CD37 with the antibody having the CDR sequences of 37.3.
Such bispecific antibodies described here may in further embodiments comprise an Fc-Fc interaction enhancing substitution in both Fc regions (i.e. the Fc regions obtained from the first and second parental antibody) of the bispecific antibody where the substitution corresponds to E430G in IgG1 when using EU numbering and which substitution enhances the Fc-Fc interaction of two or more bispecific antibodies of the invention upon binding to membrane-bound target. In another embodiment the Fc-Fc interaction enhancing substitution corresponds to E345K in IgG1 when using EU numbering.
In another embodiment of the invention the bispecific antibody comprises a second antigen binding region which binds to the same epitope on human CD37 as a CD37 antibody comprising the CDR sequences selected from the group comprising:
(i) VH CDR1 sequence set forth in SEQ ID NO: 23,
VH CDR2 sequence set forth in SEQ ID NO: 24,
VH CDR3 sequence set forth in SEQ ID NO: 25,
VL CDR1 sequence set forth in SEQ ID NO: 27,
VL CDR2 sequence: YAS, and
VL CDR3 sequence set forth in SEQ ID NO: 28; [016]
(ii) VH CDR1 sequence set forth in SEQ ID NO: 2,
VH CDR2 sequence set forth in SEQ ID NO: 3,
VH CDR3 sequence set forth in SEQ ID NO: 4,
VL CDR1 sequence set forth in SEQ ID NO: 6,
VL CDR2 sequence: EAS, and
VL CDR3 sequence set forth in SEQ ID NO: 7; [004]
(iii) VH CDR1 sequence set forth in SEQ ID NO: 40,
VH CDR2 sequence set forth in SEQ ID NO: 41,
VH CDR3 sequence set forth in SEQ ID NO: 42,
VL CDR1 sequence set forth in SEQ ID NO: 44,
VL CDR2 sequence: FAK, and
VL CDR3 sequence set forth in SEQ ID NO: 45; [G28.1] and
(iv) VH CDR1 sequence set forth in SEQ ID NO: 47,
VH CDR2 sequence set forth in SEQ ID NO: 48,
VH CDR3 sequence set forth in SEQ ID NO: 49,
VL CDR1 sequence set forth in SEQ ID NO: 51,
VL CDR2 sequence: VAT and
VL CDR3 sequence set forth in SEQ ID NO: 52. [37.3]
In another embodiment of the invention the bispecific antibody comprises a second antigen binding region which binds to the same epitope on human CD37 as a CD37 antibody comprising the CDR sequences selected from the group consisting of:
a. VH CDR1 sequence set forth in SEQ ID NO: 23,
VH CDR2 sequence set forth in SEQ ID NO: 24,
VH CDR3 sequence set forth in SEQ ID NO: 25,
VL CDR1 sequence set forth in SEQ ID NO: 27,
VL CDR2 sequence: YAS, and
VL CDR3 sequence set forth in SEQ ID NO: 28; [016]
b. VH CDR1 sequence set forth in SEQ ID NO: 2,
VH CDR2 sequence set forth in SEQ ID NO: 3,
VH CDR3 sequence set forth in SEQ ID NO: 4,
VL CDR1 sequence set forth in SEQ ID NO: 6,
VL CDR2 sequence: EAS, and
VL CDR3 sequence set forth in SEQ ID NO: 7; [004]
c. VH CDR1 sequence set forth in SEQ ID NO: 40,
VH CDR2 sequence set forth in SEQ ID NO: 41,
VH CDR3 sequence set forth in SEQ ID NO: 42,
VL CDR1 sequence set forth in SEQ ID NO: 44,
VL CDR2 sequence: FAK, and
VL CDR3 sequence set forth in SEQ ID NO: 45; [G28.1] and
d. VH CDR1 sequence set forth in SEQ ID NO: 47,
VH CDR2 sequence set forth in SEQ ID NO: 48,
VH CDR3 sequence set forth in SEQ ID NO: 49,
VL CDR1 sequence set forth in SEQ ID NO: 51,
VL CDR2 sequence: VAT and
VL CDR3 sequence set forth in SEQ ID NO: 52. [37.3]
In one embodiment of the invention the second antigen binding region of the bispecific antibody has a functional epitope comprising one or more of the amino acids E124, F162, Q163, V164, L165 and H175 of SEQ ID No:62 (CD37).
In one embodiment of the invention said second antigen binding region binds to a functional epitope comprising one or more of the amino acids selected from the group consisting of: E124, F162, Q163, V164, L165 and H175 of SEQ ID No:62 (CD37)
In one embodiment of the invention the second antigen binding region of the bispecific antibody binds to a functional epitope on CD37, wherein binding to a mutant CD37 in which any one or more of the amino acid residues at positions corresponding to positions E124, F162, Q163, V164, L165 and H175 of SEQ ID No:62 (CD37). has/have been substituted with alanines, is reduced as compared to wild type CD37 having the amino acid sequence set forth in SEQ ID NO: 62; reduced binding being determined as zscore (fold change) in binding of said antibody being lowed that −1.5, wherein zscore (fold change) in binding is calculated as set forth in Example 17.
Accordingly, in one embodiment the invention provides a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 010 and wherein the second antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 016.
In another embodiment the invention provides a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 010 and wherein the second antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 004.
In another embodiment the invention provides a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 010 and wherein the second antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody G28.1.
In another embodiment the invention provides a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 010 and wherein the second antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 37.3.
In another embodiment the invention provides a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 005 and wherein the second antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 016.
In yet another embodiment the invention provides a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 005 and wherein the second antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 004.
In another embodiment the invention provides a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 005 and wherein the second antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody G28.1.
In another embodiment the invention provides a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 005 and wherein the second antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 37.3.
In a further embodiment of the invention the second antigen binding region of the bispecific antibody comprises the CDR sequences selected from the group comprising:
(i) VH CDR1 sequence set forth in SEQ ID NO: 23,
VH CDR2 sequence set forth in SEQ ID NO: 24,
VH CDR3 sequence set forth in SEQ ID NO: 25,
VL CDR1 sequence set forth in SEQ ID NO: 27,
VL CDR2 sequence: YAS, and
VL CDR3 sequence set forth in SEQ ID NO: 28; [016]
(ii) VH CDR1 sequence set forth in SEQ ID NO: 2,
VH CDR2 sequence set forth in SEQ ID NO: 3,
VH CDR3 sequence set forth in SEQ ID NO: 4,
VL CDR1 sequence set forth in SEQ ID NO: 6,
VL CDR2 sequence: EAS, and
VL CDR3 sequence set forth in SEQ ID NO: 7; [004]
(iii) VH CDR1 sequence set forth in SEQ ID NO: 40,
VH CDR2 sequence set forth in SEQ ID NO: 41,
VH CDR3 sequence set forth in SEQ ID NO: 42,
VL CDR1 sequence set forth in SEQ ID NO: 44,
VL CDR2 sequence: FAK, and
VL CDR3 sequence set forth in SEQ ID NO: 45; [G28.1] and
(iv) VH CDR1 sequence set forth in SEQ ID NO: 47,
VH CDR2 sequence set forth in SEQ ID NO: 48,
VH CDR3 sequence set forth in SEQ ID NO: 49,
VL CDR1 sequence set forth in SEQ ID NO: 51,
VL CDR2 sequence: VAT and
VL CDR3 sequence set forth in SEQ ID NO: 52. [37.3]
In a further embodiment of the invention the second antigen binding region of the bispecific antibody comprises the CDR sequences selected from the group consisting of:
(i) VH CDR1 sequence set forth in SEQ ID NO: 23,
VH CDR2 sequence set forth in SEQ ID NO: 24,
VH CDR3 sequence set forth in SEQ ID NO: 25,
VL CDR1 sequence set forth in SEQ ID NO: 27,
VL CDR2 sequence: YAS, and
VL CDR3 sequence set forth in SEQ ID NO: 28; [016]
(ii) VH CDR1 sequence set forth in SEQ ID NO: 2,
VH CDR2 sequence set forth in SEQ ID NO: 3,
VH CDR3 sequence set forth in SEQ ID NO: 4,
VL CDR1 sequence set forth in SEQ ID NO: 6,
VL CDR2 sequence: EAS, and
VL CDR3 sequence set forth in SEQ ID NO: 7; [004]
(iii) VH CDR1 sequence set forth in SEQ ID NO: 40,
VH CDR2 sequence set forth in SEQ ID NO: 41,
VH CDR3 sequence set forth in SEQ ID NO: 42,
VL CDR1 sequence set forth in SEQ ID NO: 44,
VL CDR2 sequence: FAK, and
VL CDR3 sequence set forth in SEQ ID NO: 45; [G28.1] and
(iv) VH CDR1 sequence set forth in SEQ ID NO: 47,
VH CDR2 sequence set forth in SEQ ID NO: 48,
VH CDR3 sequence set forth in SEQ ID NO: 49,
VL CDR1 sequence set forth in SEQ ID NO: 51,
VL CDR2 sequence: VAT and
VL CDR3 sequence set forth in SEQ ID NO: 52. [37.3]
Accordingly, the present invention also provides in another embodiment a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody comprises the CDR sequences of antibody 010 (i.e. SEQ ID NOs 16-18 and 20-21) and wherein the second antigen binding region of the bispecific antibody comprises the CDR sequences of antibody 016 (i.e. SEQ ID NOs 23-25 and 27-28). As also described above, such bispecific antibody of the invention further comprises an Fc-Fc interaction enhancing mutation in the Fc region of the antibody. In one embodiment this mutation corresponds to a mutation in position E430 or E345 in IgG1 when using the EU numbering system. In one embodiment the mutation is an E430G substitution. In another embodiment it is an E345K substitution.
The present invention also provides in another embodiment a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody comprises the CDR sequences of antibody 010 (i.e. SEQ ID NOs 16-18 and 20-21) and wherein the second antigen binding region of the bispecific antibody comprises the CDR sequences of antibody 004 (i.e. SEQ ID NOs 2-4 and 6-7).
The present invention also provides in another embodiment a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody comprises the CDR sequences of antibody 010 (i.e. SEQ ID NOs 16-18 and 20-21) and wherein the second antigen binding region of the bispecific antibody comprises the CDR sequences of antibody G28.1 (i.e. SEQ ID NOs 40-42 and 44-45).
The present invention also provides in another embodiment a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody comprises the CDR sequences of antibody 010 (i.e. SEQ ID NOs 16-18 and 20-21) and wherein the second antigen binding region of the bispecific antibody comprises the CDR sequences of antibody 37.3 (i.e. SEQ ID NOs 47-49 and 51-52).
In a further embodiment of the invention the second antigen binding region of the bispecific antibody comprise the VH and VL sequences selected from the group comprising:
(i) VH sequence set forth in SEQ ID NO: 22 and VL sequence set forth in SEQ ID NO: 26 [016] or
(ii) VH sequence set forth in SEQ ID NO: 1 and VL sequence set forth in SEQ ID NO: 5 or
(iii) VH sequence set forth in SEQ ID NO: 39 and VL sequence set forth in SEQ ID NO: 43 [G28.1] or
(iv) VH sequence set forth in SEQ ID NO: 46 and VL sequence set forth in SEQ ID NO: 50 [37.3] or
a VH sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity and a VL sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity with the VH sequence and VL sequence, respectively, as set forth in any one of (i) to (iv).
In a further embodiment of the invention the second antigen binding region of the bispecific antibody comprise the VH and VL sequences selected from the group consisting of:
(i) VH sequence set forth in SEQ ID NO: 22 and VL sequence set forth in SEQ ID NO: 26 [016],
(ii) VH sequence set forth in SEQ ID NO: 1 and VL sequence set forth in SEQ ID NO: 5 [004],
(iii) VH sequence set forth in SEQ ID NO: 39 and VL sequence set forth in SEQ ID NO: 43 [G28.1],
(iv) VH sequence set forth in SEQ ID NO: 46 and VL sequence set forth in SEQ ID NO: 50 [37.3] and
a VH sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity and a VL sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity with the VH sequence and VL sequence, respectively, as set forth in any one of (i) to (iv).
Thus, in another embodiment the present invention also provides a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 010 (i.e. SEQ ID NOs 15 and 19) and wherein the second antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 016 (i.e. SEQ ID NOs 22 and 26).
In another embodiment the present invention also provides a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 010 (i.e. SEQ ID Nos 15 and 19) and wherein the second antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 004 (i.e. SEQ ID NOs 1 and 5).
In another embodiment the present invention also provides a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 010 (i.e. SEQ ID NOs 15 and 19) and wherein the second antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody G28.1 (i.e. SEQ ID NOs 39 and 43).
In another embodiment the present invention also provides a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 010 (i.e. SEQ ID Nos 15 and 19) and wherein the second antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 37.3 (i.e. SEQ ID Nos 46 and 50).
In yet another embodiment the present invention provides a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 005 (i.e. SEQ ID NOs 8 and 12) and wherein the second antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 016 (i.e. SEQ ID NOs 22 and 26).
In another embodiment the present invention also provides a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 005 (i.e. SEQ ID NOs 8 and 12) and wherein the second antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 004 (i.e. SEQ ID NOs 1 and 5).
In another embodiment the present invention also provides a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 005 (i.e. SEQ ID NOs 8 and 12) and wherein the second antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody G28.1 (i.e. SEQ ID NOs 39 and 43).
In another embodiment the present invention also provides a bispecific antibody comprising a first and a second antigen binding region wherein the first antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 005 (i.e. SEQ ID NOs 8 and 12) and wherein the second antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 37.3 (i.e. SEQ ID NOs 46 and 50).
In yet other embodiments of the present invention the VH and VL sequences disclosed above may vary within 90% sequence identity.
In yet a different embodiment the present invention provides a CD37 binding molecule comprising one antigen binding region described herein, wherein the CDR sequences are the CDR sequences of one of the antibodies 004, 005, 010, 016, 28.1 or 37.3. in one embodiment such molecule only has one antigen binding region. Hereby the binding molecule has monovalent binding for CD37. Preferably such molecule comprises an intact Fc region of an immunoglobulin. In one embodiment the CD37-binding molecule comprise a second antigen binding region for an irrelevant target which e.g. may be b12.
Fc-Fc Enhancing Mutations
In one embodiment of the invention the one or more Fc-Fc interaction enhancing mutations in said first and second Fc regions of the bispecific antibody are amino acid substitutions. The Fc region of the bispecific antibody can be said to comprise two different Fc regions, one from each parental anti-CD37 antibody. Alternatively, the bispecific antibody may comprise one or more Fc-Fc interaction enhancing mutations in each half-molecule. It is to be understood that the Fc-Fc interaction enhancing mutations are symmetrical, i.e., identical mutations are made in the two Fc regions.
In one embodiment the invention provides a bispecific antibody wherein the one or more Fc-Fc interaction enhancing mutations in said first and second Fc regions are amino acid substitutions at one or more positions corresponding to amino acid positions 430, 440 and 345 in human IgG1 when using the EU numbering system. In one embodiment the invention provides a bispecific antibody wherein the one or more Fc-Fc interaction enhancing mutations in said first and second Fc regions are amino acid substitutions at one or more positions corresponding to amino acid positions 430, 440 and 345 in human IgG1 when using the EU numbering system, with the proviso that the substitution in 440 is 440Y or 440W
In another embodiment the invention provides a bispecific antibody comprising at least one substitution in said first and second Fc regions selected from the group comprising: E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a particular preferred embodiment the bispecific antibody comprises at least one substitution in said first and second Fc regions selected from E430G or E345K, preferably E430G. Hereby bispecific antibodies are provided which will have enhanced Fc-Fc interaction between different antibodies having said mutation. It is believed that this mutation cause the antibodies to form oligomers on the target cell and thereby enhancing CDC.
In another embodiment the bispecific antibody contains one further mutation in said Fc regions which mutation is selected from K439E, S440K and S440R. A bispecific antibody having an additional mutation of K439E and a second different antibody or bispecific antibody having an additional S440K or S440R mutation will form oligomers in alternating patterns of the first and the second antibodies. This is thought to be because the additional mutations will cause a preference for interaction between the first and second antibodies rather than interaction between first and first or second and second antibodies due to non-covalent binding between said Fc regions.
It is preferred that the Fc-Fc interaction enhancing mutations in said first and second Fc regions are identical substitutions in said first and second Fc regions. Accordingly, in one preferred embodiment the bispecific antibodies have the same Fc-Fc interaction enhancing mutation in both Fc regions. The Fc region can also be described as Fc chains so that an antibody has two Fc chains which make up a common Fc region of the antibody. Accordingly, in a preferred embodiment the two Fc chains each comprise a substitution of a position selected from the group of positions corresponding to amino acid positions 430, 440 and 345 in human IgG1 when using the EU numbering system. In one embodiment the two Fc chains each comprise an E430G substitution so that a bispecific antibody of the invention comprises two E430G substitutions. In another embodiment the two Fc chains each comprise an E345K substitution so that the bispecific antibody of the invention comprises two E345K substitutions.
In an embodiment of the invention the bispecific antibody is an IgG1 isotype.
In an embodiment of the invention the bispecific antibody is an IgG2 isotype.
In an embodiment of the invention the bispecific antibody is an IgG3 isotype.
In an embodiment of the invention the bispecific antibody is an IgG4 isotype.
In an embodiment of the invention the bispecific antibody is an IgG isotype.
In an embodiment of the invention the bispecific antibody is a combination of the isotypes IgG1, IgG2, IgG3 and IgG4. For example the first half antibody obtained from the first parental antibody may be an IgG1 isotype and the second half antibody obtained from the second parental antibody may be an IgG4 isotype so that the bispecific antibody is a combination of IgG1 and IgG4. In another embodiment it is a combination of IgG1 and IgG2. In another embodiment it is a combination of IgG1 and IgG3. In another embodiment it is a combination of IgG2 and IgG3. In another embodiment it is a combination of IgG2 and IgG4. In another embodiment it is a combination of IgG3 and IgG4. Typically the core hinge will be an IgG1 type core hinge having the sequence CPPC but it could be other hinges which are stable and do not allow Fab arm exchange in vivo which is the case for the IgG4 core hinge having the sequence CPSC.
In a preferred embodiment the bispecific antibody of the invention is a full length antibody.
In yet another embodiment of the invention the bispecific antibody is a human antibody. In yet another embodiment of the invention the bispecific antibody is a humanized antibody. In yet another embodiment of the invention the bispecific antibody is a chimeric antibody. In an embodiment of the invention the bispecific antibody is a combination of human, humanized and chimeric. For example the first half antibody obtained from the first parental antibody may be a human antibody and the second half antibody obtained from the second parental antibody may be a humanized antibody so that the bispecific antibody is a combination of human and humanized. In a preferred embodiment of the invention the bispecific antibody binds both human and cynomolgus monkey CD37, having the sequences set forth in SEQ ID Nos 62 and 63, respectively. This is an advantage as this will allow preclinical toxicology studies to be performed in the cynomolgus monkey with the same bispecific molecule that will later be tested in humans. In cases where the antibodies against a human target do not also bind the target in an animal model it is very difficult to perform the preclinical toxicology studies and the non-clinical safety profile of the molecules, which is a requirement by regulatory authorities.
Bispecific Antibody Formats
The present invention provides bispecific CD37×CD37 antibodies which efficiently promote CDC- and/or ADCC-mediated killing of CD37-expressing tumor cells such as e.g. B-cell derived tumors. Depending on the desired functional properties for a particular use, particular antigen-binding regions can be selected from the set of antibodies or antigen-binding regions provided by the present invention. Many different formats and uses of bispecific antibodies are known in the art, and were reviewed by Kontermann; Drug Discov Today, 2015 July; 20(7):838-47 and; MAbs, 2012 March-April; 4(2):182-97.
A bispecific antibody according to the present invention is not limited to any particular bispecific format or method of producing it, however, a bispecific antibody of the invention should have an intact Fc domain in order to induce enhanced Fc-Fc interactions.
Examples of bispecific antibody molecules which may be used in the present invention comprise (i) a single antibody that has two arms comprising different antigen-binding regions; (ii) a dual-variable-domain antibody (DVD-Ig) where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (iii) a so-called “dock and lock” molecule, based on the “dimerization and docking domain” in Protein Kinase A.
In one embodiment, the bispecific antibody of the present invention is a cross-body or a bispecific antibody obtained via a controlled Fab-arm exchange (such as described in WO2011131746 (Genmab)).
Examples of different classes of bispecific antibodies include but are not limited to (i) IgG-like molecules with complementary CH3 domains to force heterodimerization; (ii) recombinant IgG-like dual targeting molecules, wherein the two sides of the molecule each contain the Fab fragment or part of the Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, wherein full length IgG antibodies are fused to extra Fab fragment or parts of Fab fragment; (iv) Fc fusion molecules, wherein single chain Fv molecules or stabilized diabodies are fused to heavy-chain constant-domains, Fc-regions or parts thereof; (v) Fab fusion molecules, wherein different Fab-fragments are fused together, fused to heavy-chain constant-domains, Fc-regions or parts thereof; and (vi) scFv- and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) wherein different single chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g. domain antibodies, nanobodies) are fused to an Fc-.
Examples of IgG-like molecules with complementary CH3 domain molecules include but are not limited to the Triomab/Quadroma molecules (Trion Pharma/Fresenius Biotech; Roche, WO2011069104), the so-called Knobs-into-Holes molecules (Genentech, WO9850431), CrossMAbs (Roche, WO2011117329) and the electrostatically-steered molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), the LUZ-Y molecules (Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52): 43331-9, doi: 10.1074/jbc.M112.397869. Epub 2012 Nov. 1), DIG-body and PIG-body molecules (Pharmabcine, WO2010134666, WO2014081202), the Strand Exchange Engineered Domain body (SEEDbody) molecules (EMD Serono, WO2007110205), the Biclonics molecules (Merus, WO2013157953), FcAAdp molecules (Regeneron, WO201015792), hinge engineered bispecific IgG1 and IgG2 molecules (Pfizer/Rinat, WO11143545), Azymetric scaffold molecules (Zymeworks/Merck, WO2012058768), mAb-Fv molecules (Xencor, WO2011028952), bivalent bispecific antibodies (WO2009080254) and the DuoBody® molecules (Genmab A/S, WO2011131746).
Examples of recombinant IgG-like dual targeting molecules include but are not limited to Dual Targeting (DT)-Ig molecules (WO2009058383), Two-in-one Antibody (Genentech; Bostrom, et al 2009. Science 323, 1610-1614.), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013 March-April; 5(2):208-18), approaches with common light chain (Crucell/Merus, U.S. Pat. No. 7,262,028), KABodies (NovImmune, WO2012023053) and CovX-body (CovX/Pfizer; Doppalapudi, V. R., et al 2007. Bioorg. Med. Chem. Lett. 17,501-506.).
Examples of IgG fusion molecules include but are not limited to Dual Variable Domain (DVD)-Ig molecules (Abbott, U.S. Pat. No. 7,612,181), Dual domain double head antibodies (Unilever; Sanofi Aventis, WO20100226923), IgG-like Bispecific molecules (ImClone/Eli Lilly, Lewis et al. Nat Biotechnol. 2014 February; 32(2):191-8), Ts2Ab (MedImmune/AZ; Dimasi et al. J Mol Biol. 2009 Oct. 30; 393(3):672-92) and BsAb molecules (Zymogenetics, WO2010111625), HERCULES molecules (Biogen Idec, US007951918), scFv fusion molecules (Novartis), scFv fusion molecules (Changzhou Adam Biotech Inc, CN 102250246) and TvAb molecules (Roche, WO2012025525, WO2012025530).
Examples of Fc fusion molecules include but are not limited to ScFv/Fc Fusions (Pearce et al., Biochem Mol Biol Int. 1997 September; 42(6):1179-88), SCORPION molecules (Emergent BioSolutions/Trubion, Blankenship μW, et al. AACR 100 th Annual meeting 2009 (Abstract #5465); Zymogenetics/BMS, WO2010111625), Dual Affinity Retargeting Technology (Fc-DART) molecules (MacroGenics, WO2008157379, WO2010080538) and Dual(ScFv)2-Fab molecules (National Research Center for Antibody Medicine—China).
Examples of Fab fusion bispecific antibodies include but are not limited to F(ab)2 molecules (Medarex/AMGEN; Deo et al J Immunol. 1998 Feb. 15; 160(4):1677-86.), Dual-Action or Bis-Fab molecules (Genentech, Bostrom, et al 2009. Science 323, 1610-1614.), Dock-and-Lock (DNL) molecules (ImmunoMedics, WO2003074569, WO2005004809), Bivalent Bispecific molecules (Biotecnol, Schoonjans, J Immunol. 2000 Dec. 15; 165(12):7050-7.) and Fab-Fv molecules (UCB-Celltech, WO 2009040562 A1).
Examples of scFv-, diabody-based and domain antibodies include but are not limited to Dual Affinity Retargeting Technology (DART) molecules (MacroGenics, WO2008157379, WO2010080538), COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 August; 88(6):667-75.), and dual targeting nanobodies (Ablynx, Hmila et al., FASEB J. 2010).
In one aspect, the bispecific antibody of the invention comprises a first Fc-region comprising a first CH3 region, and a second Fc-region comprising a second CH3 region, wherein the sequences of the first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. More details on these interactions and how they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are hereby incorporated by reference.
As described further herein, a stable bispecific CD37×CD37 antibody can be obtained at high yield using a particular method on the basis of one homodimeric starting CD37 antibody and another homodimeric starting CD37 antibody containing only a few, fairly conservative, asymmetrical mutations in the CH3 regions. Asymmetrical mutations mean that the sequences of said first and second CH3 regions contain amino acid substitutions at non-identical positions so that the first and second CH3 regions have different amino acid sequences.
In one aspect, the bispecific antibody as defined in any of the embodiments disclosed herein comprises first and second Fc region, wherein each of said first and second Fc region comprises at least a hinge region, a CH2 and a CH3 region, wherein in said first Fc region at least one of the amino acids in the positions corresponding to a positions selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain has been substituted, and in said second Fc region at least one of the amino acids in the positions corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain has been substituted, and wherein said first and said second Fc regions are not substituted in the same positions.
Accordingly, in a preferred embodiment of the invention the first Fc region of the bispecific antibody comprises a mutation of the amino acid corresponding to position F405 in human IgG1 and the second Fc region of the bispecific antibody comprises a further mutation of the amino acid corresponding to position K409 in human IgG1. Accordingly, these mutations are asymmetric compared to the above mentioned Fc-Fc interaction enhancing mutations.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc-region has an amino acid substitution at position 366, and said second Fc-region has an amino acid substitution at a position selected from the group consisting of: 368, 370, 399, 405, 407 and 409. In one embodiment the amino acid at position 366 is selected from Ala, Asp, Glu, His, Asn, Val, or Gln.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc-region has an amino acid substitution at position 368, and said second Fc-region has an amino acid substitution at a position selected from the group consisting of: 366, 370, 399, 405, 407 and 409.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc-region has an amino acid substitution at position 370, and said second Fc-region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 399, 405, 407 and 409.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc-region has an amino acid substitution at position 399, and said second Fc-region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 405, 407 and 409.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc-region has an amino acid substitution at position 405, and said second Fc-region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 407 and 409.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc-region has an amino acid substitution at position 407, and said second Fc-region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, and 409.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc-region has an amino acid substitution at position 409, and said second Fc-region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, and 407.
Accordingly, in one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the sequences of said first and second Fc-region contain asymmetrical mutations, i.e. mutations at different positions in the two Fc-regions, e.g. a mutation at position 405 in one of the Fc-regions and a mutation at position 409 in the other Fc-region.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc-region has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region has an amino-acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405 and 407. In one such embodiment, said first Fc-region has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region has an amino acid other than Phe, e.g. Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Cys, Lys, or Leu, at position 405. In a further embodiment hereof, said first Fc-region has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region has an amino acid other than Phe, Arg or Gly, e.g. Leu, Ala, Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 405.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region comprises a Phe at position 405 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region comprises an amino acid other than Phe, e.g. Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Leu, Met, or Cys, at position 405 and a Lys at position 409. In a further embodiment hereof, said first Fc-region comprises a Phe at position 405 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region comprises an amino acid other than Phe, Arg or Gly, e.g. Leu, Ala, Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 405 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region comprises a Phe at position 405 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region comprises a Leu at position 405 and a Lys at position 409. In a further embodiment hereof, said first Fc-region comprises a Phe at position 405 and an Arg at position 409 and said second Fc-region comprises an amino acid other than Phe, Arg or Gly, e.g. Leu, Ala, Val, Ile, Ser, Thr, Lys, Met, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 405 and a Lys at position 409. In another embodiment, said first Fc-region comprises Phe at position 405 and an Arg at position 409 and said second Fc-region comprises a Leu at position 405 and a Lys at position 409.
In a further embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region comprises an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405. In a further embodiment, said first Fc-region comprises an Arg at position 409 and said second Fc-region comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405.
In an even further embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region comprises a Lys at position 370, a Phe at position 405 and an Arg at position 409 and said second Fc-region comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region comprises an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region comprises a Lys at position 409 and: a) an Ile at position 350 and a Leu at position 405, or b) a Thr at position 370 and a Leu at position 405.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region comprises an Arg at position 409 and said second Fc region comprises a Lys at position 409 and: a) an Ile at position 350 and a Leu at position 405, or b) a Thr at position 370 and a Leu at position 405.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region comprises a Thr at position 350, a Lys at position 370, a Phe at position 405 and an Arg at position 409 and said second Fc region comprises a Lys at position 409 and: a) an Ile at position 350 and a Leu at position 405, or b) a Thr at position 370 and a Leu at position 405.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region comprises a Thr at position 350, a Lys at position 370, a Phe at position 405 and an Arg at position 409 and said second Fc-region comprises an Ile at position 350, a Thr at position 370, a Leu at position 405 and a Lys at position 409.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region has an amino acid other than Lys, Leu or Met at position 409 and said second Fc-region has an amino acid other than Phe at position 405, such as other than Phe, Arg or Gly at position 405; or said first CH3 region has an amino acid other than Lys, Leu or Met at position 409 and said second CH3 region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region having an amino acid other than Lys, Leu or Met at position 409 and a second Fc-region having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region having a Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409 and a second Fc-region having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and a Lys at position 409.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region having a Tyr at position 407 and an Arg at position 409 and a second Fc-region having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and a Lys at position 409.
In another embodiment, said first Fc-region has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr, e.g. Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, at position 407. In another embodiment, said first Fc-region has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region has a Gly, Leu, Met, Asn or Trp at position 407.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region has a Tyr at position 407 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr, e.g. Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, at position 407 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region has a Tyr at position 407 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region has a Tyr at position 407 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second Fc-region has a Gly, Leu, Met, Asn or Trp at position 407 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region has a Tyr at position 407 and an Arg at position 409 and said second Fc-region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr, e.g. Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, at position 407 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region has a Tyr at position 407 and an Arg at position 409 and said second Fc-region has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc-region has a Tyr at position 407 and an Arg at position 409 and said second Fc-region has a Gly, Leu, Met, Asn or Trp at position 407 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc-region has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409, and the second Fc-region has
(i) an amino acid other than Phe, Leu and Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 368, or
(ii) a Trp at position 370, or
(iii) an amino acid other than Asp, Cys, Pro, Glu or Gln, e.g. Phe, Leu, Met, Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asn, Trp, Tyr, or Cys, at position 399 or
(iv) an amino acid other than Lys, Arg, Ser, Thr, or Trp, e.g. Phe, Leu, Met, Ala, Val, Gly, Ile, Asn, His, Asp, Glu, Gln, Pro, Tyr, or Cys, at position 366.
In one embodiment, the first Fc-region has an Arg, Ala, His or Gly at position 409, and the second Fc region has
(i) a Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or
(ii) a Trp at position 370, or
(iii) an Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr at position 399, or
(iv) an Ala, Asp, Glu, His, Asn, Val, Gln, Phe, Gly, Ile, Leu, Met, or Tyr at position 366.
In one embodiment, the first Fc-region has an Arg at position 409, and the second Fc region has
(i) an Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or
(ii) a Trp at position 370, or
(iii) a Phe, His, Lys, Arg or Tyr at position 399, or
(iv) an Ala, Asp, Glu, His, Asn, Val, Gln at position 366.
In addition to the above-specified amino-acid substitutions, said first and second Fc regions may contain further amino-acid substitutions, deletion or insertions relative to wild-type Fc sequences.
In a preferred embodiment of the invention the second Fc region of the bispecific antibody comprises a mutation corresponding to F405 in human IgG1 and the first Fc region comprises a mutation corresponding to K409 in human IgG1 when using EU numbering.
In one embodiment the mutations at position F405 and K409 are substitutions. In a particular embodiment the substitution at position F405 is an F405L substitution. In another embodiment the substitution at position K409 is a K409R substitution.
In embodiments where the bispecific antibody is an IgG4 isotype the first Fc region may further comprise an F405L substitution and an R409K substitution. In such embodiments the second Fc region is not substituted in any of 405 and 409 amino acid positions.
It is to be understood that except expressly stated otherwise all the mentioned amino acid mutations at the disclosed positions are mutations relative to a human IgG1 and using human IgG1 for numbering using the EU numbering system.
In one embodiment, neither said first nor said second Fc-region comprises a Cys-Pro-Ser-Cys sequence in the core hinge region.
In a further embodiment, both said first and said second Fc-region comprise a Cys-Pro-Pro-Cys sequence in the core hinge region.
Hereby bispecific antibodies are provided which can be produced in high yields and which are stable in vivo.
In another embodiment the bispecific antibody of the invention has increased CDC and/or ADCC effector functions compared to an identical bispecific antibody which does not have the Fc-Fc interaction enhancing mutations. In another embodiment the bispecific antibody of the invention has increased CDC and/or ADCC effector functions compared to a monoclonal parental antibody having a binding region of either the first or the second binding region of the bispecific antibody and having identical Fc-Fc enhancing mutations as the bispecific antibody of the invention.
Method of Preparing Bispecific Antibodies of the Invention
Traditional methods such as the hybrid hybridoma and chemical conjugation methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649) can be used in the preparation of the bispecific antibodies of the invention. Co-expression in a host cell of two antibodies, consisting of different heavy and light chains, leads to a mixture of possible antibody products in addition to the desired bispecific antibody, which can then be isolated by, e.g., affinity chromatography or similar methods.
Strategies favoring the formation of a functional bispecific product, upon co-expression of different antibody constructs can also be used, e.g., the method described by Lindhofer et al. (1995 J Immunol 155:219). Fusion of rat and mouse hybridomas producing different antibodies leads to a limited number of heterodimeric proteins because of preferential species-restricted heavy/light chain pairing. Another strategy to promote formation of heterodimers over homodimers is a “knob-into-hole” strategy in which a protuberance is introduced on a first heavy-chain polypeptide and a corresponding cavity in a second heavy-chain polypeptide, such that the protuberance can be positioned in the cavity at the interface of these two heavy chains so as to promote heterodimer formation and hinder homodimer formation. “Protuberances” are constructed by replacing small amino-acid side-chains from the interface of the first polypeptide with larger side chains. Compensatory “cavities” of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino-acid side-chains with smaller ones (U.S. Pat. No. 5,731,168). EP1870459 (Chugai) and WO2009089004 (Amgen) describe other strategies for favoring heterodimer formation upon co-expression of different antibody domains in a host cell. In these methods, one or more residues that make up the CH3-CH3 interface in both CH3 domains are replaced with a charged amino acid such that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable. WO2007110205 (Merck) describe yet another strategy, wherein differences between IgA and IgG CH3 domains are exploited to promote heterodimerization.
Another in vitro method for producing bispecific antibodies has been described in WO2008119353 (Genmab), wherein a bispecific antibody is formed by “Fab-arm” or “half-molecule” exchange (swapping of a heavy chain and attached light chain) between two monospecific IgG4- or IgG4-like antibodies upon incubation under reducing conditions. The resulting product is a bispecific antibody having two Fab arms which may comprise different sequences.
A preferred method for preparing the bispecific CD37×CD37 antibodies of the present invention includes the methods described in WO2011131746 and WO2013060867 (Genmab) comprising the following steps:
a) providing a first antibody comprising an Fc region, said Fc region comprising a first CH3 region;
b) providing a second antibody comprising a second Fc region, said Fc region comprising a second CH3 region, wherein the first antibody is a CD37 antibody and the second antibody is a different CD37 antibody;
wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions;
c) incubating said first antibody together with said second antibody under reducing conditions; and
d) obtaining said bispecific antibody.
In one embodiment, said first antibody together with said second antibody are incubated under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide-bond isomerization, wherein the heterodimeric interaction between said first and second antibodies in the resulting heterodimeric antibody is such that no Fab-arm exchange occurs at 0.5 mM GSH after 24 hours at 37° C.
Without being limited to theory, in step c), the heavy-chain disulfide bonds in the hinge regions of the parent antibodies are reduced and the resulting cysteines are then able to form inter heavy-chain disulfide bond with cysteine residues of another parent antibody molecule (originally with a different specificity). In one embodiment of this method, the reducing conditions in step c) comprise the addition of a reducing agent, e.g. a reducing agent selected from the group consisting of: 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-ca rboxyethyl)phosphine (TCEP), L-cysteine and beta-mercapto-ethanol, preferably a reducing agent selected from the group consisting of: 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine. In a further embodiment, step c) comprises restoring the conditions to become non-reducing or less reducing, for example by removal of a reducing agent, e.g. by desalting.
For this method any of the CD37 antibodies described herein may be used including first and second CD37 antibodies, comprising a first and/or second Fc region. Examples of such first and second Fc regions, including combination of such first and second Fc regions may include any of those described herein.
In one embodiment of this method, said first and/or second antibodies are full-length antibodies.
The Fc regions of the first and second antibodies may be of any isotype, including, but not limited to, IgG1, IgG2, IgG3 or IgG4. In one embodiment of this method, the Fc regions of both said first and said second antibodies are of the IgG1 isotype. In another embodiment, one of the Fc regions of said antibodies is of the IgG1 isotype and the other of the IgG4 isotype. In the latter embodiment, the resulting bispecific antibody comprises an Fc region of an IgG1 and an Fc region of IgG4 and may thus have interesting intermediate properties with respect to activation of effector functions.
In a further embodiment, one of the antibody starting proteins has been engineered to not bind Protein A, thus allowing to separate the heterodimeric protein from said homodimeric starting protein by passing the product over a protein A column.
As described above, the sequences of the first and second CH3 regions of the homodimeric starting antibodies (the parental antibodies) are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. More details on these interactions and how they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are hereby incorporated by reference in their entirety.
In particular, a stable bispecific CD37×CD37 antibody can be obtained at high yield using the above method of the invention on the basis of two homodimeric starting antibodies which bind different epitopes of CD37 and contain only a few, fairly conservative, asymmetrical mutations in the CH3 regions. Asymmetrical mutations mean that the sequences of said first and second CH3 regions contain amino acid substitutions at non-identical positions.
The bispecific antibodies of the invention may also be obtained by co-expression of constructs encoding the first and second polypeptides in a single cell. Thus, in a further aspect, the invention relates to a method for producing a bispecific antibody, said method comprising the following steps:
a) providing a first nucleic-acid construct encoding a first polypeptide comprising a first Fc region and a first antigen-binding region of a first antibody heavy chain, said first Fc region comprising a first CH3 region,
b) providing a second nucleic-acid construct encoding a second polypeptide comprising a second Fc region and a second antigen-binding region of a second antibody heavy chain, said second Fc region comprising a second CH3 region,
wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions,
optionally wherein said first and second nucleic acid constructs encode light chain sequences of said first and second antibodies
c) co-expressing said first and second nucleic-acid constructs in a host cell, and
d) obtaining said heterodimeric protein from the cell culture.
Thus, the present invention also relates to a recombinant eukaryotic or prokaryotic host cell which produces a bispecific antibody of the present invention.
In one embodiment of the present invention, the bispecific antibody is obtained by any of the methods according to the present invention.
Suitable expression vectors, including promoters, enhancers, etc., and suitable host cells for the production of antibodies are well-known in the art. Examples of host cells include yeast, bacterial and mammalian cells, such as CHO or HEK cells.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a second Fc-region, wherein neither said first nor said second Fc-region comprises a Cys-Pro-Ser-Cys sequence in the hinge region.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a second Fc-region, wherein both of said first and said second Fc-region comprise a Cys-Pro-Pro-Cys sequence in the hinge region.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a second Fc-region, wherein the first and second Fc-regions are human antibody Fc-regions.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a second Fc-region, wherein the first and second antigen-binding regions comprise human antibody VH sequences and, optionally, human antibody VL sequences.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a second Fc-region, wherein the first and second antigen-binding regions are from heavy-chain antibodies.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a second Fc-region, wherein the first and second antigen-binding regions comprise a first and second light chain.
In further embodiments, the co-expression method according to the invention comprises any of the further features described under the in vitro method above.
Parental Antibodies
In another embodiment the invention relates to the parental antibodies which are used to prepare the bispecific antibodies of the invention.
Thus, in an embodiment the invention relates to an anti-CD37 antibody binding to the same epitope on human CD37 as an anti-CD37 antibody which antibody comprises:
(i) a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 16, a CDR2 sequence set forth in SEQ ID NO: 17 and a CDR3 sequence set forth in SEQ ID NO: 18, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 20, and CDR2 sequence: KAS, and CDR3 sequence set forth in SEQ ID NO: 21[010]; or
(ii) a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 9, a CDR2 sequence set forth in SEQ ID NO:10 and a CDR3 sequence set forth in SEQ ID NO: 11, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 113, and CDR2 sequence: AAS, and CDR3 sequence set forth in SEQ ID NO: 14[005].
In another embodiment the invention relates to an anti-CD37 antibody which competes for binding with an anti-CD37 antibody comprising:
(i) a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 16, a CDR2 sequence set forth in SEQ ID NO: 17 and a CDR3 sequence set forth in SEQ ID NO: 18, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 20, and CDR2 sequence: KAS, and CDR3 sequence set forth in SEQ ID NO: 21 [010]; or
(ii) a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 9, a CDR2 sequence set forth in SEQ ID NO:10 and a CDR3 sequence set forth in SEQ ID NO: 11, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 113, and CDR2 sequence: AAS, and CDR3 sequence set forth in SEQ ID NO: 14 [005].
In another embodiment the invention relates to an anti-CD37 antibody comprising:
(i) a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 16, a CDR2 sequence set forth in SEQ ID NO: 17 and a CDR3 sequence set forth in SEQ ID NO: 18, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 20, and CDR2 sequence: KAS, and CDR3 sequence set forth in SEQ ID NO: 21 [010]; or
(ii) a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 9, a CDR2 sequence set forth in SEQ ID NO:10 and a CDR3 sequence set forth in SEQ ID NO: 11, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 113, and CDR2 sequence: AAS, and CDR3 sequence set forth in SEQ ID NO: 14 [005].
In another embodiment the invention relates to an anti-CD37 antibody which comprises:
(i) a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 23, a CDR2 sequence set forth in SEQ ID NO: 24 and a CDR3 sequence set forth in SEQ ID NO: 25, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 27, and CDR2 sequence: YAS, and CDR3 sequence set forth in SEQ ID NO: 28; [016] or
(ii) a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 2, a CDR2 sequence set forth in SEQ ID NO: 3 and a CDR3 sequence set forth in SEQ ID NO: 4, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 6, and CDR2 sequence: EAS, and CDR3 sequence set forth in SEQ ID NO: 7. [004]
In another embodiment the invention relates to an antibody as defined above which comprises one or more amino acid mutations in the Fc region of the antibody which mutation(s) enhances the Fc-Fc interaction between the antibodies upon target binding compared to the Fc-Fc interaction between antibodies not having said mutation(s).
It is believed that said enhanced Fc-Fc interaction has the effect that the antibodies form oligomers such as e.g. hexamers on the target cells which oligomers formation causes the effect of enhanced CDC. In a preferred embodiment the one or more amino acid mutations in the Fc region of the antibody is amino acid substitutions at one or more positions corresponding to amino acid positions 430, 440 and 345 in human IgG1 when using the EU numbering system and where the substitution is relative to the amino acid sequence of human IgG1. In one embodiment the one or more amino acid mutations in the Fc region of the antibody is an amino acid substitution at one or more positions corresponding to amino acid positions 430,345 and 440 in human IgG1 when using the EU numbering system, with the proviso that the substitution in 440 is 440Y or 440W. In an embodiment the at least one amino acid substitution in the Fc region is selected from the group comprising: E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W.
In a preferred embodiment the at least one substitution in said Fc region is selected from E430G or E345K, preferably E430G. These Fc-Fc interaction enhancing mutations are symmetrical mutations so that the two Fc chains of the antibody have identical mutations/substitutions.
In a further embodiment the antibody may further comprise a substitution at a position corresponding to 366, 368, 370, 399, 405, 407 and 409 in human IgG1. An antibody having a substitution in one of these amino acid positions is a stable antibody; however, it can, under reducing conditions in a so called “fab arm exchange” reaction, form bispecific antibodies with an antibody having a substitution in another of these amino acid positions and having a different antigen binding region. Under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide-bond isomerization an antibody of the invention will form half molecules each comprising a single antigen binding site and an Fc region. The substitutions at non-identical positions in any of positions corresponding to 366, 368, 370, 399, 405, 407 and 409 in human IgG1 will cause the half molecules of the first antibody to favor dimerization with half molecules of the second antibody so that bispecific (heterodimeric) antibodies will form when the reducing conditions are removed and the disulfide bonds in the hinge region re-forms.
Thus, two antibodies of the present invention having different antigen binding regions and binding different epitopes on CD37 and containing a substitution in both Fc chains (Fc regions) in any of the amino acid positions corresponding to 366, 368, 370, 399, 405, 407 and 409 in human IgG1 but in non-identical positions may be suitable for preparing a bispecific antibody of the invention.
In one embodiment, the first antibody has an amino acid substitution at position 366, and said second homodimeric protein has an amino acid substitution at a position selected from the group consisting of: 368, 370, 399, 405, 407 and 409. In one embodiment the amino acid at position 366 is selected from Arg, Lys, Asn, Gln, Tyr, Glu and Gly.
In one embodiment, the first antibody has an amino acid substitution at position 368, and said second antibody has an amino acid substitution at a position selected from the group consisting of: 366, 370, 399, 405, 407 and 409.
In one embodiment, the first antibody has an amino acid substitution at position 370, and said second antibody has an amino acid substitution at a position selected from the group consisting of: 366, 368, 399, 405, 407 and 409.
In one embodiment, the first antibody has an amino acid substitution at position 399, and said second antibody has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 405, 407 and 409.
In one embodiment, the first antibody has an amino acid substitution at position 405, and said second antibody has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 407 and 409.
In one embodiment, the first antibody has an amino acid substitution at position 407, and said second antibody has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, and 409.
In one embodiment, the first antibody has an amino acid substitution at position 409, and said second antibody has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, and 407.
In one embodiment, the first antibody has an amino acid other than Lys, Leu or Met at position 409 such as an amino acid selected from the group comprising: Gly, Ala, Val, Ile, Ser, Thr, Arg, His, Asp, Asn, Glu, Gln, Trp, Phe, or Tyr at position 409 and said second antibody has an amino-acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405 and 407.
In one such embodiment, said first antibody has an amino acid other than Lys, Leu or Met at position 409 such as an amino acid selected from the group comprising: Gly, Ala, Val, Ile, Ser, Thr, Arg, His, Asp, Asn, Glu, Gln, Trp, Phe, or Tyr at position 409 and said second antibody has an amino acid other than Phe at position 405 such as an amino acid selected from the group comprising: Gly, Ala, Val, Leu, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Trp, Met or Tyr at the position corresponding to 405 in IgG1. In a further embodiment hereof, said first antibody has an amino acid other than Lys, Leu or Met at position 409 and said second antibody has an amino acid other than Phe, Arg or Gly at position 405.
In another embodiment, said first antibody comprises a Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409 and said second antibody comprises an amino acid other than Phe at position 405 and a Lys at position 409.
In another embodiment, said first antibody comprises Phe at position 405 and an Arg at position 409 and said second antibody comprises a Leu at position 405 and a Lys at position 409. In embodiments where the antibodies are of IgG1, IgG2 or IgG3 isotypes the first antibody may comprise an F405L substitution and the second antibody may comprise a K409R substitution or vice versa. However, in embodiments where the antibodies are both of the IgG4 isotypes the amino acid in position 409 is naturally an Arg (R). Thus, in such embodiments the first antibody is not substituted in position 409 but naturally has an R409 and the second antibody comprises F405L and R405K substitutions, or vice versa; the second antibody is not substituted in position 409 but naturally has an R409 and the first antibody comprises F405L and R405K substitutions. In embodiments where one or both of the first and the second antibodies are of the IgG4 isotype
Accordingly, in one embodiment the antibody of the invention may comprise a substitution corresponding to F405L in human IgG1. In another embodiment an antibody of the invention may comprise a substitution corresponding to K409R in human IgG1. Such two different antibodies are suitable for preparing a bispecific antibody of the invention. In a particularly preferred embodiment a first antibody of the invention comprises an F405L and an E430G substitution and a second antibody of the invention comprises a K409R and an E430G substitution. Hereby antibodies are provided which may form bispecific antibodies of the invention which bispecific antibodies comprise a first half molecule comprising F405L+E430G substitutions and a second half molecule comprising K409R+E430G when using IgG1 for numbering. In embodiments where the isotype is IgG4 the first half molecule comprises F405L+R409K+E430G substitutions and the second half molecule comprises an E430G when using IgG4 for numbering. Preferably, in such IgG4 embodiments the core hinge region is substituted from a ‘CPSC’ amino acid sequence to a ‘CPPC’ sequence to make the bispecific antibody more stable in vivo and/or in vitro compared to an IgG4 antibody having the CPSC core hinge.
In one embodiment of the invention the first antibody comprises a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 16, a CDR2 sequence set forth in SEQ ID NO: 17 and a CDR3 sequence set forth in SEQ ID NO: 18, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 20, and CDR2 sequence: KAS, and CDR3 sequence set forth in SEQ ID NO: 21 [010]; and an Fc region comprising an F405L substitution and on or more Fc-Fc interaction enhancing mutations. In a preferred embodiment the Fc-Fc interaction enhancing mutations are substitutions at one or more amino acid positions selected from the group comprising: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment it is E430G.
In another embodiment of the invention the first antibody comprises a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 16, a CDR2 sequence set forth in SEQ ID NO: 17 and a CDR3 sequence set forth in SEQ ID NO: 18, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 20, and CDR2 sequence: KAS, and CDR3 sequence set forth in SEQ ID NO: 21 [010]; and an Fc region comprising an K409R substitution and on or more Fc-Fc interaction enhancing mutations. In a preferred embodiment the Fc-Fc interaction enhancing mutations are substitutions at one or more amino acid positions selected from the group comprising: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment it is E430G.
In another embodiment of the invention the first antibody comprises a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 9, a CDR2 sequence set forth in SEQ ID NO:10 and a CDR3 sequence set forth in SEQ ID NO: 11, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 113, and CDR2 sequence: AAS, and CDR3 sequence set forth in SEQ ID NO: 14 [005]; and an Fc region comprising an F405L substitution and on or more Fc-Fc interaction enhancing mutations. In a preferred embodiment the Fc-Fc interaction enhancing mutations are substitutions at one or more amino acid positions selected from the group comprising: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment it is E430G.
In yet another embodiment of the invention the first antibody comprises a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 9, a CDR2 sequence set forth in SEQ ID NO:10 and a CDR3 sequence set forth in SEQ ID NO: 11, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 113, and CDR2 sequence: AAS, and CDR3 sequence set forth in SEQ ID NO: 14 [005]; and an Fc region comprising an K409R substitution and on or more Fc-Fc interaction enhancing mutations. In a preferred embodiment the Fc-Fc interaction enhancing mutations are substitutions at one or more amino acid positions selected from the group comprising: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment it is E430G.
In one embodiment of the invention the second antibody comprises a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 23, a CDR2 sequence set forth in SEQ ID NO: 24 and a CDR3 sequence set forth in SEQ ID NO: 25, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 27, and CDR2 sequence: YAS, and CDR3 sequence set forth in SEQ ID NO: 28 [016]; and an Fc region comprising an F405L substitution and on or more Fc-Fc interaction enhancing mutations. In a preferred embodiment the Fc-Fc interaction enhancing mutations are substitutions at one or more amino acid positions selected from the group comprising: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment it is E430G.
In another embodiment of the invention the second antibody comprises a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 23, a CDR2 sequence set forth in SEQ ID NO: 24 and a CDR3 sequence set forth in SEQ ID NO: 25, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 27, and CDR2 sequence: YAS, and CDR3 sequence set forth in SEQ ID NO: 28 [016]; and an Fc region comprising an K409R substitution and on or more Fc-Fc interaction enhancing mutations. In a preferred embodiment the Fc-Fc interaction enhancing mutations are substitutions at one or more amino acid positions selected from the group comprising: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment it is E430G.
In another embodiment of the invention the second antibody comprises a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 2, a CDR2 sequence set forth in SEQ ID NO: 3 and a CDR3 sequence set forth in SEQ ID NO: 4, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 6, and CDR2 sequence: EAS, and CDR3 sequence set forth in SEQ ID NO: 7 [004]; and an Fc region comprising an F405L substitution and on or more Fc-Fc interaction enhancing mutations. In a preferred embodiment the Fc-Fc interaction enhancing mutations are substitutions at one or more amino acid positions selected from the group comprising: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment it is E430G.
In yet another embodiment of the invention the second antibody comprises a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 2, a CDR2 sequence set forth in SEQ ID NO: 3 and a CDR3 sequence set forth in SEQ ID NO: 4, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 6, and CDR2 sequence: EAS, and CDR3 sequence set forth in SEQ ID NO: 7 [004]; and an Fc region comprising an K409R substitution and on or more Fc-Fc interaction enhancing mutations. In a preferred embodiment the Fc-Fc interaction enhancing mutations are substitutions at one or more amino acid positions selected from the group comprising: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment it is E430G.
In another embodiment of the invention the second antibody comprises a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 40, a CDR2 sequence set forth in SEQ ID NO: 41 and a CDR3 sequence set forth in SEQ ID NO: 42, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 44, and CDR2 sequence: FAK, and CDR3 sequence set forth in SEQ ID NO: 45 [G28.1]; and an Fc region comprising an F405L substitution and on or more Fc-Fc interaction enhancing mutations. In a preferred embodiment the Fc-Fc interaction enhancing mutations are substitutions at one or more amino acid positions selected from the group comprising: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment it is E430G.
In yet another embodiment of the invention the second antibody comprises a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 40, a CDR2 sequence set forth in SEQ ID NO: 41 and a CDR3 sequence set forth in SEQ ID NO: 42, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 44, and CDR2 sequence: FAK, and CDR3 sequence set forth in SEQ ID NO: 45 [G28.1]; and an Fc region comprising an K409R substitution and on or more Fc-Fc interaction enhancing mutations. In a preferred embodiment the Fc-Fc interaction enhancing mutations are substitutions at one or more amino acid positions selected from the group comprising: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment it is E430G.
In another embodiment of the invention the second antibody comprises a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 47, a CDR2 sequence set forth in SEQ ID NO: 48 and a CDR3 sequence set forth in SEQ ID NO: 49, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 51, and CDR2 sequence: VAT, and CDR3 sequence set forth in SEQ ID NO: 52 [37.3]; and an Fc region comprising an F405L substitution and on or more Fc-Fc interaction enhancing mutations. In a preferred embodiment the Fc-Fc interaction enhancing mutations are substitutions at one or more amino acid positions selected from the group comprising: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment it is E430G.
In yet another embodiment of the invention the second antibody comprises a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 47, a CDR2 sequence set forth in SEQ ID NO: 48 and a CDR3 sequence set forth in SEQ ID NO: 49, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 51, and CDR2 sequence: VAT, and CDR3 sequence set forth in SEQ ID NO: 52 [37.3]; and an Fc region comprising an K409R substitution and on or more Fc-Fc interaction enhancing mutations. In a preferred embodiment the Fc-Fc interaction enhancing mutations are substitutions at one or more amino acid positions selected from the group comprising: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment it is E430G.
Accordingly, the invention also relates to an anti-CD37 antibody which binds to human CD37 which antibody comprises:
(i) a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 40, a CDR2 sequence set forth in SEQ ID NO: 41 and a CDR3 sequence set forth in SEQ ID NO: 42, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 44, and CDR2 sequence: FAK, and CDR3 sequence set forth in SEQ ID NO: 45 [G28.1] or
(ii) a VH region comprising a CDR1 sequence set forth in SEQ ID NO: 47, a CDR2 sequence set forth in SEQ ID NO: 48 and a CDR3 sequence set forth in SEQ ID NO: 49, and a VL region comprising a CDR1 sequence set forth in SEQ ID NO: 51, and CDR2 sequence: VAT, and CDR3 sequence set forth in SEQ ID NO: 52 [37.3];
(iii) and wherein the antibody of (i) or (ii) comprises an Fc region comprising at least one amino acid substitution selected from the group comprising: E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W; and
(iv) wherein optionally the Fc region further comprises a mutation of either K409R or F405L.
As mentioned above the anti-CD37 antibodies of the invention may be an IgG1, IgG2, IgG3 or IgG4 isotype. In one embodiment the anti-CD37 antibody of the invention is of an IgG isotype.
In one embodiment, the antibody of the invention is human, humanized or chimeric.
In one embodiment, the antibody of the invention binds to both the human and the cynomolgos CD37 antigens.
Further Embodiments of the Invention
In another embodiment the invention relates to a composition comprising a bispecific antibody of the invention and further comprising a monospecific anti-CD37 antibody, preferably an anti-CD37 antibody having the antigen binding region of either the first or second antigen binding region of the bispecific antibody.
In one embodiment the invention relates to a pharmaceutical composition comprising a bispecific antibody of the invention or an anti-CD37 antibody of the invention and a pharmaceutically acceptable carrier.
In another embodiment the invention relates to a bispecific antibody of the invention or an antibody of the invention or a composition of the invention for use as a medicament.
In one embodiment of the invention the bispecific antibody of the invention is for use in the treatment of cancer, autoimmune disease or inflammatory disorders.
In one embodiment of the invention the anti-CD37 antibody of the invention is for use in the treatment of cancer, autoimmune disease or inflammatory disorders.
In one embodiment of the invention the composition of the invention is for use in the treatment of cancer, autoimmune disease or inflammatory disorders.
In another embodiment the invention relates to a bispecific antibody of the invention for use in the treatment of allergy, transplantation rejection or a B-cell malignancy, such as non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL), or acute lymphoblastic leukemia (ALL).
In another embodiment the invention relates to a bispecific antibody of the invention for use in the treatment of rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylids) systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosis disseminates, multiple sclerosis, inflammatory bowel disease (IBD) which includes ulcerative colitis and Crohn's disease, Chronic obstructive pulmonary disease (COPD), psoriasis, IgA nephropathy, IgM polyneuropathies, myasthenia gravis, diabetes mellitus, Reynaud's syndrome, and glomerulonephritis, pustulosis palmoplantaris (PPP), erosive lichen planus, pemphigus bullosa, epidermolysis bullosa, contact dermatitis and atopic dermatitis, polyradiculitis including Guillain-Barre syndrome.
In another embodiment the invention relates to an anti-CD37 antibody of the invention for use in the treatment of allergy, transplantation rejection or a B-cell malignancy.
In another embodiment the invention relates to an anti-CD37 antibody of the invention for use in the treatment of allergy, transplantation rejection or a B-cell malignancy, such as non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL), or acute lymphoblastic leukemia (ALL).
In another embodiment the invention relates to an anti-CD37 antibody of the invention for use in the treatment of rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylids) systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosis disseminates, multiple sclerosis, inflammatory bowel disease (IBD) which includes ulcerative colitis and Crohn's disease, Chronic obstructive pulmonary disease (COPD), psoriasis, IgA nephropathy, IgM polyneuropathies, myasthenia gravis, diabetes mellitus, Reynaud's syndrome, and glomerulonephritis, pustulosis palmoplantaris (PPP), erosive lichen planus, pemphigus bullosa, epidermolysis bullosa, contact dermatitis and atopic dermatitis, polyradiculitis including Guillain-Barre syndrome.
In another embodiment the invention relates to the bispecific antibody of the invention for use in combination with one or more further therapeutic agents. In another embodiment of the invention the anti-CD37 antibodies of the invention is for use in combination with one or more further therapeutic agents. The one or more further therapeutic agent may e.g. be selected from the group comprising: doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib and an anti-CD 20 antibody such as rituximab, ofatumumab, Obinutuzumab, Veltuzumab, Ocaratuzumab, Ocrelizumab or TRU-015.
In a preferred embodiment of the invention the further therapeutic agent is an anti-CD20 antibody.
In one embodiment of the invention the anti-CD20 antibody is capable of binding to human CD20 having the sequences set forth in SEQ ID No: 72.
In one embodiment of the invention the anti-CD20 antibody is capable of binding to cynomolgus monkey CD20 having the sequences set forth in SEQ ID No: 73.
In one embodiment of the invention the anti-CD20 antibody is capable of binding to human and cynomolgus monkey CD20 having the sequences set forth in SEQ ID Nos 72 and 73, respectively.
In one embodiment of the invention the anti-CD20 antibody is capable of binding to an epitope on human CD20, which does not comprise or require the amino acid residues alanine at position 170 or proline at position 172, but which comprises or requires the amino acid residues asparagine at position 163 and asparagine at position 166 of SEQ ID No. 72. Examples of such antibodies are the antibodies denoted 2F2 and 7D8 as disclosed in WO2004035607 (Genmab) and the antibody denoted 2C6 as disclosed in WO2005103081 (Genmab). The CDR sequences of 7D8 are disclosed in Table 1.
In one embodiment of the invention the anti-CD20 antibody is capable of binding to an epitope on human CD20, which does not comprise or require the amino acid residues alanine at position 170 or proline at position 172 of SEQ ID No. 72. An example of such an antibody is 11B8 as disclosed in WO2004035607 (Genmab). The CDR sequences of 11B8 are disclosed in Table 1.
In one embodiment of the invention the anti-CD20 antibody is capable of binding to a discontinuous epitope on human CD20, wherein the epitope comprises part of the first small extracellular loop and part of the second extracellular loop.
In one embodiment of the invention the anti-CD20 antibody is capable of binding to a discontinuous epitope on human CD20, wherein the epitope has residues AGIYAP of the small first extracellular loop and residues MESLNFIRAHTPY of the second extracellular loop.
Anti-CD20 antibodies may characterize as type-I and type II anti-CD20 antibodies. Type I anti-CD20 antibodies, have high CDC and ADCC activity, but low apoptosis activity, such as ofatumumab (2F2) and rituximab, whereas type II anti-CD20 antibodies, having low or no CDC activity, but high ADCC and apoptosis activity, such as obinutuzumab and 11B8. Also, type I antibodies induce CD20 to redistribute into large detergent resistant microdomains (rafts), whereas type II antibodies do not.
In one embodiment of the invention the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20, wherein the antigen-binding region competes for binding to human CD20 with an anti-CD20 antibody comprising the variable heavy chain (VH) sequence and variable light chain (VL) as set forth in SEQ ID No 74 and SEQ ID No 78 respectively.
In one embodiment of the invention the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20, wherein the antigen-binding region competes for binding to human CD20 with an anti-CD20 antibody comprising the variable heavy chain (VH) sequence and variable light chain (VL) as set forth in SEQ ID No 81 and SEQ ID No 109 respectively.
In one embodiment of the invention the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20, wherein the antigen-binding region competes for binding to human CD20 with an anti-CD20 antibody comprising the variable heavy chain (VH) sequence and variable light chain (VL) as set forth in SEQ ID No 94 and SEQ ID No 98 respectively.
In one embodiment of the invention the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20, wherein the antigen-binding region competes for binding to human CD20 with an anti-CD20 antibody comprising the variable heavy chain (VH) sequence and variable light chain (VL) as set forth in SEQ ID No 87 and SEQ ID No 91 respectively.
In one embodiment of the invention the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20, wherein the antigen-binding region competes for binding to human CD20 with an anti-CD20 antibody comprising the variable heavy chain (VH) sequence and variable light chain (VL) as set forth in SEQ ID No 101 and SEQ ID No 105 respectively.
In one embodiment of the invention the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20 comprising the CDR sequences:
VH CDR1 sequence set forth in SEQ ID NO:75,
VH CDR2 sequence set forth in SEQ ID NO:76,
VH CDR3 sequence set forth in SEQ ID NO:77,
VL CDR1 sequence set forth in SEQ ID NO:79
VL CDR2 sequence DAS, and
VL CDR3 sequence set forth in SEQ ID NO: 80. [7D8]
In one embodiment of the invention the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20 comprising the CDR sequences:
VH CDR1 sequence set forth in SEQ ID NO:82,
VH CDR2 sequence set forth in SEQ ID NO:83,
VH CDR3 sequence set forth in SEQ ID NO:84,
VL CDR1 sequence set forth in SEQ ID NO:85
VL CDR2 sequence DAS, and
VL CDR3 sequence set forth in SEQ ID NO: 86. [1188]
In one embodiment of the invention the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20 comprising the CDR sequences:
VH CDR1 sequence set forth in SEQ ID NO:95,
VH CDR2 sequence set forth in SEQ ID NO:96,
VH CDR3 sequence set forth in SEQ ID NO:97,
VL CDR1 sequence set forth in SEQ ID NO:99,
VL CDR2 sequence ATS, and
VL CDR3 sequence set forth in SEQ ID NO: 100. [Rituximab]
In one embodiment of the invention the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20 comprising the CDR sequences:
VH CDR1 sequence set forth in SEQ ID NO:88,
VH CDR2 sequence set forth in SEQ ID NO:89,
VH CDR3 sequence set forth in SEQ ID NO:90,
VL CDR1 sequence set forth in SEQ ID NO:92
VL CDR2 sequence DAS, and
VL CDR3 sequence set forth in SEQ ID NO: 93. [ofatumumab]
In one embodiment of the invention the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20 comprising the CDR sequences:
VH CDR1 sequence set forth in SEQ ID NO:102,
VH CDR2 sequence set forth in SEQ ID NO:103,
VH CDR3 sequence set forth in SEQ ID NO:104,
VL CDR1 sequence set forth in SEQ ID NO:106
VL CDR2 sequence QMS, and
VL CDR3 sequence set forth in SEQ ID NO: 107. [obinutuzumab]
In one embodiment of the invention the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20 comprising the CDR sequences selected form the group consisting of:
i) VH CDR1 sequence set forth in SEQ ID NO:75,
VH CDR2 sequence set forth in SEQ ID NO:76,
VH CDR3 sequence set forth in SEQ ID NO:77,
VL CDR1 sequence set forth in SEQ ID NO:79
VL CDR2 sequence DAS, and
VL CDR3 sequence set forth in SEQ ID NO: 80. [7D8];
ii) VH CDR1 sequence set forth in SEQ ID NO:82,
VH CDR2 sequence set forth in SEQ ID NO:83,
VH CDR3 sequence set forth in SEQ ID NO:84,
VL CDR1 sequence set forth in SEQ ID NO:85
VL CDR2 sequence DAS, and
VL CDR3 sequence set forth in SEQ ID NO: 86. [1188];
iii) VH CDR1 sequence set forth in SEQ ID NO:95,
VH CDR2 sequence set forth in SEQ ID NO:96,
VH CDR3 sequence set forth in SEQ ID NO:97,
VL CDR1 sequence set forth in SEQ ID NO:99,
VL CDR2 sequence ATS, and
VL CDR3 sequence set forth in SEQ ID NO: 100. [Rituximab];
iv) VH CDR1 sequence set forth in SEQ ID NO:88,
VH CDR2 sequence set forth in SEQ ID NO:89,
VH CDR3 sequence set forth in SEQ ID NO:90,
VL CDR1 sequence set forth in SEQ ID NO:92
VL CDR2 sequence DAS, and
VL CDR3 sequence set forth in SEQ ID NO: 93. [ofatumumab]; and
v) VH CDR1 sequence set forth in SEQ ID NO:102,
VH CDR2 sequence set forth in SEQ ID NO:103,
VH CDR3 sequence set forth in SEQ ID NO:104,
VL CDR1 sequence set forth in SEQ ID NO:106
VL CDR2 sequence QMS, and
VL CDR3 sequence set forth in SEQ ID NO: 107. [obinutuzumab].
In another embodiment the invention relates to use of the bispecific antibody of the invention or the anti-CD37 antibody of the invention for the manufacture of a medicament. In another embodiment hereof the use is for the manufacture of a medicament for the treatment of cancer, autoimmune diseases or an inflammatory diseases such as allergy, transplantation rejection or a B-cell malignancy, such as non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL), or acute lymphoblastic leukemia (ALL), rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylids) systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosis disseminates, multiple sclerosis, inflammatory bowel disease (IBD) which includes ulcerative colitis and Crohn's disease, Chronic obstructive pulmonary disease (COPD), psoriasis, IgA nephropathy, IgM polyneuropathies, myasthenia gravis, diabetes mellitus, Reynaud's syndrome, and glomerulonephritis, pustulosis palmoplantaris (PPP), erosive lichen planus, pemphigus bullosa, epidermolysis bullosa, contact dermatitis and atopic dermatitis, polyradiculitis including Guillain-Barre syndrome.
In another embodiment the invention relates to a method of inducing cell death, or inhibiting growth and/or proliferation of a tumor cell expressing CD37 comprising administering to an individual in need thereof an effective amount of a bispecific antibody of the invention or an anti-CD37 antibody of the invention. In certain embodiments the method is for treating an individual having allergy, transplantation rejection or a B-cell malignancy, such as non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL), or acute lymphoblastic leukemia (ALL), comprising administering to said individual an effective amount of the bispecific antibody of the invention or the anti-CD37 antibody of the invention. In certain embodiments the method comprises administering one or more further therapeutic agents in combination with said antibody or said bispecific antibody such as e.g. doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib or an anti-CD20 antibody such as rituximab, ofatumumab, obinutuzumab, veltuzumab, ocaratuzumab, ocrelizumab or TRU-015.
In one embodiment of the invention the further therapeutic agent is selected from the group comprising: cyclophosphamide, chlorambucil, bendamustine, ifosfamide, cisplatin, carboplatin, oxaliplatin, carmustine, prednisone, dexamethasone, fludarabine, pentostatin, cladribine, fluorouracil, gemcitabine, cytarabine, methotrexate, pralatrexate, gemcitabine, vincristine, paclitaxel, docetaxel, doxorubicin, mitoxantrone, etoposide, topotecan, irinotecan, bleomycin, CD20-specific rituximab, obinutuzumab and ofatumumab, CD52-specific alemtuzumab, CD30-specific brentuximab, JNJ-63709178, JNJ-64007957, HuMax-IL8, anti-DRS, anti-VEGF, anti-CD38, anti-PD-1, anti-PD-L1, anti-CTLA4, anti-CD40, anti-CD137, anti-GITR, anti-VISTA, antibodies specific for other immunomodulatory targets, brentuximab vedotin, HuMax-TAC-ADC, Interferon, thalidomide, lenalidomide, Axicabtagene ciloleucel, bortezomib, romidepsin, belinostat, vorinostat, ibrutinib, acalabrutinib, idelalisib, copanlisib, sorafenib, sunitinib, everolimus, recombinant human TRAIL, birinapant, and venetoclax.
In one embodiment of the invention the further therapeutic agent is selected from the group comprising: ibrutinib, rituximab, venetoclax, CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone), bendamustine, fludarabine, cyclophosphamide, and chlorambucil.
In one embodiment of the invention the further therapeutic agent is selected from the group comprising: ibrutinib, rituximab and venetoclax.
In a further aspect, the invention relates to a nucleic acid construct encoding one or more sequences set out in Table 1. In a further aspect the invention relates to a nucleic acid construct encoding one or more sequences selected from the group comprising SEQ ID NOs: 1, 2, 3, 4, 5, 6, 6a, 7, 8, 9, 10, 11, 12, 13, 13a, 14, 15, 16, 17, 18, 19, 20, 20a, 21, 22, 23, 24, 25, 26, 27, 27a, 28, 29, 30, 30a and 31.
The invention further relates to a nucleic acid construct encoding the VH and/or VL region of the bispecific antibody or the anti-CD37 antibody of any of the embodiments herein.
The invention further relates to a nucleic acid construct encoding the bispecific antibody or the anti-CD37 antibody of any of the embodiments herein.
In a further embodiment the invention relates to an expression vector comprising one or more nucleic acid constructs specified above. In another embodiment the invention relates to a host cell comprising an expression vector as defined above.
An expression vector in the context of the present invention may be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (a nucleic acid sequence comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, a CD37 antibody-encoding nucleic acid is comprised in a naked DNA or RNA vector, including, for example, a linear expression element (as described in for instance Sykes and Johnston, Nat Biotech 17, 355 59 (1997)), a compacted nucleic acid vector (as described in for instance U.S. Pat. No. 6,077,835 and/or WO 00/70087), a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sized nucleic acid vector (as described in for instance Schakowski et al., Mol Ther 3, 793 800 (2001)), or as a precipitated nucleic acid vector construct, such as a CaPO4-precipitated construct (as described in for instance WO200046147, Benvenisty and Reshef, PNAS USA 83, 9551 55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such nucleic acid vectors and the usage thereof are well known in the art (see for instance U.S. Pat. Nos. 5,589,466 and 5,973,972).
In one embodiment, the vector is suitable for expression of the CD37 antibodies in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503 5509 (1989), pET vectors (Novagen, Madison Wis.) and the like).
An expression vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be employed. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), and Grant et al., Methods in Enzymol 153, 516 544 (1987)).
An expression vector may also or alternatively be a vector suitable for expression in mammalian cells, e.g. a vector comprising glutamine synthetase as a selectable marker, such as the vectors described in Bebbington (1992) Biotechnology (NY) 10:169-175.
A nucleic acid and/or vector may also comprises a nucleic acid sequence encoding a secretion/localization sequence, which can target a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or into cell culture media. Such sequences are known in the art, and include secretion leader or signal peptides.
The expression vector may comprise or be associated with any suitable promoter, enhancer, and other expression-facilitating elements. Examples of such elements include strong expression promoters (e. g., human CMV IE promoter/enhancer as well as RSV, SV40, SL3 3, MMTV, and HIV LTR promoters), effective poly (A) termination sequences, an origin of replication for plasmid product in E. coli, an antibiotic resistance gene as selectable marker, and/or a convenient cloning site (e.g., a polylinker). Nucleic acids may also comprise an inducible promoter as opposed to a constitutive promoter such as CMV IE.
In one embodiment, the CD37 antibody-encoding expression vector may be positioned in and/or delivered to the host cell or host animal via a viral vector.
Thus the present invention also relates to a recombinant eukaryotic or prokaryotic host cell which produces a bispecific antibody of the present invention, such as a transfectoma.
The invention further relates to an anti-idiotypic antibody, which binds to the antigen binding region of the antibody or the bispecific antibody of the invention.
An in vitro method for detecting the presence of a human CD37 antigen or a cell expressing human CD37 in a sample, said method comprising:
(i) contacting the sample with the bispecific antibody of any of the above embodiments or the antibody of any of the embodiments herein under conditions that allow for formation of a complex between the antibody or the bispecific antibody and CD37; and
(ii) detecting the formation of a complex.
An in vivo method for detecting the presence of a human CD37 antigen, or a cell expressing human CD37 in a subject, said method comprising:
(i) administering the bispecific antibody of any of the above embodiments or the antibody of any of the embodiments herein under conditions that allow for formation of a complex between the antibody or the bispecific antibody and CD37; and
(ii) detecting the formed complex.
Sequences
TABLE 1
SEQ ID NO:
LABEL
SEQUENCE
1
VH-004-H5L2
EVQLVESGGGLVQPGGSLRLSCAASGFSLSTYDMSWVRQAPGKGLE
WVSIIYSSVGAYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVY
YCAREYGASSSDYIFSLWGQGTLVTVSS
2
VH-004-H5L2-
GFSLSTYD
CDR1
3
VH-004-H5L2-
IYSSVGA
CDR2
4
VH-004-H5L2-
AREYGASSSDYIFSL
CDR3
5
VL-004-H5L2
AQVLTQSPSPLSASVGDRVTITCQASQSVYNSQNLAWYQQKPGKAP
KLLIYEASKLASGVPSRFKGSGSGTEFTLTISSLQPDDFATYYCQGEFS
CISADCTAFGGGTKVEIK
6
VH-004-H5L2-
QSVYNSQN
CDR1
VH-004-H5L2-
EAS
CDR2
7
VH-004-H5L2-
QGEFSCISADCTA
CDR3
8
VH-005-H1L2
EQSVVESGGGLVQPGGSLRLSCTVSGFSLSSNAMNWVRQAPGKGLE
WIGLIYASGNTDYASWAKGRFTISKTSTTVYLKITSPTAEDTATYFCA
REGSVWGAAFDPWGQGTLVTVSS
9
VH-005-H1L2-
GFSLSSNA
CDR1
10
VH-005-H1L2-
IYASGNT
CDR2
11
VH-005-H1L2-
AREGSVWGAAFDP
CDR3
12
VL-005-H1L2
AYDMTQSPSSVSASVGDRVTITCQASQSISNWLAWYQQKPGKAPK
QLIYAASTLASGVPSRFKGSGSGTDFTLTISSLQPEDFATYYCQQGYS
NSNIDNTFGGGTKVEIK
13
VL-005-H1L2-
QSISNW
CDR1
VL-005-H1L2-
AAS
CDR2
14
VL-005-H1L2-
QQGYSNSNIDNT
CDR3
15
VH-010-H5L2
EVQLVESGGGLVQPGGSLRLSCAASGFSLSYNAMNWVRQAPGKGLE
WVSIIFASGRTDYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVY
YCAREGSTWGDALDPWGQGTLVTVSS
16
VH-010-H5L2-
GFSLSYNA
CDR1
17
VH-010-H5L2-
IFASGRT
CDR2
18
VH-010-H5L2-
AREGSTWGDALDP
CDR3
19
VL-010-H5L2
AYDMTQSPSTLSASVGDRVTITCQASQNIIDYLAWYQQKPGKAPKLL
IHKASTLASGVPSRFKGSGSGTEFTLTISSLQPDDFATYYCQQGYSNS
NIDNTFGGGTKVEIK
20
VL-010-H5L2-
QNIIDY
CDR1
VL-010-H5L2-
KAS
CDR2
21
VL-010-H5L2-
QQGYSNSNIDNT
CDR3
22
VH-016-H5L2
EVQLVESGGGLVQPGGSLRLSCAASGFSLSNYNMGWVRQAPGKGLE
WVSVIDASGTTYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTATY
YCARELLYFGSSYYDLWGQGTLVTVSS
23
VH-016-H5L2-
GFSLSNYN
CDR1
24
VH-016-H5L2-
IDASGTT
CDR2
25
VH-016-H5L2-
ARELLYFGSSYYDL
CDR3
26
VL-016-H5L2
DVVMTQSPSTLSASVGDRVTITCQASQNIDSNLAWYQQKPGKAPKF
LIYYASNLPFGVPSRFKGSGSGTEFTLTISSLQPDDFATYYCQCADVG
STYVAAFGGGTKVEIK
27
VL-016-H5L2-
QNIDSN
CDR1
VL-016-H5L2-
YAS
CDR2
28
VL-016-H5L2-
QCADVGSTYVAA
CDR3
29
VL-016-H5L2-
DVVMTQSPSTLSASVGDRVTITCQASQNIDSNLAWYQQKPGKAPKF
C905
LIYYASNLPFGVPSRFKGSGSGTEFTLTISSLQPDDFATYYCQSADVG
STYVAAFGGGTKVEIK
30
VL-016-H5L2-
QNIDSN
C905-CDR1
VL-016-H5L2-
YAS
C905-CDR2
31
VL-016-H5L2-
QSADVGSTYVAA
C905-CDR3
32
VH-b12
QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIHWVRQAPGQRFE
WMGWINPYNGNKEFSAKFQDRVTFTADTSANTAYMELRSLRSADTA
VYYCARVGPYSWDDSPQDNYYMDVWGKGTTVIVSS
33
VH-b12-CDR1
GYRFSNFV
34
VH-b12-CDR2
INPYNGNK
35
VH-b12-CDR3
ARVGPYSWDDSPQDNYYMDV
36
VL-b12
EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQHKPGQAPRL
VIHGVSNRASGISDRFSGSGSGTDFTLTITRVEPEDFALYYCQVYGAS
SYTFGQGTKLERK
37
VL-b12-CDR1
HSIRSRR
VL-b12-CDR2
GVS
38
VL-b12-CDR3
QVYGASSYT
39
VH-G28.1
AVQLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQNNGKSLE
WIGNIDPYYGGTTYNRKFKGKATLTVDKSSSTAYMQLKSLTSEDSAV
YYCARSVGPMDYWGQGTSVTVSS
40
VH-G28.1-CDR1
GYSFTGYN
41
VH-G28.1-CDR2
IDPYYGGT
42
VH-G28.1-CDR3
ARSVGPMDY
43
VL-G28.1
DIQMTQSPASLSASVGETVTITCRTSENVYSYLAWYQQKQGKSPQLL
VSFAKTLAEGVPSRFSGSGSGTQFSLKISSLQPEDSGSYFCQHHSDN
PWTFGGGTELEIK
44
VL-G28.1-CDR1
ENVYSY
VL-G28.1-CDR2
FAK
45
VL-G28.1-CDR3
QHHSDNPWT
46
VH-37.3
QVQVKESGPGLVAPSQSLSITCTVSGFSLTTSGVSWVRQPPGKGLE
WLGVIWGDGSTNYHSALKSRLSIKKDHSKSQVFLKLNSLQTDDTAT
YYCAKGGYSLAHWGQGTLVTVSA
47
VH-37.3-CDR1
GFSLTTSG
48
VH-37.3-CDR2
IWGDGST
49
VH-37.3-CDR3
AKGGYSLAH
50
VL-37.3
DIQMTQSPASLSVSVGETVTITCRASENIRSNLAWYQQKQGKSPQLL
VNVATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHYWG
TTWTFGGGTKLEIK
51
VL-37.3-CDR1
ENIRSN
VL-37.3-CDR2
VAT
52
VL-37.3-CDR3
QHYWGTTWT
53
IgG1-Fc
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
54
IgG1-Fc-delK
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.
55
IgG1-E430G-Fc
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHGALHNHYTQKSLSLSPGK
56
IgG1-E345R-Fc
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRRPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
K
57
IgG1-F405L-Fc
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
58
IgG1-K409R-Fc
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
59
IgG1-F405L-
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
E430G-Fc
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFLLYSKLTVDKSRWQQGNVFSCSVMHGALHNHYTQKSLSLSPGK
60
IgG1-K409R-
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
E430G-Fc
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQQGNVFSCSVMHGALHNHYTQKSLSLSPGK
61
Kappa-C
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC
62
Human CD37
MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLA
FVPLQIWSKVLAISGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFA
TQITLGILISTQRAQLERSLRDVVEKTIQKYGTNPEETAAEESWDYVQ
FQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTIL
DKVILPQLSRLGHLARSRHSADICAVPAESHIYREGCAQGLQKWLHN
NLISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR
63
Cynomolgus
MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLA
CD37 (mfCD37)
FVPLQIWSKVLAISGVFTMGLALLGCVGALKELRCLLGLYFGMLLLLFA
TQITLGILISTQRAQLERSLQDIVEKTIQKYHTNPEETAAEESWDYVQF
QLRCCGWHSPQDWFQVLTLRGNGSEAHRVPCSCYNLSATNDSTILD
KVILPQLSRLGQLARSRHSTDICAVPANSHIYREGCARSLQKWLHNN
LISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR
64
CD37EC2-FcHis
MWWRLWWLLLLLLLLWPMVWARAQLERSLRDVVEKTIQKYGTNPEE
TAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSC
YNLSATNDSTILDKVILPQLSRLGHLARSRHSADICAVPAESHIYREG
CAQGLQKWLHNNPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTAPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGKHHHHHHHH
65
CD37MfEC2-FcHis
MWWRLWWLLLLLLLLWPMVWARAQLERSLQDIVEKTIQKYHTNPEE
TAAEESWDYVQFQLRCCGWHSPQDWFQVLTLRGNGSEAHRVPCSC
YNLSATNDSTILDKVILPQLSRLGQLARSRHSTDICAVPANSHIYREG
CARSLQKWLHNNPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR
EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTAPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGKHHHHHHHH
66
IgG1-F405L-
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
E345R
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRRPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
K
67
IgG1-F405L-
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
E345K
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRKPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
68
IgG1-F405L-
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
E430S
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFLLYSKLTVDKSRWQQGNVFSCSVMHSALHNHYTQKSLSLSPGK
69
IgG1-K409R-
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
E345R
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRRPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
K
70
IgG1-K409R-
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
E345K
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRKPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
71
IgG1-K409R-
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
E430S
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQQGNVFSCSVMHSALHNHYTQKSLSLSPGK
72
Human CD20
MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMR
ESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYII
SGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISH
FLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILS
VMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKE
EVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIEN
DSSP
73
Cynomolgus
MTTPRNSVNGTFPAEPMKGPIAMQPGPKPLLRRMSSLVGPTQSFFMR
monkey CD20
ESKALGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYII
SGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISH
FLKMESLNFIRVHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILS
VMLIFAFFQELVIAGIVENEWRRTCSRPKSSVVLLSAEEKKEQVIEIKE
EVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIEN
DSSP
74
VH CD20-7D8
EVQLVESGGGLVQPDRSLRLSCAASGFTFHDYAMHWVRQAPGKGL
EWVSTISWNSGTIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDT
ALYYCAKDIQYGNYYYGMDVWGGTTVTVSS
75
VH CD20-7D8
GFTFHDYA
CDR1
76
VH CD20-7D8
ISWNSGTI
CDR2
77
VH CD20-7D8
AKDIQYGNYYYGMDV
CDR3
78
VL CD20-7D8
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL
IYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNW
PITFGQGTRLEIK
79
VL CD20-7D8
QSVSSY
CDR1
VL CD20-7D8
DAS
CDR2
80
VL CD20-7D8
QQRSNWPIT
CDR3
81
VH CD20-11B8
EVQLVQSGGGLVHPGGSLRLSCTGSGFTFSYHAMHWVRQAPGKGL
EWVSIIGTGGVTYYADSVKGRFTISRDNVKNSLYLQMNSLRAEDMA
VYYCARDYYGAGSFYDGLYGMDVWGGTTVTVSS
82
VH CD20-11B8
GFTFSYHA
CDR1
83
VH CD20-11B8
IGTGGVT
CDR2
84
VH CD20-11B8
ARDYYGAGSFYDGLYGMDV
CDR3
85
VL CD20-11B8
QSVSSY
CDR1
VL CD20-11B8
DAS
CDR2
86
VL CD20-11B8
QQRSDWPLT
CDR3
87
VH CD20-
EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGL
ofatumumab
EWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDT
ALYYCAKDIQYGNYYYGMDVWGGTTVTVSS
88
VL CD20-
GFTFNDYA
ofatumumab
CDR1
89
VH CD20-
ISWNSGSI
ofatumumab
CDR2
90
VH CD20-
AKDIQYGNYYYGMDV
ofatumumab
CDR3
91
VL CD20-
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLL
ofatumumab
IYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNW
PITFGQGTRLEIK
92
VL CD20-
QSVSSY
ofatumumab
CDR1
VL CD20-
DAS
ofatumumab
93
VL CD20-
QQRSNWPIT
ofatumumab
CDR3
94
VH CD20-
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRG
rituximab
LEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSED
SAVYYCARST
YYGGDWYFNVWGAGTTVTVSA
95
VH CD20-
GYTFTSYN
rituximab CDR1
96
VH CD20-
IYPGNGDT
rituximab CDR2
97
VH CD20-
ARSTYYGGDWYFNV
rituximab CDR3
98
VL CD20-
QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWI
rituximab
YATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSN
PPTFGGGTKLEIK
99
VL CD20-
SSVSY
rituximab CDR1
VL CD20-
ATS
rituximab CDR2
100
VL CD20-
QQWTSNPPT
rituximab CDR3
101
VH CD20-
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQG
obinutuzumab
LEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSED
TAVYYCARNVFDGYWLVYWGQGTLVTVSS
102
VH CD20-
GYAFSYSW
obinutuzumab
CDR1
103
VH CD20-
IFPGDGDT
obinutuzumab
CDR2
104
VH CD20-
ARNVFDGYWLVY
obinutuzumab
CDR3
105
VL CD20-
DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQ
obinutuzumab
SPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCA
QNLELPYTFGGGTKVEIK
106
VL CD20-
KSLLHSNGITY
obinutuzumab
CDR1
VL CD20-
QMS
obinutuzumab
CDR2
107
VL CD20-
AQNLELPYT
obinutuzumab
CDR3
108
IgG1-
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
S239D-I332E
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP
DVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSRE
EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
109
VL CD20 1188
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPAR
FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDWPLTFGGGTKVEIK
110
VH CD37-004
QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYDMSWVRQAPGKGLEWI
GIIYSSVGAYYASWAKGRFTFSKTSTTVDLKITSPTTEDTATYFCAREY
GASSSDYIFSLWGQGTLVTVSS
2
VH CD37-004
GFSLSTYD
CDR1
3
VH CD37-004
IYSSVGA
CDR2
4
VH CD37-004
AREYGASSSDYIFSL
CDR3
111
VL CD37-004
AQVLTQTPSPVSAAVGGTVTINCQASQSVYNSQNLAWYQQKPGQPP
KLLIYEASKLASGVPSRFKGSGSGTQFTLTISGVQSDDAATYYCQGEF
SCISADCTAFGGGTEVVVK
6
VL CD37-004
QSVYNSQN
CDR1
VL CD37-004
EAS
CDR2
7
VL CD37-004
QGEFSCISADCTA
CDR3
112
VH CD37-005
QSVEESGGRLVTPGTPLTLTCTVSGFSLSSNAMNWVRQAPGKGLEW
IGLIYASGNTDYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARE
GSVWGAAFDPWGPGTLVTVSS
9
VH CD37-005
GFSLSSNA
CDR1
10
VH CD37-005
IYASGNT
CDR2
11
VH CD37-005
AREGSVWGAAFDP
CDR3
113
VL CD37-005
AYDMTQTPASVEVAVGGTVTIKCQASQSISNWLAWYQQKPGQPPKQ
LIYAASTLASGVPSRFKGSGSGTQFTLTISGVESADAATYYCQQGYSN
SNIDNTFGGGTEVVVK
13
VL CD37-005
QSISNW
CDR1
VL CD37-005
AAS
CDR2
14
VL CD37-005
QQGYSNSNIDNT
CDR3
114
VH CD37-010
QSVEESGGRLVTPGTPLTLTCTVSGFSLSYNAMNWVRQAPGKGLEWI
GIIFASGRTDYASWAKGRFTISKTSTTVELKITSPTTEDTATYFCAREG
STWGDALDPWGPGTLVTVSS
16
VH CD37-010
GFSLSYNA
CDR1
17
VH CD37-010
IFASGRT
CDR2
18
VH CD37-010
AREGSTWGDALDP
CDR3
115
VL CD37-010
AYDMTQTPSSVEAAVGGTVTIKCQASQNIIDYLAWYQQKPGQPPQLL
IHKASTLASGVPSRFKGSGSGTQFTLTISGVQSDDAATYYCQQGYSN
SNIDNTFGGGTEVVVK
20
VL CD37-010
QNIIDY
CDR1
VL CD37-010
KAS
CDR2
21
VL CD37-010
QQGYSNSNIDNT
CDR3
116
VH CD37-016
QSVEESGGRLVTPGTPLTLTCTVSGFSLSNYNMGWVRQAPGKGLEW
IGVIDASGTTYYATWAKGRFTCSKTSSTVELKMTSLTTEDTATYFCAR
ELLYFGSSYYDLWGQGTLVTVSS
23
VH CD37-016
GFSLSNYN
CDR1
24
VH CD37-016
IDASGTT
CDR2
25
VH CD37-016
ARELLYFGSSYYDL
CDR3
117
VL CD37-016
DVVMTQTPASVSEPVGGTVTIKCQASQNIDSNLAWYQQKPGQPPKF
LIYYASNLPFGVSSRFKGSGSGTQFTLTISDLESADAATYYCQCADVG
STYVAAFGGGTEVVVK
27
VL CD37-016
QNIDSN
CDR1
VL CD37-016
YAS
CDR2
28
VL CD37-016
QCADVGSTYVAA
CDR3
EXAMPLES
Example 1: Generation of CD37 Specific Antibodies in Rabbits
Expression Constructs for CD37
The following codon-optimized constructs for expression of full-length CD37 variants were generated: human (Homo sapiens) CD37 (Genbank accession no. NP_001765) (SEQ ID NO: 62), cynomolgus monkey (Macaca fascicularis) CD37 ((mfCD37) (SEQ ID NO: 63). In addition, the following codon-optimized constructs for expression of various CD37 ECD variants were generated: a signal peptide encoding sequence followed by the second extracellular domain (EC2) of human CD37 (aa 112-241), fused to the Fc (CH2-CH3) domain of human IgG with a C-terminal His tag (CD37EC2-FcHis, SEQ ID NO: 64), and a similar construct for mfCD37 (CD37mfEC2-FcHis, SEQ ID NO: 65). The constructs contained suitable restriction sites for cloning and an optimal Kozak (GCCGCCACC) sequence [Kozak et al. (1999) Gene 234: 187-208]. The constructs were cloned in the mammalian expression vector pcDNA3.3 (Invitrogen) or an equivalent vector.
Transient Expression in CHO and HEK Cells
Membrane proteins were transiently transfected in Freestyle 293-F (HEK293F) cells (Life technologies, USA) using 293fectin (Life technologies) essentially as described by the manufacturer, or in Freesyle CHO-S cells (CHO) (Life technologies) by using the Freestyle Max reagent (Life technologies) essentially as described by the manufacturer. Soluble proteins were transiently expressed in Expi293 cells (Life technologies) by using the ExpiFectamine 293 reagent (Life technologies), essentially as described by the manufacturer.
The Fc fusion proteins (CD37mfEC2-FcHis and CD37EC2-FcHis) were purified from cell culture supernatant using protein A affinity chromatography.
Immunization of Rabbits
Immunization of rabbits was performed at MAB Discovery GMBH (Neuried, Germany). Rabbits were repeatedly immunized with a mixture of CD37EC2-FcHis and CD37mfEC2-FcHis or HEK293F cells transiently expressing human or mfCD37. The blood of these animals was collected and B lymphocytes were isolated. Using a MAB Discovery proprietary process, single B-cells were sorted into wells of microtiter plates and further propagated. The supernatants of these single B-cells were analyzed for specific binding to CHO-S cells transiently expressing CD37 (CHO-CD37) and mfCD37 (CHO-mfCD37).
Recombinant Antibody Production
Upon analyzing the primary screening results, primary hits were selected for sequencing, recombinant mAb production and purification. Unique variable heavy chain (VH) and light chain (VL) encoding regions were gene synthesized and cloned into mammalian expression vectors containing the human IgG1 constant region encoding sequences (Ig Kappa chain and IgG1 allotype G1m (f) containing an E430G mutation (EU numbering) heavy chain). During this process an unfavorable, unpaired cysteine in some antibody light chains was replaced by a serine.
Recombinant chimeric antibodies were produced in HEK 293 cells by transiently cotransfecting the heavy chain (HC) and light chain (LC) encoding expression vectors using an automated procedure on a Tecan Freedom Evo platform. Immunoglobulins were purified from the cell supernatant using affinity purification (Protein A) on a Dionex Ultimate 3000 HPLC system.
The reactivity of the produced chimeric (VH rabbit, Fc human) monoclonal antibodies (mAbs) containing a mutation E430G was re-analyzed for binding to CHO-CD37 or CHO-mfCD37 cells. In addition, binding to the human lymphoma cell line Daudi and functionality in the CDC assay on Daudi cells was analyzed.
Example 2: Humanization of Rabbit Chimeric Antibodies
Generation of Humanized Antibody Sequences
Humanized antibody sequences from rabbit antibodies rabbit-anti-CD37-004, -005, -010 and -016 were generated at Antitope (Cambridge, UK). Humanized antibody sequences were generated using germline humanization (CDR-grafting) technology. Humanized V region genes were designed based upon human germline sequences with closest homology to the VH and Vκ amino acid sequences of the rabbit and murine antibodies. A series of four to six VH and four or five VK (VL) germline humanized V-region genes were designed for each of the rabbit antibodies.
Structural models of the rabbit antibody V regions were produced using Swiss PDB and analyzed in order to identify amino acids in the V region frameworks that may be important for the binding properties of the antibody. These amino acids were noted for incorporation into one or more variant CDR-grafted antibodies.
The heavy and light chain V region amino acid sequence were compared against a database of human germline V and J segment sequences in order to identify the heavy and light chain human sequences with the greatest degree of homology for use as human variable domain frameworks. The germline sequences used as the basis for the humanized designs are shown in Table 2.
TABLE 2
Closest matching human germline V segment and J segment
sequences.
Heavy chain
Light chain (κ)
Human V
Human J
Human V
Human J
region
region
region
region
Rabbit anti-
germline
germline
germline
germline
CD37-
segment
segment
segment
segment
004
IGHV3-53*04
IGHJ4
IGKV1-5*01
IGKJ4
005
IGHV3-53*04
IGHJ4
IGKV1-12*01
IGKJ4
010
IGHV3-53*04
IGHJ4
IGKV1-5*03
IGKJ4
016
IGHV3-53*04
IGHJ4
IGKV1-12*01
IGKJ4
A series of humanized heavy and light chain V regions were then designed by grafting the CDRs onto the frameworks and, if necessary, by back-mutating residues which may be critical for the antibody binding properties, as identified in the structural modelling, to rabbit residues. Variant sequences with the lowest incidence of potential T cell epitopes were then selected using Antitope's proprietary in silico technologies, iTope™ and TCED™ (T Cell Epitope Database) (Perry, L. C. A, Jones, T. D. and Baker, M. P. New Approaches to Prediction of Immune Responses to Therapeutic Proteins during Preclinical Development (2008). Drugs in R&D 9 (6): 385-396; Bryson, C. J., Jones, T. D. and Baker, M. P. Prediction of Immunogenicity of Therapeutic Proteins (2010). Biodrugs 24 (1):1-8). Finally, the nucleotide sequences of the designed variants have been codon-optimized.
For antibody IgG1-016-H5L2 a variant with a point mutation in the variable domain was generated to replace a free cysteine: IgG1-016-H5L2-LC90S (also generated with additional F405L and E430G mutations). This mutant was generated by gene synthesis (Geneart).
The variable region sequences of the humanized CD37 antibodies are shown in the Sequence Listing herein and in Table 1 above.
Example 3: Generation of Bispecific Antibodies
Bispecific IgG1 antibodies were generated by Fab-arm-exchange under controlled reducing conditions. The basis for this method is the use of complementary CH3 domains, which promote the formation of heterodimers under specific assay conditions as described in WO2011/131746. The F405L and K409R (EU numbering) mutations were introduced in CD37 antibodies to create antibody pairs with complementary CH3 domains. The F405L and K409R mutations were in certain cases combined with E430G mutation.
To generate bispecific antibodies, the two parental complementary antibodies, each antibody at a final concentration of 0.5 mg/mL, were incubated with 75 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 μL TE at 31° C. for 5 hours. The reduction reaction was stopped by removing the reducing agent 2-MEA using spin columns (Microcon centrifugal filters, 30 k, Millipore) according to the manufacturer's protocol.
Example 4: Expression Constructs for Antibodies, Transient Expression and Purification
For antibody expression the VH and VL sequences were cloned in expression vectors (pcDNA3.3) containing, in case of the VH, the relevant constant heavy chain (HC), in certain cases containing a F405L or K409R mutation and/or an E345R or E430G mutation, and, in case of the VL, light chain (LC) regions.
Antibodies were expressed as IgG1,κ. Plasmid DNA mixtures encoding both heavy and light chains of antibodies were transiently transfected in Expi293F cells (Life technologies, USA) using 293fectin (Life technologies) essentially as described by Vink et al. (Vink et al., Methods, 65 (1), 5-10 2014). Next, antibodies were purified by immobilized protein G chromatography.
The following antibodies were used in the examples:
Wild-Type IgG1 Antibodies:
IgG1-004-H5L2 (having the VH and VL sequences set forth in SEQ ID NO:1 and SEQ ID NO:5)
IgG1-005-H1L2 (having the VH and VL sequences set forth in SEQ ID NO:8 and SEQ ID NO:12)
IgG1-010-H5L2 (having the VH and VL sequences set forth in SEQ ID NO:15 and SEQ ID NO:19)
IgG1-016-H5L2 (having the VH and VL sequences set forth in SEQ ID NO:22 and SEQ ID NO:26)
IgG1-G28.1 (having the VH and VL sequences set forth in SEQ ID NO:39 and SEQ ID NO:43—based on SEQ ID No 1 and 3 in EP2241577)
IgG1-G28.1-K409R-delK (also containing a C-terminal heavy chain mutation 445-PG-446)
IgG1-37.3 (having the VH and VL sequences set forth in SEQ ID NO:46 and SEQ ID NO:50—based on SEQ ID No 55 and 72 in WO2011/112978)
IgG1-b12 ((having the VH and VL sequences set forth in SEQ ID NO:32 and SEQ ID NO:36—based on the gp120 specific antibody b12 [Barbas, CF. J Mol Biol. 1993 Apr. 5; 230(3):812-23])
IgG1 Antibodies with Fc-Fc Interaction-Enhancing Mutation E430G:
IgG1-004-H5L2-E430G
IgG1-005-H1L2-E430G
IgG1-010-H5L2-E430G
IgG1-016-H5L2-E430G
IgG1-G28.1-E430G
IgG1-37.3-E430G
IgG1-b12-E430G
IgG1-005-H1L2-K409R-E430G
IgG1-010-H5L2-K409R-E430G
IgG1-016-H5L2-F405L-E430G
IgG1-016-H5L2-LC90S-F405L-E430G
IgG1-004-E430G
IgG1-005-E430G
IgG1-010-E430G
IgG1-016-E430G
IgG1 Antibodies with Fc-Fc Interaction-Enhancing Mutation E430S:
IgG1-010-H5L2-K409R-E430S
IgG1-016-H5L2-F405L-E430S
IgG1 Antibodies with Fc-Fc Interaction Enhancing Mutation E345K:
IgG1-010-H5L2-K409R-E345K
IgG1-016-H5L2-F405L-E345K
IgG1 Antibodies with Fc-Fc Interaction Enhancing Mutation E345R:
IgG1-G28.1-E345R
IgG1-b12-E345R
IgG1-010-H5L2-K409R-E345R
IgG1-016-H5L2-F405L-E345R
Bispecific Antibodies
bsIgG1-016-H5L2-F405LxIgG1-IgG1-005-H1L2-K409R
bsIgG1-016-H5L2-F405LxIgG1-010-H5L2-K409R
Bispecific Antibodies with Fc-Fc Interaction Enhancing Mutation E430G:
bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G
bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G
bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G
bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G
bsIgG1-b12-F405L-E430Gx005-H1L2-K409R-E430G
IgG1 antibody with FcγR-interaction enhancing mutation 5239D-I332E:
IgG1-G28.1-5239D-I332E
Example 5: Introduction of an Fc-Fc Interaction Enhancing Mutation into CD37 Antibodies Results in Enhanced, De Novo Capacity to Induce Complement Dependent Cytotoxicity (CDC)
Determination of Complement Dependent Cytotoxicity (CDC)
In a first experiment, tumor cells derived from an untreated CLL patient (AllCells, California, USA), were resuspended in RPMI containing 0.2% BSA (bovine serum albumin) and plated into polystyrene 96-well round-bottom plates (Greiner bio-one Cat #650101) at a density of 0.2×105 cells/well (40 μL/well) and 40 μL of a concentration series of IgG1-G28.1-K409R-delK, IgG1-G28.1-6345R or IgG1-b12-6345R (0.003-10 μg/mL final antibody concentration). IgG1-b12-E345R (based on the gp120 specific antibody b12 [Barbas, CF. J Mol Biol. 1993 Apr. 5; 230(3):812-23]) was used as negative control. For IgG1-G28.1-K409R-delK, it should be noted that the K409R mutation has no effect on binding capacity or capacity to induce CDC. Similarly, the delK (445-PG-446) mutation, which had been introduced into the antibody to facilitate biochemical analysis, did not affect target binding or capacity to induce CDC (see below).
After incubation (RT, 10 min while shaking), 20 μL of pooled normal human serum (NHS Cat #M0008 Sanquin, Amsterdam, The Netherlands) was added to each well as a source of complement and plates were incubated at 37° C. for 45 minutes. The reaction was stopped by cooling the plates on ice. Next, propidium iodide (PI; 10 μL of a 10 μg/mL solution; Sigma-Aldrich Chemie B.V., Zwijndrecht, The Netherlands) was added and lysis was detected by measurement of the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry (FACS Canto II; BD Biosciences). Graphs were generated using best-fit values of a non-linear dose-response fit with log-transformed concentrations in GraphPad Prism V6.04 software (GraphPad Software, San Diego, Calif., USA).
In a second experiment, tumor cells from another untreated CLL patient (AllCells, California, USA) were resuspended in RPMI containing 0.2% BSA, were plated into polystyrene 96-well round-bottom plates (Greiner bio-one Cat #650101) at a density of 0.5×105 cells/well (30 μL/well) and 50 μL of a concentration series of IgG1-G28.1, IgG1-G28.1-6430G or IgG1-b12 was added (0.003-10 μg/mL final antibody concentration in 3.33× serial dilutions). After incubation (RT, 15 min), 20 μL of pooled normal human serum (NHS Cat #M0008 Sanquin, Amsterdam, The Netherlands) was added to each well as a source of complement and plates were incubated at 37° C. for 45 minutes. The reaction was stopped by cooling the plates on ice. Next, propidium iodide (PI; 20 μL of a 10 μg/mL solution; Sigma-Aldrich Chemie B.V., Zwijndrecht, The Netherlands) was added and lysis was detected by measurement of the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry (FACS Canto II; BD Biosciences). Graphs were generated using best-fit values of a non-linear dose-response fit with log-transformed concentrations in GraphPad Prism V6.04 software (GraphPad Software, San Diego, Calif., USA).
FIGS. 1A and B show that CD37 antibody G28.1 without the Fc-Fc interaction enhancing E345R or E430G mutation (IgG1-G28.1 or IgG1-G28.1-K409R-delK) did not induce CDC on primary tumor cells from CLL patients, whereas G28.1 with the Fc-Fc interaction enhancing mutations E345R or E430G (IgG1-G28.1-E345R or IgG1-G28.1-E430G) induced profound, dose-dependent CDC of primary CLL cells.
Quantitative Determination of Cell Surface Antigens by Flow Cytometry (Qifi)
The CD37 and membrane complement regulatory proteins (mCRP; CD46, CD55 and CD59) expression levels on CLL tumor cells were determined using the Human IgG Calibrator Kit (Biocytix Cat #CP010). Briefly, tumor cells derived from a CLL patient (as in first experiment described above), resuspended in RPMI containing 0.2% BSA, were plated into polystyrene 96-well round-bottom plates (Greiner bio-one Cat #650101) at a density of 0.5×105 cells/well (30 μL/well), centrifuged and 50 μL of CD37 (Abcam, cat. no. 76522) or control mouse antibody (Purified Mouse IgG1,κ Isotype Control, Clone MOPC-21; BD cat. no. 555746) was added. After incubation (4° C., 30 min), 50 μL of calibration beads were added into separate wells. After washing the beads and cells twice (150 μL FACS buffer, centrifuging for 3 minutes at 300×g at 4° C. in between wash steps), 50 μL/well secondary antibody (FITC-conjugated) dilution, as provided in the Human IgG Calibrator Kit, was added. After incubation in the dark (4° C., 45 min) cells were washed twice with FACS buffer and cells were resuspended in 35 μL FACS buffer and analyzed by flow cytometry (Intellicyt iQue™ screener). The antigen quantity was determined by calculating the antibody-binding capacity based on the calibration curve, according to the manufacturer's guidelines.
FIG. 2 shows that CD37 was highly expressed on primary tumor cells from this CLL patient. The patient showed normal expression levels of mCRP's.
Example 6: Binding of CD37 Antibodies and Variants Thereof to Cell Surface Expressed CD37
Binding to cell surface expressed CD37 (Daudi cells, CHO cells expressing cynomolgus CD37) was determined by flow cytometry. Cells, resuspended in RPMI containing 0.2% BSA, were seeded at 100,000 cells/well in polystyrene 96 well round-bottom plates (Greiner bio-one Cat #650101) and centrifuged for 3 minutes at 300×g, 4° C. Serial dilutions (0.003-10 μg/mL final antibody concentration in 3.33× serial dilutions) of CD37 or control antibodies were added and cells were incubated for 30 minutes at 4° C. Plates were washed/centrifuged twice using FACS buffer (PBS/0.1% BSA/0.01% Na-Azide). Next, cells were incubated for 30 minutes at 4° C. with R-Phycoerythrin (PE)-conjugated goat-anti-human IgG F(ab′)2 (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.; cat #: 109-116-098) diluted 1/100 in PBS/0.1% BSA/0.01% Na-Azide. Cells were washed/centrifuged twice using FACS buffer, resuspended in 30 μL FACS buffer and analyzed by determining mean fluorescent intensities using an Intellicyt iQue™ screener (Westburg). Binding curves were generated using non-linear regression (sigmoidal dose-response with variable slope) analyses within GraphPad Prism V6.04 software (GraphPad Software, Sand Diego, Calif., USA).
Binding to Daudi Cells
FIG. 3 shows that humanized CD37 antibodies IgG1-004-H5L2, IgG1-005-H1L2, IgG1-010-H5L2 and IgG1-016-H5L2 showed dose-dependent binding to Daudi cells. Introduction of the Fc-Fc interaction enhancing E430G mutation, and for IgG1-005-H1L2 also the K409R mutation, into these antibodies did not affect the binding.
FIG. 4 shows that introduction of the E430G mutation into IgG1-G28.1 or IgG1-37.3 did not affect the binding to Daudi cells.
For antibody IgG1-016-H5L2 a variant with a point mutation in the variable domain was generated to replace a free cysteine in the light chain: IgG1-016-H5L2-LC90S. This variant was also generated with additional F405L and E430G mutations that were previously shown to not affect target binding characteristics. FIG. 5 shows that the IgG1-016-H5L2, IgG1-016-H5L2-E430G, IgG1-016-H5L2-F405L-E430G and IgG1-016-H5L2-LC90S-F405L-E430G all showed comparable binding to Daudi cells, thus that the LC90S mutation did not affect binding.
Binding to CHO Cells Expressing Cynomolgus Monkey CD37
Binding to CHO cells expressing cynomolgus monkey CD37 was determined by flow cytometry using a method as described above. FIG. 6 shows that IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G and IgG1-016-H5L2-E430G showed dose-dependent binding to CHO cells expressing cynomolgus monkey CD37. IgG1-G28.1 and IgG1-28.1-E430G did not bind to CHO cells expressing cynomolgus CD37.
Example 7: Identification of CD37 Antibodies that do not Compete for Binding to CD37
(Lack of) Binding Competition—Determined by Flow Cytometry
CD37 antibodies were labeled with Alexa Fluor 488 NHS Ester (Succinimidyl Ester). 1 mg of CD37 antibody (dissolved in PBS) was transferred to a 1 ml micro-centrifuge vial (reaction vial). The pH was raised by addition of a 10% volume of 1 M sodium bicarbonate buffer (pH 9). Immediately before use, 1 mg Alexa Fluor 488 NHS Ester (adjusted to room temperature) was dissolved in 100 μL DMSO. The labeling reaction was initiated by addition of 10 μL of the fresh Alexa dye solution per mg antibody. Reaction vials were capped and mixed gently by inversion. After 1 hour incubation at room temperature, the reaction was quenched by addition of 50 μL 1M Tris to each reaction vial. Unreacted dye was removed from the Alexa-labeled antibody by gel filtration using BioRad PDP10 columns equilibrated with borate saline buffer, according to the manufacturer's directions. Alexa-labeled antibodies were stored at 4° C. and protected from light.
Binding competition between different CD37 antibodies was determined by flow cytometry. Raji cells (ATCC, CCL-86) were resuspended in Raji medium (RPMI 1640, 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mM HEPES and 1 mM pyruvate) at a concentration of 1×107 cells/mL. Next, 30 μL aliquots of the cell suspension were transferred into FACS tubes together with 30 μL aliquots (40 μg/mL final concentration) of unlabeled antibody solutions. The mixture was incubated at 37° C. for 15 min while shaking gently. Next, A488-labeled antibody dilutions were prepared and after incubation, 10 μL of the labeled antibodies (4 μg/mL final antibody concentration) was transferred to the FACS tubes containing the unlabeled antibodies and cells. The mixture was incubated at 37° C. for 15 min while shaking gently. After incubation, samples were quenched by adding 4 mL of ice cold PBS, centrifuged for 3 min at 4° C. at 2000 rpm, aspirated twice and subsequently resuspended in 125 μL of PBS. Binding competition was analyzed by determining mean fluorescent intensities using a BD FACSCalibur (BD Biosciences). Fluorescence intensities were converted to Molecules of Equivalent Soluble Fluorochome (MESF) for quantitation.
FIG. 7A and FIG. 8 show that pre-incubation of Raji cells with IgG1-005-H1L2-E430G and IgG1-010-H5L2-E430G blocked subsequent binding of IgG1-005-H1L2-E430G and IgG1-010-H5L2E430G, but not of IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G and IgG1-016-H5L2-E430G.
Pre-incubation of Raji cells with IgG1-004-H5L2-E430G substantially reduced subsequent binding of IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G and IgG1-016-H5L2-E430G, but not of IgG1-005-H1L2-E430G and IgG1-010-H5L2-E430G.
Pre-incubation of Raji cells with IgG1-016-H5L2-E430G blocked subsequent binding of IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G and IgG1-016-H5L2-E430G, but not of IgG1-005-H1L2-E430G and IgG1-010-H5L2-E430G.
Pre-incubation of cells with IgG1-37.3-E430G blocked the subsequent binding of all tested antibodies. However, as discussed above pre-incubating with either of IgG1-005-H1L2-E430G or IgG1-010-H5L2-E430G did not block the binding of IgG1-37.3-E430G.
Pre-incubation of cells with IgG1-G28.1-E430G blocked the subsequent binding of IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G and IgG1-016-H5L2-E430G, but not of IgG1-005-H1L2-E430G and IgG1-010-H5L2-E430G.
(Lack of) Binding Competition—Determined by Functional Screening Using a CDC Assay
To determine whether non-cross-blocking CD37 antibodies show enhanced CDC when combined, and to confirm the potential to functionally combine non-cross-blocking CD37 antibodies, a CDC assay using individual CD37 antibodies and combinations thereof was performed.
Raji cells, resuspended in RPMI containing 0.2% BSA, were plated into polystyrene 96-well round-bottom plates (Greiner bio-one Cat #650101) at a density of 1×105 cells/well (30 μL/well) and 50 μL of humanized CD37 antibodies, variants thereof, combinations thereof or control antibody IgG1-b12 was added (10 μg/mL final antibody concentration, combinations 5+5 μg/mL). After incubation (RT, 15 min, while shaking), 20 μL of pooled normal human serum (NHS Cat #M0008 Sanquin, Amsterdam, The Netherlands) was added to each well and plates were incubated at 37° C. for 45 minutes. Plates were centrifuged (3 minutes, 1200 rpm) and supernatant was discarded. Propidium iodide (PI; 30 μL of a 1.67 μg/mL solution; Sigma-Aldrich Chemie B.V., Zwijndrecht, The Netherlands) was added and lysis was detected by measurement of the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry (Intellicyt iQue™ screener, Westburg). Data was analyzed using GraphPad Prism software (Graphpad software, San Diego, Calif., USA).
FIGS. 7B and C show that the combination of IgG1-004-H5L2 plus IgG1-010-H5L2 (with or without E430G mutation) and the combination of IgG1-005-H1L2 plus IgG1-016-H5L2 (with or without E430G mutation induced enhanced CDC compared to their individual counterparts. The combination of IgG1-004-H5L2 plus IgG1-016-H5L2 (with or without E430G mutation) did not induce enhanced CDC compared to their individual counterparts.
FIGS. 7D and E show that the combination of IgG1-004-H5L2 plus IgG1-005-H1L2 (with or without E430G mutation) and the combination of IgG1-010-H5L2 plus IgG1-016-H5L2 (with or without E430G mutation induced enhanced CDC compared to their individual counterparts. The combination of IgG1-005-H1L2 plus IgG1-010-H5L2 (with or without E430G mutation) did not induce enhanced CDC compared to their individual counterparts.
FIGS. 7F and G show that the combination of IgG1-37.3 plus IgG1-005-H1L2 (with or without E430G mutation) and the combination of IgG1-37.3 plus IgG1-010-H5L2 (with or without E430G mutation induced enhanced CDC compared to their individual counterparts.
Hence, functional combination studies confirmed the results of the binding competition studies for described CD37 antibodies and showed that non-cross-blocking CD37 antibodies can functionally be combined.
Example 8: Introducing an Fc-Fc Interaction Enhancing Mutation into Humanized CD37 Antibodies Results in Enhanced, De Novo Capacity to Induce Complement Dependent Cytotoxicity (CDC)
Daudi cells, resuspended in RPMI containing 0.2% BSA, were plated into polystyrene 96-well round-bottom plates (Greiner bio-one Cat #650101) at a density of 1×105 cells/well (30 μL/well) and 50 μL of a concentration series of humanized CD37 antibodies and variants thereof, or control antibody IgG1-b12, was added (0.003-10 μg/mL final antibody concentration in 3.33× serial dilutions). After incubation (RT, 15 min), 20 μL of pooled normal human serum (NHS, Cat #M0008 Sanquin, Amsterdam, The Netherlands) was added to each well and plates were incubated at 37° C. for 45 minutes. Plates were centrifuged (3 minutes, 1200 rpm) and supernatant was discarded. Propidium iodide (PI; 30 μL of a 1.67 μg/mL solution; Sigma-Aldrich Chemie B.V., Zwijndrecht, The Netherlands) was added and lysis was detected by measurement of the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry (Intellicyt iQue™ screener, Westburg). Graphs were generated using best-fit values of a non-linear dose-response fit with log-transformed concentrations in GraphPad Prism V6.04 software (GraphPad Software, San Diego, Calif., USA).
FIG. 9 shows that IgG1-004-H5L2, IgG1-005-H1L2, IgG1-010-H5L2 and IgG1-016-H5L2 did not induce CDC in Daudi cells. Upon introduction of the Fc-Fc interaction enhancing E430G mutation, these antibodies (IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G and IgG1-016-H5L2-E430G) induced profound, dose-dependent CDC of Daudi cells.
FIG. 10A shows that IgG1-G28.1 and IgG1-37.3 did not induce CDC on Daudi cells. Upon introduction of the Fc-Fc interaction enhancing E430G mutation, these antibodies (IgG1-G28.1-E430G and IgG1-37.3-E430G) induced profound, dose-dependent CDC of Daudi cells.
For antibody IgG1-016-H5L2 a variant with a point mutation in the variable domain was generated to replace a free cysteine in the light chain: IgG1-016-H5L2-LC90S. In addition, this variant was also generated with an F405L mutation (previously shown not to affect target binding or CDC) and an Fc-Fc interaction enhancing E430G mutation. FIG. 11 shows that the IgG1-016-H5L2-E430G, IgG1-016-H5L2-F405L-E430G and IgG1-016-H5L2-LC90S-F405L-E430G all showed comparable activity in an in vitro CDC assay, thus that the LC90S mutation did not affect the capacity to induce CDC. IgG1-016-H5L2 did not induce CDC on Daudi cells.
Also, introduction of other Fc-Fc interaction enhancing mutations, E345K, E345R, E430S and RRGY, in IgG1-010-H5L2 and IgG1-016-H5L2 resulted in profound CDC of Daudi cells. FIGS. 10B and C show that maximum lysis of Daudi cell was comparable for all tested Fc-Fc interaction enhancing mutations.
Example 9: Bispecific CD37 Antibodies with an Fc-Fc Interaction Enhancing Mutation are More Potent in Inducing CDC than Monospecific Bivalent CD37 Antibodies with an Fc-Fc Interaction Enhancing Mutation Due to Monovalent Binding and Dual Epitope Targeting
F405L or K409R mutations were introduced into humanized CD37 antibodies containing the E430G mutation, to allow for the generation of bispecific antibodies (bsIgG1) with two CD37-specific Fab-arms that do not compete for binding to CD37. The capacity of bispecific CD37 antibodies containing the E430G mutation to induce CDC was determined as described above, and compared to that of CD37 monospecific bivalent antibodies containing the E430G mutation, a combination of two CD37 monospecific bivalent antibodies containing the E430G mutation that do not compete for binding to CD37 (with the end concentration of the combined antibodies together identical to the concentration of the individual bispecific antibodies), monovalent CD37 antibodies containing the E430G mutation (i.e. bispecific antibodies containing one CD37-specific Fab arm and one non-binding Fab-arm derived from IgG1-b12, and containing the E430G mutation) or a combination of two monovalent CD37 antibodies containing the E430G mutation that do not compete for binding to CD37.
CDC on Daudi Cells
FIG. 12A shows that bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G was more potent than either IgG1-005-H1L2-E430G or IgG1-016-H5L2-E430G in inducing CDC on Daudi cells. The bispecific bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G was also more potent than a combination of IgG1-005-H1L2-K409R-E430G plus IgG1-016-H5L2-F405L-E430G. Monovalent CD37-binding antibodies bsIgG1-b12-F405L-E430Gx005-H1L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G also induced CDC on Daudi cells, but were less efficient in doing so than bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G.
FIG. 12B shows that bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G was more potent than either IgG1-010-H5L2-E430G or IgG1-016-H5L2-E430G in inducing CDC on Daudi cells. The bispecific bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G was also more potent than a combination of IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G. Monovalent binding antibodies bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G also induced CDC on Daudi cells, with bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G being less potent and bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G being equally potent compared to bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G.
The capacity to induce CDC by bispecific CD37 antibodies containing the E430G mutation was also compared to that of bispecific CD37 antibodies without the E430G mutation. FIG. 13 shows that bsIgG1-016-H5L2-F405Lx005-H1L2-K409R as well as bsIgG1-016-H5L2-F405Lx010-H5L2-K409R were capable of inducing CDC on Daudi cells, but were less potent in doing so compared to their E430G containing counterparts bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G.
CDC on OCI-Ly-7 Cells
FIG. 12C shows that monovalent binding antibodies bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G were more potent in inducing CDC on OCI-Ly-7 cells compared to their monospecific bivalent binding counterparts, IgG1-016-H5L2-E430G and IgG1-010-H5L2-E430G. The combination of monovalent binding antibodies (bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G plus bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G) was more potent than the combination of bivalent antibodies (IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G), as demonstrated by a consistent lower EC50 in two independent experiments (FIG. 12D). Also, bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G was more potent in inducing CDC on OCI-Ly-7 cells than the combination of bivalent antibodies (IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G), as demonstrated by a consistent lower EC50 in three independent experiments (FIG. 12E).
The potency of the combination of monovalent binding antibodies (bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G plus bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G) and of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G in inducing CDC in OCI-Ly-7 cells was comparable.
CDC on Primary CLL Tumor Cells
The capacity of bispecific CD37 antibodies containing the E430G mutation to induce CDC on tumor cells derived from a CLL patient was determined as described above, and compared to that of CD37 antibodies containing the E430G mutation or a combination of CD37 antibodies containing the E430G mutation or monovalent CD37 antibodies containing the E430G mutation.
FIG. 14A shows that bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G was more potent than either IgG1-005-H1L2-K409R-E430G or IgG1-016-H5L2-F405L-E430G in inducing CDC on primary CLL tumor cells. The bispecific bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G was also more potent than a combination of IgG1-005-H1L2-K409R-E430G plus IgG1-016-H5L2-F405L-E430G. Monovalent binding antibodies bsIgG1-b12-F405L-E430Gx005-H1L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G also induced CDC on primary CLL tumor cells, but were less efficient in doing so than bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G.
FIG. 14B shows that bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G was more potent than either IgG1-010-H5L2-E430G or IgG1-016-H5L2-E430G in inducing CDC on primary CLL tumor cells. The bispecific bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G was also more potent than a combination of IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G. Monovalent binding antibodies bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G also induced CDC on primary CLL tumor cells, with bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G being less potent and bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G being equally potent compared to bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G.
Example 10: Bispecific CD37 Antibodies with an Fc-Fc Interaction Enhancing Mutation Induce CDC on a Variety of B Cell Lymphoma Cell Lines with a Wide Range of CD37 Expression
The capacity of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, at a concentration of 10 μg/mL, to induce CDC was determined (as described above) on a range of B cell lymphoma cell lines, derived from a variety of B cell lymphoma subtypes. The expression levels of CD37 molecules on the cell surface of these cell lines were determined by quantitative flow cytometry as described above.
Table 3 gives an overview of the cell lines tested.
TABLE 3
B cell lymphoma cell lines.
Cell line
Lymphoma type
Source
JVM-2
MCL
DSMZ; ACC 12
JVM-13
MCL
ATCC; CRL-3003
Jeko-1
MCL
DSMZ; ACC 553
Z-138
MCL
ATCC; CRL-3001
Daudi
Burkitt's
ATCC; CCL-213
Raji
Burkitt's
ATCC; CCL-86
Wien-133
Burkitt's
BioAnaLab, Oxford, U.K
SU-DHL-8
DLBCL
DSMZ; ACC 573
OCI-Ly19
DLBCL
DSMZ; ACC 528
OCI-Ly7
DLBCL
DSMZ; ACC 688
SU-DHL-4
DLBCL
DSMZ; ACC 495
RC-K8
DLBCL
DSMZ; ACC 561
U-2932
DLBCL
DSMZ; ACC 633
WIL-2S
Plasmablastic
ATCC; CRL-8885
RI-1
DLBCL
DSMZ; ACC 585
WSU-DLCL2
DLBCL
DSMZ; ACC 575
FIG. 15 shows that bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G induced CDC on a wide range of B cell lymphoma cell lines, derived from various B cell lymphoma types.
Example 11: Bispecific CD37 Antibodies with an Fc-Fc Interaction Enhancing Mutation are More Potent in Inducing Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
Labeling of Target Cells
The capacity of CD37 antibodies to induce ADCC was determined by a chromium release assay. Daudi or Raji cells were collected (5×106 cells/mL) in 1 mL culture medium (RPMI 1640 supplemented with 10% Donor Bovine Serum with Iron (DBSI; ThermoFischer, Cat #10371029) and Penicillin Streptomycin mixture (pen/strep), to which 100 μCi 51Cr (Chromium-51; PerkinElmer, Cat #NEZ030005MC) had been added. Cells were incubated in a water bath at 37° C. for 1 hour while shaking. After washing of the cells (twice in PBS, 1500 rpm, 5 min), the cells were resuspended in RPMI 1640/10% DBSI/pen/strep and counted by trypan blue exclusion. Cells were diluted to a density of 1×105 cells/mL.
Preparation of Effector Cells
Peripheral blood mononuclear cells from healthy volunteers (Sanquin, Amsterdam, The Netherlands) were isolated from 45 mL of freshly drawn heparin blood (buffy coats) by Ficoll density centrifugation (Bio Whittaker; lymphocyte separation medium, cat 17-829E) according to the manufacturer's instructions. After resuspension of cells in RPMI 1640/10% DBSI/pen/strep, cells were counted by trypan blue exclusion and diluted to a density of 1×107 cells/mL.
ADCC Assay Procedure
50 μL of 51Cr-labeled targets cells were pipetted into 96-well round-bottom microtiter plates (Greiner Bio-One; Cat #650101), and 50 μL of a concentration series of (1.5-5,000 ng/mL final concentrations in 3-fold dilutions) CD37 or control antibodies, diluted in RPMI 1640/10% DBSI/pen/strep was added. Cells were incubated at room temperature (RT) for 15 min and 50 μL effector cells were added, resulting in an effector to target ratio of 100:1. Cells were incubated for 4 hours at 37° C. and 5% CO2. For determination of maximal lysis, 50 μL 51Cr-labeled Daudi cells (5.000 cells) were incubated with 100 μL 5% Triton-X100; for determination of spontaneous lysis (background lysis), 5,000 51Cr-labeled Daudi cells were incubated in 150 μL medium without any antibody or effector cells. The level of antibody-independent cell lysis was determined by incubating 5,000 Daudi cells with 500,000 PBMCs without antibody. Plates were centrifuged (1200 rpm, 10 min) and 25 μL of supernatant was transferred to 100 μL Microscint-40 solution (Packard, Cat #6013641) in 96-Wells plates. Plates were sealed and shaken for 15 minutes at 800 rpm and released 51Cr was counted using a scintillation counter (TopCount®, PerkinElmer). The percentage specific lysis was calculated as follows:
% specific lysis=(cpm sample−cpm spontaneous lysis)/(cpm maximal lysis−cpm spontaneous lysis) wherein cpm is counts per minute.
FIG. 16A shows that bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G was more potent than either IgG1-005-H1L2-K409R-E430G or IgG1-016-H5L2-F409L-E430G or than a combination of IgG1-005-H1L2-K409R-E430G plus IgG1-016-H5L2-F405L-E430G in inducing ADCC on Daudi cells.
FIG. 16B shows that bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G was more potent than either IgG1-010-H5L2-E430G or IgG1-016-H5L2-E430G or a combination of IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G in inducing ADCC on Daudi cells.
FIG. 16C shows similar results as FIG. 16B for PBMCs from a different donor, and in addition shows that bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G was more potent than monovalent binding antibodies bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G in inducing ADCC on Raji cells.
Example 12: Bispecific CD37 Antibodies with an Fc-Fc Interaction Enhancing Mutation Induce Potent Ex Vivo CDC in Primary Tumor Cells from Patients with Various B Cell Malignancies
The CDC efficacy of bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G was analyzed using primary patient-derived tumor cells from five different B cell malignancies: chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL) and Non-Hodgkin's lymphoma (not further specified). All patient samples were obtained after written informed consent and stored using protocols approved by the VUmc Medical Ethical Committee in accordance with the declaration of Helsinki. Patient bone marrow mononuclear cells (BMNCs) or peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation (Ficoll-Paque PLUS, GE Healthcare) from bone marrow aspirates or peripheral blood samples of patients. Cells were either used directly or stored in liquid nitrogen until further use.
Patient lymph node tissue was dissected into small fragments and collected in α-MEM medium (ThermoFischer Scientific, Waltham, Mass.) containing 1% Penicillin-Streptomycin, 0.2% heparin and 5% platelet lysate and left overnight at 37° C. After incubation, the supernatant (non-stromal cell compartment including tumor cells) was collected and cells were filtered using a 70 μM Easy Strainer (Greiner Bio-one). Cells were counted, resuspended in RPMI 1640 medium containing 25% heat-inactivated FBS and 10% DMSO, and frozen in liquid nitrogen until further use.
The CD37 and membrane complement regulatory proteins (mCRP; CD46, CD55 and CD59) expression levels on isolated patient cells were determined using a QifiKit (DAKO, cat. no. K007811). Cells were incubated with the purified antibodies CD37 (BD, cat. no. 555456), CD46 (BioLegend, cat. no. 352404), CD55 (BioLegend, cat. no. 311302), CD59 (BioLegend, cat. no. 304702), and b12 (Genmab) at 4° C. for 30 min. After this the method as provided by the QifiKit manufacturer was used. After the final step of Qifi kit procedure, cells were incubated with lymphoma cell specific markers to enable tumor cell identification. FIG. 17 shows the expression levels per indication.
The patient-derived tumor cells were opsonized with 10 μg/mL or 100 μg/mL bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G and CDC induction was assessed in the presence of 20% pooled NHS. The following cell markers were used to identify different cell populations: CD45-KO (Beckman Coulter B36294), CD19-PC7 (Beckman Coulter, cat. no. IM3628), CD3-V450 (BD, cat. no. 560365), CD5-APC (BD, cat. no. 345783), CD5-PE (DAKO, cat. no. R084201), CD10-APC-H7 (BD, cat. no. 655404), CD10-PE (DAKO, cat. no. R084201), CD23-FITC (Biolegend, cat. no. 338505), lambda-APC-H7 (BD, cat. no. 656648), kappa-PE (DAKO, cat. no. R043601) and lambda-FITC (Emelca Bioscience CYT-LAMBF). Within the CD45+ cell population, malignant B cells were defined by different markers depending on the indication: CD3−/CD19+/CD5+ (CLL), CD3−/CD19+/CD10+ (FL, DLBCL), CD3−/CD19+/CD5+/CD23− (MCL). In case malignant B cells could not be identified based on these markers, malignant cells were identified based on clonality using kappa/lambda staining. In a few samples, malignant B cells could also not be identified based on clonality; in these cases the total B cell population was assessed, without distinction between normal and malignant B cells. Killing was calculated as the fraction of 7-amino actinomycin D (7-AAD; BD, cat. no. 555816) positive malignant B cells (%) determined by an LSRFortessa flow cytometer (BD Biosciences, San Jose, Calif.).
FIG. 18 shows that bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G was highly potent (lysis of more than 50%) in inducing CDC in tumor cells derived patients with CLL, FL, MCL, DLBCL or B-NHL (not further specified). In cells from one patient with relapsed/refractory FL, bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G was less capable of inducing CDC.
Example 13: Binding of a Bispecific CD37 Antibody with an Fc-Fc Interaction Enhancing Mutation to Human or Cynomolgus Monkey B Cells in Whole Blood, and Induction of Cytotoxicity in B Cells in Whole Blood
Binding to Human or Cynomolgus Monkey B Cells
Binding to human or cynomolgus monkey B cells was determined in a whole blood binding assay. Heparin-treated human blood from healthy volunteers was derived from UMC Utrecht (Utrecht, The Netherlands), hirudin-treated blood from cynomolgus monkeys was derived from Covance (Munster, Germany). Blood was aliquoted to wells of a 96-well round-bottom plate (Greiner Bio-one, cat. no. 65010; 35 μL/well). Red blood cells (RBC) were lysed by addition of 100 μL RBC lysis buffer (10 mM KHCO3 [Sigma P9144], 0.1 mM EDTA [Fluka 03620] and 0.15 mM NH4CL [Sigma A5666]) and incubated on ice until RBC lysis was complete. After centrifugation for 3 minutes at 300×g, cells were incubated for 30 minutes at 4° C. with serial dilutions (0.014-30 μg/mL final antibody concentration in 3× serial dilutions) of Alexa-488 labeled bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or Alexa-488 labeled control IgG1 (IgG1-b12) and a directly labeled antibody to identify B cells (among a mixture of antibodies to further identify blood cell subsets):
For human blood B cells the following antibody was used
Target
protein
Clone
Label
Target cells
Company
Cat. no.
CD19
HIB19
BV711
B cells
Biolegend
302245
For cynomolgus monkey blood B cells the following antibody was used
Target
protein
Clone
Label
Target cells
Company
Cat. no.
CD19
J3-119
PE
B cells
Beckman
A07769
Coulter
Cells were pelleted and washed twice in 150 μL FACS buffer and resuspended in 150 μL TO-PRO-3 (end concentration 0.2 μM; Molecular Probes, cat no. T3605). Samples were measured by flow cytometry using an LSRFortessa flow cytometer. Binding is expressed as geometric mean of A488 fluorescence intensity for viable TO-PRO-3−/CD14−/CD19+ B-cells (human) or viable TO-PRO-3−/CD14−/CD19+/CD20+ B-cells (cynomolgus monkey). Log-transformed data were analyzed using best-fit values of a non-linear dose-response fit in GraphPad PRISM.
FIG. 19 shows the concentration dependent binding of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to B cells in (A) human and (B) cynomolgus monkey blood, for one representative donor/animal. The average EC50 values for binding to human and cynomolgus monkey B cells were in the same range ([0.85 μg/mL±0.284 based on binding to B cells in blood from 6 human donors] and [0.63 μg/mL±0.228 based on binding to B cells in blood from 4 animals], respectively), indicating that bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G shows comparable binding to human and cynomolgus monkey CD37.
Cytotoxicity to Human or Cynomolgus Monkey B Cells
Cytotoxicity towards human or cynomolgus monkey B cells was determined in a whole blood cytotoxicity assay. Hirudin-treated human blood from healthy volunteers was derived from UMC Utrecht (Utrecht, The Netherlands), hirudin-treated blood from cynomolgus monkeys was derived from Covance (Munster, Germany). Blood was aliquoted to wells of a 96-well round-bottom plate, 35 μL/well.
Serial dilutions (0.0005-10 μg/mL final antibody concentration in 3× serial dilutions; final volume 100 μL/well) of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or IgG1-b12 were added. In cytotoxicity assays using human whole blood, the monoclonal FcγR-interaction enhanced CD37 specific antibody IgG1-G28.1-5239D-I332E was included as reference. Samples were incubated at 37° C. for 4 hours. Thereafter, red blood cells were lysed as described above and samples were stained to identify B cells as described above. Cells were pelleted and washed twice in 150 μL FACS buffer and resuspended in 150 μL TO-PRO-3 (end concentration 0.2 μM; Molecular Probes, cat no. T3605). Samples were measured by flow cytometry using an LSRFortessa flow cytometer. After exclusion of doublets the percentage viable TO-PRO-3−/CD14−/CD19+ B-cells (human) or viable TO-PRO-3−/CD14−/CD19+/CD20+ B cells (cynomolgus monkey) was determined. The percentage B-cell depletion was calculated as follows: % B cell depletion=100*[(% B-cells no Ab control-% B cells sample)/(% B cells no Ab control)]. Log-transformed data were analyzed using best-fit values of a non-linear dose-response fit in GraphPad PRISM.
FIG. 20 shows the concentration dependent cytotoxicity of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to B cells in (A) human and (B) cynomolgus monkey blood, for one representative donor/animal.
Based on EC50, the capacity of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to induce cytotoxicity in human and cynomolgus monkey B cells was comparable: the average EC50 for cytotoxicity to human B cells (in blood from 6 donors) was 0.077 μg/mL±0.039; the average EC50 for cytotoxicity to cynomolgus monkey B cells (in blood from 4 animals) was 0.043 μg/mL±0.019.
FIG. 20A also shows the cytotoxicity of the FcγR-interaction enhanced monoclonal CD37 antibody IgG1-G28.1-5239D-I332E to human B cells for a representative responding donor, which showed lower cytotoxicity than bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G. In B cells from 3 responding donors, a maximum B-cell depletion of 50% by IgG1-G28.1-5239D-I332E was measured, whereas in 3 other donors no cytotoxicity to B cells by this antibody was measured. BsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G induced cytotoxicity in 93-99% of B cells in 6/6 donors. Binding of IgG1-G28.1-S239D-I332E to CD37 expressed on Daudi cells was comparable to that of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G (data not shown).
Example 14: Potent CDC Activity by a Combination of a Bispecific CD37 Antibody with an Fc-Fc Interaction Enhancing Mutation with a CD20-Specific Antibody
The capacity to induce CDC was tested for a combination of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and an anti-CD20 antibody (IgG1-CD20-ofa; ofatumumab) on patient derived CLL tumor cells obtained from ConversantBio (Huntsville, Ala., USA). Patient derived PBMCs were resuspended in RPMI containing 0.2% BSA (bovine serum albumin) and plated into polystyrene 96-well round-bottom plates (Greiner bio-one Cat #650101) at a density of 0.1×106 cells/well (30 μL/well) and 50 μL of a concentration series of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G (0.0625-0.05 μg/mL) and IgG1-CD20-ofa (1-8 μg/mL) was added in 2-fold dilutions. BsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-CD20-ofa were combined at antibody concentrations that were based on relative potency (differences in EC50s) of each of the antibodies, by mixing two concentrations that would, on average, separately reach the same effect. IgG1-b12 was used as negative control.
After incubation (RT, 15 min while shaking), 20 μL of pooled normal human serum (NHS Cat #M0008 Sanquin, Amsterdam, The Netherlands) was added to each well as a source of complement and plates were incubated at 37° C. for 45 minutes. The reaction was stopped by cooling the plates on ice. After centrifugation for 3 minutes at 300×g, cells were washed twice with 150 μL FACS buffer and incubated for 30 minutes at 4° C. with an R-Phycoerythrin (PE) labeled mouse-anti-human IgG1-CD19 antibody (clone J3-119, Beckman Coulter, cat no. A07769, 1:50 diluted from stock) to determine the tumor B cells and TO-PRO-3 (end concentration 0.2 μM; Molecular Probes, cat no. T3605) for the identification of dead cells. Cells were pelleted and washed twice in 150 μL FACS buffer and measured by flow cytometry using an LSRFortessa flow cytometer. The percentage of viable cells was calculated as follows: % viable cells=100*(#TO-PRO-3 negative events)/(# total events).
FIGS. 21A-D show that both bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and ofatumumab induced CDC in tumor cells derived from 2 CLL patients, with CDC activity increasing with increasing dose levels. Combining bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G with ofatumumab resulted in enhanced CDC activity at all tested concentrations for both CLL patients tested, although these effects were less evident at higher antibody concentrations, where almost complete cell kill was induced by the single agents (FIGS. 21A and B). These results indicate that the addition of ofatumumab to bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G can improve CDC-mediated tumor cell kill in malignant B cells obtained from CLL patients.
Example 15: Anti-Tumor Activity of a Bispecific CD37 Antibody with an Fc-Fc Interaction Enhancing Mutation in Xenograft Models of B Cell Malignancies
Anti-Tumor Activity in a Subcutaneous JVM-3 Human Chronic B Cell Leukemia Xenograft Model
JVM-3 cells (1×107) were inoculated into the right flank of CB17.SCID mice and antibody treatment (3 weekly doses of 0.1, 0.3, 1, 3 or 10 mg/kg, injected intravenously; IgG1-b12 was used as negative control, dosed at 10 mg/kg) was initiated when tumors reached a mean volume of approximately 158 mm3. Tumor volumes were measured twice weekly in two dimensions using a caliper, and the volume was expressed in mm3 using the formula: V=(L×W×W)/2, where V is tumor volume, L is tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L).
FIG. 22A shows the tumor volume per dose group over time, FIG. 22B shows the tumor volumes per mouse per dose group on day 25 when all groups were still complete. Three weekly doses of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G at 1, 3 or 10 mg/kg significantly reduced the JVM-3 cell tumor growth, whereas dosing at 0.1 or 0.3 mg/kg did not affect tumor growth (Mann Whitney test, p<0.01).
Anti-Tumor Activity in an Intravenous Daudi-Luc Burkitt's Lymphoma Xenograft Model
On day 0, SCID mice (C.B-17/IcrHan®Hsd-Prkdcscid; Harlan) were intravenously injected with Daudi-luc cells (luciferase transfected Daudi cell, 2.5×106 cells/mouse). At day 14, 21 and 28, mice were injected intraperitoneally with 0.1, 0.3, 1, 3 or 10 mg/kg of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G. IgG1-b12 was used as negative control antibody, dosed at 10 mg/kg. Tumor growth was evaluated weekly (starting at day 2) by bioluminescence imaging (BLI). Mice were injected intraperitoneally with 100 μL firefly D-luciferin (30 mg/mL; Caliper LifeSciences, cat. no. 119222) and bioluminescence (radiance in p/s/cm2/sr [photons per second per cm2 per square radian]) was measured under isoflurane anesthesia using a Biospace Bioluminescence Imaging System (PerkinElmer; mice were imaged from the dorsal site).
FIG. 23A shows luciferase activity (bioluminescence, as a measure of tumor volume) per dose group over time, FIG. 23B shows the luciferase activity per mouse per dose group on day 36 when all groups were still complete. Three weekly doses of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G at 0.1, 0.3, 1, 3 or 10 mg/kg significantly reduced the in vivo growth of Daudi-luc cells (One Way Anova, Uncorrected Fisher's LSD).
Example 16: Evaluation of Plasma Clearance of a Bispecific CD37 Antibody with an Fc-Fc Interaction Enhancing Mutation in SCID Mice
11-12 week old, female SCID mice (C.B-17/IcrHan®Hsd-Prkdcscid; Harlan) (3 mice per group) were injected intravenously (i.v.) injected with a single dose of 100 μg (5 mg/kg) or 500 μg (25 mg/kg) of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or IgG1-b12. The experiment was set up to study antibody clearance in absence of target-mediated clearance as neither bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G nor IgG1-b12 show cross-reactivity with mouse.
50-100 μL blood samples were collected from the saphenous vein at 10 minutes, 4 hours, 24 hours, 2 days, 7 or 8 days, 14 days and 21 days after antibody administration. Blood was collected into heparin containing vials and centrifuged for 5 minutes at 10,000 g. Plasma samples were diluted 1:50 for mice dosed with 5 mg/kg (20 μL sample in 980 μL PBSA (PBS supplemented with 0.2% bovine serum albumin (BSA)) and 1:20 for mice dosed with 25 mg/kg (20 μL sample in 380 μL PBSA) and stored at −20° C. until determination of mAb concentrations.
Human IgG concentrations were determined using a sandwich ELISA. Mouse mAb anti-human IgG-kappa clone MH16 (CLB Sanquin, The Netherlands; cat. no. M1268), coated in 100 μL overnight at 4° C. to 96-well Microlon ELISA plates (Greiner, Germany) at a concentration of 2 μg/mL, was used as capturing antibody. After blocking plates with PBSA for 1 hour at room temperature (RT), samples were added, serially diluted in PBSA, and incubated on a plate shaker for 1 hour at RT. Plates were washed three times with 300 μL PBST (PBS supplemented with 0.05% Tween 20) and subsequently incubated for 1 hour at RT with goat anti-human IgG immunoglobulin (Jackson, West Grace, Pa.; cat. no. 109-035-098; 1:10.000 in PBST supplemented with 0.2% BSA). Plates were washed again three times with 300 μL PBST before incubation with 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany) protected from light. The reaction was stopped by adding 100 μL 2% oxalic acid. Absorbance was measured in a microplate reader (Biotek, Winooski, Vt.) at 405 nm. Human IgG concentration was calculated by using the injected material as a reference curve. As a plate control, purified human IgG1 (The binding site, cat. no. BP078) was included. Human IgG concentrations (in μg/mL) were plotted (FIGS. 24A and C) and the area under the curve (AUC) was calculated using Graphpad prism 6.0. IgG clearance until the last day of blood sampling (day 21) was determined by the formula D*1.000/AUC, in which D is the dose of injection (1 mg/kg) (FIGS. 24B and D).
There were no substantial differences between plasma clearance rates of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-b12, demonstrating that bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G showed a comparable pharmacokinetic profile as wild type human IgG1 in absence of target binding.
Example 17: Determination of the Contribution of CD37 Amino Acid Residues to Binding of CD37 Antibodies Using Alanine Scanning
Library Design
A CD37 single residue alanine library was synthesized (Geneart) in which all amino acid (aa) residues in the extracellular domains of human CD37 (Uniprot P11049) were individually mutated to alanines except for positions already containing alanines or cysteines. Cysteines were not mutated to minimize the chance of structural disruption of the antigen. The library was cloned in the pMAC expression vector containing a CMV/TK-polyA expression cassette, an Amp resistance gene and a pBR322 replication origin.
Library Production and Screening
The wild type CD37 and alanine mutants were expressed individually in FreeStyle HEK293 cells according to the manufacturer's instructions (Thermo Scientific). One day post transfection the cells were harvested. Approximately 100,000 cells were incubated with 20 μL Alexa488 conjugated bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G (monovalent binding 010) or Alexa488 conjugated bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G (monovalent binding 016) at a concentration of 3 μg/mL in FACS buffer (PBS+0.1% (w/v) bovine serum albumin (BSA)+0.02% (w/v) sodium azide). Cells were incubated for 1 hour at room temperature. Subsequently, cells were washed twice by adding 150 μL FACS buffer and removing the supernatant after centrifugation. Cells were resuspended in 20 μL fresh FACS buffer and stored at 4° C. until analysis by flow cytometry using an iQue screener (IntelliCyt). The entire experiment was performed 2 times.
Data Analysis
For every sample, the average antibody binding per cell was determined as the geometric mean of the fluorescence intensity (gMFI) for the ungated cell population. The gMFI is influenced by the affinity of the antibody for the CD37 mutant and the expression level of the CD37 mutant per cell. Since specific alanine mutations can impact the surface expression level of the mutant CD37, and to correct for expression differences for each CD37 mutant in general, data were normalized against the binding intensity of a non-competing CD37 specific control antibody (in this example antibodies monovalent binding 010 and monovalent binding 016 were non-competing antibodies and one antibody was used as control for the other antibody), using the following equation:
Normalized
gMFI
aa
posititon
=
Log
1
0
(
gMFI
Test
Ab
gMFI
Control
Ab
)
In which ‘aa position’ refers to either a particular alanine mutant position in CD37 or wild type (wt) CD37.
To express loss or gain of binding of the antibodies the standard score was determined according to the following calculation:
zscore
(
fold
change
)
=
Normalized
gMFJ
aa
position
-
μ
σ
Where μ and σ are the mean and standard deviation (SD) of the Normalized gMFI of all mutants.
Gain of binding in most cases will be caused by loss of binding of the reference antibody to specific ala mutants. Using these calculations, amino acid positions for which, upon replacing the amino acid with alanine, there is no loss or gain of binding by a particular antibody will give a zscore of ‘0’, gain of binding will result in ‘zscore>0’ and loss of binding will result in ‘zscore<0’. To correct for sample variation, only CD37 amino acid residues where the zscore was lower than −1.5 were considered ‘loss of binding mutants’. In case the gMFI of the control antibody for a particular CD37 mutant was lower than the mean gMFI−2.5×SD of the mean gMFIControl Ab, data were excluded from analysis (as for those CD37 mutants it was assumed expression levels were not sufficient).
FIG. 25 shows the ‘zscore (fold change)’ of the CD37 antibodies to CD37 variants with ala mutations at positions 42 to 131 (according to SEQ ID No 94). The results indicate that:
binding of antibody 010 is at least dependent on aa Y182, D189, T191, I192, D194, K195, V196, I197 and P199 of human CD37,
binding of antibody 016 is at least dependent on aa E124, F162, Q163, V164, L165 and H175 of human CD37.
In Summary
In summary, bispecific antibodies composed of two CD37-specific antibodies that do not compete for target binding with an Fc-Fc interaction enhancing mutation, showed the most favorable combination of CDC potency and ADCC potency in CD37-positive tumor cells. For both effector mechanisms, the bispecific antibodies with the Fc-Fc interaction enhancing mutation showed superior potency compared to the combination of two non-competing CD37 antibodies containing the Fc-Fc interaction enhancing mutation or to the single CD37 antibodies with the Fc-Fc interaction enhancing mutation.
Example 18: In Vitro Evaluation of CDC Activity of Mixtures of Novel Hexamerization-Enhanced CD37 Antibodies with Clinically Established CD20 Antibody Products on Raji Cells
The CDC activity of mixtures of CD37 antibodies with an Fc-Fc interaction enhancing mutation, IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-E430G, IgG1-005-E430G, IgG1-010-E430G and IgG1-016-E430G (the latter 4 being chimeric rabbit/human), plus the clinically established CD20-targeting monoclonal antibody products MabThera (rituximab; Roche, H01241308), Arzerra (ofatumumab; Novartis; C656294) and Gazyva (obinutuzumab, GA101; Roche, D287-41A GACD20) was tested in vitro using Burkitt's lymphoma Raji cells. Raji cells (ATCC, Cat No. CCL-86) were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 1 U/mL penicillin, 1 μg/mL streptomycin, and 4 mM L-glutamine. 0.1×106 Raji cells were pre-incubated with antibodies in a total volume of 80 μL RPMI/0.2% BSA per well for 15 min on a shaker at RT. Next, NHS was added to the pre-incubated cells to a final volume of 100 μL (final antibody concentrations 10 μg/mL; 20% NHS) and incubated for 45 minutes at 37° C. For all tested total antibody concentrations, different ratios of the two antibodies in the mixtures were tested (1:0-3:1-1:1-1:3-0:1). Plates were centrifuged and cells were resuspended in 30 μL PI (2 μg/mL). Killing was calculated as the fraction PI-positive cells (%) determined by flow cytometry on an iQue screener (Intellicyt). Data were analyzed and plotted using GraphPad Prism software.
The mixtures of the tested CD37 antibodies with an Fc-Fc interaction enhancing mutation and clinically established CD20 antibody products showed enhanced dose-dependent CDC activity compared to the same concentration of the single antibodies on Raji cells (FIG. 8). There was little difference in CDC activity at the different tested ratios of the two antibodies in the mixtures (1:3, 1:1 or 3:1). These data indicate that the mixture of a hexamerization-enhanced CD37 antibody with an Fc-Fc interaction enhancing mutation plus a clinically established CD20 antibody product, such as MabThera, Arzerra (type I CD20 antibodies) or Gazyva (type II CD20 antibody), may improve the therapeutic potential for patients with B cell malignancies, which frequently become refractory to standard CD20 targeted therapies alone.
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17066190
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genmab holding b.v.
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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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Apr 1st, 2022 06:04PM
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Apr 1st, 2022 06:04PM
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Genmab A/S
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Health Care
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Pharmaceuticals & Biotechnology
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cph:gen
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Genmab A/S
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Jul 7th, 2020 12:00AM
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Jan 31st, 2017 12:00AM
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https://www.uspto.gov?id=US10703818-20200707
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Human monoclonal antibodies against CD25
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Isolated human monoclonal antibodies which bind to and inhibit human CD25, and related antibody-based compositions and molecules, are disclosed. The human antibodies can be produced by a hybridoma, a transfectoma or in a nonhuman transgenic animal, e.g., a transgenic mouse, capable of producing multiple isotypes of human monoclonal antibodies by undergoing V-D-J recombination and isotype switching. Also disclosed are pharmaceutical compositions comprising the human antibodies, nonhuman transgenic animals, hybridomas and transfectomas which produce the human antibodies, and therapeutic and diagnostic methods for using the human antibodies.
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10703818
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1. A method of treating a lymphoid neoplasm involving T cells expressing CD25 in a patient, comprising administering to the patient an antibody that binds to human CD25 in an amount effective to treat the lymphoid neoplasm, wherein the antibody is selected from the group consisting of:
(a) an antibody comprising heavy and light chain variable region sequences set forth in SEQ ID NOs: 6 and 8, respectively;
(b) an antibody comprising heavy and light chain variable region sequences set forth in SEQ ID NOs: 14 and 16, respectively;
(c) an antibody comprising heavy and light chain variable region sequences set forth in SEQ ID NOs: 2 and 4, respectively;
(d) an antibody comprising heavy and light chain variable region sequences set forth in SEQ ID NOs: 10 and 12, respectively;
(e) an antibody comprising heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 35, 36, and 37, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 38, 39, and 40, respectively;
(f) an antibody comprising heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 17, 18, and 19, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 20, 21, and 22, respectively;
(g) an antibody comprising heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 26, 27, and 28, respectively; and
(h) an antibody comprising heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 29, 30, and 31, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 32, 33, and 34, respectively.
2. The method of claim 1, wherein the cells expressing CD25 are activated T cells.
3. The method of claim 1, wherein the lymphoid neoplasm is selected from the group consisting of Hodgkin's disease, hairy cell leukemia, and cutaneous T cell lymphoma.
4. The method of claim 1, further comprising separately administering another therapeutic agent and/or therapy to the patient.
5. The method of claim 4, wherein the therapeutic agent is an immunosuppressant selected from the group consisting of cyclosporine, azathioprine, mycophenolic acid, mycophenolate mofetil, corticosteroids, prednisone, methotrexate, gold salts, sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine, 15-deoxyspergualine, 6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus, OKT3, and anti-thymocyte globulin.
6. The method of claim 4, wherein the therapeutic agent is an anti-inflammatory agent selected from the group consisting of a steroidal drug, a nonsteroidal anti-inflammatory drug, and a disease modifying anti-rheumatic drug.
7. The method of claim 4, wherein the therapeutic agent is an agent or therapy for treating an inflammatory or hyperproliferative skin disorder selected from the group consisting of coal tar, A vitamin, anthralin, calcipotrien, tarazotene, corticosteroids, methotrexate, retinoids, cyclosporine, etanercept, alefacept, efaluzimab, 6-thioguanine, mycophenolate mofetil, tacrolimus, hydroxyurea, and phototherapy.
8. The method of claim 4, wherein the therapeutic agent is selected from the group consisting of doxorubicin, cisplatin, bleomycin, carmustine, chlorambucil, and cyclophosphamide.
9. The method of claim 1, wherein the antibody is an IgG1 or IgG4 antibody.
10. The method of claim 1, wherein the antibody is linked to a cytotoxic agent, a radioisotope, or a drug.
11. The method of claim 1, wherein the antibody is a human, monoclonal IgG1 antibody, a bispecific antibody or a multispecific antibody.
12. The method of claim 11, wherein the bispecific or the multispecific antibody further comprises a binding specificity for CD3, CD4, IL-15R, membrane bound or receptor bound TNF-α, or receptor bound IL-15.
13. A method of treating cutaneous T cell lymphoma in a patient, comprising administering to the patient an antibody that binds to human CD25 in an amount effective to treat the cutaneous T cell lymphoma, wherein the antibody comprises heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 35, 36, and 37, respectively, and light chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 38, 39, and 40, respectively, and wherein the antibody is linked to a cytotoxic agent, a radioisotope, or a drug.
14. The method of claim 13, wherein the antibody comprises heavy and light chain variable region sequences set forth in SEQ ID NOs: 14 and 16, respectively.
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14
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RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 14/592,558, filed on Jan. 8, 2015 (now U.S. Pat. No. 9,598,493), which is a continuation of U.S. patent application Ser. No. 13/465,181, filed on May 7, 2012 (now U.S. Pat. No. 8,961,968), which is a continuation of U.S. patent application Ser. No. 12/283,775, filed on Sep. 16, 2008 (now U.S. Pat. No. 8,182,812), which is divisional of U.S. patent application Ser. No. 10/714,353, filed on Nov. 14, 2003 (now U.S. Pat. No. 7,438,907), which claims the benefit of U.S. Provisional Application No. 60/426,690, filed on Nov. 15, 2002. The contents of the aforementioned applications are hereby incorporated by reference.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 31, 2017, is named GMI_059DVCN2DV_Sequence_Listing.txt and is 35,213 bytes in size.
BACKGROUND OF THE INVENTION
The high affinity interleukin-2 receptor (IL-2R) is a heterotrimeric cell surface receptor composed of α, β and γc-polypeptide chains (KD 10−11 M). The 55 kDa α-chain, also known as IL-2Rα, CD25, p55, and Tac (T cell activation) antigen, is unique to the IL-2R. The β (CD122; P75) and γc (CD132) chains are part of a cytokine receptor superfamily (hematopoietin receptors) and are functional components of other cytokine receptors, such as IL-15R (Waldmann (1993) Immunol. Today 14(6):264-70; Ellery et al. (2002) Cytokine Growth Factor Rev. 13(1): 27-40). The intermediate affinity receptor is a dimer composed of a β- and a γc-chain (KD 10−9 M) while the low affinity receptor consists of a monomeric α-subunit that has no signal transduction capacity (KD 10−8 M) (Waldmann (1993) Immunol. Today 14(6):264-70).
Resting T cells, B cells, and monocytes express few CD25 molecules. However, the receptor is rapidly transcribed and expressed upon activation (Ellery et al. (2002) Cytokine Growth Factor Rev. 13(1): 27-40; Morris et al. (2000) Ann. Rheum. Dis. 59 (Suppl. 1):i109-14). Cells expressing the high affinity IL-2R express CD25 (the CD25-subunit) in excess which leads to both high and low affinity IL-2 binding profiles (Waldmann et al. (1993) Blood 82(6):1701-12; de Jong et al. (1996) J. Immunol. 156(4):1339-48). CD25 is highly expressed by T cells in some autoimmune diseases, such as rheumatoid arthritis, scleroderma, and uveitis, as well as skin disorders, e.g., psoriasis and atopic dermatitis, and a variety of lymphoid neoplasms, e.g., T cell leukemia, and Hodgkin's disease (Waldmann (1993) Immunol. Today 14(6):264-70; Kuttler et al. (1999) J. Mol. Med. 77(1):226-9). In addition, CD25 expression is associated with allograft rejection and graft-versus-host responses (Jones et al. (2002) J. Immunol. 168(3):1123-1130; Anasetti et al. (1994) Blood 84(4): 1320-7).
Accordingly, CD25 is an important target for antibody-mediated therapy, for example, to reduce inflammation in autoimmune diseases, treat tumors, and prevent transplant rejection. However, while the results obtained and clinical experience to date clearly establish CD25 as a useful target for immunotherapy, they also show that currently available murine and chimeric antibodies do not constitute ideal therapeutic agents. Therefore, the need exists for further therapeutic antibodies against CD25 which are effective in preventing and/or treating a range of diseases involving cells expressing CD25.
SUMMARY OF THE INVENTION
The present invention provides novel antibody therapeutics for treating and/or preventing diseases associated with cells expressing CD25, including organ, tissue and cell transplant rejection, including allograft and xenograft rejection, graft-versus-host disease, autoimmune diseases, inflammatory and hyperproliferative skin disorders, and lymphoid neoplasms, among others. The antibodies encompassed by the invention are improved in that they are fully human and, thus, are potentially less immunogenic in patients. The antibodies are also advantageous based on their superior functional (e.g., therapeutic) properties.
As shown herein, the human antibodies of the invention bind to CD25 when tested by ELISA or flow cytometry.
The human antibodies of the invention typically bind to CD25 with a dissociation equilibrium constant (KD) of approximately 10−8 M or less, such as 10−9 M or less, 10−10 M or less, or 10−11M or even less when determined by surface plasmon resonance (SPR) technology in a BIAcore® 3000 instrument using recombinant human IL-2Rα as the ligand and the antibody as the analyte. Such antibodies typically do not cross-react with related cell-surface antigens and do thus not inhibit their function.
Furthermore, the human antibodies of the present invention inhibit (e.g., block) the interaction of CD25 with its ligand, IL-2. For example, binding can be inhibited by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Examples of cells which express CD25 and, the cellular function of which therefore, can be inhibited by the human antibodies of the present invention include, among others T cells, B cells and monocytes. For example, as shown herein, the human antibodies of the invention can inhibit IL-2 binding to CD25. Such inhibition of IL-2 binding to CD25 concomitantly inhibits various cellular mechanisms induced by IL-2 binding. As also shown herein, human antibodies of the invention can inhibit anti-CD3 antibody-induced T cell proliferation in a dose-dependent manner. As also shown herein, human antibodies of the invention can inhibit mixed lymphocyte reaction (MLR) in a dose-dependent manner. Inhibition of proliferation in such experiments may be monitored by a decrease in the accumulation of cell mass as measured in ELISA or by a decrease in the incorporation of BrdU in the cell's DNA.
Human antibodies of the invention include IgG1 (e.g., IgG1,κ and IgG1,λ), and IgG4 (e.g., IgG4,κ and IgG4,λ) antibodies. However, other antibody isotypes are also encompassed by the invention, including IgG2, IgG3, IgM, IgA1, IgA2, secretory IgA, IgD, and IgE. The antibodies can be whole antibodies or antigen-binding fragments thereof including, for example, Fab, Fab′, F(ab)2, F(ab′)2, Fv, single chain Fv (scFv) fragments or bispecific antibodies. Furthermore, the antigen-binding fragments include binding-domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide (such as a heavy chain variable region or a light chain variable region) that is fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. Such binding-domain immunoglobulin fusion proteins are further disclosed in US 2003/0118592 and US 2003/0133939. Particular human antibodies of the present invention include those referred to as AB1, AB7, AB11, and AB12, encoded by human heavy chain and human kappa light chain nucleic acids comprising nucleotide sequences in their variable regions as set forth in SEQ ID NOs:1, 5, 9, or 13 and SEQ ID NOs:3, 7, 11, or 15, respectively, and conservative sequence modifications thereof. In another embodiment, the human antibodies are characterized by having human heavy chain and human kappa light chain variable regions comprising the amino acid sequences as set forth in SEQ ID NOs:2, 6, 10, or 14 and SEQ ID NOs:4, 8, 12, or 16, respectively, and conservative sequence modifications thereof.
Other particular human antibodies of the invention include those which comprise a CDR (complementarity determining region) having a human heavy and light chain CDR1, a human heavy and light chain CDR2, and a human heavy and light chain CDR3, wherein
(a) the human heavy chain CDR1, CDR2, and CDR3 comprise an amino acid sequence selected from the group consisting of the CDR1, CDR2, and CDR3 amino acid sequences shown in FIGS. 1-10 (SEQ ID NOs:17-19, 23-25, 29-31, or 35-37), and conservative sequence modifications thereof, and
(b) the human light chain CDR1, CDR2, and CDR3 comprise an amino acid sequence selected from the group consisting of the CDR1, CDR2, and CDR3 amino acid sequences shown in FIGS. 1-10 (SEQ ID NOs: 20-22, 26-28, 32-34, or 38-40), and conservative sequence modifications thereof.
In another embodiment, human anti-CD25 antibodies of the present invention can be characterized by one or more of the following properties:
a) specificity for human CD25;
b) a binding affinity to CD25 corresponding to a KD of approximately 10−8 M or less, such as 10−9M or less, 10−10 M or less, or 10−11M or even less, when determined by surface plasmon resonance (SPR) technology in a BIAcore® 3000 instrument using recombinant IL-2Rα as the ligand and the antibody as the analyte;
c) the ability to tolerize T cells;
d) the ability to block the interaction of CD25 with its ligand, IL-2;
e) the ability to eliminate T cells expressing CD25;
f) the ability to inhibit proliferation of T cells expressing CD25;
g) the ability to inhibit anti-CD3 antibody-induced T cell proliferation of peripheral blood mononuclear cells (PBMCs);
h) the ability to block mixed lymphocyte reaction (MLR); and/or
i) internalization of CD25 expressed on T cells.
The term “tolerized”, as used herein, means that the T cells are not capable to react to an antigen after a rechallenge with this antigen.
Human anti-CD25 antibodies of the present invention can be derivatized, linked to or co-expressed with other binding specificities. In a particular embodiment, the antibodies are linked to one or more binding specificities for a different target antigen, such as an antigen on an effector cell.
Accordingly, the present invention also includes bispecific and multispecific molecules that bind to both human CD25 and to one or more different target antigens, such as CD3, CD4, IL-15R, membrane bound or receptor bound TNF-α, or membrane bound or receptor bound IL-15.
In another embodiment, human anti-CD25 antibodies of the invention are derivatized, linked to or co-expressed with another functional molecule, e.g., another peptide or protein (e.g., a Fab fragment). For example, the antibody can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., to produce a bispecific or a multispecific antibody), a cytotoxin, cellular ligand or antigen (e.g., to produce an immunoconjugate, such as an immunotoxin). The antibody also can be linked to other therapeutic moieties, e.g., a radioisotope, a small molecule anti-cancer drug, an anti-inflammatory agent, or an immunosuppressive agent. Accordingly, the present invention encompasses a large variety of antibody conjugates, bispecific and multispecific molecules, and fusion proteins, all of which bind to CD25-expressing cells and which can be used to target other molecules to such cells.
Human antibodies, immunoconjugates, bispecific and multispecific molecules and compositions of the present invention can be used in a variety of methods for inhibiting, killing and/or modulating activity and/or growth (e.g., proliferation) of cells expressing CD25. In one embodiment, the method includes inhibiting the proliferation of T cells expressing CD25. In another embodiment, the method includes inhibiting graft-versus-host responses, e.g., MLR. In still another embodiment, the method includes killing of cells expressing CD25 (e.g., by complement-mediated lysis or by linking the antibody to a cytotoxin). The cells are preferably killed or inhibited without killing or inhibiting the activity of cells which do not express CD25 but which may, for example, express a structurally related cell-surface antigen (i.e., without cross-reactivity to related but functionally distinct cell surface antigens). Cells expressing CD25 which can be inhibited or killed using the human antibodies of the invention include, for example, activated T lymphocytes, B lymphocytes, monocytes, macrophages, Kuppfer cells of the liver, and Langerhans' cells of the skin expressing CD25.
Accordingly, human antibodies of the present invention can be used to treat and/or prevent a variety of diseases and conditions wherein activated cells expressing CD25 play an active role in the pathogenesis by administering the antibodies to patients suffering from such diseases and conditions. Exemplary diseases that can be treated (e.g., ameliorated) or prevented include, but are not limited to, transplant rejection, including allograft and xenograft rejection, in patients undergoing or who have undergone organ or tissue transplantation, such as heart, lung, combined heart-lung, trachea, kidney, liver, pancreas, oesophagus, bowel, skin, limb, umbilical cord, stem cell, islet cell transplantation, etc. Antibodies of the present invention may thus be used as prophylactics in allograft and xenograft rejection, or be used to reverse, treat, or otherwise ameliorate acute allograft or xenograft rejection episodes.
Further diseases than can be treated include graft-versus-host disease, e.g. blood transfusion graft-versus-host disease and bone marrow graft-versus-host disease; inflammatory, immune or autoimmune diseases, such as rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, type 1 diabetes, insulin-requiring type 2 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, dermato-polymyositis, Sjögren's syndrome, arteritides, including giant cell arteritis, aplastic anemia, asthma, scleroderma, and uveitis; inflammatory or hyperproliferative skin disorders, e.g., psoriasis, including plaque psoriasis, pustulosis palmoplantaris (PPP), erosive lichen planus, pemphigus bullosa, epidermolysis bullosa, contact dermatitis and atopic dermatitis; and a variety of lymphoid neoplasms, e.g., T cell leukemia, Hodgkin's disease, hairy cell leukemia, or cutaneous T cell lymphoma, including mycosis fungoides, and Sezary's syndrome.
Further diseases that can be treated are
malignancies wherein an inhibition of infiltrating CD25+ regulatory T cells is beneficial, such as gastric cancer, esophageal cancers, malignant melanoma, colorectal cancer, pancreas cancer, breast cancer, small cell lung cancer, non-small cell lung cancer, cervical cancer, ovarian cancer, and renal cell carcinoma;
hematological disorders, such as adult T cell leukemia/lymphoma, anaplastic large cell lymphoma, chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), peripheral T cell lymphoma, and secondary amyloidosis;
skin disorders, such as pyoderma gangraenosum, granuloma annulare, allergic contact dermatitis, cicatricial pemphigoid, and herpes gestationis;
hepato-gastrointestinal disorders, such as collagen colitis, sclerosing cholangitis, chronic active hepatitis, lupoid hepatitis, autoimmune hepatitis, alcoholic hepatitis, chronic pancreatis, and acute pancreatitis;
cardiac disorders, such as myocarditis, and pericarditis;
vascular disorders, such as arteriosclerosis, giant cell arteritis/polymyalgia rheumatica, Takayasu arteritis, polyarteritis nodosa, Kawasaki syndrome, Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, leukocytoclastic angiitis, and secondary leukocytoclastic vasculitis;
renal disorders, such as acute glomerulonphritis, chronic glomerulonephritis, minimal change nephritis, and Goodpasture's syndrome;
pulmonary disorders, such as alveolitis, bronchiolitis obliterans, silicosis, and berylliosis;
neurological disorders, such as multiple sclerosis, Alzheimer's disease, myasthenia gravis, chronic demyelinating polyneuropathy, and polyradiculitis including Guillain-Barré syndrome;
connective tissue disorders, such as relapsing polychondritis, sarcoidosis, systemic lupus erythematosus, CNS lupus, discoid lupus, lupus nephritis, chronic fatigue syndrome, and fibromyalgia;
endocrinological disorders, such as Graves' disease, Hashimoto's thyroiditis, and subacute thyroiditis; and
viral infections, such as tropical spastic paraparesis.
In a particular embodiment of the invention, the subject being administered the antibody is additionally treated with one or more further therapeutic agents, such as immunosuppressive agents, anti-inflammatory agents, chemotherapeutic agents, cytotoxic agents or other agents which serve to enhance the therapeutic effect of the antibody.
In yet another aspect, the present invention provides a method for detecting in vitro or in vivo the presence of CD25 in a sample or individual, e.g., for diagnosing a CD25-related disease, preferably at an early stage. This can also be useful for monitoring the disease and effect of treatment with an anti-CD25 antibody and for determining and adjusting the dose of the antibody to be administered. In one embodiment, detecting the presence of CD25 in a sample is achieved by contacting a sample to be tested, optionally along with a control sample, with a human monoclonal antibody of the invention under conditions that allow for formation of a complex between the antibody and CD25. Complex formation is then detected (e.g., using ELISA, flow cytometry or Western blotting). When using a control sample along with the test sample, complex is detected in both samples and any statistically significant difference in the formation of complexes between the samples is indicative of the presence of CD25 in the test sample. The in vivo method can be performed using imaging technique such as PET (positron emission tomography) or SPECT (single photon emission computed tomography).
In a further aspect, the invention relates to anti-idiotypic antibodies which bind to the human monoclonal antibodies of the invention. These anti-idiotypic antibodies can be used as an immunodiagnostic tool to detect and quantify levels of human monoclonal antibodies against CD25 in laboratory or patient samples. This may be useful for examining pharmacokinetics of the anti-CD25 antibody or for determining and adjusting the dosage of the anti-CD25 antibody and for monitoring the disease and the effect of treatment in a patient.
Mouse anti-idiotypic antibodies can be made e.g. by immunizing Balb/C mice with the human monoclonal antibodies according to the invention, and generating hybridomas from spleens of these mice by fusion with myeloma cells such as NS1 using standard techniques.
In yet another aspect, the invention provides a transgenic non-human animal, such as a transgenic mouse, which express human monoclonal antibodies that bind to CD25. In a particular embodiment, the transgenic non-human animal is a transgenic mouse having a genome comprising a human heavy chain transgene or transchromosome and a human light chain transgene or transchromosome encoding all or a portion of an antibody of the invention. The transgenic non-human animal can be immunized with a purified or enriched preparation of CD25 antigen and/or cells expressing CD25. Preferably, the transgenic non-human animal, e.g., the transgenic mouse, is capable of producing multiple isotypes of human monoclonal antibodies to CD25 (e.g., IgG, IgA and/or IgM) by undergoing V-D-J recombination and isotype switching. Isotype switching may occur by, e.g., classical or non-classical isotype switching.
Accordingly, in yet another aspect, the invention provides isolated B cells from a transgenic non-human animal as described above, e.g., a transgenic mouse, which expresses human anti-CD25 antibodies. The isolated B cells can then be immortalized by fusion to an immortalized cell to provide a source (e.g., a hybridoma) of human anti-CD25 antibodies. Such hybridomas (i.e., which produce human anti-CD25 antibodies) are also included within the scope of the invention.
As exemplified herein, human antibodies of the invention can be obtained directly from hybridomas which express the antibody, or can be cloned (e.g., from hybridomas or phage which display antigen-binding portions of the antibodies) and recombinantly expressed in a host cell (e.g., a CHO (Chinese Hamster Ovary) cell, or a NS/0 cell). Further examples of host cells are microorganisms, such as E. coli, and fungi, such as yeast. Alternatively, they can be produced recombinantly in a transgenic non-human animal or plant. Accordingly, in another aspect, the present invention provides methods for producing human monoclonal antibodies which bind to human CD25. In one embodiment, the method includes immunizing a transgenic non-human animal, e.g., a transgenic mouse, as previously described (e.g., having a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or a portion of an anti-CD25 antibody), with a purified or enriched preparation of human CD25 antigen and/or cells expressing human CD25. B cells (e.g., splenic B cells) of the animal are then obtained and fused with myeloma cells to form immortal, hybridoma cells that secrete human monoclonal antibodies against CD25.
In yet another aspect, the invention provides nucleic acid molecules encoding human anti-CD25 antibodies (e.g., variable regions thereof), as well as recombinant expression vectors which include the nucleic acids of the invention, and host cells transfected with such vectors. Methods of producing the antibodies by culturing these host cells are also encompassed by the invention. Particular nucleic acids provided by the invention comprise the nucleotide sequences shown in SEQ ID NOs:1, 5, 9, or 13 and SEQ ID NOs:3, 7, 11, or 15 encoding the heavy and light chains, respectively, of human anti-CD25 antibodies AB1, AB7, AB11, and AB12.
Other features and advantages of the instant invention will be apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the amino acid sequences of the light (kappa) chain VJ regions of human monoclonal antibodies AB1, AB7, AB11, and AB12 (SEQ ID NOs: 4, 8, 12, and 16, respectively) with CDRs designated.
FIG. 2 shows the amino acid sequences of the heavy chain VDJ regions of human monoclonal antibodies AB1, AB7, AB11, and AB12 (SEQ ID NOs: 2, 6, 10, and 14, respectively) with CDRs designated.
FIG. 3 shows the amino acid sequence (SEQ ID NO: 2) and the corresponding nucleotide sequence (SEQ ID NO: 1) of the heavy chain VDJ region of human monoclonal antibody AB1 with CDRs designated.
FIG. 4 shows the amino acid sequence (SEQ ID NO: 4) and the corresponding nucleotide sequence (SEQ ID NO: 3) of the light (kappa) chain VJ region of human monoclonal antibody AB1 with CDRs designated.
FIG. 5 shows the amino acid sequence (SEQ ID NO: 6) and the corresponding nucleotide sequence (SEQ ID NO: 5) of the heavy chain VDJ region of human monoclonal antibody AB7 with CDRs designated.
FIG. 6 shows the amino acid sequence (SEQ ID NO: 8) and the corresponding nucleotide sequence (SEQ ID NO: 7) of the light (kappa) chain VJ region of human monoclonal antibody AB7 with CDRs designated.
FIG. 7 shows the amino acid sequence (SEQ ID NO: 10) and the corresponding nucleotide sequence (SEQ ID NO: 9) of the heavy chain VDJ region of human monoclonal antibody AB11 with CDRs designated.
FIG. 8 shows the amino acid sequence (SEQ ID NO: 12) and the corresponding nucleotide sequence (SEQ ID NO: 11) of the light (kappa) chain VJ region of human monoclonal antibody AB11 with CDRs designated.
FIG. 9 shows the amino acid sequence (SEQ ID NO: 14) and the corresponding nucleotide sequence (SEQ ID NO: 13) of the heavy chain VDJ region of human monoclonal antibody AB12 with CDRs designated.
FIG. 10 shows the amino acid sequence (SEQ ID NO: 16) and the corresponding nucleotide sequence (SEQ ID NO: 15) of the light (kappa) chain VJ region of human monoclonal antibody AB12 with CDRs designated.
FIG. 11 is a graph showing inhibition of IL-2 binding to its receptor, CD25, by supernatants of human monoclonal antibodies AB1, AB7, AB11, and AB12, compared to inhibition of IL-2 binding by ZENAPAX® antibody (daclizumab, recombinant humanized IgG1 anti-CD25 antibody, Roche).
FIG. 12 is a graph showing inhibition of ZENAPAX® antibody binding to CD25 by human monoclonal antibodies AB1, AB7, AB11, and AB12.
FIG. 13 is a graph showing inhibition of anti-CD3 antibody-induced T cell proliferation (using PBMCs) by human monoclonal antibodies AB1, AB7, AB12, compared to inhibition by a control antibody (hIgG1/κ) and ZENAPAX® antibody.
FIG. 14 is a graph showing inhibition of MLR by human monoclonal antibodies AB1, AB7, AB12, compared to inhibition by a control antibody (hIgG1/κ) and ZENAPAX® antibody.
FIGS. 15A-15C show photographs using FITC filter visualizing the internalization of CD25 by FITC-labeled AB12. FIG. 15A shows the result for T cell blasts pre-incubated with FITC-labeled AB12 after 18 hours incubation at 37° C., and FIG. 15B shows the result for T cell blasts cultured for 18 hours at 37° C. in the presence of FITC-labeled AB12. For comparison FIG. 15C shows the result for T cell blasts cultured for 18 hours at 37° C. in the presence of FITC-labeled isotype control antibody (anti-KHL).
FIGS. 16A and 16B show the internalization of CD25 by FITC-labeled AB12 as measured by flow cytometry where a ratio of mean fluorescence intensitity (MFI) of above 1 indicates that internalization has taken place. FIG. 16A shows the result for T cell blasts pre-incubated with FITC-labeled AB12 at 4° C. and 37° C., respectively, and FIG. 16B shows the result of T cell blasts cultured in the presence of FITC-labeled AB12 at 4° C. and 37° C., respectively.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides antibody-based therapies for treating and diagnosing a variety of disorders involving cells expressing CD25. Therapies of the invention employ isolated human monoclonal antibodies which specifically bind to an epitope present on CD25. Such antibodies include all known isotypes, e.g., IgA, IgG1-4, IgE, IgM, and IgD antibodies.
In one embodiment the antibody is an IgG1 antibody, more particularly an IgG1,κ or IgG1,λ isotype. In another embodiment the antibody is an IgG3 antibody, more particularly an IgG3,κ or IgG3,λ isotype. In yet another embodiment the antibody is an IgG4 antibody, more particularly an IgG4,κ or IgG4,λ isotype. In still another embodiment the antibody is an IgA1 or IgA2 antibody. In still a further embodiment the antibody is an IgM antibody.
In one embodiment, the human antibodies are produced in a nonhuman transgenic animal, e.g., a transgenic mouse, capable of producing multiple isotypes of human monoclonal antibodies to CD25 by undergoing V-D-J recombination and isotype switching. Such transgenic animal can also be a transgenic rabbit for producing polyclonal antibodies such as disclosed in US 2003/0017534. Accordingly, the invention also encompasses human polyclonal antibodies which specifically bind to CD25. Accordingly, aspects of the invention include not only antibodies, antibody fragments, and pharmaceutical compositions thereof, but also nonhuman transgenic animals, B cells, host cell transfectomas, and hybridomas which produce monoclonal antibodies. Methods of using the antibodies of the invention to block or inhibit cells expressing CD25 are also provided and are useful in the treatment of disorders associated with CD25. Methods of using the antibodies of the invention to detect a cell expressing CD25 are encompassed by the invention.
In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.
The terms “CD25” and “CD25 antigen” are used interchangeably herein, and include any variants, isoforms and species homologs of human CD25 which are naturally expressed by cells or are expressed on cells transfected with the CD25 gene. Synonyms of CD25, as recognized in the art, include CD25, p55, and Tac (T cell activation) antigen. Binding of an antibody of the invention to the CD25 antigen inhibits and/or blocks CD25 from binding to its ligand, IL-2, and, concomitantly, the resultant cellular function thereof. For example, in one embodiment, the human antibodies of the invention inhibit anti-CD3 antibody-induced T cell proliferation. In another embodiment, the human monoclonal antibodies inhibit MLR.
As used herein, the term “inhibits growth” (e.g., referring to cells) is intended to include any measurable decrease in the cell growth when contacted with an anti-CD25 antibody as compared to the growth of the same cells not in contact with an anti-CD25 antibody, e.g., the inhibition of growth of a cell culture by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
As used herein, the terms “inhibits binding” and “blocks binding” (e.g., referring to inhibition/blocking of binding of IL-2 to CD25) are used interchangeably and encompass both partial and complete inhibition/blocking. The inhibition/blocking of binding of IL-2 to CD25 preferably reduces or alters the normal level or type of cell signaling that occurs when IL-2 binds to CD25 without inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in the binding affinity of IL-2 to CD25 when in contact with an anti-CD25 antibody as compared to the ligand not in contact with an anti-CD25 antibody, e.g., the blocking of binding of IL-2 to CD25 by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
The term “antibody” as referred to herein includes intact antibodies and any antigen binding fragment (i.e., “antigen-binding portion”) or single chain thereof. An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
The term “antigen-binding portion” of an antibody (or simply “antibody portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD25). It has been shown that the antigen-binding function of an antibody can be performed by fragments of an intact or full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of a VL and VH domain; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; (vi) an isolated complementarity determining region (CDR), and (vii) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. A further example is binding-domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide that is fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. The binding-domain immunoglobulin fusion proteins are further disclosed in US 2003/0118592 and US 2003/0133939. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
The term “epitope” means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost by treatment with denaturing solvents.
The term “bispecific molecule” is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has two different binding specificities. For example, the molecule may bind to, or interact with, (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell.
The term “multispecific molecule” or “heterospecific molecule” is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has more than two different binding specificities. For example, the molecule may bind to, or interact with, (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Accordingly, the invention includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules which are directed to CD25 and to other targets, such as Fc receptors on effector cells.
The term “bispecific antibodies” also includes diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).
The term “human antibody derivatives” refers to any modified form of the antibody, e.g., a conjugate of the antibody and another agent or antibody.
The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may also include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term “human monoclonal antibody” refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic or transchromosomal nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, fused to an immortalized cell.
The term “recombinant human antibody”, as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further in Section I, below), (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
The term “transfectoma”, as used herein, includes recombinant eukaryotic host cell expressing the antibody, such as CHO cells, NS/0 cells, HEK293 cells, plant cells, or fungi, including yeast cells.
As used herein, a “heterologous antibody” is defined in relation to the transgenic nonhuman organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic nonhuman animal, and generally from a species other than that of the transgenic nonhuman animal.
An “isolated antibody,” as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to CD25 is substantially free of antibodies that specifically bind antigens other than CD25). An isolated antibody that specifically binds to an epitope, isoform or variant of human CD25 may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., CD25 species homologs). Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment of the invention, a combination of “isolated” monoclonal antibodies having different specificities are combined in a well defined composition.
As used herein, “specific binding” refers to antibody binding to a predetermined antigen. Typically, the antibody binds with an affinity corresponding to a KD of about 10−8 M or less, such as about 10−9 M or less, about 10−10 M or less, or about 10−11M or even less when determined by surface plasmon resonance (SPR) technology in a BIAcore® 3000 instrument using recombinant IL-2Rα as the ligand and the antibody as the analyte, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, preferably at least 100 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen”.
The term “kd” (sec−1), as used herein, is intended to refer to the dissociation equilibrium rate constant of a particular antibody-antigen interaction. Said value is also referred to as the koff value.
The term “ka” (M−1×sec−1), as used herein, is intended to refer to the association equilibrium rate constant of a particular antibody-antigen interaction.
The term “KD” (M), as used herein, is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction.
The term “KA” (M−1), as used herein, is intended to refer to the association equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing the ka by the kd.
As used herein, “isotype” refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.
As used herein, “isotype switching” refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes.
As used herein, “nonswitched isotype” refers to the isotypic class of heavy chain that is produced when no isotype switching has taken place; the CH gene encoding the nonswitched isotype is typically the first CH gene immediately downstream from the functionally rearranged VDJ gene. Isotype switching has been classified as classical or non-classical isotype switching. Classical isotype switching occurs by recombination events which involve at least one switch sequence region in the transgene. Non-classical isotype switching may occur by, for example, homologous recombination between human σμ and human Σμ (δ-associated deletion). Alternative non-classical switching mechanisms, such as intertransgene and/or interchromosomal recombination, among others, may occur and effectuate isotype switching.
As used herein, the term “switch sequence” refers to those DNA sequences responsible for switch recombination. A “switch donor” sequence, typically a μ switch region, will be 5′ (i.e., upstream) of the construct region to be deleted during the switch recombination. The “switch acceptor” region will be between the construct region to be deleted and the replacement constant region (e.g., γ, ε, etc.).
As used herein, “glycosylation pattern” is defined as the pattern of carbohydrate units that are covalently attached to a protein, more specifically to an immunoglobulin (antibody) protein. A glycosylation pattern of a heterologous antibody can be characterized as being substantially similar to glycosylation patterns which occur naturally on antibodies produced by the species of the nonhuman transgenic animal, when one of ordinary skill in the art would recognize the glycosylation pattern of the heterologous antibody as being more similar to said pattern of glycosylation in the species of the nonhuman transgenic animal than to the species from which the CH genes of the transgene were derived.
The term “naturally-occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
The term “rearranged” as used herein refers to a configuration of a heavy chain or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete VH or VL domain, respectively. A rearranged immunoglobulin (antibody) gene locus can be identified by comparison to germline DNA; a rearranged locus will have at least one recombined heptamer/nonamer homology element.
The term “unrearranged” or “germline configuration” as used herein in reference to a V segment refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment.
The term “nucleic acid molecule”, as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
The term “isolated nucleic acid molecule,” as used herein in reference to nucleic acids encoding whole antibodies or antibody portions (e.g., VH, VL, CDR3) that bind to CD25, is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the intact antibody or antibody portion are free of other nucleotide sequences encoding whole antibodies or antibody portions that bind antigens other than CD25, which other sequences may naturally flank the nucleic acid in human genomic DNA. In one embodiment, the human anti-CD25 antibody includes the heavy chain (VH) and light chain (VL) variable amino acid regions of AB1, AB7, AB11, or AB12 encoded by the nucleotide sequences shown in SEQ ID NOs: 1, 5, 9, or 13 and SEQ ID NOs: 3, 7, 11, or 15, respectively.
As disclosed and claimed herein, the sequences set forth in SEQ ID NOs: 1-40 include “conservative sequence modifications,” i.e., nucleotide and amino acid sequence modifications which do not significantly affect or alter the binding characteristics of the antibody encoded by the nucleotide sequence or containing the amino acid sequence. Such conservative sequence modifications include nucleotide and amino acid substitutions, additions and deletions. Modifications can be introduced into SEQ ID NOs:1-40 by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a human anti-CD25 antibody is preferably replaced with another amino acid residue from the same side chain family.
The present invention also encompasses “derivatives” of the amino acid sequences as set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 17-40 and conservative sequence modifications thereof, wherein one or more of the amino acid residues have been derivatised, e.g. by acylation or glycosylation, without significantly affecting or altering the binding characteristics of the antibody to CD25.
Furthermore, the present invention comprises antibodies in which one or more alterations have been made in the Fc region in order to change functional or pharmacokinetic properties of the antibodies. Such alterations may result in a decrease or increase of C1q binding and CDC or of FcγR binding and antibody-dependent cellular cytotoxicity (ADCC). Substitutions can for example be made in one or more of the amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 of the heavy chain constant region, thereby causing an alteration in an effector function while retaining binding to antigen as compared with the unmodified antibody, cf. U.S. Pat. Nos. 5,624,821 and 5,648,260. Further reference may be had to WO 00/42072 disclosing antibodies with altered Fc regions that increase ADCC, and WO 94/29351 disclosing antibodies having mutations in the N-terminal region of the CH2 domain that alter the ability of the antibodies to bind to FcRI and thereby decreases the ability of the antibodies to bind to C1q which in turn decreases the ability of the antibodies to fix complement. Furthermore, Shields et al., J. Biol. Chem. (2001) 276:6591-6604 teaches combination variants, e.g. T256A/S298A, S298A/E333A, and S298A/E333A/K334A, that improve FcγRIII binding.
The in vivo half-life of the antibodies can also be improved by modifying the salvage receptor epitope of the Ig constant domain or an Ig-like constant domain such that the molecule does not comprise an intact CH2 domain or an intact Ig Fc region, cf. U.S. Pat. Nos. 6,121,022 and 6,194,551. The in vivo half-life can furthermore be increased by making mutations in the Fc region, e.g. by substituting threonine for leucine at position 252, threonine for serine at position 254, or threonine for phenylalanine at position 256, cf. U.S. Pat. No. 6,277,375.
Furthermore, the glycosylation pattern of the antibodies can be modified in order to change the effector function of the antibodies. For example, the antibodies can be expressed in a transfectoma which does not add the fucose unit normally attached to the carbohydrate attached to Asn at position 297 of Fc in order to enhance the affinity of Fc for FcγRIII which in turn will result in an increased ADCC of the antibodies in the presence of NK cells, cf. Shield et al. (2002) J. Biol. Chem., 277:26733. Furthermore, modification of galactosylation can be made in order to modify CDC. Further reference may be had to WO 99/54342 and Umana et al., Nat. Biotechnol. (1999) 17:176 disclosing a CHO cell line engineered to express GntIII resulting in the expression of monoclonal antibodies with altered glycoforms and improved ADCC activity.
Furthermore, the antibody fragments, e.g. Fab fragments; of the invention can be pegylated to increase the half-life. This can be carried out by pegylation reactions known in the art, as described, for example, in Focus on Growth Factors (1992) 3:4-10; EP 154 316 and EP 401 384.
Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a anti-CD25 antibody coding sequence, such as by saturation mutagenesis, and the resulting modified anti-CD25 antibodies can be screened for binding activity.
Accordingly, antibodies encoded by the (heavy and light chain variable region) nucleotide sequences disclosed herein and/or containing the (heavy and light chain variable region) amino acid sequences disclosed herein (i.e., SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 17-40) include substantially similar antibodies encoded by or containing similar sequences which have been conservatively modified. Further discussion as to how such substantially similar antibodies can be generated based on the partial (i.e., heavy and light chain variable regions) sequences disclosed herein as SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 17-40 is provided below.
For nucleotide and amino acid sequences, the term “homology” indicates degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate insertions or deletions. Alternatively, substantial homology exists when the DNA segments will hybridize under selective hybridization conditions, to the complement of the strand. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (on the internet at the website at gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available on the internet at the website at gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See the internet at the website of ncbi.nlm.nih.gov.
The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).
The nucleic acid compositions of the present invention, while often in a native sequence (except for modified restriction sites and the like), from either cDNA, genomic or mixtures thereof, may be mutated in accordance with standard techniques to provide gene sequences. For coding sequences, these mutations, may affect amino acid sequence as desired. In particular, DNA sequences substantially homologous to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated (where “derived” indicates that a sequence is identical or modified from another sequence).
A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcription of regulatory sequences, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. For switch sequences, operably linked indicates that the sequences are capable of effecting switch recombination.
The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells and NS/0 cells.
As used herein, the term “subject” includes any human or nonhuman animal. The term “nonhuman animal” includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.
The term “transgenic, nonhuman animal” refers to a nonhuman animal having a genome comprising one or more human heavy and/or light chain transgenes or transchromosomes (either integrated or non-integrated into the animal's natural genomic DNA) and which is capable of expressing fully human antibodies. For example, a transgenic mouse can have a human light chain transgene and either a human heavy chain transgene or human heavy chain transchromosome, such that the mouse produces human anti-CD25 antibodies when immunized with CD25 antigen and/or cells expressing CD25. The human heavy and light chain transgene and/or transchromosome can be integrated into the chromosomal DNA of the mouse or maintained extrachromosomally. Such transgenic and transchromosomal mice (collectively referred to herein as “transgenic mice”) are capable of producing multiple isotypes of human monoclonal antibodies to CD25 (e.g., IgG, IgA, IgM, IgD and/or IgE) by undergoing V-D-J recombination and isotype switching. Transgenic, nonhuman animal can also be used for production of a specific anti-CD25 antibody by introducing genes encoding such specific anti-CD25 antibody, for example by operatively linking the genes to a gene which is expressed in the milk of the animal. Various aspects of the invention are described in further detail in the following subsections.
I. Production of Human Antibodies to CD25
Human monoclonal antibodies of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed, e.g., viral or oncogenic transformation of B lymphocytes or phage display techniques using libraries of human antibody genes.
The preferred animal system for preparing hybridomas that secrete human monoclonal antibodies is the murine system. Hybridoma production in the mouse is a very well established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.
In a preferred embodiment, human monoclonal antibodies directed against CD25 can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include mice referred to herein as HuMAb mice, e.g. HCo7 and HCo12 mice, and KM mice, respectively, and are collectively referred to herein as “transgenic mice.”
The HuMAb mouse contains a human immunoglobulin gene miniloci that encodes unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg, N. et al. (1994) Nature 368 (6474):856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or κ light chain and in response to immunization, the introduced human heavy and light chain transgenes, undergo class switching and somatic mutation to generate high affinity human IgG,κ monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13:65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536-546). The preparation of HuMAb mice is described in detail in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al (1993) International Immunology 5:647-656; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Lonberg N. et al., (1994) Nature 368(6474):856-859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Taylor, L. et al. (1994) International Immunology 6:579-591; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13:65-93; Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536-546; Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851. See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay, as well as U.S. Pat. No. 5,545,807 to Surani et al.; WO 98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.
The HCo7 mice have a JKD disruption in their endogenous light chain (kappa) genes (as described in Chen et al. (1993) EMBO J. 12: 821-830), a CMD disruption in their endogenous heavy chain genes (as described in Example 1 of WO 01/14424), a KCo5 human kappa light chain transgene (as described in Fishwild et al. (1996) Nature Biotechnology 14:845-851), and a HCo7 human heavy chain transgene (as described in U.S. Pat. No. 5,770,429).
The HCo12 mice have a JKD disruption in their endogenous light chain (kappa) genes (as described in Chen et al. (1993) EMBO J. 12: 821-830), a CMD disruption in their endogenous heavy chain genes (as described in Example 1 of WO 01/14424 by Korman et al.), a KCo5 human kappa light chain transgene (as described in Fishwild et al. (1996) Nature Biotechnology 14:845-851), and a HCo12 human heavy chain transgene (as described in Example 2 of WO 01/14424 by Korman et al.). In the KM mouse strain, the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al. (1993) EMBO J. 12:811-820 and the endogenous mouse heavy chain gene has been homozygously disrupted as described in Example 1 of WO 01/09187. This mouse strain carries a human kappa light chain transgene, KCo5, as described in Fishwild et al. (1996) Nature Biotechnology 14:845-851. This mouse strain also carries a human heavy chain transchromosome composed of chromosome 14 fragment hCF (SC20) as described in WO 02/43478.
Immunizations
To generate fully human monoclonal antibodies to CD25, transgenic or transchromosomal mice containing human immunoglobulin genes (e.g., HCo12, HCo7 or KM mice) can be immunized with an enriched preparation of CD25 antigen, recombinant CD25, and/or cells expressing CD25, as described, for example, by Lonberg et al. (1994), supra; Fishwild et al. (1996), supra, and WO 98/24884. Alternatively, mice can be immunized with DNA encoding human CD25. Preferably, the mice will be 6-16 weeks of age upon the first infusion. For example, an enriched preparation of the CD25 antigen or recombinant CD25 antigen can be used to immunize the HuMAb mice intraperitoneally. In the event that immunizations using a purified or enriched preparation of the CD25 antigen do not result in antibodies, mice can also be immunized with cells expressing CD25, e.g., a cell line, to promote immune responses.
Cumulative experience with various antigens has shown that the HuMAb transgenic mice respond best when initially immunized intraperitoneally (IP) or subcutaneously (SC) with CD25 expressing cells in complete or incomplete Freund's adjuvant, followed by IP immunizations (up to a total of 10) with CD25 expressing cells, e.g. in phosphate buffered saline (PBS). The immune response can be monitored over the course of the immunization protocol with serum samples being obtained by retroorbital bleeds. The serum can be screened by FACS analysis (as described below), and mice with sufficient titers of anti-CD25 human immunoglobulin can be used for fusions. Mice can be boosted intravenously with CD25 expressing cells before, for example 3 and 2 days before, sacrifice and removal of the spleen and lymph nodes.
Generation of Hybridomas Producing Human Monoclonal Antibodies to CD25
To generate hybridomas producing human monoclonal antibodies to human CD25, splenocytes and lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can then be screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice can be fused to SP2/0-Ag14 myeloma cells (ATCC, CRL 1581) with 50% PEG (w/v). Cells can be plated at approximately 1×105 per well in flat bottom microtiter plate, followed by a two-week incubation in selective medium containing besides usual reagents 10% fetal Clone Serum, 5 Origen Hybridoma Cloning Factor (IGEN) and 1×HAT (Sigma). After approximately two weeks, cells can be cultured in medium in which the HAT is replaced with HT (Sigma). Individual wells can then be screened by ELISA for human kappa-light chain containing antibodies and by FACS analysis using CD25 expressing cells for CD25 specificity. Once extensive hybridoma growth occurs, the clones can be screened for IgG production, usually after 7-10 days. The antibody-secreting hybridomas can be replated, screened again, and if still positive for human IgG, anti-CD25 monoclonal antibodies can be subcloned at least twice by limiting dilution. The stable subclones can then be cultured in vitro to generate antibody in tissue culture medium for characterization.
Generation of Transfectomas Producing Human Monoclonal Antibodies to CD25
Human antibodies of the invention also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (Morrison, S. (1985) Science 229:1202).
For example, to express the antibodies, or antibody fragments thereof, DNAs encoding partial or full-length light and heavy chains, can be obtained by standard molecular biology techniques (e.g., PCR amplification, site directed mutagenesis) and can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term “operatively linked” is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). The light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term “regulatory sequence” is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter or β-globin promoter.
In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, and most preferably mammalian host cells, is the most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.
Preferred mammalian host cells for expressing the recombinant antibodies of the invention include CHO cells (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NS/0 myeloma cells, COS cells, HEK293 cells and SP2 cells. In particular for use with NS/0 myeloma cells, another preferred expression system is the GS (glutamine synthetase) gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338 841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
Further Recombinant Means for Producing Human Monoclonal Antibodies to CD25
Alternatively the cloned antibody genes can be expressed in other expression systems, including prokaryotic cells, such as microorganisms, e.g. E. coli for the production of scFv antibodies, algi, as well as insect cells. Furthermore, the antibodies can be produced in transgenic nonhuman animals, such as in milk from sheep and rabbits or eggs from hens, or in transgenic plants. See e.g. Verma, R., et al. (1998). Antibody engineering: Comparison of bacterial, yeast, insect and mammalian expression systems. J. Immunol. Meth. 216:165-181; Pollock, et al. (1999). Transgenic milk as a method for the production of recombinant antibodies. J. Immunol. Meth. 231:147-157; and Fischer, R., et al. (1999). Molecular farming of recombinant antibodies in plants. Biol. Chem. 380:825-839.
Use of Partial Antibody Sequences to Express Intact Antibodies
Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain CDRs. For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V(D)J joining during B cell maturation. Germline gene sequences will also differ from the sequences of a high affinity secondary repertoire antibody which contains mutations throughout the variable gene but typically clustered in the CDRs. For example, somatic mutations are relatively infrequent in the amino terminal portion of framework region 1 and in the carboxy-terminal portion of framework region 4. Furthermore, many somatic mutations do not significantly alter the binding properties of the antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody in order to recreate an intact recombinant antibody having binding properties similar to those of the original antibody (see WO 99/45962). Partial heavy and light chain sequence spanning the CDR regions is typically sufficient for this purpose. The partial sequence is used to determine which germline variable and joining gene segments contributed to the recombined antibody variable genes. The germline sequence is then used to fill in missing portions of the variable regions. Heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized as a set of short, overlapping, oligonucleotides and combined by PCR amplification to create an entirely synthetic variable region clone. This process has certain advantages such as elimination or inclusion or particular restriction sites, or optimization of particular codons.
The nucleotide sequences of heavy and light chain transcripts from hybridomas are used to design an overlapping set of synthetic oligonucleotides to create synthetic V sequences with identical amino acid coding capacities as the natural sequences. The synthetic heavy and kappa chain sequences can differ from the natural sequences in three ways: strings of repeated nucleotide bases are interrupted to facilitate oligonucleotide synthesis and PCR amplification; optimal translation initiation sites are incorporated according to Kozak's rules (Kozak (1991) J. Biol. Chem. 266:19867-19870); and HindIII sites are engineered upstream of the translation initiation sites.
For both the heavy and light chain variable regions, the optimized coding, and corresponding non-coding, strand sequences are broken down into 30-50 nucleotide approximately the midpoint of the corresponding non-coding oligonucleotide. Thus, for each chain, the oligonucleotides can be assembled into overlapping double stranded sets that span segments of 150-400 nucleotides. The pools are then used as templates to produce PCR amplification products of 150-400 nucleotides. Typically, a single variable region oligonucleotide set will be broken down into two pools which are separately amplified to generate two overlapping PCR products. These overlapping products are then combined by PCR amplification to form the complete variable region. It may also be desirable to include an overlapping fragment of the heavy or light chain constant region (including the BbsI site of the kappa light chain, or the AgeI site of the gamma heavy chain) in the PCR amplification to generate fragments that can easily be cloned into the expression vector constructs.
The reconstructed heavy and light chain variable regions are then combined with cloned promoter, leader sequence, translation initiation, constant region, 3′ untranslated, polyadenylation, and transcription termination sequences to form expression vector constructs. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected, or separately transfected into host cells which are then fused to form a host cell expressing both chains.
A similar procedure may be followed to graft novel antigen-specificity into an existing mature antibody. Preferably, an acceptor antibody is chosen which originates from the same variable germ-line gene as the CDR-donor antibody. One or more CDRs from the donor antibody are then transferred using the techniques described above.
Exemplary plasmids for use in construction of expression vectors for human IgGκ are described below. The plasmids were constructed so that PCR amplified V heavy and V kappa light chain cDNA sequences could be used to reconstruct complete heavy and light chain minigenes. These plasmids can be used to express completely human IgG1,κ or IgG4,κ antibodies. Similar plasmids can be constructed for expression of other heavy chain isotypes, or for expression of antibodies comprising lambda light chains.
Accordingly, in another embodiment, the invention provides various methods for preparing human anti-CD25 antibodies. In one embodiment, the method involves:
preparing an antibody comprising (1) human heavy chain framework regions and human heavy chain CDRs, wherein at least one of the human heavy chain CDRs comprises an amino acid sequence selected from the amino acid sequences of CDRs shown in FIGS. 1-10 (or corresponding amino acid residues in SEQ ID NOs:17-19, 23-25, 29-31, or 35-37); and (2) human light chain framework regions and human light chain CDRs, wherein at least one of the human light chain CDRs comprises an amino acid sequence selected from the amino acid sequences of CDRs shown in FIGS. 1-10 (or corresponding amino acid residues in SEQ ID NOs: 20-22, 26-28, 32-34, or 38-40); wherein the antibody retains the ability to bind to CD25.
The ability of the antibody to bind to CD25 can then be determined using standard binding assays, such as those set forth in the Examples (e.g., a FACS analysis).
Since it is well known in the art that antibody heavy and light chain CDR3 domains play a particularly important role in the binding specificity/affinity of an antibody for an antigen, the recombinant antibodies of the invention prepared as set forth above preferably comprise the heavy and light chain CDR3s of AB1, AB7, AB11, or AB12. The antibodies further can comprise the CDR2s of AB1, AB7, AB11, or AB12. The antibodies further can comprise the CDR1s of AB1, AB7, AB11, or AB12. Accordingly, the invention further provides anti-CD25 antibodies comprising: (1) human heavy chain framework regions, a human heavy chain CDR1 region, a human heavy chain CDR2 region, and a human heavy chain CDR3 region, wherein the human heavy chain CDR3 region is the CDR3 of AB1, AB7, AB11, or AB12 as shown in FIGS. 1-10 (or corresponding amino acid residues as shown in SEQ ID NOs: 19, 25, 31, or 37); and (2) human light chain framework regions, a human light chain CDR1 region, a human light chain CDR2 region, and a human light chain CDR3 region, wherein the human light chain CDR3 region is the CDR3 of AB1, AB7, AB11, or AB12 as shown in FIGS. 1-10 (or corresponding amino acid residues as shown in SEQ ID NOs:22, 28, 34, or 40), wherein the antibody binds to CD25. The antibody may further comprise the heavy chain CDR2 and/or the light chain CDR2 of AB1, AB7, AB11, or AB12. The antibody may further comprise the heavy chain CDR1 and/or the light chain CDR1 of AB1, AB7, AB11, or AB12.
Preferably, the CDR1, 2, and/or 3 of the engineered antibodies described above comprise the exact amino acid sequence(s) as those of AB1, AB7, AB11, or AB12 disclosed herein. However, the ordinarily skilled artisan will appreciate that some deviation from the exact CDR sequences of AB1, AB7, AB11, or AB12 may be possible while still retaining the ability of the antibody to bind CD25 effectively (e.g., conservative substitutions). Accordingly, in another embodiment, the engineered antibody may be composed of one or more CDRs that are, for example, 90%, 95%, 98% or 99.5% homologous to one or more CDRs of AB1, AB7, AB11, or AB12.
In addition or alternative to simply binding CD25, engineered antibodies such as those described above may be selected for their retention of other functional properties of antibodies of the invention, such as:
(1) high affinity binding to CD25;
(2) inhibition or blocking of CD25 binding to IL-2;
(3) elimination of T cells expressing CD25;
(4) tolerization of T cells;
(5) inhibition of proliferation of T cells expressing CD25;
(6) inhibition of anti-CD3 antibody-induced T cell proliferation of PBMCs;
(7) inhibition of MLR; and/or
(8) internalization of CD25 expressed on T cells.
Characterization of Binding of Human Monoclonal Antibodies to CD25
Human anti-CD25 antibodies of the invention can be isolated and characterized in a number of different ways. For example, selected hybridomas can be grown in suitable flasks for monoclonal antibody purification. Supernatants can then be filtered and concentrated before affinity chromatography with protein A-sepharose (for IgG1 isotype antibodies) (Pharmacia, Piscataway, N.J.) or anti-human IgG coated sepharose or protein G-sepharose in case of IgG3 isotype antibodies. Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using 1.43 extinction coefficient. The monoclonal antibodies can be aliquoted and stored at −80° C.
To determine if the selected human anti-CD25 monoclonal antibodies bind to unique epitopes, site-directed or multi-site directed mutagenesis can be used.
To determine the isotype of purified antibodies, isotype ELISAs can be performed. Wells of microtiter plates can be coated with 10 μg/ml of anti-human Ig overnight at 4° C. After blocking with 5% BSA (bovine serum albumin), the plates are reacted with 10 μg/ml of monoclonal antibodies or purified isotype controls, at ambient temperature for two hours. The wells can then be reacted with either human IgG1, IgG2, IgG3 or IgG4, IgE, IgA1, IgA2, or human IgM-specific alkaline phosphatase-conjugated probes. After washing, the plates are developed with pNPP substrate (1 mg/ml) and analyzed by OD at 405 nm.
In order to demonstrate the presence of anti-CD25 antibodies in sera of immunized mice or the binding of monoclonal antibodies to live cells expressing the CD25, flow cytometry can be used. Briefly, cell lines expressing CD25 (grown under standard growth conditions) are mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA and 0.02% sodium-azide, and incubated at 4° C. for 30 minutes. After washing, the cells are reacted with fluorescein-labeled anti-human IgG antibody under the same conditions as the primary antibody staining. The samples can be analyzed by flow cytometry with a flow cytometer (e.g., Becton Dickinson FACS instrument) using light and side scatter properties to gate on single, living cells. An alternative assay using fluorescence microscopy may be used (in addition to or instead of) the flow cytometry assay. Cells can be stained exactly as described above and examined by fluorescence microscopy. This method allows visualization of individual cells, but may have diminished sensitivity depending on the density of the antigen.
Anti-CD25 human IgGs can be further tested for reactivity with CD25 antigen by Western blotting. Briefly, cell extracts from cells expressing CD25 can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes, blocked with 20% non-fat milk, and probed with the monoclonal antibodies to be tested. Human IgG binding can be detected using anti-human IgG alkaline phosphatase and developed with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).
Inhibition of Activity of Cells Expressing CD25
In addition to binding specifically to CD25, human monoclonal anti-CD25 antibodies can be tested for their ability to inhibit various activities of cells, such as T cell and other lymphocytes, expressing CD25. For example, T cell proliferation assays can be carried out using known techniques. In one technique, human PBMCs are diluted in a suitable medium and then stimulated with, for example, an anti-CD3 antibody, before adding varying concentrations of the experimental antibodies to determine the effect they have on T cell proliferation. T cell proliferation of purified T cells can also be assessed in the presence of anti-CD3 and anti-CD28 monoclonal antibodies.
Assays for MLR can also be conducted using known techniques. For example, PBMCs from a first donor can be irradiated and mixed with PBMCs from a second donor. Varying concentrations of antibody can then be added to the cells, followed by measurement of the MLR response.
II. Production of Transgenic Non-Human Animals which Generate Human Monoclonal Anti-CD25 Antibodies
In yet another aspect, the invention provides transgenic and transchromosomal nonhuman animals, such as transgenic or transchromosomal mice, which are capable of expressing human antibodies that specifically bind to CD25. In a particular embodiment, the invention provides a transgenic or transchromosomal mouse having a genome comprising a human heavy chain transgene, such that the mouse produces human anti-CD25 antibodies when immunized with cells expressing CD25. The human heavy chain transgene can be integrated into the chromosomal DNA of the mouse, as is the case for transgenic, e.g., HuMAb mice, as described in detail herein and exemplified. Alternatively, the human heavy chain transgene can be maintained extrachromosomally, as is the case for transchromosomal (e.g., KM) mice as described in WO 02/43478. Such transgenic and transchromosomal animals are capable of producing multiple isotypes of human monoclonal antibodies to CD25 (e.g., IgG, IgA and/or IgE) by undergoing V-D-J/V-J recombination and isotype switching. The design of a transgenic or transchromosomal nonhuman animal that responds to foreign antigen stimulation with a heterologous antibody repertoire, requires that the heterologous immunoglobulin transgenes contained within the transgenic animal function correctly throughout the pathway of B cell development. This includes, for example, isotype switching of the heterologous heavy chain transgene. Accordingly, transgenes are constructed so that isotype switching can be induced and one or more of the following characteristics of antibody genes: (1) high level and cell-type specific expression, (2) functional gene rearrangement, (3) activation of and response to allelic exclusion, (4) expression of a sufficient primary repertoire, (5) signal transduction, (6) somatic hypermutation, and (7) domination of the transgene antibody locus during the immune response.
Not all of the foregoing criteria need be met. For example, in those embodiments wherein the endogenous immunoglobulin loci of the transgenic animal are functionally disrupted, the transgene need not activate allelic exclusion. Further, in those embodiments wherein the transgene comprises a functionally rearranged heavy and/or light chain immunoglobulin gene, the second criteria of functional gene rearrangement is unnecessary, at least for that transgene which is already rearranged. For background on molecular immunology, see, Fundamental Immunology, 2nd edition (1989), Paul William E., ed. Raven Press, N.Y.
In certain embodiments, the transgenic or transchromosomal nonhuman animals used to generate the human monoclonal antibodies of the invention contain rearranged, unrearranged or a combination of rearranged and unrearranged heterologous immunoglobulin heavy and light chain transgenes in the germline of the transgenic animal. Each of the heavy chain transgenes comprises at least one CH gene. In addition, the heavy chain transgene may contain functional isotype switch sequences, which are capable of supporting isotype switching of a heterologous transgene encoding multiple CH genes in the B cells of the transgenic animal. Such switch sequences may be those which occur naturally in the germline immunoglobulin locus from the species that serves as the source of the transgene CH genes, or such switch sequences may be derived from those which occur in the species that is to receive the transgene construct (the transgenic animal). For example, a human transgene construct that is used to produce a transgenic mouse may produce a higher frequency of isotype switching events if it incorporates switch sequences similar to those that occur naturally in the mouse heavy chain locus, as presumably the mouse switch sequences are optimized to function with the mouse switch recombinase enzyme system, whereas the human switch sequences are not. Switch sequences may be isolated and cloned by conventional cloning methods, or may be synthesized de novo from overlapping synthetic oligonucleotides designed on the basis of published sequence information relating to immunoglobulin switch region sequences (Mills et al., Nucl. Acids Res. 15:7305-7316 (1991); Sideras et al., Intl. Immunol. 1:631-642 (1989)). For each of the foregoing transgenic animals, functionally rearranged heterologous heavy and light chain immunoglobulin transgenes are found in a significant fraction of the B cells of the transgenic animal (at least 10%).
The transgenes used to generate the transgenic nonhuman animals of the invention include a heavy chain transgene comprising DNA encoding at least one variable gene segment, one diversity gene segment, one joining gene segment and at least one constant region gene segment. The immunoglobulin light chain transgene comprises DNA encoding at least one variable gene segment, one joining gene segment and at least one constant region gene segment. The gene segments encoding the light and heavy chain gene segments are heterologous to the transgenic animal in that they are derived from, or correspond to, DNA encoding immunoglobulin heavy and light chain gene segments from a species not consisting of the transgenic nonhuman animal. In one aspect of the invention, the transgene is constructed such that the individual gene segments are unrearranged, i.e., not rearranged so as to encode a functional immunoglobulin light or heavy chain. Such unrearranged transgenes support recombination of the V, D, and J gene segments (functional rearrangement) and preferably support incorporation of all or a portion of a D region gene segment in the resultant rearranged immunoglobulin heavy chain within the transgenic animal when exposed to CD25 antigen.
In an alternate embodiment, the transgenes comprise an unrearranged “mini-locus”. Such transgenes typically comprise a substantial portion of the C, D, and J segments as well as a subset of the V gene segments. In such transgene constructs, the various regulatory sequences, e.g. promoters, enhancers, class switch regions, splice-donor and splice-acceptor sequences for RNA processing, recombination signals and the like, comprise corresponding sequences derived from the heterologous DNA. Such regulatory sequences may be incorporated into the transgene from the same or a related species of the nonhuman animal used in the invention. For example, human immunoglobulin gene segments may be combined in a transgene with a rodent immunoglobulin enhancer sequence for use in a transgenic mouse. Alternatively, synthetic regulatory sequences may be incorporated into the transgene, wherein such synthetic regulatory sequences are not homologous to a functional DNA sequence that is known to occur naturally in the genomes of mammals. Synthetic regulatory sequences are designed according to consensus rules, such as, for example, those specifying the permissible sequences of a splice-acceptor site or a promoter/enhancer motif. For example, a minilocus comprises a portion of the genomic immunoglobulin locus having at least one internal (i.e., not at a terminus of the portion) deletion of a non-essential DNA portion (e.g., intervening sequence; intron or portion thereof) as compared to the naturally-occurring germline Ig locus.
Preferred transgenic and transchromosomal nonhuman animals, e.g., mice, will exhibit immunoglobulin production with a significant repertoire, ideally substantially similar to that of a human after adjusting for volume.
The repertoire will ideally approximate that shown in a human when adjusted for volume, usually with a diversity at least about 10% as great, preferably 25 to 50% or more. Generally, at least about a thousand different immunoglobulins (ideally IgG), preferably 104 to 106 or more, will be produced, depending on the number of different V, J and D regions introduced into the mouse genome and driven by the additional diversity generated by V(-D-)J gene segment rearrangements and random nucleotide additions at the joining regions. Typically, the immunoglobulins will exhibit an affinity (KD) for preselected antigens of below 10−8 M, such as of below 10−9 M, 10−10 M or 10−11 M or even lower.
Transgenic and transchromosomal nonhuman animals, e.g., mice, as described above can be immunized with, for example, cells expressing CD25. Alternatively, the transgenic animals can be immunized with DNA encoding human CD25. The animals will then produce B cells which undergo class-switching via switch recombination (cis-switching) and express immunoglobulins reactive with CD25. The immunoglobulins will be human antibodies (also referred to as “human sequence antibodies”), wherein the heavy and light chain polypeptides are encoded by human transgene sequences, which may include sequences derived by somatic mutation and V region recombinatorial joints, as well as germline-encoded sequences; these human antibodies can be referred to as being substantially identical to a polypeptide sequence encoded by a human VL and JL or VH, DH and JH gene segments, even though other non-germline sequences may be present as a result of somatic mutation and differential V-J and V-D-J recombination joints. The variable regions of each antibody chain are typically at least 80 percent similar to human germline V, and J gene segments, and, in the case of heavy chains, human germline V, D, and J gene segments; frequently at least 85 percent similar to human germline sequences present on the transgene; often 90 or 95 percent or more similar to human germline sequences present on the transgene. However, since non-germline sequences are introduced by somatic mutation and VJ and VDJ joining, the human sequence antibodies will frequently have some variable region sequences which are not encoded by human V, D, or J gene segments as found in the human transgene(s) in the germline of the mice. Typically, such non-germline sequences (or individual nucleotide positions) will cluster in or near CDRs, or in regions where somatic mutations are known to cluster.
Another aspect of the invention includes B cells derived from transgenic or transchromosomal nonhuman animals as described herein. The B cells can be used to generate hybridomas expressing human monoclonal antibodies which bind with high affinity (e.g., a dissociation equilibrium constant (KD) of lower than 10−8 M) to human CD25. Thus, in another embodiment, the invention provides a hybridoma which produces a human antibody having an affinity (KD) of below 10−8 M, such as of below 10−9 M, 10−10 M or 10−11 M or even lower when determined by scatchard analysis of CD25 expressing cells using a radio-actively labeled monoclonal antibody or by determination of the half-maximal binding concentration using FACS analysis, or by analysis using surface plasmon resonance as measured on a BIAcore® instrument.
Herein the monoclonal antibody comprises a human sequence light chain composed of (1) a light chain variable region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human VL gene segment and a human JL segment, and (2) a light chain constant region encoded by a human CL gene segment; and a human sequence heavy chain composed of a (1) a heavy chain variable region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human VH gene segment, a D region, and a human JH segment, and (2) a constant region encoded by a human CH gene segment. It should be noted that human D genes may be substantially altered by recombination and somatic mutation events such that the original human germ-line sequence may not be readily recognized.
The development of high affinity human monoclonal antibodies against CD25 can be facilitated by a method for expanding the repertoire of human variable region gene segments in a transgenic nonhuman animal having a genome comprising an integrated human immunoglobulin transgene, said method comprising introducing into the genome a V gene transgene comprising V region gene segments which are not present in said integrated human immunoglobulin transgene. Often, the V region transgene is a yeast artificial chromosome (YAC) comprising a portion of a human VH or VL (VK) gene segment array, as may naturally occur in a human genome or as may be spliced together separately by recombinant methods, which may include out-of-order or omitted V gene segments. Often at least five or more functional V gene segments are contained on the YAC. In this variation, it is possible to make a transgenic animal produced by the V repertoire expansion method, wherein the animal expresses an immunoglobulin chain comprising a variable region sequence encoded by a V region gene segment present on the V region transgene and a C region encoded on the human Ig transgene. By means of the V repertoire expansion method, transgenic animals having at least 5 distinct V genes can be generated; as can animals containing at least about 24 V genes or more. Some V gene segments may be non-functional (e.g., pseudogenes and the like); these segments may be retained or may be selectively deleted by recombinant methods available to the skilled artisan, if desired.
Once the mouse germline has been engineered to contain a functional YAC having an expanded V segment repertoire, substantially not present in the human Ig transgene containing the J and C gene segments, the trait can be propagated and bred into other genetic backgrounds, including backgrounds where the functional YAC having an expanded V segment repertoire is bred into a nonhuman animal germline having a different human Ig transgene. Multiple functional YACs having an expanded V segment repertoire may be bred into a germline to work with a human Ig transgene (or multiple human Ig transgenes). Although referred to herein as YAC transgenes, such transgenes when integrated into the genome may substantially lack yeast sequences, such as sequences required for autonomous replication in yeast; such sequences may optionally be removed by genetic engineering (e.g., restriction digestion and pulsed-field gel electrophoresis or other suitable method) after replication in yeast is no longer necessary (i.e., prior to introduction into a mouse ES cell or mouse prozygote). Methods of propagating the trait of human sequence immunoglobulin expression, include breeding a transgenic animal having the human Ig transgene(s), and optionally also having a functional YAC having an expanded V segment repertoire. Both VH and VL gene segments may be present on the YAC. The transgenic animal may be bred into any background desired by the practitioner, including backgrounds harboring other human transgenes, including human Ig transgenes and/or transgenes encoding other human lymphocyte proteins. The invention also provides a high affinity human sequence immunoglobulin produced by a transgenic mouse having an expanded V region repertoire YAC transgene. Although the foregoing describes a preferred embodiment of the transgenic animal of the invention, other embodiments are contemplated which have been classified in three categories:
I. Transgenic animals containing an unrearranged heavy and rearranged light chain immunoglobulin transgene;
II. Transgenic animals containing an unrearranged heavy and unrearranged light chain immunoglobulin transgene; and
III. Transgenic animal containing rearranged heavy and an unrearranged light chain immunoglobulin transgene.
Of these categories of transgenic animal, the preferred order of preference is as follows II>I>III where the endogenous light chain genes (or at least the κ gene) have been knocked out by homologous recombination (or other method) and I>II>III where the endogenous light chain genes have not been knocked out and must be dominated by allelic exclusion.
III. Bispecific/Multispecific Molecules which Bind to CD25
In yet another embodiment of the invention, human monoclonal antibodies to CD25 can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., an Fab′ fragment) to generate a bispecific or multispecific molecule which binds to multiple binding sites or target epitopes. For example, an antibody of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, peptide or binding mimetic.
Accordingly, the present invention includes bispecific and multispecific molecules comprising at least one first binding specificity for CD25 and a second binding specificity for a second target epitope. In a particular embodiment of the invention, the second target epitope is CD3, CD4, IL-15R, membrane bound or receptor bound TNF-α, or membrane bound or receptor bound IL-15. In another embodiment, the second target epitope is an Fc receptor, e.g., human FcγRI (CD64) or human FcαRI (CD89), or a T cell receptor. Therefore, the invention includes bispecific and multispecific molecules capable of binding both to FcγR, FcαR or FcεR expressing effector cells (e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)), and to target cells expressing CD25. These bispecific and multispecific molecules target CD25 expressing cells to effector cell and, like the human monoclonal antibodies of the invention, trigger Fc receptor-mediated effector cell activities, such as phagocytosis of a CD25 expressing cells, ADCC, cytokine release, or generation of superoxide anion.
Bispecific and multispecific molecules of the invention can further include a third binding specificity, in addition to an anti-Fc binding specificity and the anti-CD25 binding specificity. In one embodiment, the third binding specificity is an anti-enhancement factor (EF) portion, e.g., a molecule which binds to a surface protein involved in cytotoxic activity and thereby increases the immune response against the target cell. The “anti-enhancement factor portion” can be an antibody, a functional antibody fragment or a ligand that binds to a given molecule, e.g., an antigen or a receptor, and thereby results in an enhancement of the effect of the binding determinants for the Fc receptor or target cell antigen. The “anti-enhancement factor portion” can bind an Fc receptor or a target cell antigen. Alternatively, the anti-enhancement factor portion can bind to an entity that is different from the entity to which the first and second binding specificities bind. For example, the anti-enhancement factor portion can bind a cytotoxic T cell (e.g. via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell that results in an increased immune response against the target cell).
In one embodiment, the bispecific and multispecific molecules of the invention comprise as a binding specificity at least one antibody, including, e.g., an Fab, Fab′, F(ab′)2, Fv, or scFv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in Ladner et al. U.S. Pat. No. 4,946,778. The antibody may also be a binding-domain immunoglobulin fusion protein as disclosed in US 2003/0118592 and US 2003/0133939.
In one embodiment, the binding specificity for an Fc receptor is provided by a human monoclonal antibody, the binding of which is not blocked by human immunoglobulin G (IgG). As used herein, the term “IgG receptor” refers to any of the eight γ-chain genes located on chromosome 1. These genes encode a total of twelve transmembrane or soluble receptor isoforms which are grouped into three Fcγ receptor classes: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). In one preferred embodiment, the Fcγ receptor is a human high affinity FcγRI.
The production and characterization of these preferred monoclonal antibodies are described by Fanger et al. in WO 88/00052 and in U.S. Pat. No. 4,954,617. These antibodies bind to an epitope of FcγRI, FcγRII or FcγRIII at a site which is distinct from the Fcγ binding site of the receptor and, thus, their binding is not blocked substantially by physiological levels of IgG. Specific anti-FcγRI antibodies useful in this invention are MAb 22, MAb 32, MAb 44, MAb 62 and MAb 197. In other embodiments, the anti-FcγRI antibody is a humanized form of MAb 22 (H22). The production and characterization of the H22 antibody is described in Graziano, R. F. et al. (1995) J. Immunol. 155 (10): 4996-5002 and WO 94/10332. The H22 antibody producing cell line was deposited at the American Type Culture Collection on Nov. 4, 1992 under the designation HA022CL1 and has the accession No. CRL 11177.
In still other preferred embodiments, the binding specificity for an Fc receptor is provided by an antibody that binds to a human IgA receptor, e.g., FcαRI (CD89), the binding of which is preferably not blocked by human immunoglobulin A (IgA). The term “IgA receptor” is intended to include the gene product of one α-gene (FcαRI) located on chromosome 19. This gene is known to encode several alternatively spliced transmembrane isoforms of 55 to 110 kDa. FcαRI (CD89) is constitutively expressed on monocytes/macrophages, eosinophilic and neutrophilic granulocytes, but not on non-effector cell populations. FcαRI has medium affinity for both IgA1 and IgA2, which is increased upon exposure to cytokines such as G-CSF or GM-CSF (Morton, H. C. et al. (1996) Critical Reviews in Immunology 16:423-440). Four FcαRI-specific monoclonal antibodies, identified as A3, A59, A62 and A77, which bind FcαRI outside the IgA ligand binding domain, have been described (Monteiro, R. C. et al., 1992, J. Immunol. 148:1764).
FcαRI and FcγRI are preferred trigger receptors for use in the invention because they are (1) expressed primarily on immune effector cells, e.g., monocytes, PMNs, macrophages and dendritic cells; (2) expressed at high levels (e.g., 5,000-100,000 per cell); (3) mediators of cytotoxic activities (e.g., ADCC, phagocytosis); and (4) mediating enhanced antigen presentation of antigens, including self-antigens, targeted to them.
An “effector cell specific antibody” as used herein refers to an antibody or functional antibody fragment that binds the Fc receptor of effector cells. Preferred antibodies for use in the subject invention bind the Fc receptor of effector cells at a site which is not bound by endogenous immunoglobulin.
As used herein, the term “effector cell” refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, e.g., lymphocytes (e.g., B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Some effector cells express specific Fc receptors and carry out specific immune functions. In preferred embodiments, an effector cell is capable of inducing ADCC, e.g., a neutrophil capable of inducing ADCC. For example, monocytes, macrophages, which express FcR are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. In other embodiments, an effector cell can phagocytose a target antigen, target cell, or microorganism. The expression of a particular FcR on an effector cell can be regulated by humoral factors such as cytokines. For example, expression of FcγRI has been found to be up-regulated by interferon gamma (IFN-γ). This enhanced expression increases the cytotoxic activity of FcγRI-bearing cells against targets. An effector cell can phagocytose or lyse a target antigen or a target cell.
“Target cell” shall mean any cell in a subject (e.g., a human or animal) that can be targeted by a composition (e.g., a human monoclonal antibody, a bispecific or a multispecific molecule) of the invention. In preferred embodiments, the target cell is a cell expressing or overexpressing CD25. Cells expressing CD25 typically include activated T cells, monocytes and B cells.
Bispecific and multispecific molecules of the present invention can be made using chemical techniques (see e.g., D. M. Kranz et al. (1981) Proc. Natl. Acad. Sci. USA 78:5807), “polydoma” techniques (See U.S. Pat. No. 4,474,893, to Reading), or recombinant DNA techniques.
In particular, bispecific and multispecific molecules of the present invention can be prepared by conjugating the constituent binding specificities, e.g., the anti-FcR and anti-CD25 binding specificities, using methods known in the art and described in the examples provided herein. For example, each binding specificity of the bispecific and multispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus, Behring Ins. Mitt. (1985) No. 78, 118-132; Brennan et al., Science (1985) 229:81-83, and Glennie et al., J. Immunol. (1987) 139:2367-2375. Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).
When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.
Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific and multispecific molecule is a MAb×MAb, MAb×Fab, Fab×F(ab′)2 or ligand x Fab fusion protein. A bispecific and multispecific molecule of the invention, e.g., a bispecific molecule can be a single chain molecule, such as a single chain bispecific antibody, a single chain bispecific molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific and multispecific molecules can also be single chain molecules or may comprise at least two single chain molecules. Methods for preparing such bi- and multispecific molecules are described for example in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858.
Binding of the bispecific and multispecific molecules to their specific targets can be confirmed by enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), FACS analysis, a bioassay (e.g., growth inhibition), BIAcore® analysis, or a Western Blot Assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, the FcR-antibody complexes can be detected using e.g., an enzyme-linked antibody or antibody fragment which recognizes and specifically binds to the antibody-FcR complexes. Alternatively, the complexes can be detected using any of a variety of other immunoassays. For example, the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986). The radioactive isotope can be detected by such means as the use of a γ counter or a scintillation counter or by autoradiography.
IV. Immunoconjugates
In another aspect of the invention, human anti-CD25 antibodies are conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radioisotope. Such conjugates are referred to herein as “immunoconjugates”. Immunoconjugates which include one or more cytotoxins are referred to as “immunotoxins.” A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-de-hydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
Suitable therapeutic agents for forming immunoconjugates of the invention include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisplatin (cis-dichlorodiamine platinum (II) (DDP)), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). Other examples of therapeutic cytotoxins that can be conjugated to an antibody of the invention include calicheamicins and duocarmycins.
Antibodies of the present invention also can be conjugated to a radioisotope, e.g., iodine-131, yttrium-90 or indium-111, to generate cytotoxic radiopharmaceuticals for treating a CD25-related disorder, such as a cancer. The antibody conjugates of the invention can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin A, or diphtheria toxin, or an agent active at the cell surface, such as phospholipase enzymes, e.g. phospholipase C.
Techniques for conjugating such therapeutic moieties to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985); and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58 (1982).
In a further embodiment, the human monoclonal antibodies according to the invention are attached to a linker-chelator, (e.g. tiuxetan), which allows for the antibody to be conjugated to a radioisotope.
V. Pharmaceutical Compositions
In another aspect, the present invention provides compositions, including, pharmaceutical compositions, containing one or a combination of human monoclonal antibodies of the present invention. The compositions may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995.
Compositions of the invention also can be administered in combination therapy, i.e., combined with other agents relevant for the disease or condition to be treated. For example, the combination therapy can include a composition of the present invention with at least one immunosuppressive agent, at least one anti-inflammatory agent, at least one psoriasis agent, or at least one chemotherapeutic agent.
In one embodiment, such therapeutic agents include one or more immunosuppressive agents, such as cyclosporine, azathioprine, mycophenolic acid, mycophenolate mofetil, corticosteroids, such as prednisone, methotrexate, gold salts, sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine, 15-deoxyspergualine, 6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, anti-thymocyte globulin, etc.
In a further embodiment, the compositions of the invention are administered in combination with two or more immunosuppressive agents, such as prednisone and cyclosporine; prednisone, cyclosporine and azathioprine; or prednisone, cyclosporine and mycophenolate mofetil.
In a further embodiment, such therapeutic agents include one or more anti-inflammatory agents, such as a steroidal drug or a NSAID (nonsteroidal anti-inflammatory drug). Preferred agents include, for example, aspirin and other salicylates, Cox-2 inhibitors, such as rofecoxib and celecoxib, NSAIDs such as ibuprofen, fenoprofen, naproxen, sulindac, diclofenac, piroxicam, ketoprofen, diflunisal, nabumetone, etodolac, oxaprozin, and indomethacin.
In another embodiment, such therapeutic agents include one or more DMARDs, such as methotrexate, hydroxychloroquine, sulfasalazine, pyrimidine synthesis inhibitors, e.g. leflunomide, IL-1 receptor blocking agents, e.g. anakinra, and TNF-α blocking agents, e.g. etanercept, infliximab and adalimumab. Further suitable DMARDs are anti-IL-6R antibodies, CTLA4Ig, and anti-IL-15 antibodies.
In another embodiment, such therapeutic agents include one or more agents for treating inflammatory or hyperproliferative skin disorders, such as topical medications, including coal tar, A vitamin, anthralin, calcipotrien, tarazotene, and corticosteroids, oral or injected medications, such as corticosteroids, methotrexate, retinoids, e.g. acicretin, cyclosporine, etanercept, alefacept, efaluzimab, 6-thioguanine, mycophenolate mofetil, tacrolimus (FK-506), and hydroxyurea. Other examples are CTLA4Ig and infliximab. Other treatments may include exposure to sunlight or phototherapy, including UVB (broad-band and narrow-band ultraviolet B), UVA (ultraviolet A) and PUVA (psoralen methoxalen plus ultraviolet A).
In a further embodiment, the compositions of the invention are administered in combination with two or more of the above therapies, such as methotrexate+phototherapy (PUVA or UVA); methotrexate+acitretin; acitretin+phototherapy (PUVA or UVA); methotrexate+acitretin+phototherapy (PUVA or UVB); hydroxyurea+phototherapy (PUVA or UVB); hydroxyurea+acitretin; cyclosporine+methotrexate; or calcipotrien+phototherapy (UVB).
In yet another embodiment, such therapeutic agents include one or more chemotherapeutics, such as doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, vindesine, vincristine, and chlorambucil.
In yet another embodiment, the present antibodies may be administered in conjunction with radiotherapy and/or bone marrow transplantation.
In still another embodiment, the present antibodies may be administered in combination with other antibodies, e.g. other immunosuppressive human monoclonal antibodies, such as antibodies binding to p75 of the IL-2 receptor, or antibodies binding to e.g. MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40, CD45, IFN-γ, TNF-α, IL-4, IL-5, IL-6R, IL-7, IL-8, IL-10, CD11a, CD20 or CD58, or antibodies binding to their ligands; or in combination with other immunomodulatory compounds, e.g., soluble IL-15R or IL-10.
As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
A “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous acids and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
Compositions of the present invention, including pharmaceutical (therapeutic) compositions, can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
To administer compositions of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
In one embodiment the human monoclonal antibodies of the invention are administered in crystalline form by subcutaneous injection, cf. Yang et al. (2003) PNAS, 100(12):6934-6939.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Therapeutic compositions of the present invention can be formulated for particular routes of administration, such as oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01% to about 99% of active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%.
Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forms for the topical or transdermal administration of compositions of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.01 to 99.5% (more preferably, 0.1 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a compositions of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the site of the target. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). The dosage can be determined or adjusted by measuring the amount of circulating monoclonal anti-CD25 antibodies at different time points following administration in a biological sample by making use of anti-idiotypic antibodies targeting the anti-CD25 antibodies.
Human monoclonal antibodies of the invention may be administered for prevention of transplant rejection by induction treatment, i.e. as a prophylactic short-term therapy for single or multiple administration before transplantation and in the very early phase following transplantation, e.g. shortly before the transplantation and up to 3 months after transplantation.
In one embodiment, monoclonal antibodies of the invention may e.g. be administered for prevention of transplant rejection in total dosages of about 20 to 100 mg e.g. administered as 15 or 20 mg intravenous infusions with the first dose given pre-operatively and the subsequent doses given within the first 10 days post-operatively. Alternatively, the dosages may be administered by bolus injections. In another embodiment monoclonal antibodies of the invention may be administered for prevention of transplant rejection in a dosage of from 0.5-1.5 mg/kg intravenously, every other week for up to five doses with the first dose given pre-operatively. Such administration may be combined with immunosuppressive therapy, e.g. steroids, such as prednisone or methylprednisolone, and cyclosporine; steroids, such as prednisone or methylprednisolone, cyclosporine and azathioprine; or steroids, such as prednisone or methylprednisolone, cyclosporine and mycophenolate mofetil. Administration of the human antibodies may advantageously be steroid sparing or result in rapid steroid withdrawal.
In another embodiment, human monoclonal antibodies of the invention may be administered for treating or preventing transplant rejection by a two-dose intravenous infusion regimen (about 20 mg per dose on the day of transplantation and about 20 mg at day 4 post-transplantation). Such administration may be combined with immuno-suppressive therapy, e.g. as disclosed above. For example, 1 g mycophenolate mofetil may be administered orally before surgery, and 500 mg methylprednisolone at the time of anesthesia induction. Cyclosporine may be introduced on the second day after transplantation and mycophenolate mofetil may be continued at 1 g after transplantation. Steroids may be tapered to prednisone 20 mg orally on the fourth postoperative day.
In yet another embodiment, human monoclonal antibodies of the invention may be administered for treating or preventing transplant rejection by a two-dose induction therapy the first 1 mg/kg dose given 1 hour before surgery and the second dose 4 days after transplantation. Such administration may be combined with immunosuppressive therapy, e.g. as disclosed above.
Human monoclonal antibodies of the invention may be administered for prevention of transplant rejection by long-term therapy, e.g. by administration of a dose in the range of 10 to 150 mg, such as 20 to 40 mg, on a weekly basis or monthly basis, for example 3 to 8 weekly administrations, optionally followed by one or more monthly administrations. By long-term therapy cyclosporine maintenance therapy may be reduced or avoided.
Therapeutic antibody compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or U.S. Pat. No. 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.
In certain embodiments, human monoclonal antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention can cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134), different species of which may comprise the formulations of the inventions, as well as components of the invented molecules; p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273. In one embodiment of the invention, the therapeutic compounds of the invention are formulated in liposomes; in a more preferred embodiment, the liposomes include a targeting moiety. In a most preferred embodiment, the therapeutic compounds in the liposomes are delivered by bolus injection to a site proximal to the desired area, e.g., the site of inflammation or infection, or the site of a tumor. The composition must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
The efficient dosages and the dosage regimens for the human monoclonal antibodies of the invention depend on the disease or condition to be treated and can be determined by the persons skilled in the art.
A “therapeutically effective dosage” for preventing transplant rejection preferably will reduce the number and severity of early transplant rejection episodes.
A “therapeutically effective dosage” for rheumatoid arthritis preferably will result in an ACR20 Preliminary Definition of Improvement in the patients, more preferred in an ACR50 Preliminary Definition of Improvement and even more preferred in an ACR70 Preliminary Definition of Improvement.
ACR20 Preliminary Definition of Improvement is defined as: ≥20% improvement in: Tender Joint Count (TJC) and Swollen Joint Count (SJC) and ≥20% improvement in 3 of following 5 assessments: Patient Pain Assessment (VAS), Patient Global assessment (VAS), Physician Global Assessment (VAS), Patient Self-Assessed Disability (HAQ), and Acute Phase Reactant (CRP or ESR).
ACR50 and ACR70 are defined in the same way with ≥50% and ≥70% improvements, respectively. For further details see Felson et al. in American College of Rheumatology Preliminary Definition of Improvement in Rheumatoid Arthritis; Arthritis Rheumatism (1995) 38:727-735.
Alternatively, a therapeutically effective dosage for rheumatoid arthritis can be measured by DAS (disease activity score), including DAS28 and, more preferably, DAS56, as defined by EULAR.
A “therapeutically effective dosage” for psoriasis preferably will result in a PASI50, more preferably a PASI75, and even more preferably a PASI90 in the patients or a reduction in the overall psoriasis evaluation comparing impression of improvement after drug treatment when compared to pretreatment condition. PASI (Psoriasis Area and Severity Index) is a score system used for evaluation of the area and severity of the disease. PASI50 is defined as ≥50% improvement of the score. In the same way, PASI75 and PASI90 are defined as ≥75% and ≥90% improvement of the score, respectively.
A “therapeutically effective dosage” for tumor therapy can be measured by objective tumor responses which can either be complete or partial. A complete response (CR) is defined as no clinical, radiological or other evidence of disease. A partial response (PR) results from a reduction in aggregate tumor size of greater than 50%. Median time to progression is a measure that characterizes the durability of the objective tumor response.
A “therapeutically effective dosage” for tumor therapy can also be measured by its ability to stabilize the progression of disease. The ability of a compound to inhibit cancer can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit cell growth or to induce apoptosis by in vitro assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
VI. Uses and Methods of the Invention
Human antibodies of the present invention, as well as derivatives/conjugates and compositions thereof, have numerous utilities involving the treatment of CD25 mediated disorders or disorders involving cells expressing CD25.
In one embodiment, human antibodies of the present invention can be administered in vivo to a subject to block or inhibit binding of CD25 to its ligand (IL-2). This, in turn, can be used to prevent or inhibit a variety of diseases associated with CD25 bearing cells.
Exemplary diseases that can be treated (e.g., ameliorated) or prevented include, but are not limited to, transplant rejection, including allograft and xenograft rejection, in patients undergoing or who have undergone organ or tissue transplantation, such as heart, lung, combined heart-lung, trachea, kidney, liver, pancreas, oesophagus, bowel, skin, limb transplantation, umbilical cord transplantation, stem cell transplantation, islet cell transplantation, etc. Such patients includes adults but can also be pediatric patients.
Antibodies of the present invention may thus be used in prophylaxis of allograft and xenograft rejection or be used to reverse, treat, or otherwise ameliorate acute allograft or zenograft rejection episodes.
Further diseases that can be treated include graft-versus-host disease, e.g. blood transfusion graft-versus-host disease and bone marrow graft-versus-host disease; inflammatory, immune or autoimmune diseases, such as rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, type 1 diabetes, insulin-requiring type 2 diabetes, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, dermato-polymyositis, Sjögren's syndrome, arteritides, including giant cell arteritis, aplastic anemia, asthma, scleroderma, and uveitis; inflammatory or hyperproliferative skin disorders, e.g., psoriasis, including plaque psoriasis, pustulosis palmoplantaris (PPP), erosive lichen planus, pemphigus bullosa, epidermolysis bullosa, contact dermatitis and atopic dermatitis; and a variety of lymphoid neoplasms, e.g., T cell leukemia, Hodgkin's disease, hairy cell leukemia, or cutaneous T cell lymphoma, including mycosis fungoides and Sezary's syndrome.
Further diseases that can be treated are
malignancies wherein an inhibition of infiltrating CD25+ regulatory T cells is beneficial, such as gastric cancer, esophageal cancers, malignant melanoma, colorectal cancer, pancreas cancer, breast cancer, small cell lung cancer, non-small cell lung cancer, cervical cancer, ovarian cancer, and renal cell carcinoma;
hematological disorders, such as adult T cell leukemia/lymphoma, anaplastic large cell lymphoma, chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), peripheral T cell lymphoma, and secondary amyloidosis;
skin disorders, such as pyoderma gangraenosum, granuloma annulare, allergic contact dermatitis, cicatricial pemphigoid, and herpes gestationis;
hepato-gastrointestinal disorders, such as collagen colitis, sclerosing cholangitis, chronic active hepatitis, lupoid hepatitis, autoimmune hepatitis, alcoholic hepatitis, chronic pancreatis, and acute pancreatitis;
cardiac disorders, such as myocarditis, and pericarditis;
vascular disorders, such as arteriosclerosis, giant cell arteritis/polymyalgia rheumatica, Takayasu arteritis, polyarteritis nodosa, Kawasaki syndrome, Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, leukocytoclastic angiitis, and secondary leukocytoclastic vasculitis;
renal disorders, such as acute glomerulonphritis, chronic glomerulonephritis, minimal change nephritis, and Goodpasture's syndrome;
pulmonary disorders, such as alveolitis, bronchiolitis obliterans, silicosis, and berylliosis;
neurological disorders, such as multiple sclerosis, Alzheimer's disease, myasthenia gravis, chronic demyelinating polyneuropathy, and polyradiculitis including Guillain-Barré syndrome;
connective tissue disorders, such as relapsing polychondritis, sarcoidosis, systemic lupus erythematosus, CNS lupus, discoid lupus, lupus nephritis, chronic fatigue syndrome, and fibromyalgia;
endocrinological disorders, such as Graves' disease, Hashimoto's thyroiditis, and subacute thyroiditis; and
viral infections, such as tropical spastic paraparesis.
Suitable routes of administering the antibody compositions (e.g., human antibodies and immunoconjugates) of the invention in vivo and in vitro are well known in the art and can be selected by those of ordinary skill. For example, the antibody compositions can be administered by injection (e.g., intravenous or subcutaneous). Suitable dosages of the molecules used will depend on the age and weight of the subject and the concentration and/or formulation of the antibody composition.
The antibody can be administered alone or along with another therapeutic agent, such as an immunosuppressive agent, an anti-inflammatory agent, an agent for treating inflammatory or hyperproliferative skin disorders, a chemotherapeutic agent, or a cytotoxin which acts in conjunction with or synergistically with the antibody composition to treat or prevent diseases associated with cells expressing CD25, especially activated T cells.
As previously described, human anti-CD25 antibodies of the invention can be co-administered with one or other more therapeutic agents, e.g., an immunosuppressive agent or an anti-inflammatory agent to increase the overall anti-inflammatory effect. The antibody can be linked to the agent (as an immunocomplex) or can be administered separate from the agent. In the latter case (separate administration), the antibody can be administered before, after or concurrently with the agent.
Also within the scope of the present invention are kits comprising the antibody compositions of the invention (e.g., human antibodies and immunoconjugates) and instructions for use. The kit can further contain one ore more additional agents, such as an immunosuppressive agent, or one or more additional human antibodies of the invention.
Accordingly, patients treated with antibody compositions of the invention can be additionally administered (prior to, simultaneously with, or following administration of a human antibody of the invention) with another therapeutic agent, such as an immunosuppressive agent, an anti-inflammatory agent, an agent for treating inflammatory or hyperproliferative skin disorders, or a chemotherapeutic agent, which enhances or augments the therapeutic effect of the human antibodies.
In yet another embodiment, immunoconjugates of the invention can be used to target compounds (e.g., therapeutic agents, labels, cytotoxins, immunosuppressants, etc.) to cells which have CD25 bound to their surface by linking such compounds to the antibody. Thus, the invention also provides methods for localizing ex vivo or in vitro cells expressing CD25 (e.g., with a detectable label, such as a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor). In another embodiment, the invention provides methods for killing cells which have CD25 bound to their surface by administering immunotoxins of the present invention.
In a further embodiment, the antibodies of the invention can be used in vivo or in vitro for diagnosing diseases wherein activated cells expressing CD25 play an active role in the pathogenesis by detecting levels of CD25, or levels of cells which contain CD25 on their membrane surface. This can be achieved, for example, by contacting a sample to be tested, optionally along with a control sample, with the human antibody under conditions that allow for formation of a complex between the antibody and CD25. Complex formation is then detected (e.g., using an ELISA). When using a control sample along with the test sample, complex is detected in both samples and any statistically significant difference in the formation of complexes between the samples is indicative of the presence of CD25 in the test sample.
The present invention is further illustrated by the following examples which should not be construed as further limiting.
EXAMPLES
Example 1 Production of Human Antibodies Against CD25
Antigen:
A transfectant cell line expressing cell surface CD25 was developed to use as a reagent for immunizing HuMAb mice and characterizing anti-CD25 antibodies. This cell line was a CHO cell line engineered to express the extra-cellular domains of CD25 coupled to the transmembrane domain of platelet-derived growth factor receptor. The CD25 sequences were amplified from cDNA prepared from HUT102 cells and the platelet derived-growth factor receptor sequences were obtained from the pDISPLAY vector (Invitrogen Corporation). An expression construct encoding the fusion protein was engineered in an expression vector. The CHO transfectant cell line underwent 2 rounds of methotrexate amplification in 5 nM and 50 nM methotrexate to increase the expression levels of CD25.
CHO-CD25 Transfectoma Culture:
CHO-CD25 transfectoma cells (Medarex Inc., NJ, USA) were cultured in CHO-S-SFM II medium (Gibco BRL), without hypoxanthine, without thymidine, with penicillin (5000 U/ml), streptamycin (5000 mg/ml; BioWhittaker, Belgium), and methotrexate (final concentration 50 nM, Sigma). Cells were refreshed every two to three days.
Transgenic Mice:
HCo7 and HCo12 mice were housed in filter cages and were evaluated to be in good physical condition on dates of immunization, bleeds, and the day of the fusion. The mice that produced the selected hybridomas were all males. Mouse ID's 23185, 23196, 23197, and 23198 have the (CMD)++; (HCo7) 11952+; (JKD)++; (KCo5) 9272+ genotype. Mouse ID 23175 was of (CMD)++; (HCo12) 15087+; (JKD)++; (KCo5) 9272+ genotype. Individual transgene designations are in parentheses, followed by line numbers for randomly integrated transgenes. The symbols ++ and + indicate homozygous or hemizygous; however, because the mice are routinely screened using a PCR-based assay, it was not possible to distinguish between heterozygosity and homozygosity for the randomly integrated human Ig transgenes. A + designation may be given to mice that are actually homozygous for these elements.
Immunization Procedure and Schedule:
Mice were immunized with antigen in two forms: Live cells (the CD25 transfected CHO cells described above) and purified protein (recombinant human CD25 (rhCD25), an NS/0-expressed recombinant protein from R&D Systems, (cat #223-2A/CF), Minneapolis, Minn.). Soluble rhCD25 was mixed with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (WA). Freund's adjuvant was obtained from Gibco-BRL, Rockville, Md. Mice were injected with 0.2 ml prepared antigen into the intraperitoneal cavity. Final tail vein immunizations were performed with soluble CD25 in sterile PBS or saline (0.9% NaCl). Immunizations with transfected cells were administered into the intraperitoneal cavity (i.p.) at 0.2 ml in sterile saline at 1.0-2.0×107 cells per mouse. All immunizations were injected into the intraperitoneal cavity. Three and two days prior to fusion, intravenous (i.v.) boosts were performed. The immunization schedule is described in Table 1. All mice were included among a cohort of twelve (12) mice from HCo7 and HCo12 genotypes.
TABLE 1
Bleed and
Date of activity
Immunization: adjuvant, Antigen
titer*/Fusions
Day 1
1.5 × 107 live CD25 transfected cells,
IP in saline
Day 12
CFA, rhCD25 (20 μg)
Day 21
1.5 × 107 live CD25 transfected cells,
IP in saline
Day 28
CFA, rhCD25 (20 μg)
Day 35
Titer
Day 42
Fusion
23175/23197
Day 42
1.5 × 107 live CD25 transfected cells,
IP in saline
Day 56
CFA, rhCD25 (20 μg)
Day 63
Titer
Day 68
1.5 × 107 live CD25 transfected cells,
IP in saline
Day 71
Fusion
23196/23198
Day 81
Titer
Day 85
Fusion 23185
*For titers, please see Table 2.
Mouse Titers:
The titers for mouse # s 23175, 23185, 23196, 23197, and 23198 are shown below in Table 2. The titers shown in Table 2 indicate serum dilutions which showed positive in CD25 specific tests. The response to the antigen after repeated immunizations show a robust response level and the mouse was prepared for fusion.
TABLE 2
Mouse #
Titer Day 35
Titer Day 63
Titer Day 81
23175
12800
23185
6400
12800
12800
23196
6400
25600
23197
50000
23198
3200
25600
Fusion Procedure:
The SP2/0-ag14 myeloma cell line (ATCC CRL 1581, lot F-15087) was used for the fusions. The original ATCC vial was thawed and expanded in culture. A seed stock of frozen vials was prepared from this expansion. Cells were maintained in culture for 6-8 weeks, passed twice a week.
High Glucose DMEM: (Mediatech Cellgro, #1001233) containing 10% FBS (Hyclone cat # SH30071), antibiotic-antimycotic (100×) (Gibco, #15240062), and 0.1% L-glutamine was used to culture myeloma cells. Additional media supplements were added to the hybridoma growth media, including 5% Origen-Hybridoma Cloning Factor (Igen), 4.5×10−4 M sodium pyruvate, HAT (1.0×10−4 M hypoxanthine, 4.0×10−4 M aminopterin, 1.6×10−5 M thymidine; Sigma), and fetal bovine serum (Hyclone, Logan, Utah).
The spleen from mouse number #23197 was normal in size and yielded 4.0×108 viable cells. The spleen from mouse number #23175 was normal in size and yielded 2.6×108 viable cells. Spleens from mouse #23196 and #23198 were normal in size and yielded 2.4×108 and 2.0×108 viable cells, respectively. The last spleen from mouse #23185 was normal in size and yielded 1.9×108 viable cells. The splenocytes were fused according to standard procedure.
Media Used for Hybridoma Generation (Fusion):
High glucose DMEM (Mediatech, lot #10013264) containing 10% fetal bovine serum (FBS); (Hyclone, Logan, Utah, SH30071 lot # AJE10321) antibiotic-antimycotic (GibcoBRL, lot #15240062), and 0.1% L-glutamine (Gibco, lot #1013845) were used to culture the myeloma cells. Additional media supplements were added to the hybridoma growth media, which included: 5% Origen-Hybridoma Cloning Factor (Igen, lot #36600 and 36782 and 36684), 4.5×10−4 M sodium pyruvate, HAT (Sigma, H 0262): 1.0×10−4 M hypoxanthine, 4.0×10−7 M aminopterin, 1.6×10−5 M thymidine, or HT (Sigma, H0137): 1.0×10−4 M hypoxanthine, 1.6×10−5 M thymidine.
The spleen and lymph nodes were removed from the immunized mice and these organs were placed into a tube containing DMEM+10% FBS. The tube was transferred to a tissue culture room and a single cell suspension was made from the spleen and lymph nodes and the cells were counted. An appropriate volume of SP2/0 cells (ATCC CRL 1581, lot F-15087; 6 spleen or lymph node cells per 1 cell of SP2/0) was transferred and the cells were mixed and resuspended. Approximately 1.2 ml of PEG was added (1 minute while gently swirling the tube in a beaker containing 37° C. water). The tube was left for 90 seconds, 15 ml of DMEM was added and washing with medium was performed. After spinning the cells down, the supernatant was removed and the cells were resuspended. Ten (10) ml of HAT-containing medium was added to the tube. After incubating for 30-60 minutes in a CO2 incubator, the cells were plated into 96-well culture plates, 200 μl/well (about 1×107 cells per 96-well plate). On day 7, cells were fed with HT-containing medium, 250 μl/well (HT medium is HAT medium with aminopterin removed).
The initial ELISA screen for human IgG,κ antibodies was performed 7-10 days post fusion. Human IgG,κ positive wells were then screened on soluble CD25 coated ELISA plates. Antigen positive hybridomas were then transferred to 24-well plates, and eventually to tissue culture flasks. Antigen positive hybridomas were preserved at several stages in the development process by freezing cells in Origen DMSO freeze medium (Fisher Cat # IG-50-0715).
ELISA Protocol for IgG/κ Detection (Used for Screening the Fusions):
ELISA plates were coated overnight with anti-human-κ, 1 μg/ml (Immunotech, lot #0173) or anti-human-γ, 1 μg/ml (Jackson, lot #109-006-098), 50 μg/well. Plates were emptied and residual binding sites were blocked with PBS supplemented with tween-20 (0.05%) and 5% chicken serum (PBSTC) for 1 hour at room temperature (RT). Plates were washed 3 times with PBS supplemented with 0.05% tween-20 (PBST). Supernatants derived from the fusions and subclones were generally tested diluted 1:2 in PBSTC. As a positive control, human IgG (Calbiochem) was used. After incubating the samples for about 2 hours, the plates were washed with PBST and a secondary antibody, anti-human-IgG-Fc-HRP conjugated (Jackson, lot #109-036-098), 1:5000 diluted in PBSTC was added to the wells (100 μl). After incubation of 1 hour at RT, the ELISA was developed using ABTS (Sigma) according to the manufacturer's recommendations.
Isotype Determination by ELISA:
96-well ELISA plates (Greiner, Germany) were coated overnight (100 μl/well, room temperature (RT)) with mouse-anti-human IgG1 (CLB, Netherlands, dilute 1:5,000 from stock) or with mouse-anti-human IgG3 (CLB, dilute 1:10,000 from stock). After washing the plates 3× with PBST (150 μl/well), plates were incubated with PBSTC for 1 hour at RT. Supernatants of human CD25 monoclonal antibody clones were then added (100 μl/well; 2 hours at RT). Anti-KLH IgG1 (1 μg/ml) and anti-KLH IgG3 (1 μg/ml) supernatants served as positive controls. Culture medium and PBSTC served as negative controls. After washing in PBST (3×), goat-anti-hIgG-HRP (Fc specific; Jackson Labs, Maine, USA) was added (1 hour at RT). For detection of IgG1, the conjugate was diluted 1:500, whereas for detection of IgG3 the conjugate was diluted 1:2000. After washing in PBST (3×), 10 mg ABTS (Roche) per 10 ml ABTS buffer (Roche) was made and 100 μl added to each well. After 20 minutes, absorption was read at 405 nm with an ELISA reader (EL 808, Bio-Tek Instruments, Vermont, USA).
Based on the immunization procedure 4 antigen specific hybridomas were selected which were all derived from HCo mice: AB1, AB7, AB11 and AB12. Isotypes of these four clones were found to be IgG1,κ.
The antibodies of the invention can be recombinantly expressed as other isotypes, for example IgG2, IgG3, IgG4, IgM, and IgA.
Media Used for Maintaining the Hybridomas after Selection:
All human CD25 monoclonal antibody hybridoma cell lines were cultured in Dulbecco's Modified Eagle Medium (Biowhittaker, lot # BE12-709F) supplemented with 10% FCS (Wisent Multicell optimum C241), 2 mM L-glutamine (Glutamax-II), 50 IU/ml penicillin, 50 μg/ml streptomycin (pen/strep), 2 μM β-ME (all derived from Gibco BRL, Life Technologies, Scotland), 24% HCF (Origen, Igen International Inc., Gaithersburg, USA).
Purification of Antibodies:
Before purification or concentration of the human CD25 specific antibodies from the culture supernatant, the cell culture supernatant must be filtered through a vacuum driven disposable bottle top filter to remove gross material such as cell rests or other impurities. The sample can be concentrated if the volume of the sample is above 500 ml to a volume beneath 500 ml with a PREP/SCALE™ TFF, 1 ft2 cartridge (Millipore, USA).
Protein A purification of the CD25 specific antibodies was performed using affinity chromatography.
After equilibration of the 5 ml Protein A column (ProtA 5 ml SP, version 041201, Amersham Pharmacia Biotech AB, Sweden) with PBS, pH 7.4, and priming of the sample-pump A with PBS, pH 7.4, the supernatant containing CD25 specific antibodies was loaded onto the column, unbound sample washed out, and the system-pump B rinsed with 0.1 M citric acid, pH 5, (elution buffer 1). Thereafter, bovine IgG (present in the culture supernatant) was eluted with elution buffer 1 via the system pump B. After rinsing the system-pump A with 0.1 M citric acid, pH 3, (elution buffer 2), human CD25 specific antibodies were eluted via the system-pump A with elution buffer 2. System-pump B was then rinsed with 0.1 M citric acid, pH 2, (elution buffer 3) and all remaining IgG bound to the column was eluted via system-pump B with elution buffer 3. The eluted CD25 specific antibodies were neutralized with 10% (v/v) 2 M Tris-HCl (Sigma), pH 9, and the peak-fractions were then pooled.
Pooled peak-fractions from elution-step 2 were dialyzed to PBS (30 ml purified material to 51 PBS), for 18 hours at 4° C. To preserve and store the purified material, the samples were concentrated. The concentration of human IgG was determined using Nephelometric assay (Dade-Behring, BNII) using polyclonal anti-IgG antibodies (CLB, Amsterdam, The Netherlands, lot # M1090). The antibodies were aliquoted, snap frozen, and stored at −80° C.
Example 2 Antibody Sequencing of Human Antibodies Against CD25
Sequencing of the VL and VH Regions of the Antibodies
Sequencing:
The VDJ-regions were sequenced after cloning in the pGEMT-Vector System II. Sequencing was performed at Baseclear (Leiden, Netherlands). The sequences were aligned to germline V-gene sequences in Vbase available on the internet at the website mrc-cpe.cam.ac.uk.
RNA Preparation:
Total RNA was prepared from 5×106 cells of four (4) different human CD25 hybridoma cell lines (AB1, AB7, AB11, AB12) with Rneasy kit (Qiagen, Westburg, Leusden, Netherlands) according to the manufacturer's protocol.
cDNA Preparation:
Complementary DNA (cDNA) of RNA from human CD25 hybridoma cells was prepared from 3 μg total RNA with AMV Reverse Transcriptase with buffer (Roche Diagnostics GmbH, Mannheim, Germany), oligo d(T)15 (Promega, Madison, Wis., USA), dNTP (Roche Diagnostics GmbH, Mannheim, Germany) and RNAsin (Promega) according to the manufacturer's protocol (2000, version 3).
VH and VL regions were amplified using the following PCR primers:
VH:
FR1 5′ primers
(SEQ ID NO: 41)
AB62
CAg gTK CAg CTg gTg CAg TC
(SEQ ID NO: 42)
AB63
SAg gTg CAg CTg KTg gAg TC
(SEQ ID NO: 43)
AB65
gAg gTg CAg CTg gTg CAg TC
VH leader 5′ primers
(SEQ ID NO: 44)
AB85
ATg gAC Tgg ACC Tgg AgC ATC
(SEQ ID NO: 45)
AB86
ATg gAA TTg ggg CTg AgC Tg
(SEQ ID NO: 46)
AB87
ATg gAg TTT ggR CTg AgC Tg
(SEQ ID NO: 47)
AB88
ATg AAA CAC CTg Tgg TTC TTC
(SEQ ID NO: 48)
AB89
ATg ggg TCA ACC gCC ATC CT
VH 3′ primer
(SEQ ID NO: 49)
AB90
TgC CAg ggg gAA gAC CgA Tgg
VK:
FR1 5′ primers
(SEQ ID NO: 50)
AB8
RAC ATC CAg ATg AYC CAg TC
(SEQ ID NO: 51)
AB9
gYC ATC YRg ATg ACC CAg TC
(SEQ ID NO: 52)
AB10
gAT ATT gTg ATg ACC CAg AC
(SEQ ID NO: 53)
AB11
gAA ATT gTg TTg ACR CAg TC
(SEQ ID NO: 54)
AB12
gAA ATW gTR ATg ACA CAg TC
(SEQ ID NO: 55)
AB13
gAT gTT gTg ATg ACA CAG TC
(SEQ ID NO: 56)
AB14
gAA ATT gTg CTg ACT CAg TC
VK leader 5′ primers:
(SEQ ID NO: 57)
AB123
CCC gCT Cag CTC CTg ggg CTC CTg
(SEQ ID NO: 58)
AB124
CCC TgC TCA gCT CCT ggg gCT gC
(SEQ ID NO: 59)
AB125
CCC AgC gCA gCT TCT CTT CCT CCT gC
(SEQ ID NO: 60)
AB126
ATg gAA CCA Tgg AAg CCC CAg CAC AgC
VK 3′ primer
(SEQ ID NO: 61)
AB16
Cgg gAA gAT gAA gAC AgA Tg
In the above primer sequences, K, S, R, Y and W have the following meanings:
K=G or T
S=C or G
R=A or G
Y=C or T
W=A or T
PCR Conditions Used to Amplify VH and VL Regions for Cloning:
Polymerase chain reactions (PCR) were performed with AmpliTaq polymerase (Perkin Elmer) on a T1 Thermocycler 96 (Biometra, Westburg, Leusden, Netherlands).
PCR Cycling Protocol:
94° C. 2 min
11cycles
94° C. 30 sec
65° C. 30 sec, minus 1° per cycle
72° C. 30 sec
30 cycles
94° C. 30 sec
55° C. 30 sec
72° C. 30 sec
72° C. 10 min
cool down to 4° C.
Cloning of VH and VL in pGEMT-Vector System II:
After analysing the PCR products on an agarose gel, the products were purified with the QIAEX II Gel Extraction Kit (Qiagen, Westburg, Leusden, Netherlands). Always 2 independently amplified PCR products, using FR1 or leader primers, of each VH and VL region were cloned in pGEMT-Vector System II (Promega) according to manufacturer's protocol (1999, version 6).
After transformation to E. coli JM109, individual colonies were screened by colony PCR using T7 and SP6 primers, 30 annealing cycles at 55° C. Plasmid DNA from colonies was purified using Qiaprep Spin miniprep kit (Qiagen). To further analyse the VH and VL regions a Ncol/Notl (NE Biolabs, Westburg, Leusden, Netherlands) digestion was performed and analysed on agarose gel.
The four selected hybridoma cell lines expressed the following antibody sequences:
AB1: a human monoclonal IgG1,κ antibody with the amino acid sequences: SEQ ID NOs: 2 and 4;
AB7: a human monoclonal IgG1,κ antibody with the amino acid sequences: SEQ ID NOs: 6 and 8;
AB11: a human monoclonal IgG1,κ antibody with the amino acid sequences: SEQ ID NOs: 10 and 12; and
AB12: a human monoclonal IgG1,κ antibody with the amino acid sequences: SEQ ID NOs: 14 and 16.
The sequences obtained are shown in FIGS. 1-10.
Example 3 Binding Characteristics of Human Antibodies Against CD25
Binding of Supernatants of Human CD25 Monoclonal Antibodies to CD25 Constitutively Expressed on CHO Cells:
AB1, AB7, AB11 and AB12 all bound to CD25 expressed on transfected CHO cells when determined by flow cytometry (see Table 3).
Binding of Supernatants of Human CD25 Monoclonal Antibodies to hrCD25 in ELISA Assay:
AB1, AB7, AB11, and AB12 all bound CD25 when tested in an ELISA using hrCD25 as the coating antigen. 96-well plates (Greiner) were coated overnight at RT with rhCD25 (100 ng/ml; R&D), wereupon non-specific binding was blocked by coating the plates with PBSTC for 1 hour at RT. After washing (3×) the plates with PBST, 100 μl of sample antibody was added. After washing the plates 3× (PBST), plates were incubated with streptavidin-poly-HRP (1:10,000) in PBS and 100 μl added to each well (1 hour, RT). After washing the plates (3× in PBST), 10 mg ABTS (Roche) per 10 ml ABTS buffer (Roche) was made and 100 μl added to each well. After 20 minutes, absorption was read at 405 nm with an ELISA reader (EL 808, Bio-Tek Instruments).
TABLE 3
Clone names, isotypes, and binding to CD25
Clone
Subclass
CD25-binding1
CHO-CD252
AB1
IgG1
+
+
AB7
IgG1
+
+
AB11
IgG1
+
+
AB12
IgG1
+
+
1Binding of clone culture supernatants as determined by rhCD25 ELISA
2Binding to CD25 expressed in transfected CHO cells and determined by flow cyometry
Inhibition of Binding of Biotinylated IL-2 to its Receptor by Supernatants of Human CD25 Monoclonal Antibodies:
In order to examine the extent of which human monoclonal antibodies block or inhibit IL-2 binding to CD25 96-well plates (Greiner) were coated overnight at RT with rhCD25 (100 ng/ml; R&D systems, MN, USA), whereupon non-specific binding was blocked by coating the plates with PBSTC for 1 hour at RT. After washing (3×) the plates with PBST, 100 of sample antibody (concentration range: 10, 33, and 100 ng/ml) was added. For comparison ZENAPAX® antibody was also added. After 10 minutes, rIL-2-biotin (50 ng/ml) was added (1.5 hours, RT). After washing the plates 3× (in PBST), plates were incubated with streptavidin-poly-HRP (dilute 1:10,000 from stock) in PBS, and 100 μl was added to each well (1 hour, RT). After washing the plates (3× in PBST), 10 mg ABTS (Roche) per 10 ml ABTS buffer (Roche) was made and 100 μl added to each well. After 20 minutes, absorption was read at 405 nm with an ELISA reader (EL 808, Bio-Tek Instruments). Data show one out of two representative experiments. As shown in FIG. 11, supernatants of human CD25 monoclonal antibodies AB1, AB7, AB11 and AB12 were able to inhibit binding of biotinylated IL-2 to CD25 more efficiently than ZENAPAX® antibody.
Inhibition of Binding of ZENAPAX® Antibody to CD25 by Supernatants of Human CD25 Monoclonal Antibodies:
In order to examine the extent of which human monoclonal antibodies block or inhibit binding of ZENAPAX® antibody to CD25, 96-well plates (Greiner) were coated overnight at RT with rhCD25 (100 ng/ml; R&D systems, MN, USA), whereupon non-specific binding was blocked by coating the plates with PBSTC for 1 hour at RT. After washing (3×) the plates with PBST, 100 μl of sample (concentration range: 10, 33, and 100 ng/ml) was added. After 10 minutes, biotinylated ZENAPAX® antibody (5 ng/ml) was added (1.5 hours, RT). After washing the plates 3× (in PBST), plates were incubated with streptavidin-poly-HRP (dilute 1:10,000 from stock) in PBS, and 100 μl was added to each well (1 hour, RT). After washing the plates (3× in PBST), 10 mg ABTS (Roche) per 10 ml ABTS buffer (Roche) was made and 100 μl added to each well. After 20 minutes, absorption was read at 405 nm with an ELISA reader (EL 808, Bio-Tek Instruments). Data show one out of two representative experiments. As shown in FIG. 12, supernatants of human monoclonal antibodies AB1, AB7, AB11, and AB12 block ZENAPAX® antibody binding to CD25.
Example 4 Human Monoclonal Antibodies Against CD25 Inhibit Anti-CD3 Antibody-Induced T Cell Proliferation
Human antibodies were tested for their ability to inhibit T cell proliferation using the T cell proliferation assay. For comparison ZENAPAX® antibody as well as an isotype control antibody (hIgG1/κ) were also tested.
PBMC Isolation:
Human blood cells (obtained in buffy coats from Dutch Red Cross Blood Bank, Utrecht, Netherlands) were put on a ficoll gradient (Pharmacia, 2500 rpm, 25 minutes). With a pipette, PBMCs were collected in RPMI 1640 (supplemented with 10% FCS (Wisent Multicell optimum C241), 2 mM L-glutamine, 50 IU/ml penicillin, 50 μg/ml streptomycin, 25 mM HEPES (all derived from Bio Whittaker, Europe)).
T Cell Proliferation Assay:
Human PBMCs were diluted in RPMI 1640 (supplemented with 10% FCS (Wisent Multicell optimum C241), 2 mM L-glutamine, 50 IU/ml penicillin, 50 μg/ml streptomycin, 25 mM HEPES (all derived from Bio Whittaker, Europe)) to 1.5×105 cells/well (in triplet) in 96-well flat bottom plates (Greiner). The cells were stimulated with anti-CD3 antibody (CLB-T3/4.E, cat #M1654, 10 ng/ml). Then, 50 μl of increasingly diluted experimental antibodies were added to the cells (ranging from 500 ng/ml to 7.8 ng/ml, in two-step dilutions). After five days (37° C., 5% CO2) proliferation was quantified by using BrdU (end concentration: 10 μM, Roche) according to the method described below.
BrdU Labeling Assay (Roche BrdU-Staining Kit, Cat No 1 647 229):
BrdU labeling solution (100 μM) was added to the wells and cells were incubated overnight (37° C., 5% CO2). Cells were resuspended in wells and centrifuged (10 minutes, 300 g). Supernatant was discarded and cell pellet was dried (1 hour, 60° C.). The pellet was then incubated with FixDenat (200 μl/well; 30 minutes, RT). After incubation, FixDenat was discarded and 100 μl/well anti-BrdU-POD (add 100 μl anti-BrdU stock solution to 10 ml Ab-dilution solution) was added to the pellet (1 hour, RT). After discarding the supernatant, plates were washed (3×) with washing solution (200 μl/well). Finally, 100 ml/well substrate solution was added to the pellet (5 minutes, RT). Coloring reaction was stopped by H2SO4 (25 μl/well, 1M) and optical density was read by ELISA reader at 450 nm (Bio-Tek Instruments).
As shown in FIG. 13, human monoclonal antibodies AB1, AB7, and AB12 inhibited anti-CD3 antibody-induced T cell proliferation in a dose-dependent manner. The inhibition by the human antibodies was more efficient than by ZENAPAX® antibody. Data show one out of three representative experiments.
Example 5 Human Monoclonal Antibodies Against CD25 Inhibit MLR
Human antibodies were tested for their ability to inhibit MLR using the MLR assay. For comparison ZENAPAX® antibody as well as an isotype control antibody (hIgG1/κ) were also tested. Human PBMCs (obtained in buffy coats from Dutch Red Cross Blood Bank, Utrecht, Netherlands) from two non-MHC-matching donors were diluted in RPMI 1640 (supplemented with 10% FCS (Wisent Multicell optimum C241), 2 mM L-glutamine, 50 IU/ml penicillin, 50 μg/ml streptomycin (all derived from Gibco BRL, Life Technologies, Paisley, Scotland)) to 2.0×106 cells/ml. PBMCs from the first donor were irradiated (2000 rads) and mixed (1.0×105 cells/well) with PBMCs from the second donor (1.0×105 cells/well) in 96-well flat bottom plates (Greiner) in triplet. Then, 50 μl of increasingly diluted experimental antibodies were added to the cells (ranging from 50 ng/ml to 0.8 ng/ml, in two-step dilutions). After six days of culture, (37° C., 5% CO2) proliferation was quantified by using BrdU (end concentration: 10 μM, Roche) according to the method described above.
As shown in FIG. 14, human monoclonal antibodies AB1, AB7, and AB12 inhibited the MLR in a dose-dependent manner. Inhibition of MLR by AB1, AB7, and AB12 (at doses between about 1 and 3 ng/ml) was more efficient than inhibition by ZENAPAX® antibody. Data show one out of three representative experiments.
Example 6 Kinetic Analysis of AB12 on BIAcore® 3000 instrument
Affinity analyses were assessed by monitoring changes in surface plasmon resonance using a BIAcore® 3000 instrument. A BIAcore® 3000 and BIAcore® 3000 software control (BIAcore®, Uppsala, Sweden, lot #BR-1100-43) was used. Human CD25 (R&D Systems, lot #223-2A/CF0) was immobilized to a CM-5 sensor chip at low-density (BIAcore®, lot #BR-1000-14) using amine-coupling chemistry according to the manufacturer's recommendations. After blocking the residual binding sites of the activated sensor chip using ethanol-amine-HCl, a kinetic analysis was performed at 25° C. (according to the manufacturer's recommendations) using human monoclonal antibody AB12 and for comparison ZENAPAX® antibody. Samples containing AB12 and ZENAPAX® antibody, respectively, were flowed over the surface of the coated sensor chip allowing AB12 and ZENAPAX® antibody to associate with rhCD25. The association and dissociation of AB12 and ZENAPAX® antibody, respectively, were monitored using surface plasmon resonance (SPR) on the sensor chip. The results were visualized using a BIAcore® 3000 (Bio-tek Instruments) and analyzed using the BIAevaluation Software 3.1 (BIAcore®, Uppsala, Sweden) and Languir binding 1:1 was used as pre-fixed model.
The KD of AB12 for the binding to rhCD25 determined by BIAcore® analysis: 4.74×10−11±0.43×10−11.
The KD of ZENAPAX® antibody for the binding to rhCD25 determined by BIAcore® analysis: 1.52×10−10±0.27×10−10.
Example 7 AB12-Treatment of T Cell Blasts Results in Internalization of CD25
AB12 was tested for its ability to induce internalization of CD25. Anti-KLH (human IgG1/κ isotype antibody, specific for keyhole limpet hemocyanin) was included as isotype control antibody.
Induction of T Cell Blasts:
After isolation of peripheral blood mononuclear cells (PBMCs) from heparin-blood samples using lymphocyte separation medium gradient, PBMCs were stimulated for three to four days with 5 μg/ml phytohemagglutinin (PHA; Difco, cat #211796) in culture medium (37° C., 5% CO2).
Stimulation of T Cell Blasts to Examine Internalization:
After harvesting cells and washing in PBS, cells were counted with trypan blue. One part of T-cell blasts (1×106 cells/ml) was pre-incubated (4° C., for 15 min) with FITC-labeled AB12 (2 μg/ml AB12-FITC), or FITC-labeled anti-KLH (2 μg/ml) as isotype control, or without addition of antibodies. After pre-incubation, cells were washed in PBS, and 1×106 cells (in 1 ml culture medium) were added to 24-well plates, and incubated for 18 hours (37° C., 5% CO2). The remainder of T cell blasts was incubated in the absence or presence of FITC-labeled AB12 (2 μg/ml), or FITC-labeled anti-KLH (2 μg/ml) for 18 hours (37° C., 5% CO2).
After incubation, cells were harvested, and labeled with rhodamine-labeled wheat-germ agglutinin (1 μg/ml, membrane labeling; Molecular Probes, cat No. W-849), at 4° C. for 15 min. Thereafter, cells were washed with PBS, and resuspended in 25 μl Vectashield DAPI (Vector Laboratories, Burlingame, Calif., USA). Then, 10 μl of the cell suspension was pipetted on tissue slides, covered, and analyzed by fluorescence microscopy (Carl Zeiss), and photographs taken with TRITC filter for rhodamine-staining (filter set 15, Zeiss), or FITC filter for the FITC-staining (filter set 09, Zeiss). The membrane staining, obtained with the rhodamine-labeled wheat-germ agglutinin, is not shown.
As shown in FIGS. 15A and 15B, after 18 hours of culture, the AB12-FITC signal can be found inside the cells. FIG. 15A shows the result after culturing the cells for 18 hours following pre-incubation (15 min) with AB12-FITC and washing, and FIG. 15B shows the result after culturing the cells for 18 hours in the presence of AB12-FITC. A control experiment with the irrelevant FITC-conjugated anti-KLH antibody (FIG. 15C) shows no internalization of FITC-labeled antibody.
Example 8 AB12-Treatment of T Cell Blasts Results in Internalization of CD25 as Measured by Flow Cytometry
In another experiment flow cytometry was used to determine internalization of FITC-labeled AB12 in T cell blasts at different time intervals.
After isolation of peripheral blood mononuclear cells (PBMCs) from heparin-blood samples using lymphocyte separation medium gradient (Ficoll), PBMCs were stimulated for three to four days with 5 μg/ml phytohemagglutinin (PHA; Difco, cat No 211796) in culture medium (37° C., 5% CO2).
After three days of culture the T cell blasts were harvested, washed with PBS, and counted with trypan blue. To the T cell blasts (2.5×106 cells in 2 ml culture medium), 2 μg/ml FITC-labeled AB12 or FITC-labeled anti-KLH (isotype control antibody) was added. After pre-incubation of cells (4° C., 1 hour), cells were split into two portions. One portion was washed in culture medium, whereas the other portion was not washed. After washing, the pre-incubation samples were resuspended in culture medium. Both portions were incubated at 4° C. or 37° C.
After 0, 0.5, 1, or 4.5 hours of incubation (either at 4° C. or 37° C.), 3 ml of FACS buffer (PBS supplemented with 0.05% BSA and 0.01 μg/ml sodium azide) was added to the cells, and the cells were spun down at 300 g (4° C.). In one portion, cells were resuspended in 200 μl FACS buffer, whereas in the other portion, cells were resuspended in 200 μl FACS buffer and 1 mg/ml ethidium bromide (Sigma, cat No. E8751). The ethidium bromide was added immediately before cell acquisition by flow cytometry.
Ethidium bromide was used to quench the fluorescence signal on the cell surface. As shown in FIG. 16, the fluorescence ratio of the samples incubated at 4° C. measured with or without ethidium bromide was approximately one. This indicates that no internalization has taken place. Cells cultured at 37° C. showed an increase of this fluorescence ratio over time. This indicates that internalization of AB12-FITC has occurred. As expected, incubation of cells in the continued presence of FITC-labeled AB12 results in higher levels of internalization (FIG. 16B) as compared to cells only pre-incubated with FITC-labeled AB12 (FIG. 16A). FIG. 16A shows the ratio of mean fluorescence intensity (MFI) for cells pre-incubated for 1 hour and excess FITC-labeled-AB12 washed away. FIG. 16B shows the result after culturing the cells in the presence of FITC-labeled AB12. The ratio of MFI is determined by dividing MFI of test samples by MFI of sample at 0 hours. No staining was observed with the isotype control antibody (anti-KLH-FITC, data not shown).
This internalization characteristic of the antibodies of the invention will make them suitable for conjugating with a toxin for the treatment of for example adult T cell leukemia/lymphoma, anaplastic large cell lymphoma, cutaneous T cell lymphoma (including mycosis fungoides and Sezary's syndrome), peripheral T cell lymphomas, Hodgkin's lymphoma, hairy cell leukemia, and chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL).
In another setting, the antibodies of the invention are subjected to radiolabeling with a suitable radioisotope for the treatment of for example adult T cell leukemia/lymphoma, anaplastic large cell lymphoma, cutaneous T cell lymphoma (including mycosis fungoides and Sezary's syndrome), peripheral T cell lymphomas, Hodgkin's lymphoma, hairy cell leukemia, and chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL).
Furthermore, the antibodies may be labeled with 111In for determining the tumor burden and thereby adjusting the dosage of radiolabeled antibody to be administered.
EQUIVALENTS
Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. Any combination of the embodiments disclosed in the dependent claims are also contemplated to be within the scope of the invention.
INCORPORATION BY REFERENCE
All patents, pending patent applications and other publications cited herein are hereby incorporated by reference in their entirety.
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15420824
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genmab a/s
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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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Apr 1st, 2022 06:04PM
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Apr 1st, 2022 06:04PM
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Genmab A/S
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Health Care
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Pharmaceuticals & Biotechnology
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cph:gen
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Genmab A/S
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Aug 10th, 2021 12:00AM
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Apr 22nd, 2020 12:00AM
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https://www.uspto.gov?id=US11084882-20210810
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Multispecific antibodies against CD40 and CD137
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Multispecific antibodies binding to human CD40 and human CD137, methods for preparing such multispecific antibodies, and methods of using such multispecific antibodies for therapeutic or other purposes.
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11084882
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1. A multispecific antibody comprising a first heavy chain and a first light chain comprising a first antigen-binding region capable of binding to human CD40, and a second heavy chain and a second light chain comprising a second antigen-binding region capable of binding to human CD137,
wherein the first antigen-binding region capable of binding to human CD40 comprises a first heavy chain variable (VH) region comprising an HCDR1 sequence comprising the amino sequence as set forth in SEQ ID NO: 1, an HCDR2 sequence comprising the amino sequence as set forth in SEQ ID NO: 2, and an HCDR3 sequence comprising the amino sequence as set forth in SEQ ID NO: 3, and a first light chain variable (VL) region comprising a LCDR1 sequence comprising the amino sequence as set forth in SEQ ID NO: 4, a LCDR2 sequence comprising the amino sequence YTS, and a LCDR3 sequence comprising the amino sequence as set forth in SEQ ID NO: 5;
wherein the second antigen-binding region capable of binding to human CD137 comprises a second VH region comprising an HCDR1 sequence comprising the amino sequence as set forth in SEQ ID NO: 64, an HCDR2 sequence comprising the amino sequence as set forth in SEQ ID NO: 65, and an HCDR3 sequence comprising the amino sequence as set forth in SEQ ID NO: 66, and a second VL region comprising a LCDR1 sequence comprising the amino sequence as set forth in SEQ ID NO: 67, a LCDR2 sequence comprising the amino sequence GAS, and a LCDR3 sequence comprising the amino sequence as set forth in SEQ ID NO: 68;
wherein the first and second heavy chains each comprise an amino acid substitution at one or more positions corresponding to EU index positions T366, L368, K370, D399, F405, Y407, and/or K409 in a human IgG1 heavy chain, and wherein the first and second heavy chains are not substituted in the same positions;
wherein the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, in the first and second heavy chains; and
wherein (i) the amino acid in the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the first heavy chain, and the amino acid position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the second heavy chain, or (ii) the amino acid in the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the first heavy chain, and the amino acid position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the second heavy chain.
2. The multispecific antibody according to claim 1, wherein the first VH region comprises the amino acid sequence as set forth in SEQ ID NO:117, and the first VL region comprises the amino acid sequence as set forth in SEQ ID NO:121, and wherein the second VH region comprises the amino acid sequence as set forth in SEQ ID NO:123, and the second VL region comprises the amino sequence as set forth in SEQ ID NO: 127.
3. The multispecific antibody according to claim 1, wherein the amino acid in the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the first heavy chain, and the amino acid position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the second heavy chain.
4. The multispecific antibody according to claim 1, wherein the amino acid in the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is L in the first heavy chain, and the amino acid position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is R in the second heavy chain.
5. The multispecific antibody of claim 1, wherein the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:119 and the first light chain comprises the amino acid sequence as set forth in SEQ ID NO:122, and wherein the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:125 and the second light chain comprises the amino acid sequence as set forth in SEQ ID NO:128.
6. The multispecific antibody of claim 1, wherein the first heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:119 and the first light chain comprises the amino acid sequence as set forth in SEQ ID NO:122.
7. The multispecific antibody of claim 1, wherein the second heavy chain comprises the amino acid sequence as set forth in SEQ ID NO:125 and the second light chain comprises the amino acid sequence as set forth in SEQ ID NO:128.
8. The multispecific antibody according to claim 1, wherein the first VH region comprises the amino acid sequence as set forth in SEQ ID NO:117.
9. The multispecific antibody according to claim 1, wherein the first VH region comprises the amino acid sequence as set forth in SEQ ID NO:117, and the first VL region comprises the amino acid sequence as set forth in SEQ ID NO:121.
10. The multispecific antibody according to claim 1, wherein the second VH region comprises the amino acid sequence as set forth in SEQ ID NO:123.
11. The multispecific antibody according to claim 1, wherein the second VH region comprises the amino acid sequence as set forth in SEQ ID NO:123, and the second VL region comprises the amino sequence as set forth in SEQ ID NO: 127.
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RELATED APPLICATIONS
This application is a division of U.S. patent application Ser. No. 16/316,534, filed Jan. 9, 2019, which is a national stage application under 35 U.S.C. § 371 of PCT/EP2017/067924, filed Jul. 14, 2017, which designated the U.S. and claims the benefit of PCT/EP2016/066840, filed Jul. 14, 2016. The entire contents of the foregoing applications are incorporated herein by reference.
SEQUENCE LISTING
This application contains a Sequence Listing which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on Aug. 23, 2019, is named “P101_Sequence_Listing_121660009.txt” and is 119,412 bytes in size.
FIELD OF THE INVENTION
The present invention relates to multispecific antibodies binding to CD40 and CD137, and to uses of such multispecific antibodies, in particular to the use for treatment of cancer.
BACKGROUND OF THE INVENTION
CD40 is a member of the tumor necrosis factor (TNF) receptor (TNFR) family and is known as a co-stimulatory protein found on a diversity of cell types. CD40 is constitutively expressed by antigen-presenting cells (APCs), including dendritic cells (DCs), B cells and macrophages. It can also be expressed by endothelial cells, platelets, smooth muscle cells, fibroblasts and epithelial cells. Consistent with its widespread expression on normal cells, CD40 is also expressed on a wide range of tumor cells.
The presentation of peptide antigens in the context of MHC class II molecules to antigen-specific CD4+ T cells, together with co-stimulatory signals (from CD80 and/or CD86), results in the activation of CD4+ T cells and the up-regulation of the DC licensing factors CD40 ligand (CD40L) and lymphotoxin-α1β2 (LTα1β2). Expression of CD40L and LTα1β2 on activated antigen-specific CD4+ T cells induces signaling through CD40 and the LTβ receptor (LTβR), and this licenses DCs to induce CD8+ T-cell responses. CD40 signaling results in the production of interleukin-12 (IL-12) and the up-regulation of CD70, CD86, 4-1BB ligand (4-1BBL), OX40 ligand (OX40L) and GITR ligand (GITRL), whereas LTβR signaling leads to the production of type I interferons (IFNs). The signaling system that controls the activity of nuclear factor kappaB (NF-κB) is responsive to virtually all TNFR superfamily members. Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) also contribute to these events. Priming of CD8+ T cells by MHC class I-restricted peptides results in the up-regulation of CD27, 4-1BB, OX40 and glucocorticoid-induced TNFR-related protein (GITR). Stimulation of these receptors on CD8+ T cells by their cognate TNF superfamily ligands, in combination with IL-12 and type I IFNs, results in robust CD8+ T cell activation, proliferation and effector function, as well as the formation and maintenance of CD8+ T cell memory. CD40 antibodies can exert different actions, CD40-expressing tumor cell kill by induction of antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated phagocytosis (ADCP), induction of cell signaling to induce direct apoptosis or growth arrest, but also, independent of CD40 expression on the tumor cells, through licensing of APCs to stimulate an anti-cancer immune response. Antibodies binding to CD40 can trigger CD40 on APCs to prime effector cytotoxic T lymphocytes (CTLs) and induce release of IL-2 by these cells, and indirectly activate NK cells. Antibodies stimulating CD40 have been disclosed in the prior art, and include CP-870,893, a human IgG2 antibody (WO 2003/040,170); dacetuzumab, a humanized IgG1 antibody (WO 2000/075,348) and Chi Lob 7/4, a chimeric IgG1 antibody (US 2009/0074711). Furthermore, an antagonistic CD40 antibody has been disclosed, lucatumumab, a human IgG1 antibody (WO 2002/028,481).
CD137 (4-1BB) is also a member of the TNFR family. CD137 is a co-stimulatory molecule on CD8+ and CD4+ T cells, regulatory T cells (Tregs), Natural Killer T cells (NK(T) cells), B cells and neutrophils. On T cells, CD137 is not constitutively expressed, but induced upon T-cell receptor (TCR) activation (for example, on tumor infiltrating lymphocytes (TILs) (Gros et al., J. Clin Invest 2014; 124(5):2246-59)). Stimulation via its natural ligand 4-1BBL or agonist antibodies leads to signaling using TRAF-2 and TRAF-1 as adaptors. Early signaling by CD137 involves K-63 poly-ubiquitination reactions that ultimately result in activation of the nuclear factor(NF)-kB and mitogen-activated protein(MAP)-kinase pathways. Signaling leads to increased T cell co-stimulation, proliferation, cytokine production, maturation and prolonged CD8+ T-cell survival. Agonistic antibodies against CD137 have been shown to promote anti-tumor control by T cells in various pre-clinical models (Murillo et al., Clin Cancer Res 2008; 14(21):6895-906). Antibodies stimulating CD137 can induce survival and proliferation of T cells, thereby enhancing the anti-tumor immune response. Antibodies stimulating CD137 have been disclosed in the prior art, and include urelumab, a human IgG4 antibody (AU2004279877) and utomilumab, a human IgG2 antibody (Fisher et al. 2012 Cancer Immunol. Immunother. 61: 1721-1733).
Westwood J A, et al., Leukemia Research 38 (2014), 948-954 discloses “Combination anti-CD137 and anti-CD40 antibody therapy in murine myc-driven hematological cancers”.
US20090074711 discloses “Human therapies using chimeric agonistic anti-human CD40 antibody”.
However, despite these and other advances in the art, there is a need for multispecific antibodies that can bind both CD40 and CD137, simultaneously binding to CD40-expressing APCs and CD137-expressing T cells, thereby bringing these cell types in close contact. This, in turn, can lead to activation of both cell types and efficient induction of anti-tumor immunity.
SUMMARY OF THE INVENTION
The present inventors have identified multispecific antibodies that can bind both CD40 and CD137 and elicit T cell and APC activation.
So, in one aspect, the invention relates to a multispecific antibody comprising
(i) a first antigen-binding region binding to human CD40, and (ii) a second antigen-binding region binding to human CD137.
In some embodiments, the invention relates to such a multispecific antibody wherein the first antigen-binding region comprises heavy and light chain variable region CDR1, CDR2 and CDR3 which comprise specific amino acid sequences, optionally with mutations, or the amino acid sequences of an antibody which competes with or has the specificity of an antibody comprising such specific amino acid sequences. In specific embodiments, the first antigen-binding region comprises heavy and light chain variable sequences comprising the CDR1, CDR2 and CDR3 of anti-CD40 antibody 001, or competes with or has the specificity of such an antibody.
In some embodiments, the invention relates to such a multispecific antibody wherein the second antigen-binding region comprises heavy and light chain variable sequences wherein the CDR1, CDR2 and CDR3 comprise specific amino acid sequences or provide specific amino acid sequences, optionally with mutations, or comprise the amino acid sequences of an antibody which competes with or has the specificity of an antibody comprising such specific amino acid sequences. In specific embodiments, the second antigen-binding region comprises heavy and light chain variable sequences comprising the CDR1, CDR2 and CDR3 of anti-CD137 antibody 001, 002, 003, 004, 005, 006, 007, 008, 009, 010, 011 or 012, or competes with or has the specificity of any such antibody.
These and other aspects and embodiments, including nucleic acids encoding the amino acid sequences of such multispecific antibodies; expression vectors comprising such nucleic acids; host cells comprising such nucleic acids or expression vectors; compositions comprising such multispecific antibodies; such compositions for use in treating cancer or other diseases; methods for producing such multispecific antibodies; and diagnostic methods and kits based on such multispecific antibodies, are described in further detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Sequence alignments for human, African elephant and wild boar CD137. Amino acids in African elephant or wild boar CD137 that differ from those in the human sequence are highlighted in black.
FIG. 2: CD137 shuffle constructs, containing African elephant (shuffle 5) or wild boar (shuffle 1-4, 6) CD137 domains.
FIG. 3: Expression of CD137 shuffle constructs on HEK293-T17 cells. HEK293-T17 cells were transfected with the CD137 shuffle constructs. Cell surface expression of the constructs was measured by flow cytometry, using a polyclonal anti-CD137 antibody that recognizes human, wild boar and African elephant CD137.
FIG. 4: Binding of CD137 antibody clones to CD137 shuffle constructs expressed on HEK293-T17 cells. HEK293-T17 cells were transfected with the CD137 shuffle constructs, and with human CD137 (hCD137 wt), African elephant of wild boar CD137, as indicated. Binding of the different CD137 antibody clones to these constructs expressed on HEK293-T17 cells was measured by flow cytometry. Staining with polyclonal anti-CD137 antibody is shown as a control.
FIG. 5: Matrix-like mixing grids used for automated bispecific antibody discovery. Parental antibodies can be plated as indicated. Parental antibodies can then be combined to obtain bispecific antibodies using a simple matrix-like mixing grid. Controlled Fab-arm exchange can then be performed to obtain bispecific antibodies.
FIGS. 6A-6B: Expression of CD40 and CD137 on the cell surface of stably transduced HEK293-NFK-gfp-luc and K562 cells. NF-KB/293/GFP-Luc™ (FIG. 6A) and K562 (FIG. 6B) cells were stably transduced with CD40 or CD137. Surface expression of CD40 (left panels) and CD137 (right panels) was determined by flow cytometry (white curves: control without antibody; grey curves: antibody staining).
FIGS. 7A-7L: Analysis of bispecific antibodies simultaneously targeting CD40 and CD137 (CD40xCD137). Bispecific antibodies targeting CD40 and CD137 (CD40-FEALxCD137-FEAR) were tested in the reporter assay in duplicate (FIGS. 7A-7L: CD40-001xCD137-001 until CD40-001xCD137-012). Activation of CD137 was measured by luciferase activity (relative luminescence units, RLU) of NF-KB/293/GFP-Luc™ transduced with CD137 (HEK293_NFK_CD137_gfp_luc) upon incubation with the indicated bispecific antibodies and K562 cells transduced with CD40 (K562_CD40) for trans-activation or wildtype K562 cells (K562 wt) as a control. Activation of CD40 was measured by luciferase activity (RLU) of NF-KB/293/GFP-Luc™ transfected with CD40 (HEK293_.NFK_.CD40_gfp_luc) upon incubation with the indicated bispecific antibodies and K562 transduced with CD137 (K562_CD137) for trans-activation or wildtype K562 cells (K562_wt) as a control. The two monospecific, monovalent antibodies with one irrelevant arm (b12-FEALxCD137-FEAR, b12-FEALxCD4O-FEAR) were used as control for the bispecific CD40xCD137 antibodies.
FIGS. 8A-8E: Induction of CD8+ T-cell proliferation by CD40×CD137 bispecific antibodies in a non-antigen-specific T cell assay. CFSE-labeled PBMCs were incubated with CD40×CD137 bispecific antibodies or monospecific, monovalent control antibodies, for four days. Proliferation of CD8+ cells was measured by flow cytometry. Data shown are CFSE plots showing CD8+ T-cell proliferation induced by the indicated bispecific and control antibodies at 0.02 μg/mL (FIG. 8A), percentages of divided cells and proliferation indices for CD40-001-FEAL×CD137-005-FEAR (FIG. 8B), CD40-001-FEAL×CD137-009-FEAR (FIG. 8C), CD40-001-FEAL×CD137-003-FEAR (FIG. 8D) and CD40-001-FEAL×CD137-011-FEAR (FIG. 8E), as calculated using FlowJo software.
FIGS. 9A-9E: Enhancement of CD8+ T-cell proliferation by CD40×CD137 bispecific antibodies in an antigen-specific T-cell assay. T cells transfected with a claudin-6-specific TCR and labeled with CFSE were incubated with claudin-6 IVT-RNA-electroporated immature DCs in the presence or absence of CD40×CD137 bispecific antibodies or control antibodies for five days. CD8+ T-cell proliferation was measured by flow cytometry, Data shown are CFSE plots showing CD8+ T-cell proliferation induced by the indicated bispecific antibodies and controls at 0.02 μg/mL (FIG. 9A), percentages divided cells and proliferation indices for the indicated bispecific antibodies (FIG. 9B) and for CD40-001-FEAL×CD137-005-FEAR and control antibodies (FIG. 9C) and CD40-001-FEAL×CD137-009-FEAR and control antibodies (FIG. 9D) at the indicated concentrations, as calculated using FlowJo software. Proliferation index curves for the indicated bispecific antibodies at serial dilutions ranging from 6.4×10−5 to 5 μg/mL are also shown (FIG. 9E). Curves were analyzed by non-linear regression (sigmoidal dose-response with variable slope) using GraphPad Prism 5 software (GraphPad Software, San Diego, Calif., USA). The EC50 values for induction of T-cell proliferation for CD40-001-FEAL×CD137-005-FEAR and CD40-001-FEAL×CD137-009-FEAR were 0.005 and 0.030 μg/mL, respectively.
FIG. 10: Induction of CD8+ T-cell proliferation by the humanized CD40×CD137 bispecific antibody in a non-antigen-specific T cell assay. CFSE-labeled PBMCs were incubated with humanized CD40×CD137 bispecific antibody, the parental bispecific antibody or IgG1 control antibody for four days. Proliferation of CD8+ T cells was measured by flow cytometry. Data shown are percentages of divided cells and proliferation index, as calculated by FlowJo software. (n.d.=not determined)
FIG. 11: Enhancement of CD8+ T-cell proliferation by a humanized CD40×CD137 bispecific antibody in an antigen-specific T-cell assay. T cells transfected with a claudin-6-specific TCR and labeled with CFSE were incubated with claudin-6 IVT-RNA-electroporated immature DCs in the presence or absence of a humanized CD40×CD137 bispecific antibody (BisG1-CD40-001-H6LC1-FEAL×CD137-009-HC7LC2-FEAR), the parental bispecific antibody or an IgG1 control antibody for four days. CD8+ T-cell proliferation was measured by flow cytometry. Data shown are percentages divided cells and proliferation indices for the indicated antibodies, as calculated using FlowJo software. (n.d.=not determined)
FIG. 12: Ex vivo expansion of TILs from a human melanoma tissue resection by a CD40×CD137 bispecific antibody. Tumor pieces from the resected tissue were cultured with 100 U/mL IL-2 and the indicated concentration of a CD40×CD137 bispecific antibody (BisG1-CD40-001-FEAL×CD137-009-FEAR). After 14 days of culture, cells were harvested and analyzed by flow cytometry. Relative viable TIL count per sample (normalized to 1,000 measured counting beads) is shown. Each data point refers to a single well, representing the expansion of TILs out of two tumor pieces analyzed in one FACS tube. The line indicates the mean of five measured samples.
FIG. 13: Ex vivo expansion of TILs from a human non-small cell lung cancer (NSCLC) tissue resection by a CD40×CD137 bispecific antibody. Tumor pieces from resected NSCLC tissue were cultured with 10 U/mL IL-2 and the indicated concentration of CD40×CD137 bispecific antibody (BisG1-CD40-001-FEAL×CD137-009-FEAR). After 10 days of culture, cells were harvested and analyzed by flow cytometry. Relative viable TIL count per sample (normalized to 1,000 measured counting beads) is shown. Each data point refers to a single well, representing the expansion of TILs out of two tumor pieces analyzed in one FACS tube. The line indicates the mean of five measured samples.
TABLE 1
Sequences
Type of
Sequence
Sequence name
sequence
Sequence
identifier
CD40-001
VH CDR1
GYTFTEYI
SEQ ID NO: 1
antibody (mouse)
VH CDR2
IIPNNGGT
SEQ ID NO: 2
VH CDR3
TRREVYGRNYYALDY
SEQ ID NO: 3
VL CDR1
QGINNY
SEQ ID NO: 4
VL CDR2
YTS
VL CDR3
QQYSNLPYT
SEQ ID NO: 5
VH
EVQLQQSGPDLVKPGASVKISCKTS
SEQ ID NO: 6
GYTFTEYIMHWVKQSHGKSLEWIG
GIIPNNGGTSYNQKFKDKATMTVDK
SSSTGYMELRSLTSEDSAVYYCTRRE
VYGRNYYALDYWGQGTLVTVSS
VL
DIQMTQTTSSLSASLGDRVTITCSA
SEQ ID NO: 7
SQGINNYLNWYQQKPDGTVKLLIYY
TSSLHSGVPSRFSGSGSGTDYSLTIS
NLEPEDIATYYCQQYSNLPYTFGGGT
KLEIK
CD137 antibody
VH CDR1
GFSLSSYA
SEQ ID NO: 8
clone 001 (rabbit)
VH CDR2
IWNNGAT
SEQ ID NO: 9
VH CDR3
ARSANDAWSTYSDL
SEQ ID NO: 10
VL CDR1
QTITNY
SEQ ID NO: 11
VL CDR2
KAS
VL CDR3
QNYYYGSSSGYGFV
SEQ ID NO: 12
VH
QSVEESGGRLVTPGTPLTLTCTVSGFS
SEQ ID NO: 13
LSSYAVSWVRQAPGKGLEWIGVIWN
NGATHYATWAKGRFTISKASTTVDLK
VTSPTTEDTATYFCARSANDAWSTYS
DLWGQGTLVTVSS
VL
DIVMTQTPASVEAAVGGTVTIKCQASQ
SEQ ID NO: 14
TITNYLSWYQQKPGQPPKLLIYKASTLT
SGVSSRFKGSGSGTEFTLTISDLESDDA
ATYYCQNYYYGSSSGYGFVFGGGTEVVV
K
CD137 antibody
VH CDR1
GFSLTYYA
SEQ ID NO: 15
clone 002 (rabbit)
VH CDR2
IYDNGAT
SEQ ID NO: 16
VH CDR3
ARSANNAWSTYSNL
SEQ ID NO: 17
VL CDR1
EDISSY
SEQ ID NO: 18
VL CDR2
KAS
VL CDR3
QSYYSGSISGYGFV
SEQ ID NO: 19
VH
QSVEESGGRLVTPGTPLTLTCTVSGFS
SEQ ID NO: 20
LTYYAVTWVRQPPGKGLEWIGVIYDN
GATAFATWAKGRFTMSKNSTTVALKV
TSPTTEDTATYFCARSANNAWSTYSN
LWGQGTLVTVSS
VL
DIVMTQTPSSVSAAVGGTVTINCQAS
SEQ ID NO: 21
EDISSYLSWYQQKLGQPPKLLIYKAST
LESGVPSRFKGSGSGTEYTLTISDLES
DDAATYYCQSYYSGSISGYGFVFGGGT
GVVVK
CD137 antibody
VH CDR1
GFTISSYH
SEQ ID NO: 22
clone 003 (rabbit)
VH CDR2
IYGGTATT
SEQ ID NO: 23
VH CDR3
ARARYSGGSYANYVFNL
SEQ ID NO: 24
VL CDR1
QSISSY
SEQ ID NO: 25
VL CDR2
RTS
VL CDR3
QGYDWSSSNRYDNT
SEQ ID NO: 26
VH
QSVEESGGRLVTPGTPLTLTCTAS
SEQ ID NO: 27
GFTISSYHMIWVRQAPGEGLEWI
GDIYGGTATTDYASWAKGRFTIS
KTSTTVDLKMTSLTTEDTATYFCA
RARYSGGSYANYVFNLWGQGTLV
TVSS
VL
DIVMTQTPASVEAAVGGTVTIKCQ
SEQ ID NO: 28
ASQSISSYLAWYQQKPGQPPKLLI
YRTSTLESGVPSRFKGSGSGTEFTL
TISDLESADAATYYCQGYDWSSSN
RYDNTFGGGTEVVVK
CD137 antibody
VH CDR1
GFSLSRYD
SEQ ID NO: 29
clone 004 (rabbit)
VH CDR2
ISSSGGT
SEQ ID NO: 30
VH CDR3
AREGDYWDFNL
SEQ ID NO: 31
VL CDR1
QSISNL
SEQ ID NO: 32
VL CDR2
GAS
VL CDR3
AGGFPGLDTVAA
SEQ ID NO: 33
VH
QSLEESGGRLVTPGTPLTLTCTASGF
SEQ ID NO: 34
SLSRYDMSWVRQAPGKGLEYIGVIS
SSGGTNYANWAKGRFTISKTSTTVD
LKITSPTTEDTATYFCAREGDYWDFN
LWGPGTLVTVSS
VL
AQVLTQTPSSVSAAVGGTVTINCQA
SEQ ID NO: 35
SQSISNLLAWYQQKPGQPPKLLIYG
ASTLASGVPSRFSGSGSGTEFTLTIS
DLESDDAATYYCAGGFPGLDTVAAF
GGGTEAVVT
CD137 antibody
VH CDR1
GFTISDFH
SEQ ID NO: 36
clone 005 (rabbit)
VH CDR2
IITSASTT
SEQ ID NO: 37
VH CDR3
ARSTYTDTSGYYFDF
SEQ ID NO: 38
VL CDR1
QSIYNGNR
SEQ ID NO: 39
VL CDR2
SAS
VL CDR3
LGSYDCDSADCFA
SEQ ID NO: 40
VH
QSVEESGGRLVTPGTPLTLTCTASG
SEQ ID NO: 41
FTISDFHVTWVRQAPGKGLEWIGTI
ITSASTTAYATWARGRFTISKSSTTV
NLKIVSPTTEDTATYFCARSTYTDTS
GYYFDFWGQGTLVTVSS
VL
AQVLTQTASPVSAAVGGTVIINCQSS
SEQ ID NO: 42
QSIYNGNRLSWYQQKPGQPPKLLIYS
ASTLASGVSSRFKGSGSGTQFTLAISD
VQSDDAATYYCLGSYDCDSADCFAFG
GGTEVVVE
CD137 antibody
VH CDR1
GFSLSSYA
SEQ ID NO: 43
clone 006 (rabbit)
VH CDR2
ISTSGIT
SEQ ID NO: 44
VH CDR3
ARLNGFDDYVRYFDF
SEQ ID NO: 45
VL CDR1
ESIASN
SEQ ID NO: 46
VL CDR2
AAS
VL CDR3
QSAFYVSSSDNA
SEQ ID NO: 47
VH
QSVEESGGRLVTPGTPLTLTCTVSGFS
SEQ ID NO: 48
LSSYAMSWVRQAPGKGLEWIGIISTS
GITYYASWAKGRFTISKTSTMVDLKIT
SPTTEDTATYFCARLNGFDDYVRYFDF
WGLGTLVTVSS
VL
AIELTQTPSSVSAAVGGTVTINCQASE
SEQ ID NO: 49
SIASNLAWYQQKPGQPPKLLIYAASYL
ASGVPSRFKGSGSGTEYTLTISGVQSA
DAATYYCQSAFYVSSSDNAFGGGTEVV
VK
CD137 antibody
VH CDR1
GFSLSSYD
SEQ ID NO: 50
clone 007 (rabbit)
VH CDR2
IGSDGSA
SEQ ID NO: 51
VH CDR3
ARDWNDYWAHDL
SEQ ID NO: 52
VL CDR1
QIVTSKSA
SEQ ID NO: 53
VL CDR2
KAS
VL CDR3
AGGYYNSGDLNP
SEQ ID NO: 54
VH
QSLEESGGRLVTPGTPLTLTCTASGFS
SEQ ID NO: 55
LSSYDVSWVRQAPGKGLEYIGFIGSD
GSAHYATWVKGRFTISKTSTTVDLKIT
SPTTEDTATYFCARDWNDYWAHDLW
GPGTLVTVSS
VL
AQVLTQTTSPVSAAVGGTVTINCQAS
SEQ ID NO: 56
QIVTSKSALSWYQQKPGQPPRLLIYK
ASTLASGVPSRFSGSGSGTQFTLTIS
DLESDDAATYYCAGGYYNSGDLNPF
GGGTEVVVK
CD137 antibody
VH CDR1
GFSLSSYD
SEQ ID NO: 57
clone 008 (rabbit)
VH CDR2
ISSSGNT
SEQ ID NO: 58
VH CDR3
AREGDYWDFNL
SEQ ID NO: 59
VL CDR1
QSISNL
SEQ ID NO: 60
VL CDR2
RAS
VL CDR3
AGGFPGLDTGAT
SEQ ID NO: 61
VH
QSLEESGGRLVTPGTPLTLTCTASGFSL
SEQ ID NO : 62
SSYDMSWVRQAPGKGLEYIGYISSSG
NTYYASWAKSRFTISKTSTTVDLKITS
PTTEDTATYFCAREGDYWDFNLWGPG
TLVTVSS
VL
AQVLTQTPSSVSAAVGGTVTINCQAS
SEQ ID NO: 63
QSISNLLAWYQQKPGQRPRLLIYRAS
TLASGVPSRFKGSGSGTEFTLTISDLE
SEDAATYYCAGGFPGLDTGATFGGGT
EAVVT
CD137 antibody
VH CDR1
GFSLNDYW
SEQ ID NO: 64
clone 009 (rabbit)
VH CDR2
IDVGGSL
SEQ ID NO: 65
VH CDR3
ARGGLTYGFDL
SEQ ID NO: 66
VL CDR1
EDISSY
SEQ ID NO: 67
VL CDR2
GAS
VL CDR3
HYYATISGLGVA
SEQ ID NO: 68
VH
QSLEESGGRLVTPGTPLTLTCTVSG
SEQ ID NO: 69
FSLNDYWMSWVRQAPGKGLEWIG
YIDVGGSLYYASWAKGRFTISRTST
TVDLKMTSLTTEDTATYFCARGGLT
YGFDLWGPGTLVTVSS
VL
DIVMTQTPASVSEPVGGTVTINCQA
SEQ ID NO: 70
SEDISSYLAWYQQKPGQRPKRLIYG
ASDLASGVPSRFSASGSGTEYALTIS
DLESADAATYYCHYYATISGLGVAFG
GGTEVVVK
CD137 antibody
VH CDR1
GFSLSTYA
SEQ ID NO: 71
clone 010 (rabbit)
VH CDR2
VYDNGYI
SEQ ID NO: 72
VH CDR3
ARSADGSWSTYFNL
SEQ ID NO: 73
VL CDR1
ESISNY
SEQ ID NO: 74
VL CDR2
KAS
VL CDR3
QTNYCCSSSDNGFA
SEQ ID NO: 75
VH
QSVEESGGRLVTPGTPLTLTCTVSGFSL
SEQ ID NO: 76
STYAMIWVRQAPGKGLEWIGVVYDNG
YISHATWVKGRFTISKTSTTVGLEITSP
TTEDTATYFCARSADGSWSTYFNLWG
QGTLVTVSS
VL
DIVMTQTPASVEAAVGGTVTIKCQAS
SEQ ID NO: 77
ESISNYLAWYQQKPGQPPKLLIYKAS
TLASGVSSRFKGSGSGTEFTLTISDL
ESADAATYYCQTNYCCSSSDNGFAF
GGGTEVVVK
CD137 antibody
VH CDR1
GIDLSSYH
SEQ ID NO: 78
clone 011 (rabbit)
VH CDR2
IAYGGNT
SEQ ID NO: 79
VH CDR3
ARGYSEDSYWGL
SEQ ID NO: 80
VL CDR1
QNIENY
SEQ ID NO: 81
VL CDR2
DTS
VL CDR3
QQDYGIIFVDNI
SEQ ID NO: 82
VH
QSLEESGGRLVTPGTPLTLTCTVSGIDL
SEQ ID NO: 83
SSYHMCWVRQAPGKGLEYIGYIAYGG
NTYYANWAKGRFTISKTSTTVDLRITS
PTTEDTATYFCARGYSEDSYWGLWGP
GTLVTVSS
VL
AYDMTQTPASVEAAVGGTVTIKCQAS
SEQ ID NO: 84
QNIENYLAWYQQKPGQPPKLLIYDTS
KLTSGVPSRFSGSGSGTDFTLTISGVQ
SDDAATYYCQQDYGIIFVDNIFGGGTE
VVVK
CD137 antibody
VH CDR1
GFSLSDYY
SEQ ID NO: 85
clone 012 (rabbit)
VH CDR2
MSGSGST
SEQ ID NO: 86
VH CDR3
ARDGDYAGWGYATGAFDP
SEQ ID NO: 87
VL CDR1
QSVVGNSL
SEQ ID NO: 88
VL CDR2
SAS
VL CDR3
TGRYNSDTDTFV
SEQ ID NO: 89
VH
QSVEESGGRLVTPGTPLTLTCTVSGFSL
SEQ ID NO: 90
SDYYMTWVRQAPGKGLEYIGIMSGSG
STYYASWAKGRFTISKTSSTTLELKITS
PTTEDTAIYFCARDGDYAGWGYATGAF
DPWGPGTLVTVSS
VL
AAVLTQTPSPVSAAVGGTVTISCQASQ
SEQ ID NO: 91
SVVGNSLLSWFQKKPGQPPKLLIYSAS
SLASGVPSRFKGSGSGTQFTLTISDLES
DDAATYYCTGRYNSDTDTFVFGGGTEV
VVK
Human CD137
MGNSCYNIVATLLLVLNFERTRSLQDPCS
SEQ ID NO: 92
(TNR9_Human)
NCPAGTFCDNNRNQICSPCPPNSFSSAG
GQRTCDICRQCKGVFRTRKECSSTSNAE
CDCTPGFHCLGAGCSMCEQDCKQGQELT
KKGCKDCCFGTFNDQKRGICRPWTNCSL
DGKSVLVNGTKERDVVCGPSPADLSPGA
SSVTPPAPAREPGHSPQIISFFLALTSTALL
FLLFFLTLRFSVVKRGRKKLLYIFKQPFMRP
VQTTQEEDGCSCRFPEEEEGGCEL
Human CD137
MGNSCYNIVATLLLVLNFERTRSVPDPCS
SEQ ID NO: 93
shuffle 6 (amino
NCSAGTFCGKNIQELCMPCPPNSFSSAG
acids 24-47
GQRTCDICRQCKGVFRTRKECSSTSNAE
replaced by wild
CDCTPGFHCLGAGCSMCEQDCKQGQELT
boar CD137)
KKGCKDCCFGTFNDQKRGICRPWTNCSL
DGKSVLVNGTKERDVVCGPSPADLSPGA
SSVTPPAPAREPGHSPQIISFFLALTSTALL
CCCEL
Human CD137
MGNSCYNIVATLLLVLNFERTRSLQDPCS
SEQ ID NO: 94
shuffle 5 (amino
NCPAGTFCDNNRNQICSPCPLNSFSSTGG
acids 48-88
QMNCDMCRKCEGVFKTKRACSPTRDAEC
replaced by
ECTPGFHCLGAGCSMCEQDCKQGQELTK
African elephant
KGCKDCCFGTFNDQKRGICRPWTNCSLD
CD137)
GKSVLVNGTKERDVVCGPSPADLSPGAS
SVTPPAPAREPGHSPQIISFFLALTSTALLF
LLFFLTLRFSVVKRGRKKLLYIFKQPFMRP
VQTTQEEDGCSCRFPEEEEGGCEL
Human CD137
MGNSCYNIVATLLLVLNFERTRSLQDPCS
SEQ ID NO: 95
shuffle 4 (amino
NCPAGTFCDNNRNQICSPCPPNSFSSAG
acids 89-114
GQRTCDICRQCKGVFRTRKECSSTSNAE
replaced by wild
CDCVPGFRCLGAGCAMCEEYCQQGQELT
boar CD137)
QKGCKDCCFGTFNDQKRGICRPWTNCSL
DGKSVLVNGTKERDVVCGPSPADLSPGA
SSVTPPAPAREPGHSPQIISFFLALTSTALL
FLLFFLTLRFSVVKRGRKKLLYIFKQPFMRP
VQTTQEEDGCSCRFPEEEEGGCEL
Human CD137
MGNSCYNIVATLLLVLNFERTRSLQDPCS
SEQ ID NO: 96
shuffle 3 (amino
NCPAGTFCDNNRNQICSPCPPNSFSSAG
acids 115-138
GQRTCDICRQCKGVFRTRKECSSTSNAE
replaced by wild
CDCTPGFHCLGAGCSMCEQDCKQGQELT
boar CD137)
KEGCKDCSFGTFNDEEHGVCRPWTDCSL
DGKSVLVNGTKERDVVCGPSPADLSPGA
SSVTPPAPAREPGHSPQIISFFLALTSTALL
FLLFFLTLRFSVVKRGRKKLLYIFKQPFMRP
VQTTQEEDGCSCRFPEEEEGGCEL
Human CD137
MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPA
SEQ ID NO: 97
shuffle 2 (amino
GTFCDNNRNQICSPCPPNSFSSAGGQRTCDICR
acids 139-161
QCKGVFRTRKECSSTSNAECDCTPGFHCLGAGC
replaced by wild
SMCEQDCKQGQELTKKGCKDCCFGTFND
boar CD137)
QKRGICRPWTNCSLAGKPVLMNGTKARD
VVCGPRPADLSPGASSVTPPAPAREPGHS
PQIISFFLALTSTALLFLLFFLTLRFSVVKRG
RKKLLYIFKQPFMRPVQTTQEEDGCSCRF
PEEEEGGCEL
Human CD137
MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPA
SEQ ID NO: 98
shuffle 1 (amino
GTFCDNNRNQICSPCPPNSFSSAGGQRTCDICR
acids 162-186
QCKGVFRTRKECSSTSNAECDCTPGFHCLGAGC
replaced by wild
SMCEQDCKQGQELTKKGCKDCCFGTFNDQKR
boar CD137)
GICRPWTNCSLDGKSVLVNGTKERDVVCGPSPT
DFSPGTPSTTMPVPGGEPGHTSHIISFFLALTST
ALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPV
QTTQEEDGCSCRFPEEEEGGCEL
b12
VH CDR1
GYRFSNFV
SEQ ID NO: 99
VH CDR2
INPYNGNK
SEQ ID NO: 100
VH CDR3
ARVGPYSWDDSPQDNYYMDV
SEQ ID NO: 101
VL CDR 1
HSIRSRR
SEQ ID NO: 102
VL CDR 2
CVS
VL CDR 3
QVYGASSYT
SEQ ID NO: 103
VH
QVQLVQSGAEVKKPGASVKVSCQASGYR
SEQ ID NO: 104
FSNFVIHWVRQAPGQRFEWMGWINPYN
GNKEFSAKFQDRVTFTADTSANTAYMELR
SLRSADTAVYYCARVGPYSWDDSPQDNY
YMDVWGKGTTVIVSS
VL
EIVLTQSPGTLSLSPGERATFSCRSSHSIR
SEQ ID NO: 105
SRRVAWYQHKPGQAPRLVIHGVSNRASG
ISDRFSGSGSGTDFTLTITRVEPEDFALW
CQVYGASSYTFGQGTKLERK
IgG1m(a) CH3
GQPREPQVYTLPPSRDELTKNQVSLTCLV
SEQ ID NO: 106
region
KGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPGK
IgG1m(f) CH3
GQPREPQVYTLPPSREEMTKNQVSLTCLV
SEQ ID NO: 107
region
KGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPGK
IgG1m(ax) CH3
GQPREPQVYTLPPSRDELTKNQVSLTCLV
SEQ ID NO: 108
region
KGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEGLHNHYTQKSLSLSPGK
IgG1 heavy chain
ASTKGPSVFPLAPSSKSTSGGTAALGCLV
SEQ ID NO: 109
constant region-
KDYFPEPVTVSWNSGALTSGVHTFPAVLQ
WT*
SSGLYSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPGK
IgG1 heavy chain
ASTKGPSVFPLAPSSKSTSGGTAALGCLV
SEQ ID NO: 110
constant region-
KDYFPEPVTVSWNSGALTSGVHTFPAVLQ
F405L*
SSGLYSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFLL
YSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGK
IgG1 heavy chain
ASTKGPSVFPLAPSSKSTSGGTAALGCLV
SEQ ID NO: 111
constant region-
KDYFPEPVTVSWNSGALTSGVHTFPAVLQ
K409R*
SSGLYSLSSVVTVPSSSLGTQTYICNVNH
KPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLY
SRLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPGK
Human IgG1
ASTKGPSVFPLAPSSKSTSGGTAALGCLV
SEQ ID NO: 112
heavy chain
KDYFPEPVTVSWNSGALTSGVHTFPAVLQ
constant sequence
SSGLYSLSSVVTVPSSSLGTQTYICNVNH
with FEAR*
KPSNTKVDKRVEPKSCDKTHTCPPCPAPE
FEGGPSVFLFPPKPKDTLMISRTPEVTCVV
VAVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLY
SRLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPGK
Human IgG1
ASTKGPSVFPLAPSSKSTSGGTAALGCLV
SEQ ID NO: 113
heavy chain
KDYFPEPVTVSWNSGALTSGVHTFPAVLQ
constant sequence
SSGLYSLSSVVTVPSSSLGTQTYICNVNH
with FEAL*
KPSNTKVDKRVEPKSCDKTHTCPPCPAPE
FEGGPSVFLFPPKPKDTLMISRTPEVTCVV
VAVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFLL
YSKLTVDKSRWQQGNVFSCSVMHEALH
NHYTQKSLSLSPGK
Human
Kappa
RTVAAPSVFIFPPSDEQLKSGTASVVCLLN
SEQ ID NO: 114
light chain
NFYPREAKVQWKVDNALQSGNSQESVTE
constant sequence
QDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC
Human CD40
MVRLPLQCVLWGCLLTAVHPEPPTACREK
SEQ ID NO: 115
QYLINSQCCSLCQPGQKLVSDCTEFTETE
CLPCGESEFLDTWNRETHCHQHKYCDPN
LGLRVQQKGTSETDTICTCEEGWHCTSE
ACESCVLHRSCSPGFGVKQIATGVSDTIC
EPCPVGFFSNVSSAFEKCHPWTSCETKDL
VVQQAGTNKTDVVCGPQDRLRALVVIPII
FGILFAILLVLVFIKKVAKKPTNKAPHPKQE
PQEINFPDDLPGSNTAAPVQETLHGCQPV
TQEDGKESRISVQERQ
Human IgG1
ASTKGPSVFPLAPSSKSTSGGTAALGCLV
SEQ ID NO:
heavy chain
KDYFPEPVTVSWNSGALTSGVHTFPAVLQ
116
constant sequence
SSGLYSLSSVVTVPSSSLGTQTYICNVNH
with FEA*
KPSNTKVDKRVEPKSCDKTHTCPPCPAPE
FEGGPSVFLFPPKPKDTLMISRTPEVTCVV
VAVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHN
HYTQKSLSLSPGK
VH-CD40-001-
VH
EVQLVQSGAEVKKPGASVKVSCKTSGYT
SEQ ID NO: 117
HC6
FTEYIMHWVRQAPGQGLEWMGGIIPNNG
GTSYNQKFQGRVTMTVDKSTSTGYMELS
SLRSEDTAVYYCTRREVYGRNYYALDYW
GQGTLVTVSS
CD40-001-HC6
HC, IgG1
EVQLVQSGAEVKKPGASVKVSCKTSGYT
SEQ ID NO: 118
FTEYIMHWVRQAPGQGLEWMGGIIPNNG
GTSYNQKFQGRVTMTVDKSTSTGYMELS
SLRSEDTAVYYCTRREVYGRNYYALDYW
GQGTLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLG
TQTYICNVNHKPSNTKVDKRVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPGK
CD40-001-HC6-
HC, IgG1
EVQLVQSGAEVKKPGASVKVSCKTSGYT
SEQ ID NO: 119
FEAL
FEAL
FTEYIMHWVRQAPGQGLEWMGGIIPNNG
GTSYNQKFQGRVTMTVDKSTSTGYMELS
SLRSEDTAVYYCTRREVYGRNYYALDYW
GQGTLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLG
TQTYICNVNHKPSNTKVDKRVEPKSCDKT
HTCPPCPAPEFEGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVAVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFLLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPGK
CD40-001-HC6-
HC, IgG1
EVQLVQSGAEVKKPGASVKVSCKTSGYT
SEQ ID NO: 120
FEAR
FEAR
FTEYIMHWVRQAPGQGLEWMGGIIPNNG
GTSYNQKFQGRVTMTVDKSTSTGYMELS
SLRSEDTAVYYCTRREVYGRNYYALDYW
GQGTLVTVSSASTKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLG
TQTYICNVNHKPSNTKVDKRVEPKSCDKT
HTCPPCPAPEFEGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVAVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSRLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPGK
VL-CD40-001-LC1
VL
DIQMTQSPSSLSASVGDRVTITCSASQGI
SEQ ID NO: 121
NNYLNWYQQKPGKAVKLLIYYTSSLHSGV
PSRFSGSGSGTDYTFTISSLQPEDIATYYC
QQYSNLPYTFGGGTKVEIK
CD40-001-LC1
LC, kappa
DIQMTQSPSSLSASVGDRVTITCSASQGI
SEQ ID NO: 122
NNYLNWYQQKPGKAVKLLIYYTSSLHSGV
PSRFSGSGSGTDYTFTISSLQPEDIATYYC
QQYSNLPYTFGGGTKVEIKRTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC
CD137-009 humanized antibody (HC7 and LC2):
VH-CD137-009-
VH
EVQLVESGGGLVQPGRSLRLSCTASGFSL
SEQ ID NO: 123
HC7
NDYWMSWVRQAPGKGLEWVGYIDVGGS
LYYAASVKGRFTISRDDSKSIAYLQMNSL
KTEDTAVYYCARGGLTYGFDLWGQGTLV
TVSS
CD137-009-HC7
HC, IgG1
EVQLVESGGGLVQPGRSLRLSCTASGFSL
SEQ ID NO: 124
NDYWMSWVRQAPGKGLEWVGYIDVGGS
LYYAASVKGRFTISRDDSKSIAYLQMNSL
KTEDTAVYYCARGGLTYGFDLWGQGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKRVEPKSCDKTHTCPP
CPAPELLGGPSVFLFPPKPKDTLMISRTPE
VTCVVVDVSHEDPEVKFNWYVDGVEVHN
AKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPGK
CD137-009-HC7-
HC, IgG1
EVQLVESGGGLVQPGRSLRLSCTASGFSL
SEQ ID NO: 125
FEAR
FEAR
NDYWMSWVRQAPGKGLEWVGYIDVGGS
LYYAASVKGRFTISRDDSKSIAYLQMNSL
KTEDTAVYYCARGGLTYGFDLWGQGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKRVEPKSCDKTHTCPP
CPAPEFEGGPSVFLFPPKPKDTLMISRTPE
VTCVVVAVSHEDPEVKFNWYVDGVEVHN
AKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSRLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPGK
CD137-009-HC7-
HC, IgG1
EVQLVESGGGLVQPGRSLRLSCTASGFSL
SEQ ID NO: 126
FEAL
FEAL
NDYWMSWVRQAPGKGLEWVGYIDVGGS
LYYAASVKGRFTISRDDSKSIAYLQMNSL
KTEDTAVYYCARGGLTYGFDLWGQGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYIC
NVNHKPSNTKVDKRVEPKSCDKTHTCPP
CPAPEFEGGPSVFLFPPKPKDTLMISRTPE
VTCVVVAVSHEDPEVKFNWYVDGVEVHN
AKTKPREEQYNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQP
REPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFLLYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPGK
VL-CD137-009-
VL
DIVMTQSPSSLSASVGDRVTITCQASEDI
SEQ ID NO: 127
LC2
SSYLAWYQQKPGKAPKRLIYGASDLASG
VPSRFSASGSGTDYTFTISSLQPEDIATYY
CHYYATISGLGVAFGGGTKVEIK
CD137-009-LC2
LC, kappa
DIVMTQSPSSLSASVGDRVTITCQASEDI
SEQ ID NO: 128
SSYLAWYQQKPGKAPKRLIYGASDLASG
VPSRFSASGSGTDYTFTISSLQPEDIATYY
CHYYATISGLGVAFGGGTKVEIKRTVAAP
SVFIFPPSDEQLKSGTASVVCLLNNFYPRE
AKVQWKVDNALQSGNSQESVTEQDSKD
STYSLSSTLTLSKADYEKHKVYACEVTHQ
GLSSPVTKSFNRGEC
Human CD137
Amino acids
LQDPCSNCPAGTFCDNNRNQICSP
SEQ ID NO: 129
(shuffle 6)
24-47 of
human CD137
Human CD137
Amino acids
CPPNSFSSAGGQRTCDICRQCKGVFRTR
SEQ ID NO: 130
(shuffle 5)
48-88 of
KECSSTSNAECDC
human CD137
Human CD137
Amino acids
TPGFHCLGAGCSMCEQDCKQGQELTK
SEQ ID NO: 131
(shuffle 4)
89-114 of
human CD137
Human CD137
Amino acids
KGCKDCCFGTFNDQKRGICRPWTN
SEQ ID NO: 132
(shuffle 3)
115-138 of
human CD137
Human CD137
Amino acids
CSLDGKSVLVNGTKERDVVCGPS
SEQ ID NO: 133
(shuffle 2)
139-161 of
human CD137
Human CD137
Amino acids
PADLSPGASSVTPPAPAREPGHSPQ
SEQ ID NO: 134
(shuffle 1)
162-186 of
human CD137
Wild Boar CD137
MGNGYYNIVATVLLVMNFERTRSVPDPCS
SEQ ID NO: 135
NCSAGTFCGKNIQELCMPCPSNSFSSTSG
QKACNVCRKCEGVFRTKKECSSTSNAVC
ECVPGFRCLGAGCAMCEEYCQQGQELTQ
EGCKDCSFGTFNDEEHGVCRPWTDCSLA
GKPVLMNGTKARDVVCGPRPTDFSPGTP
STTMPVPGGEPGHTSHVIIFFLALMSTAVF
VLVSYLALRFSVVQQGRKKLLYIVKQPFLK
PAQTVQEEDACSCRFPEEEEGECEL
African Elephant
MGNGYYNMVATVLLVMNFERTGAVQDSC
SEQ ID NO: 136
CD137
RDCLAGTYCVKNESQICSPCPLNSFSSTG
GQMNCDMCRKCEGVFKTKRACSPTRDAE
CECVSGFHCLGAGCTMCQQDCKQGQEL
TKEGCKDCCLGTFNDQKNGICRPWTNCS
LEGKSVLANGTKKRDVVCGPPAADSFPDT
SSVTVPAPERKPDHHPQIITFFLALISAALL
FLVFFLVVRFSVAKWGRKKLLYIFKQPFIK
PVQTAQEEDGCSCRFPEEEEGDCEL
*amino acids positions 118-447 according to EU numbering
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “CD40” as used herein, refers to CD40, also referred to as tumor necrosis factor receptor superfamily member 5 (TNFRSF5), which is the receptor for the ligand TNFSF5/CD40L. CD40 is known to transduce TRAF6- and MAP3K8-mediated signals that activate ERK in macrophages and B cells, leading to induction of immunoglobulin secretion by the B cells. Other synonyms used for CD40 include, but are not limited to, B-cell surface antigen CD40, Bp50, CD40L receptor and CDw40. In one embodiment, CD40 is human CD40, having UniProt accession number P25942. The sequence of human CD40 is also shown in SEQ ID NO:115. Amino acids 1-20 of SEQ ID NO:115 correspond to the signal peptide of human CD40; while amino acids 21-193 of SEQ ID NO:115 correspond to the extracellular domain of human CD40; and the remainder of the protein; i.e. from amino acids 194-215 and 216-277 of SEQ ID NO:115 is transmembrane and cytoplasmic domain, respectively.
The term “CD137” as used herein, refers to CD137 (4-1BB), also referred to as tumor necrosis factor receptor superfamily member 9 (TNFRSF9), which is the receptor for the ligand TNFSF9/4-1BBL. CD137 (4-1BB) is believed to be involved in T-cell activation. Other synonyms for CD137 include, but are not limited to, 4-1BB ligand receptor, CDw137, T-cell antigen 4-1BB homolog and T-cell antigen ILA. In one embodiment, CD137 (4-1BB) is human CD137 (4-1BB), having UniProt accession number Q07011. The sequence of human CD137 is also shown in SEQ ID NO:92. Amino acids 1-23 of SEQ ID NO:92 correspond to the signal peptide of human CD137; while amino acids 24-186 of SEQ ID NO:92 correspond to the extracellular domain of human CD137; and the remainder of the protein, i.e. from amino acids 187-213 and 214-255 of SEQ ID NO:92 are transmembrane and cytoplasmic domain, respectively.
The term “chimeric antibody” as used herein, refers to an antibody wherein the variable region is derived from a non-human species (e.g. derived from rodents) and the constant region is derived from a different species, such as human. Chimeric antibodies may be generated by antibody engineering. “Antibody engineering” is a term used generically for different kinds of modifications of antibodies, and processes for antibody engineering are well-known for the skilled person. In particular, a chimeric antibody may be generated by using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Thus, the chimeric antibody may be a genetically or an enzymatically engineered recombinant antibody. It is within the knowledge of the skilled person to generate a chimeric antibody, and thus, generation of the chimeric antibody may be performed by other methods than those described herein. Chimeric monoclonal antibodies for therapeutic applications in humans are developed to reduce anticipated antibody immunogenicity of non-human antibodies, e.g. rodent antibodies. They may typically contain non-human (e.g. murine or rabbit) variable regions, which are specific for the antigen of interest, and human constant antibody heavy and light chain domains. The terms “variable region” or “variable domain” as used in the context of chimeric antibodies, refer to a region which comprises the CDRs and framework regions of both the heavy and light chains of an immunoglobulin, as described below.
The term “humanized antibody” as used herein, refers to a genetically engineered non-human antibody, which contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the six non-human antibody CDRs, which together form the antigen-binding site, onto a homologous human acceptor framework region (FR) (see WO92/22653 and EP0629240). In order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e. the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modeling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back-mutations, may be applied to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties and/or additional amino acid mutations may be introduced in the constant region.
As used herein, a protein which is “derived from” another protein, e.g., a parent protein, means that one or more amino acid sequences of the protein are identical or similar to one or more amino acid sequences in the other or parent protein. For example, in an antibody, binding arm, antigen-binding region, constant region, or the like which is derived from another or a parent antibody, binding arm, antigen-binding region, or constant region, one or more amino acid sequences are identical or similar to those of the other or parent antibody, binding arm, antigen-binding region, or constant region. Examples of such one or more amino acid sequences include, but are not limited to, those of the VH and VL CDRs and/or one or more or all of the framework regions, VH, VL, CL, hinge, or CH regions. For example, a humanized antibody can be described herein as “derived from” a non-human parent antibody, meaning that at least the VL and VH CDR sequences are identical or similar to the VH and VL CDR sequences of said non-human parent antibody. A chimeric antibody can be described herein as being “derived from” a non-human parent antibody, meaning that typically the VH and VL sequences may be identical or similar to those of the non-human parent antibody. Another example is a binding arm or an antigen-binding region which may be described herein as being “derived from” a particular parent antibody, meaning that said binding arm or antigen-binding region typically comprises identical or similar VH and/or VL CDRs, or VH and/or VL sequences to the binding arm or antigen-binding region of said parent antibody. As described elsewhere herein, however, amino acid modifications such as mutations can be made in the CDRs, constant regions or elsewhere in the antibody, binding arm, antigen-binding region or the like, to introduce desired characteristics. When used in the context of one or more sequences derived from a first or parent protein, a “similar” amino acid sequence preferably has a sequence identity of at least about 50%, such as at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 97%, 98% or 99%.
Non-human antibodies can be generated in a number of different species, such as mouse, rabbit, chicken, guinea pig, llama and goat.
Monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Other techniques for producing monoclonal antibodies can be employed, e.g., viral or oncogenic transformation of B-lymphocytes or phage display techniques using libraries of antibody genes, and such methods are well known to a person skilled in the art.
Hybridoma production in such non-human species is a very well established procedure. Immunization protocols and techniques for isolation of splenocytes of immunized animals/non-human species for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.
The term “human antibody” as used herein, refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
Human monoclonal antibodies can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system.
The term “immunoglobulin” refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. See for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain (abbreviated “HC”) typically is comprised of a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain constant region (abbreviated herein as CH or CH). The heavy chain constant region typically is comprised of three domains, CH1, CH2, and CH3. The heavy chain may typically further comprise a hinge region. Each light chain (abbreviated “LC”) typically is comprised of a light chain variable region (abbreviated herein as VL or VL) and a light chain constant region (abbreviated herein as CL or CL). The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise stated or contradicted by context, CDR sequences herein are identified according to IMGT rules using DomainGapAlign (Program version: 4.9.1; 2013-12-19) (Lefranc M P., Nucleic Acids Research 1999; 27:209-212, and Ehrenmann F., Kaas Q. and Lefranc M. P. Nucleic Acids Research 2010; 38, D301-307; see also internet http address www.imgt.org/).
Unless otherwise stated or contradicted by context, reference to amino acid positions in the constant regions in the present invention is according to the EU-numbering (Edelman et al., Proc Natl Acad Sci USA. 1969 May; 63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242).
The term “antibody” (Ab) in the context of the present invention refers to a molecule comprising at least one antibody variable domain such as an immunoglobulin heavy chain variable region, or an immunoglobulin heavy chain variable region and a light chain variable region, or a fragment thereof, or a derivative of either thereof, which has the ability to specifically bind to an antigen, such as under typical physiological conditions with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity). In particular the antibody may be an immunoglobulin molecule, a fragment of an immunoglobulin molecule or a derivative thereof. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q, the first component in the classical pathway of complement activation. As indicated above, the term antibody herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that are antigen-binding fragments, i.e., retain the ability to specifically bind to the antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term “antibody” include (i) a Fab′ or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or a monovalent antibody as described in WO2007059782 (Genmab); (ii) F(ab′)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting essentially of the VH and CH1 domains; (iv) an Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends Biotechnol. 2003 November; 21(11):484-90); (vi) camelid or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 January; 5(1):111-24) and (vii) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present invention, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present invention, as well as bispecific formats of such fragments, are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. An antibody as generated can possess any isotype and/or subclass.
Regular antibodies; e.g. antibodies raised in any species are normally monospecific, bivalent antibodies, which means that they comprise two antigen-binding regions which bind to the same epitope.
The term “multispecific antibody” in the context of the present invention refers to an antibody having different antigen-binding regions defined by different antibody sequences. Thus a multispecific antibody may have two, three, four, five or more different antigen-binding regions. Examples of multispecific antibodies include antibodies having two different antigen-binding regions; i.e. a bispecific antibody.
Examples of multispecific antibodies comprising three or more different antigen-binding regions include but are not limited to (i) bispecific antibodies coupled with an additional single chain variable Fragment (scFv) at their Fc part (Weidle et al., Cancer Genomics Proteomics. 2013 January-February; 10(1):1-18), (ii) fusion proteins consisting of three or more scFv (triabody, tetrabody; Chames et al., FEMS Microbiol Lett. 2000 Aug. 1; 189(1):1-8) and (iii) fusion proteins connected to scFv (Kermer et. al. Mol Cancer Ther. 2014 January; 13(1):112-21).
The term “bispecific antibody” in the context of the present invention refers to an antibody having two different antigen-binding regions defined by different antibody sequences.
When used herein, unless contradicted by context, the term “Fab-arm” or “arm” refers to one heavy chain-light chain pair and is used interchangeably with “half molecules” herein.
The term “binding arm comprising an antigen-binding region” means an antibody molecule or fragment that comprises an antigen-binding region. Thus, a binding arm can comprise, e.g., the six VH and VL CDR sequences, the VH and VL sequences, a Fab or Fab′ fragment, or a Fab-arm.
When used herein, unless contradicted by context, the term “Fc region” refers to an antibody region comprising at least a hinge region, a CH2 domain, and a CH3 domain.
As used herein, the term “isotype” refers to the specific type of immunoglobulin encoded by the HC (for instance IgG, IgD, IgA, IgE, and IgM) or LC (kappa, κ or lambda, λ) genes. Within each isotype, there may be several subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc.
The term “monovalent antibody” means in the context of the present invention that an antibody molecule is capable of binding a single molecule of the antigen, and thus is not capable of antigen cross-linking.
A “CD40 antibody” or “anti-CD40 antibody” is an antibody as described above, which binds specifically to the antigen CD40.
A “CD137 antibody” or “anti-CD137 antibody” is an antibody as described above, which binds specifically to the antigen CD137.
A “CD40×CD137 antibody” or “anti-CD40×CD137 antibody” is a bispecific antibody, which comprises two different antigen-binding regions, one of which binds specifically to the antigen CD40 and one of which binds specifically to the antigen CD137.
The term “specifically binds”, “specifically binding”, “specific binding” or other similar wording refers to the ability of an antibody to preferentially bind to a particular antigen compared to other antigens, or to a particular part (epitope) of an antigen compared to other parts of the same antigen.
As used herein, the term “binding” in the context of the binding of an antibody to a predetermined antigen or epitope typically is a binding with an affinity corresponding to a KD of about 10−7 M or less, such as about 10−8 M or less, such as about 10−9 M or less, about 10−10 M or less, or about 10−11 M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antibody as the ligand and the antigen as the analyte (or vice versa), and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than the KD for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The amount with which the affinity is higher is dependent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is highly specific), then the amount with which the affinity for the antigen is higher than the affinity for a non-specific antigen may be at least 10,000 fold.
The term “kd” (sec−1), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the koff value.
The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.
Two antibodies have the “same specificity” if they bind to the same antigen and to the same epitope. Whether an antibody to be tested recognizes the same epitope as a certain antigen-binding antibody, i.e., the antibodies bind to the same epitope, may be tested by different methods well known to a person skilled in the art.
The competition between the antibodies can be detected by a cross-blocking assay. For example, a competitive ELISA assay may be used as a cross-blocking assay. For example, target antigen may be coated on the wells of a microtiter plate and antigen-binding antibody and candidate competing test antibody may be added. The amount of the antigen-binding antibody bound to the antigen in the well indirectly correlates with the binding ability of the candidate competing test antibody that competes therewith for binding to the same epitope. Specifically, the larger the affinity of the candidate competing test antibody is for the same epitope, the smaller the amount of the antigen-binding antibody bound to the antigen-coated well. The amount of the antigen-binding antibody bound to the well can be measured by labeling the antibody with detectable or measurable labeling substances.
An antibody competing for binding to an antigen with another antibody, e.g., an antibody comprising heavy and light chain variable regions as described herein, or an antibody having the specificity for an antigen of another antibody, e.g., an antibody comprising heavy and light chain variable regions as described herein, may be an antibody comprising variants of said heavy and/or light chain variable regions as described herein, e.g. modifications in the CDRs and/or a certain degree of identity as described herein.
An “isolated multispecific antibody” as used herein is intended to refer to a multispecific antibody which is substantially free of other antibodies having different antigenic specificities (for instance an isolated bispecific antibody that specifically binds to CD40 and CD137 is substantially free of monospecific antibodies that specifically bind to CD40 or CD137).
The term “epitope” means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The epitope may comprise amino acid residues directly involved in the binding and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked or covered by the specifically antigen-binding peptide (in other words, the amino acid residue is within the footprint of the specifically antigen-binding peptide).
The term “monoclonal antibody” as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
When used herein the term “heterodimeric interaction between the first and second CH3 regions” refers to the interaction between the first CH3 region and the second CH3 region in a first-CH3/second-CH3 heterodimeric antibody.
When used herein the term “homodimeric interactions of the first and second CH3 regions” refers to the interaction between a first CH3 region and another first CH3 region in a first-CH3/first-CH3 homodimeric antibody and the interaction between a second CH3 region and another second CH3 region in a second-CH3/second-CH3 homodimeric antibody.
When used herein the term “homodimeric antibody” refers to an antibody comprising two first Fab-arms or half-molecules, wherein the amino acid sequence of said Fab-arms or half-molecules is the same.
When used herein the term “heterodimeric antibody” refers to an antibody comprising a first and a second Fab-arm or half-molecule, wherein the amino acid sequence of said first and second Fab-arms or half-molecules are different. In particular, the CH3 region, or the antigen-binding region, or the CH3 region and the antigen-binding region of said first and second Fab-arms/half-molecules are different.
The term “reducing conditions” or “reducing environment” refers to a condition or an environment in which a substrate, such as a cysteine residue in the hinge region of an antibody, is more likely to become reduced than oxidized.
The present invention also provides multispecific antibodies, such as bispecific antibodies, comprising functional variants of the VL regions, VH regions, or one or more CDRs of the bispecific antibodies of the examples. A functional variant of a VL, VH, or CDR used in the context of a bispecific antibody still allows each antigen-binding region of the bispecific antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity and/or the specificity/selectivity of the parent bispecific antibody and in some cases such a bispecific antibody may be associated with greater affinity, selectivity and/or specificity than the parent bispecific antibody.
Such functional variants typically retain significant sequence identity to the parent bispecific antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences may e.g. be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970) algorithm.
In the context of the present invention the following notations are, unless otherwise indicated, used to describe a mutation; name of amino acid which is mutated, followed by the position number which is mutated, followed by what the mutation encompass. Thus if the mutation is a substitution, the name of the amino acid which replaces the prior amino acid is included, if the amino acid is deleted it is indicated by a *, if the mutation is an addition the amino acid being added is included after the original amino acid. Amino acid names may be one or three-letter codes. Thus for example; substitution of a Lysine in position 409 with an Arginine is referred to as K409R, substitution of Lysine in position 409 with any amino acid is referred to as K409X, deletion of Lysine in position 409 is referred to as K409* and addition of P after Lysine at position K409 is referred to as K409KP.
Exemplary variants include those which differ from the VH and/or VL and/or CDRs of the parent sequences mainly by conservative substitutions; for instance 12, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements.
In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected in the following:
Amino acid residue classes for conservative substitutions:
Acidic Residues: Asp (D) and Glu (E)
Basic Residues: Lys (K), Arg (R), and His (H)
Hydrophilic Uncharged Residues: Ser (S), Thr (T), Asn (N), and Gln (Q)
Aliphatic Uncharged Residues: Gly (G), Ala (A), Val (V), Leu (L), and Ile (I)
Non-polar Uncharged Residues: Cys (C), Met (M), and Pro (P)
Aromatic Residues: Phe (F), Tyr (Y), and Trp (W)
The first and/or second antigen-binding region of the present invention may also be a variant of a first and/or second antigen-binding region, respectively, disclosed herein.
It is well known to a person skilled in the art how to introduce modifications and that certain amino acids of the CDR sequences may be modified; e.g. by amino acid substitutions to e.g. increase affinity of an antibody to its target antigen, reduce potential immunogenicity of non-human antibodies to be used in humans and/or to increase the yield of antibodies expressed by a host cell. Such modifications can be introduced without affecting the epitope of the target antigen to which the antibody binds.
The term “recombinant host cell” (or simply “host cell” or “cell”), as used herein, is intended to refer to a cell into which a nucleic acid, such as an expression vector has been introduced, e.g. a nucleic acid, such as an expression vector encoding a multispecific antibody of the invention. Recombinant host cells include, for example, transfectomas, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F™, PER.C6® or NSO cells, and lymphocytic cells.
The term “treatment” refers to the administration of an effective amount of a therapeutically active multispecific antibody of the present invention with the purpose of easing, ameliorating, arresting or eradicating (curing) symptoms or disease states.
The term “effective amount” or “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of a multispecific antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the multispecific antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the multispecific antibody or a fragment thereof, are outweighed by the therapeutically beneficial effects.
The term “anti-idiotypic antibody” refers to an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
In the context of the present invention the term “induce Fc-mediated effector function to a lesser extent” used in relation to an antibody, including a multispecific antibody, means that the antibody induces Fc-mediated effector functions, such function in particular being selected from the list of IgG Fc receptor (FcgammaR, FcγR) binding, C1q binding, ADCC or CDC, to a lesser extent compared to a human IgG1 antibody comprising (i) the same CDR sequences, in particular comprising the same first and second antigen-binding regions, as said antibody and (ii) two heavy chains comprising human IgG1 hinge, CH2 and CH3 regions.
Fc-mediated effector function may be measured by binding to FcγRs, binding to C1q, or induction of Fc-mediated cross-linking via FcγRs.
Further Aspects and Embodiments of the Invention
The present invention relates to a molecule comprising two different antigen-binding regions, one of which has specificity for human CD40 and one of which has specificity for human CD137.
In a particular embodiment, said molecule may be a multispecific antibody.
Thus the present invention relates to a multispecific antibody comprising (i) a first antigen-binding region binding to human CD40; and (ii) a second antigen-binding region binding to human CD137.
As shown by the inventors of the present invention a bispecific antibody according to the present invention may induce intracellular signaling when binding to CD40 expressed on one cell and binding to CD137 expressed on another cell. Thus, a multispecific antibody according to the present invention is able to trans-activate two different cells. In humans, CD40 is expressed on a number of cells including antigen-presenting cells (APCs), such as dendritic cells, whereas CD137 is expressed on T cells and other cells. Thus, multispecific antibodies, such as bispecific antibodies, according to the present invention binding to CD40 and CD137 are able to bind simultaneously to APCs and T cells expressing these receptors. Without being bound by theory, multispecific antibodies, such as bispecific antibodies, according to the present invention may thus (i) mediate cell-to-cell interaction between APCs and T cells by receptor binding and (ii) activate both CD40 and CD137 at once, which is primarily induced by cross-linking and receptor clustering upon cell-to-cell interaction and not necessarily dependent on agonistic activity of the parental monospecific bivalent antibodies. Thus, these trans-activating multispecific antibodies, such as bispecific antibodies, exert co-stimulatory activity in the context of APC:T cell interactions, and can elicit a T cell response against tumor cells. As such, this mechanism of action can reflect natural T-cell activation via antigen-presentation by activated APCs, allowing for the presentation of a variety of tumor-specific antigens by the APCs to T cells. Without being limited to theory, the costimulatory activity may provide for one or more of (i) only specific T cells being activated (i.e., those that are in contact with an APC) as opposed to any T cell and (ii) re-activation of exhausted T cells, by strong co-stimulation via activated APCs and CD137 triggering and (iii) the priming of T cells by inducing antigen presentation by activated APCs and at the same time triggering CD137.
Thus, a multispecific, such as a bispecific, antibody of the present invention may be used for treatment of a disease which would benefit from activation of T cells, such as cancer.
In one embodiment, the multispecific antibody according to the present invention comprises
(I) a first antigen-binding region binding to human CD40, wherein said first antigen-binding region comprises heavy and light chain variable region CDR1, CDR2, and CDR3 selected from the group consisting of:
a) heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO:3 or a sequence wherein up to four amino acids are modified in SEQ ID NO:3, and light chain variable region CDR3 having the sequence set forth in SEQ ID NO:5 or a sequence wherein up to four amino acids are modified in SEQ ID NO:5; and
b) heavy and light chain variable region CDR3 of an antibody which (i) competes for human CD40 binding with an antibody comprising heavy and light chain variable region CDR3 according to a) and/or (ii) has the specificity for CD40 of an antibody comprising heavy and light chain variable region CDR3 according to a), and
(II) a second antigen-binding region binding to human CD137.
In a further embodiment, the first antigen-binding region may further comprise heavy chain variable region CDR1 having the sequence as set forth in SEQ ID NO:1 or a sequence wherein up to 2 amino acids are modified in SEQ ID NO:1, and/or heavy chain variable region CDR2 having the sequence as set forth in SEQ ID NO:2 or a sequence wherein up to 2 amino acids are modified in SEQ ID NO:2; and/or light chain variable region CDR1 having the sequence as set forth in SEQ ID NO:4 or a sequence wherein up to 2 amino acids are modified in SEQ ID NO:4, and/or light chain variable region CDR2 having the sequence YTS or a sequence wherein up to 2 amino acids are modified in YTS.
Thus, in one embodiment, the present invention relates to a multispecific antibody comprising
(I) a first antigen-binding region binding to human CD40, wherein said first antigen-binding region comprises heavy and light chain variable region CDR1, CDR2, and CDR3 selected from the group consisting of:
a) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1, 2 and 3, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:4, YTS and 5, respectively;
b) heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) having a total of one to twelve mutations; and
c) heavy and light chain variable region CDR1, CDR2 and CDR3 of an antibody which (i) competes for human CD40 binding with an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) or b) and/or (ii) has the specificity for CD40 of an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) or b), and
(II) a second antigen-binding region binding to human CD137.
In a further embodiment, said first antigen-binding region comprises a first heavy chain variable (VH) sequence, and a first light chain variable (VL) sequence, and said second antigen-binding region comprises a second heavy chain variable (VH) sequence, and a second light chain variable (VL) sequence, wherein said variable sequences each comprises three CDR sequences, CDR1, CDR2 and CDR3, respectively, and four framework sequences, FR1, FR2, FR3 and FR4, respectively.
In a further embodiment, said multispecific antibody comprises (I) a first binding arm comprising said first antigen-binding region, and (II) a second binding arm comprising said second antigen-binding region.
In one embodiment, the first binding arm comprises said first antigen-binding region and a first heavy chain constant sequence, and the second binding arm comprises said second-antigen-binding region and a second heavy chain constant sequence.
In a further embodiment, (i) said first binding arm comprises said first antigen-binding region, wherein the first binding arm comprises a first heavy chain comprising a first heavy chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence, and (ii) said second binding arm comprises said second antigen-binding region, wherein the second binding arm comprises a second heavy chain comprising a second heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence, and a second light chain comprising a second light chain variable (VL) sequence.
In a further embodiment, said first light chain further comprises a first light chain constant (CL) sequence, and said second light chain further comprises a second light chain constant (CL) sequence.
In one embodiment, the first binding arm comprises a first Fab-arm comprising said first antigen-binding region, and the second binding arm comprises a second Fab-arm comprising said second antigen-binding region.
In one embodiment, said first and second antigen-binding regions of the multispecific antibody according to the present invention are derived from a humanized antibody. In one embodiment, the first and second binding arm may be derived from a humanized antibody.
In one embodiment, the first and second binding arms of the multispecific antibody according to the present invention are derived from a full-length antibody.
In one embodiment, the first and second binding arm of the multispecific antibody according to the present invention are derived from a full-length IgG1,λ (lambda) or IgG1,κ (kappa) antibody.
In one embodiment, the first and second binding arms are derived from a monoclonal antibody.
In one embodiment, the first and said second heavy chains of the multispecific antibody according to the present invention are of an IgG isotype. The subclass of the first and second heavy chains may, for example, be separately selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In one embodiment, the first and second heavy chains are of the same IgG subclass, such as IgG1.
In one embodiment, the multispecific antibody according to the present invention is an isolated antibody.
In a further embodiment, each of said first and second heavy chains comprises at least a hinge region, a CH2 and a CH3 region. In a further embodiment, the CH3 regions of said first and second heavy chains comprise asymmetrical mutations.
In one embodiment, the multispecific antibody according to the present invention is a bispecific antibody.
In one embodiment, the multispecific antibody according to the present invention may cross-link one cell expressing human CD40, e.g. a first cell, and another cell expressing human CD137, e.g. a second cell.
In one embodiment, said cross-linking is determined by an assay using a first cell line expressing human CD40 and a second cell line expressing human CD137, and wherein either the first or the second cell line comprises a reporter structure resulting in the production of a measurable reporter upon NF-κB activation.
In one embodiment said first cell may be an antigen-presenting cell and said second cell may be a T-cell, such as a CD4+ or a CD8+ T-cell.
Different methods may be used to determine cross-linking a first cell expressing CD40 and a second cell expressing CD137, and the present invention is not limited to any particular method.
In one embodiment, said cross-linking may be determined by a reporter assay e.g. as described in Example 4. In brief, said assay comprises co-culturing a reporter cell line expressing a first target antigen and transduced with a reporter gene (luciferase for instance) driven by NF-κB response elements with a second cell line expressing a second target antigen, adding a multispecific antibody according to the present invention in concentrations from 100 ng/mL to 10,000 ng/mL to the cell co-culture, and measuring reporter gene expression, e.g. luciferase generation, wherein said first target antigen is human CD40 and said second target antigen is human CD137 or vice versa.
A multispecific antibody capable of inducing cross-linking of the CD40 and CD137 expressed on different cells, will in this assay result in measurable activation of the first target antigen based on the reporter gene expression upon NF-κB pathway activation.
In one embodiment, the multispecific antibody according to the present invention may be able to induce reporter gene expression produced upon NF-κB activation only upon addition of the second cell line expressing the second target antigen without the NF-κB reporter gene.
In one embodiment, the multispecific antibody according to the present invention may be able to induce higher reporter gene expression produced upon NF-κB activation when the second cell line expressing the second target antigen without the NF-κB reporter gene is added compared to addition of a second cell line not expressing the second target antigen.
In one embodiment, the multispecific antibody is a bispecific antibody, and said bispecific antibody may, in one embodiment:
(i) induce reporter gene expression when added to a co-culture of the reporter cell line expressing CD137 and the second cell line expressing CD40, or
(ii) induce a higher amount of the reporter gene expression when added to the co-culture of the reporter cell line expressing CD137 and the second cell line expressing CD40, compared to a reference bispecific antibody comprising the same second antigen-binding region binding to human CD137, but wherein the first antigen-binding region of the reference bispecific antibody binds to an irrelevant target antigen, e.g. wherein the first antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:99, 100 and 101, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:102, GVS and 103, respectively.
In one embodiment, the multispecific antibody is a bispecific antibody, and said bispecific antibody may, in one embodiment:
(i) induce reporter gene expression when added to a co-culture of the reporter cell line expressing CD40 and the second cell line expressing CD137, or
(ii) induce a higher amount of the reporter gene expression when added to the co-culture of the reporter cell line expressing CD40 and the second cell line expressing CD137, compared to a reference bispecific antibody comprising the same first antigen-binding region binding to human CD40, but wherein the second antigen-binding region of the reference bispecific antibody comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:99, 100 and 101, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:102, GVS and 103, respectively.
In one embodiment, the multispecific antibody according to the present invention induces and/or enhances proliferation of T cells, e.g. wherein said T cells are CD4+ and/or CD8+ T cells.
Different methods for determining or measuring proliferation of T cells may be used and the present invention is not limited to any particular method.
In one embodiment, said induction or enhancement of proliferation of T cells is determined by a non-antigen-specific T-cell proliferation assay, e.g. as described in Example 5. Thus induction and/or enhancement of proliferation of T cells may be determined by sub-optimal activation of T cells in a PBMC pool (peripheral blood mononuclear lymphocyte). The sub-optimal activation may be determined by titrating the concentration of anti-CD3 antibody added to a PBMC pool, measuring T cell proliferation and choosing the anti-CD3 antibody concentration which results in low T cell proliferation but allows for further enhancement of the T cell proliferation. This concentration is PBMC-donor-dependent and is determined for each donor before the assay is performed.
In one embodiment, said induction or enhancement of proliferation of T cells is determined by activating T cells in PBMCs with said sub-optimal concentration of an anti-CD3 antibody, contacting the PBMCs with the multispecific antibody and determining proliferation of the T cells. In a further embodiment, the PBMCs may be labelled with CFSE, contacting the PBMCs with the multispecific antibody may be performed by incubation for 4 days, and proliferation of the T cells may be measured by flow cytometry.
Inducing a certain reaction or effect such as “inducing proliferation of T cells” may mean that there was no such reaction or effect such as proliferation of T cells before induction, but it may also mean that there was a certain level of reaction or effect such as proliferation of T cells before induction and after induction said reaction or effect such as proliferation of T cells is enhanced. Thus, “inducing” also includes “enhancing”.
Proliferation of T cells may also be measured by an antigen-specific T cell proliferation assay, e.g., as described in Example 6, using a test antigen of interest. Thus, induction and/or enhancement of T cell proliferation may be measured by co-culturing T cells expressing a TCR specific for a peptide of the test antigen presented in major histocompatibility complex (MHC) and DCs presenting a corresponding peptide in MHC, which is then recognized by the TCR. For example, the T cells may be CD8+ T cells and the MHC may be MHC Class I, or the T cells may be CD4+ T cells and the MHC may be MHC Class II. T cells expressing a specific TCR may be generated by transduction with mRNA encoding the TCR. DCs presenting the corresponding peptide may be generated by transduction of the DCs with mRNA encoding the antigen. Co-culture of the TCR-positive T cells with the antigen-presenting cells induces T-cell proliferation; the extent of the proliferation may depend on the antigen density presented by the DCs and/or on the strength of the costimulatory signal. In one embodiment, proliferation of T cells may be measured by such an antigen-specific T-cell assay using CFSE labeled T-cells, adding antibodies to be tested and after 4 days measuring T cell proliferation by flow cytometry.
In one embodiment, said induction or enhancement of proliferation of T cells is determined using tumor-infiltrating lymphocytes (TILs) in an ex vivo expansion assay, e.g., as described in Example 11. The effect of the multispecific antibody of the invention on the induction or enhancement of proliferation of the TILs may be assessed by incubating a human tumor sample with interleukin-2 (IL-2) and said antibody, and retrieving and counting viable TILs after incubation for a period of about 10 to about 14 days. An induction or enhancement of proliferation of TILs can then be determined by comparison with a suitable control, e.g., a human tumor sample incubated without any multispecific antibody or with a reference (control) multispecific antibody. For example, a sample of human tumor tissue can be isolated, e.g., via a biopsy or from a surgical specimen, washed in serum-free medium, and tumor pieces with a diameter of about 1-2 mm placed into culture dishes or wells, e.g., 1 or 2 tumor pieces in 1 mL suitable medium, and incubated at 37° C. A suitable medium can be, for example, a serum-free medium (e.g. X-VIVO 15) supplemented with 10% Human Serum Albumin, 1% Pen/Strep, 1% Fungizone and IL-2 at a concentration ranging from 10 to 100 U/mL, e.g., 10 U/mL or 100 U/mL. The multispecific antibody can then be added at a suitable concentration in TIL medium. After a total culture period of 10-14 days, optionally splitting the cell culture if needed during this period, TILs can be harvested and counted, e.g., by flow cytometry, using, e.g., anti-CD3, anti-CD4, anti-CD8, anti-CD56 and 7-AAD antibodies to permit detection of viable CD4+ and CD8+ T cells as well as NK cells.
In one embodiment, the multispecific antibody is a bispecific antibody, which induces and/or enhances more proliferation of T cells compared to a reference bispecific antibody comprising a second antigen-binding region according to any aspect or embodiment described herein, but wherein the first antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:99, 100 and 101, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:102, GVS and 103, respectively.
In one embodiment, the multispecific antibody is a bispecific antibody, which induces and/or enhances more proliferation of T cells compared to a reference bispecific antibody comprising a first antigen-binding region according to any aspect or embodiment described herein, but wherein the second antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:99, 100 and 101, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:102, GVS and 103, respectively.
Binding to CD40
As described above, the multispecific antibody according to the present invention comprises a first antigen-binding region binding to human CD40.
In one embodiment, the multispecific antibody according to the present invention comprises a first antigen-binding region binding to human CD40, wherein said first antigen-binding region comprises heavy and light chain variable region CDR3 selected from the group consisting of:
a) heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO:3 or a sequence wherein up to four amino acids are modified in SEQ ID NO:3, and light chain variable region CDR3 having the sequence set forth in SEQ ID NO:5 or a sequence wherein up to four amino acids are modified in SEQ ID NO:5,
b) heavy and light chain variable region CDR3 of an antibody which (i) competes for human CD40 binding with an antibody comprising heavy and light chain variable region CDR3 according to a) and/or (ii) has the specificity for CD40 of an antibody comprising heavy and light chain variable region CDR3 according to a).
In a further embodiment, the first antigen-binding region may further comprise a heavy chain variable region CDR1 having the sequence as set forth in SEQ ID NO:1 or a sequence wherein up to 2 amino acids are modified in SEQ ID NO:1, and/or heavy chain variable region CDR2 having the sequence as set forth in SEQ ID NO:2 or a sequence wherein up to 2 amino acids are modified in SEQ ID NO:2; and/or light chain variable region CDR1 having the sequence as set forth in SEQ ID NO:4 or a sequence wherein up to 2 amino acids are modified in SEQ ID NO:4, and/or light chain variable region CDR2 having the sequence YTS or a sequence wherein up to 2 amino acids are modified in YTS.
In one embodiment, the multispecific antibody according to the present invention comprises a first antigen-binding region binding to human CD40, wherein said first antigen-binding region comprises heavy and light chain variable region CDR1, CDR2 and CDR3 selected from the group consisting of:
a) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1, 2 and 3, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:4, YTS and 5, respectively,
b) heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) having a total of one to twelve mutations; and
c) heavy and light chain variable region CDR1, CDR2 and CDR3 of an antibody which (i) competes for human CD40 binding with an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) or b) and/or (ii) has the specificity for CD40 of an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) or b).
In one embodiment said first antigen-binding region comprises a first heavy chain variable (VH) sequence, and a first light chain variable (VL) sequence, wherein said variable sequences each comprises three CDR sequences, CDR1, CDR2 and CDR3, respectively.
In one embodiment, said first antigen-binding region comprises a first heavy chain variable (VH) sequence, and a first light chain variable (VL) sequence, and wherein said variable sequences each comprises three CDR sequences, CDR1, CDR2 and CDR3, respectively, and four framework sequences, FR1, FR2, FR3 and FR4, respectively.
In one embodiment, said first antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1, 2 and 3, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:4, YTS and 5, respectively. Thus the first antigen-binding region may comprise heavy and light chain variable region CDR1, CDR2 and CDR3 having the sequences of the CD40 antibody as set forth in Table 1.
An example of an antibody comprising such a first antigen-binding region is the chimeric antibody Chi Lob 7/4 and CD40-001 disclosed herein.
In another embodiment, said first antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1, 2 and 3, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:4, YTS and 5, respectively having a total of one to twelve mutations, such as one to eleven mutations, one to ten mutations, one to eight mutations, one to seven mutations, one to six mutations, one to five mutations, one to four mutations, one to three mutations, or one to two mutations.
In one embodiment, said mutation may be an amino acid substitution, such as a conservative amino acid substitution.
In one embodiment, said mutations may be distributed across the VH CDR1, 2 and 3 and VL CDR1, 2 and 3 so that each of the VH and VL CDR3 comprises at the most three mutations and each of the VH and VL CDR2 and CDR1 comprises at the most two amino acid mutations.
In a further embodiment, the first antigen-binding region comprises heavy and light chain CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1, 2, 3, 4, YTS and 5, respectively, having a total of one to twelve mutations and wherein the VH and VL CDR3 each comprises up to three amino acid mutations, and the VH and VL CDR1 and CDR2 each comprises up to two amino acid mutations.
In a further embodiment, the first antigen-binding region comprises heavy and light chain CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1, 2, 3, 4, YTS and 5, respectively, having a total of one to ten mutations, such as one to eight, and wherein the VH and VL CDR1, CDR2, and CDR3 each comprises up to two amino acid mutations.
In a further embodiment, the first antigen-binding region comprises heavy and light chain CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1, 2, 3, 4, YTS and 5, respectively, having a total of one to six mutations, and wherein the VH and VL CDR1, CDR2, and CDR3 each comprises at the most one amino acid mutation.
It is well known to a person skilled in the art how to introduce mutations and that certain amino acids of the CDR sequences may be mutated; e.g. by amino acid substitutions to e.g. increase affinity of the antibody to its target antigen, reduce potential immunogenicity of non-human antibodies to be used in humans and/or to increase the yield of antibodies expressed by a host cell. Such mutations can be introduced without affecting the epitope of the target to which the antibody binds.
In another embodiment, said first antigen-binding region comprises heavy and light chain variable region CDR1, CDR2 and CDR3 of an antibody which (i) competes for human CD40 binding with an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) or b) and/or (ii) has the specificity for CD40 of an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) or b).
In a further embodiment, said first antigen-binding region comprises heavy and light chain variable regions of an antibody which (i) competes for human CD40 binding with an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) or b) and/or (ii) has the specificity for CD40 of an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) or b).
The term “competes” refers in this context to the competition between two antibodies for binding to a target antigen. If two antibodies do not block each other for binding to a target antigen, such antibodies are non-competing and this is an indication that said antibodies do not bind to the same part, i.e. epitope of the target antigen. It is well known to a person skilled in the art how to test for competition of antibodies for binding to a target antigen. An example of such a method is a so-called cross-competition assay, which may e.g. be performed as an ELISA or by flow-cytometry.
For example, an ELISA-based assay may be performed by coating ELISA plate wells with each of the antibodies; adding the competing antibody and His-tagged extracellular domain of the target antigen and incubate; detecting whether the added antibody inhibited binding of the His-tagged protein to the coated antibody may be performed by adding biotinylated anti-His antibody, followed by Streptavidin-poly-HRP, and further developing the reaction with ABTS and measuring the absorbance at 405 nm. For example a flow-cytometry assay may be performed by incubating cells expressing the target antigen with an excess of unlabeled antibody, incubating the cells with a sub-optimal concentration of biotin-labelled antibody, followed by incubation with fluorescently labeled streptavidin and analyzing by flow cytometry.
In one embodiment, said VH sequence of the first antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to at least one of SEQ ID NO:117 and SEQ ID NO:6, such as SEQ ID NO:117.
In one embodiment, said VL sequence of the first antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to at least one of SEQ ID NO:121 and SEQ ID NO:7, such as SEQ ID NO:121.
In one embodiment, said VH and VL sequence of the first antigen-binding region each comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO:6 and SEQ ID NO:7, respectively.
In one embodiment, said VH and VL sequence of the first antigen-binding region each comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO:117 and SEQ ID NO:121, respectively.
In one embodiment, the VH and VL sequences only deviate in the non-CDR sequences as set forth in SEQ ID NO:6 and 7, respectively.
In one embodiment, the VH and VL sequences only deviate in the non-CDR sequences as set forth in SEQ ID NO:117 and 121, respectively.
In one embodiment, the VH and VL sequences only deviate in the framework sequences.
In one embodiment, the respective FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the first antigen-binding region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of said VH and VL sequences.
In one embodiment, the respective FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the first antigen-binding region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:6, and VL sequence as set forth in SEQ ID NO:7.
In one embodiment, the respective FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the first antigen-binding region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:6, and VL sequence as set forth in SEQ ID NO:7, and the heavy and light chain variable region CDR1, CDR2 and CDR3 of the first antigen-binding region has a total of one to twelve mutations compared to the heavy and light chain variable region CDR1, CDR2 and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, 3, 4, YTS and 5, respectively. In a further embodiment, said mutations may be as described above.
In an even further embodiment, the respective FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the first antigen-binding region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:6, and VL sequence as set forth in SEQ ID NO:7, and the first antigen-binding region comprises heavy and light chain variable region CDR1, CDR2 and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, 3, 4, YTS and 5, respectively.
In one embodiment, the respective FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the first antigen-binding region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:117, and VL sequence as set forth in SEQ ID NO:121, and the heavy and light chain variable region CDR1, CDR2 and CDR3 of the first antigen-binding region has a total of one to twelve mutations compared to the heavy and light chain variable region CDR1, CDR2 and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, 3, 4, YTS and 5, respectively. In a further embodiment, said mutations may be as described above.
In an even further embodiment, the respective FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the first antigen-binding region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:117, and VL sequence as set forth in SEQ ID NO:121, and the first antigen-binding region comprises heavy and light chain variable region CDR1, CDR2 and CDR3 having the sequences as set forth in SEQ ID NOs:1, 2, 3, 4, YTS and 5, respectively.
In one embodiment, said VH sequence of the first antigen-binding region comprises the amino acid sequence of SEQ ID NO:117.
In one embodiment, said VL sequence of the first antigen-binding region comprises the amino acid sequence of SEQ ID NO:121.
In a further embodiment, said VH and VL sequences of the first antigen-binding region comprise the amino acid sequences of SEQ ID NO:117 and SEQ ID NO:121, respectively.
In one embodiment, said VH sequence of the first antigen-binding region comprises the amino acid sequence of SEQ ID NO:6.
In one embodiment, said VL sequence of the first antigen-binding region comprises the amino acid sequence of SEQ ID NO:7.
In a further embodiment, said VH and VL sequences of the first antigen-binding region comprises the amino acid sequences of SEQ ID NO:6 and SEQ ID NO:7, respectively.
In one embodiment, the multispecific antibody according to the present invention may comprise a first binding arm comprising said first antigen-binding region of any aspect or embodiment herein.
In one embodiment, the multispecific antibody according to the present invention comprises a first binding arm comprising said first antigen-binding region and a first heavy chain constant sequence.
In one embodiment, the multispecific antibody according to the present invention comprises a first binding arm comprising said first antigen-binding region, wherein the first binding arm comprises a first heavy chain comprising a first heavy chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence.
In one embodiment, said first light chain further comprises a first light chain constant (CL) sequence.
In a further embodiment, said first heavy chain comprises at least a hinge region, a CH2 and a CH3 region.
In a specific embodiment, the multispecific antibody according to the present invention comprises a first Fab-arm comprising said first antigen-binding region.
In one embodiment, the first antigen-binding region may be derived from a mouse antibody.
In one embodiment, the first antigen-binding region may be derived from a chimeric antibody, such as Chi Lob 7/4.
In one embodiment, the first antigen-binding region may be derived from a humanized antibody.
In one embodiment, the first binding arm may be derived from a full-length antibody.
In one embodiment, the first binding arm may be derived from a full-length IgG1,λ (lambda) or IgG1,κ (kappa) antibody.
In one embodiment, the first binding arm may be derived from a monoclonal antibody.
In one embodiment, said first heavy chain may be of an IgG isotype, optionally selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.
In one embodiment, the first binding arm may be derived from an antibody comprising a HC comprising SEQ ID NO:118 and an LC comprising SEQ ID NO:122, optionally with one or more mutations in the constant region of the HC, such as 1 to 10, such as 1 to 5, such as 1, 2, 3, 4 or 5 mutations.
In one embodiment, the first binding arm comprises a HC comprising SEQ ID NO:118, 119 or 120 and an LC comprising SEQ ID NO:122.
Binding to CD137
The multispecific antibody according to the present invention comprises a second antigen-binding region binding to human CD137.
In a further embodiment, the second antigen-binding region also binds to cynomolgus CD137.
In one embodiment, said second antigen-binding region comprises heavy and light chain variable region CDR3 selected from the group consisting of:
a) heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO:10 or a sequence wherein up to four amino acids are modified in SEQ ID NO:10, and light chain variable region CDR3 having the sequence set forth in SEQ ID NO:12 or a sequence wherein up to four amino acids are modified in SEQ ID NO:12 (CD137 clone 001),
b) heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO:17 or a sequence wherein up to four amino acids are modified in SEQ ID NO:17, and light chain variable region CDR3 having the sequence set forth in SEQ ID NO:19 or a sequence wherein up to four amino acids are modified in SEQ ID NO:19 (CD137 clone 002),
c) heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO:24 or a sequence wherein up to four amino acids are modified in SEQ ID NO:24, and light chain variable region CDR3 having the sequence set forth in SEQ ID NO:26 or a sequence wherein up to four amino acids are modified in SEQ ID NO:26 (CD137 clone 003),
d) heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO:31 or a sequence wherein up to four amino acids are modified in SEQ ID NO:31, and light chain variable region CDR3 having the sequence set forth in SEQ ID NO:33 or a sequence wherein up to four amino acids are modified in SEQ ID NO:33 (CD137 clone 004),
e) heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO:38 or a sequence wherein up to four amino acids are modified in SEQ ID NO:38, and light chain variable region CDR3 having the sequence set forth in SEQ ID NO:40 or a sequence wherein up to four amino acids are modified in SEQ ID NO:40 (CD137 clone 005),
f) heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO:45 or a sequence wherein up to four amino acids are modified in SEQ ID NO:45, and light chain variable region CDR3 having the sequence set forth in SEQ ID NO:47 or a sequence wherein up to four amino acids are modified in SEQ ID NO:47 (CD137 clone 006),
g) heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO:52 or a sequence wherein up to four amino acids are modified in SEQ ID NO:52, and light chain variable region CDR3 having the sequence set forth in SEQ ID NO:54 or a sequence wherein up to four amino acids are modified in SEQ ID NO:54 (CD137 clone 007),
h) heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO:59 or a sequence wherein up to four amino acids are modified in SEQ ID NO:59, and light chain variable region CDR3 having the sequence set forth in SEQ ID NO:61 or a sequence wherein up to four amino acids are modified in SEQ ID NO:61 (CD137 clone 008),
i) heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO:66 or a sequence wherein up to four amino acids are modified in SEQ ID NO:66, and light chain variable region CDR3 having the sequence set forth in SEQ ID NO:68 or a sequence wherein up to four amino acids are modified in SEQ ID NO:68 (CD137 clone 009),
j) heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO:73 or a sequence wherein up to four amino acids are modified in SEQ ID NO:73, and light chain variable region CDR3 having the sequence set forth in SEQ ID NO:75 or a sequence wherein up to four amino acids are modified in SEQ ID NO:75 (CD137 clone 010),
k) heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO:80 or a sequence wherein up to four amino acids are modified in SEQ ID NO:80, and light chain variable region CDR3 having the sequence set forth in SEQ ID NO:82 or a sequence wherein up to four amino acids are modified in SEQ ID NO:82 (CD137 clone 011),
l) heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO:87 or a sequence wherein up to four amino acids are modified in SEQ ID NO:87, and light chain variable region CDR3 having the sequence set forth in SEQ ID NO:89 or a sequence wherein up to four amino acids are modified in SEQ ID NO:89 (CD137 clone 012), and
m) heavy and light chain variable region CDR3 of an antibody which (i) competes for human CD137 binding with an antibody comprising heavy and light chain variable region CDR3 according to any of a) to l) and/or (ii) has the specificity for CD137 of an antibody comprising heavy and light chain variable CDR3 according to any of a) to l).
In a further embodiment, said second antigen-binding region further comprises heavy and/or light chain region CDR1 and CDR2 selected from the group consisting of:
a) heavy chain variable region CDR1 having the sequence set forth in SEQ ID NO:8 or a sequence wherein up to two amino acids are modified in SEQ ID NO:8, and/or heavy chain variable region CDR2 having the sequence set forth in SEQ ID NO:9 or a sequence wherein up to two amino acids are modified in SEQ ID NO:9, and/or light chain variable region CDR1 having the sequence set forth in SEQ ID NO:11 or a sequence wherein up to two amino acids are modified in SEQ ID NO:11, and/or light chain variable region CDR2 having the sequence KAS or a sequence wherein up to two amino acids are modified in KAS (CD137 clone 001),
b) heavy chain variable region CDR1 having the sequence set forth in SEQ ID NO:15 or a sequence wherein up to two amino acids are modified in SEQ ID NO:15, and/or heavy chain variable region CDR2 having the sequence set forth in SEQ ID NO:16 or a sequence wherein up to two amino acids are modified in SEQ ID NO:16, and/or light chain variable region CDR1 having the sequence set forth in SEQ ID NO:18 or a sequence wherein up to two amino acids are modified in SEQ ID NO:18, and/or light chain variable region CDR2 having the sequence KAS or a sequence wherein up to two amino acids are modified in KAS (CD137 clone 002),
c) heavy chain variable region CDR1 having the sequence set forth in SEQ ID NO:22 or a sequence wherein up to two amino acids are modified in SEQ ID NO:22, and/or heavy chain variable region CDR2 having the sequence set forth in SEQ ID NO:23 or a sequence wherein up to two amino acids are modified in SEQ ID NO:23, and/or light chain variable region CDR1 having the sequence set forth in SEQ ID NO:25 or a sequence wherein up to two amino acids are modified in SEQ ID NO:25, and/or light chain variable region CDR2 having the sequence RTS or a sequence wherein up to two amino acids are modified in RTS (CD137 clone 003),
d) heavy chain variable region CDR1 having the sequence set forth in SEQ ID NO:29 or a sequence wherein up to two amino acids are modified in SEQ ID NO:29, and/or heavy chain variable region CDR2 having the sequence set forth in SEQ ID NO:30 or a sequence wherein up to two amino acids are modified in SEQ ID NO:30, and/or light chain variable region CDR1 having the sequence set forth in SEQ ID NO:32 or a sequence wherein up to two amino acids are modified in SEQ ID NO:32, and/or light chain variable region CDR2 having the sequence GAS or a sequence wherein up to two amino acids are modified in GAS (CD137 clone 004),
e) heavy chain variable region CDR1 having the sequence set forth in SEQ ID NO:36 or a sequence wherein up to two amino acids are modified in SEQ ID NO:36, and/or heavy chain variable region CDR2 having the sequence set forth in SEQ ID NO:37 or a sequence wherein up to two amino acids are modified in SEQ ID NO:37, and/or light chain variable region CDR1 having the sequence set forth in SEQ ID NO:39 or a sequence wherein up to two amino acids are modified in SEQ ID NO:39, and/or light chain variable region CDR2 having the sequence SAS or a sequence wherein up to two amino acids are modified in SAS (CD137 clone 005),
f) heavy chain variable region CDR1 having the sequence set forth in SEQ ID NO:43 or a sequence wherein up to two amino acids are modified in SEQ ID NO:43, and/or heavy chain variable region CDR2 having the sequence set forth in SEQ ID NO:44 or a sequence wherein up to two amino acids are modified in SEQ ID NO:44, and/or light chain variable region CDR1 having the sequence set forth in SEQ ID NO:46 or a sequence wherein up to two amino acids are modified in SEQ ID NO:46, and/or light chain variable region CDR2 having the sequence AAS or a sequence wherein up to two amino acids are modified in AAS (CD137 clone 006),
g) heavy chain variable region CDR1 having the sequence set forth in SEQ ID NO:50 or a sequence wherein up to two amino acids are modified in SEQ ID NO:50, and/or heavy chain variable region CDR2 having the sequence set forth in SEQ ID NO:51 or a sequence wherein up to two amino acids are modified in SEQ ID NO:51, and/or light chain variable region CDR1 having the sequence set forth in SEQ ID NO:53 or a sequence wherein up to two amino acids are modified in SEQ ID NO:53, and/or light chain variable region CDR2 having the sequence KAS or a sequence wherein up to two amino acids are modified in KAS (CD137 clone 007),
h) heavy chain variable region CDR1 having the sequence set forth in SEQ ID NO:57 or a sequence wherein up to two amino acids are modified in SEQ ID NO:57, and/or heavy chain variable region CDR2 having the sequence set forth in SEQ ID NO:58 or a sequence wherein up to two amino acids are modified in SEQ ID NO:58, and/or light chain variable region CDR1 having the sequence set forth in SEQ ID NO:60 or a sequence wherein up to two amino acids are modified in SEQ ID NO:60, and/or light chain variable region CDR2 having the sequence RAS or a sequence wherein up to two amino acids are modified in RAS (CD137 clone 008),
i) heavy chain variable region CDR1 having the sequence set forth in SEQ ID NO:64 or a sequence wherein up to two amino acids are modified in SEQ ID NO:64, and/or heavy chain variable region CDR2 having the sequence set forth in SEQ ID NO:65 or a sequence wherein up to two amino acids are modified in SEQ ID NO:65, and/or light chain variable region CDR1 having the sequence set forth in SEQ ID NO:67 or a sequence wherein up to two amino acids are modified in SEQ ID NO:67, and/or light chain variable region CDR2 having the sequence GAS or a sequence wherein up to two amino acids are modified in GAS (CD137 clone 009),
j) heavy chain variable region CDR1 having the sequence set forth in SEQ ID NO:71 or a sequence wherein up to two amino acids are modified in SEQ ID NO:71, and/or heavy chain variable region CDR2 having the sequence set forth in SEQ ID NO:72 or a sequence wherein up to two amino acids are modified in SEQ ID NO:72, and/or light chain variable region CDR1 having the sequence set forth in SEQ ID NO:74 or a sequence wherein up to two amino acids are modified in SEQ ID NO:74, and/or light chain variable region CDR2 having the sequence KAS or a sequence wherein up to two amino acids are modified in KAS (CD137 clone 010),
k) heavy chain variable region CDR1 having the sequence set forth in SEQ ID NO:78 or a sequence wherein up to two amino acids are modified in SEQ ID NO:78, and/or heavy chain variable region CDR2 having the sequence set forth in SEQ ID NO:79 or a sequence wherein up to two amino acids are modified in SEQ ID NO:79, and/or light chain variable region CDR1 having the sequence set forth in SEQ ID NO:81 or a sequence wherein up to two amino acids are modified in SEQ ID NO:81, and/or light chain variable region CDR2 having the sequence DTS or a sequence wherein up to two amino acids are modified in DTS (CD137 clone 011), and
l) heavy chain variable region CDR1 having the sequence set forth in SEQ ID NO:85 or a sequence wherein up to two amino acids are modified in SEQ ID NO:85, and/or heavy chain variable region CDR2 having the sequence set forth in SEQ ID NO:86 or a sequence wherein up to two amino acids are modified in SEQ ID NO:86, and/or light chain variable region CDR1 having the sequence set forth in SEQ ID NO:88 or a sequence wherein up to two amino acids are modified in SEQ ID NO:88, and/or light chain variable region CDR2 having the sequence SAS or a sequence wherein up to two amino acids are modified in SAS (CD137 clone 012).
In one embodiment said second antigen-binding region comprises heavy and light chain variable region CDR1, CDR2 and CDR3 selected from the group consisting of:
a) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:8, 9 and 10, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:11, KAS and 12, respectively, (CD137 clone 001),
b) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:15, 16 and 17, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:18, KAS and 19, respectively, (CD137 clone 002),
c) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:22, 23, and 24, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:25, RTS and 26, respectively, (CD137 clone 003),
d) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:29, 30 and 31, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:32, GAS and 33, respectively, (CD137 clone 004),
e) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:36, 37 and 38, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS and 40, respectively, (CD137 clone 005),
f) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:43, 44 and 45, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:46, AAS, and 47, respectively, (CD137 clone 006),
g) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:50, 51 and 52, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:53, KAS and 54, respectively, (CD137 clone 007),
h) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:57, 58 and 59, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:60, RAS and 61, respectively, (CD137 clone 008),
i) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:64, 65 and 66, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:67, GAS and 68, respectively, (CD137 clone 009),
j) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:71, 72 and 73, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:74, KAS and 75, respectively, (CD137 clone 010),
k) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:78, 79 and 80, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:81, DTS and 82, respectively, (CD137 clone 011),
l) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:85, 86 and 87, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:88, SAS and 89, respectively, (CD137 clone 012),
m) heavy and light chain variable region CDR1, CDR2 and CDR3 according to any of a) to l) having a total of one to twelve mutations, and
n) heavy and light chain variable region CDR1, CDR2 and CDR3 of an antibody which (i) competes for human CD137 binding with an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to any of a) to m) and/or (ii) has the specificity for CD137 of an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to any of a) to m).
Thus, the second antigen-binding region may comprise the heavy and light chain variable region CDR1, CDR2 and CDR3 sequences of a CD137 antibody as set forth in Table 1; i.e. CD137 clone 001, CD137 clone 002, CD137 clone 003, CD137 clone 004, CD137 clone 005, CD137 clone 006, CD137 clone 007, CD137 clone 008, CD137 clone 009, CD137 clone 010, CD137 clone 011 or CD137 clone 012. In particular, the second antigen-binding region may comprise the heavy and light chain variable region CDR1, CDR2 and CDR3 sequences from the same CD137 antibody clone, optionally wherein the framework regions are primarily human framework regions, optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence.
In a further embodiment, the second antigen-binding region comprises heavy and light chain variable regions of an antibody which (i) competes for human CD137 binding with an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to any of a) to m) and/or (ii) has the specificity for CD137 of an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to any of a) to m).
In one embodiment, said second antigen-binding region binds to human CD137 (SEQ ID NO:92) to a higher degree than it binds to a mutant human CD137 (SEQ ID NO:93). The mutant human CD137 of SEQ ID NO:93 is also referred to as shuffle 6 herein.
In another embodiment, said second antigen-binding region binds to human CD137 (SEQ ID NO:92) to a higher degree than it binds to a mutant human CD137 (SEQ ID NO:94). The mutant human CD137 of SEQ ID NO:94 is also referred to as shuffle 5 herein.
In a further embodiment, said second antigen-binding region binds to human CD137 (SEQ ID NO:92) to the same degree that it binds to a mutant human CD137 (SEQ ID NO:95). The mutant human CD137 of SEQ ID NO:95 is also referred to as shuffle 4 herein.
In the context of the present invention “to a higher degree” means that the affinity of the second antigen-binding region is higher for human CD137 (SEQ ID NO:92) than for a mutant human CD137 (SEQ ID NO:93 and 94, shuffle 6 and 5 respectively). If there is no binding to the mutant CD137, the affinity for human CD137 will be infinitely higher than for said mutant CD137. However, in case of binding to said mutant CD137 the affinity may be 2-fold, such as 3-fold, or 4-fold, or 5-fold, or 6-fold higher for human CD137 than for the respective mutant CD137.
In the context of the present invention “to the same degree” means that the affinity of the second antigen-binding region is similar for human CD137 (SEQ ID NO:92) and for a mutant human CD137 (SEQ ID NO:95, shuffle 4). In particular, “similar” in this context may mean that the affinity for human CD137 and for said mutant CD137 differs at the most by 2.5-fold, such as 2.2-fold, or 2.0-fold, or 1.8-fold, or 1.75-fold or 1.5-fold.
The mutant human CD137 in SEQ ID NO:93 corresponds to the amino acid sequence of human CD137 wherein amino acids 24-47 (shuffle 6) were replaced by the corresponding amino acids from wild boar CD137.
Thus, in one embodiment, the second antigen-binding region binds to an epitope of human CD137 which comprises or requires one or more of the amino acids L, Q, D, P, C, S, N, C, P, A, G, T, F, C, D, N, N, R, N, Q, I, C, S and P at positions 24-47 of SEQ ID NO:92 (corresponding to SEQ ID NO:129).
The mutant human CD137 in SEQ ID NO:94 corresponds to the amino acid sequence of human CD137 wherein amino acids 48-88 (shuffle 5) were replaced by the corresponding amino acids from African elephant CD137.
Thus, in one embodiment, the second antigen-binding region binds to an epitope of human CD137 which comprises or requires one or more of the amino acids C, P, P, N, S, F, S, S, A, G, G, Q, R, T, C, D, I, C, R, Q, C, K, G, V, F, R, T, R, K, E, C, S, S, T, S, N, A, E, C, D and C at positions 48-88 of SEQ ID NO:92 (corresponding to SEQ ID NO:130).
The mutant human CD137 in SEQ ID NO:95 corresponds to the amino acid sequence of human CD137 wherein amino acids 59-114 (shuffle 4) were replaced by the corresponding amino acids from African elephant CD137.
Thus, in one embodiment, the second antigen-binding region does not bind to an epitope of human CD137 which comprises or requires one or more of the amino acids T, P, G, F, H, C, L, G, A, G, C, S, M, C, E, Q, D, C, K, Q, G, Q, E, L, T and K 89-114 at positions of SEQ ID NO:92 (corresponding to SEQ ID NO:131).
In one embodiment, binding to the mutant and human CD137 may be performed as the shuffle assay described in Example 2. Thus, binding to human CD137 (SEQ ID NO:92) and mutant human CD137 (SEQ ID NOs:93, 94 and 95) may be determined by preparing shuffle constructs derived from human CD137 in which protein domains of the human CD137 are replaced by the corresponding domain of CD137 from different species, using human CD137 and the different species of CD137 as reference constructs; transducing cells with plasmids encoding the reference construct or the shuffle constructs, respectively, and measuring binding of the antibody to each these CD137 constructs by flow cytometry, such as FACS.
Loss of binding to certain shuffle constructs indicates that the corresponding region is likely to be involved in the antibody epitope. Thus, protein domains of human CD137 contributing to the epitope of the anti-human CD137 antibodies may thereby be determined by the shuffle assay. The different species of CD137 used to create the shuffle constructs should be chosen so that the monoclonal anti-human CD137 antibodies do not bind to the whole CD137 protein from these different species (reference construct).
Determination of binding to human CD137 and mutants thereof may in particular be performed with a monoclonal antibody comprising two second antigen-binding regions according to the present invention.
In one embodiment, said second antigen-binding region comprises heavy and light chain variable region CDR1, CDR2 and CDR3 selected from the group consisting of:
a) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:8, 9 and 10, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:11, KAS and 12, respectively, (CD137 clone 001),
b) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:15, 16 and 17, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:18, KAS and 19, respectively, (CD137 clone 002),
c) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:36, 37 and 38, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS and 40, respectively, (CD137 clone 005),
d) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:43, 44 and 45, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:46, AAS and 47, respectively, (CD137 clone 006),
e) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:64, 65 and 66, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:67, GAS and 68, respectively, (CD137 clone 009),
f) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:71, 72 and 73, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:74, KAS and 75, respectively, (CD137 clone 010),
g) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:85, 86 and 87, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:88, SAS and 89, respectively, (CD137 clone 012),
h) heavy and light chain variable region CDR1, CDR2 and CDR3 according to any of a) to g) having a total of one to twelve mutations, and
i) heavy and light chain variable region CDR1, CDR2 and CDR3 of an antibody which (i) competes for human CD137 binding with an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to any of a) to h) and/or (ii) has the specificity for CD137 of an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to any of a) to h).
Hence, in one embodiment, said second antigen-binding region comprises heavy and light chain variable region CDR1, CDR2 and CDR3 selected from the group consisting of:
a) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:64, 65 and 66, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:67, GAS and 68, respectively, (CD137 clone 009),
b) heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) having a total of one to twelve mutations;
c) heavy and light chain variable region CDR1, CDR2 and CDR3 of an antibody which (i) competes for human CD137 binding with an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) or b) and/or (ii) has the specificity for CD137 of an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) or b).
In another embodiment, said second antigen-binding region comprises heavy and light chain variable region CDR1, CDR2 and CDR3 selected from the group consisting of:
a) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:36, 37 and 38, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS and 40, respectively, (CD137 clone 005),
b) heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) having a total of one to twelve mutations, and
c) heavy and light chain variable region CDR1, CDR2 and CDR3 of an antibody which (i) competes for human CD137 binding with an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) or b) and/or (ii) has the specificity for CD137 of an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) or b).
In a particular embodiment, said second antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:64, 65 and 66, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:67, GAS and 68, respectively. An example of such an antibody includes, but is not limited to, the antibody referred to herein as CD137 clone 009.
In another embodiment, said second antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:36, 37 and 38, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS and 40, respectively. An example of such an antibody includes, but is not limited to, the antibody referred to herein as CD137 clone 005.
In another embodiment, said second antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:64, 65 and 66, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:67, GAS and 68, respectively, (CD137 clone 009), having a total of one to twelve mutations, such as one to ten mutations, or one to eight mutations, or one to six mutations, or one to four mutations, or to two mutations.
In another embodiment, said second antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:36, 37 and 38, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS and 40, respectively, (CD137 clone 005), having a total of one to twelve mutations, such as one to ten mutations, or one to eight mutations, or one to six mutations, or one to four mutations, or one to two mutations.
In one embodiment, said mutation may be an amino acid substitution, such as a conservative amino acid substitution.
In one embodiment, said mutations may be distributed across the VH CDR1, 2 and 3 and VL CDR1, 2 and 3 so that each of the VH and VL CDR3 comprises at the most three mutations and each of the VH and VL CDR2 and CDR1 comprises at the most two amino acid modifications.
Hence, in a further embodiment, the second antigen-binding region comprises heavy and light chain CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:64, 65, 66, 67, GAS and 68, respectively, (CD137 clone 009), having a total of one to twelve mutations and wherein the VH and VL CDR3 each comprises up to three amino acid modifications, and the VH and VL CDR1 and CDR2 each comprises up to two amino acid modifications.
In a further embodiment, the second antigen-binding region comprises heavy and light chain CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:64, 65, 66, 67, GAS and 68, respectively, (CD137 clone 009), having a total of one to ten mutations, such as one to eight, and wherein the VH and VL CDR1, CDR2, and CDR3 each comprises up to two amino acid modifications.
In a further embodiment, the second antigen-binding region comprises heavy and light chain CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:64, 65, 66, 67, GAS and 68, respectively, (CD137 clone 009), having a total of one to six mutations, and wherein the VH and VL CDR1, CDR2, and CDR3 each comprises at most one amino acid modification.
In another embodiment, the second antigen-binding region comprises heavy and light chain CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:36, 37, 38, 39, SAS and 40, respectively, (CD137 clone 005), having a total of one to twelve mutations and wherein the VH and VL CDR3 each comprises up to three amino acid modifications, and the VH and VL CDR1 and CDR2 each comprises up to two amino acid modifications.
In a further embodiment, the second antigen-binding region comprises heavy and light chain CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:36, 37, 38, 39, SAS and 40, respectively, (CD137 clone 005), having a total of one to ten mutations, such as one to eight, and wherein the VH and VL CDR1, CDR2, and CDR3 each comprises up to two amino acid modifications.
In a further embodiment, the second antigen-binding region comprises heavy and light chain CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:36, 37, 38, 39, SAS and 40, respectively, (CD137 clone 005), having a total of one to six mutations, and wherein the VH and VL CDR1, CDR2, and CDR3 each comprises at most one amino acid modification.
In a further embodiment, there may be a total of one to twelve mutations; such as one to ten mutations, or one to eight mutations, or one to six mutations, or one to four mutations, or one to two mutations; and each CDR sequence comprises at the most two amino acid substitutions.
It is well known to a person skilled in the art how to introduce mutations and that certain amino acids of the CDR sequences may be mutated; e.g., by amino acid substitutions to, e.g., increase affinity of the antibody to its target antigen or reducing immunogenicity for non-human antibodies to be used for treatment of humans. Such mutations can be introduced without affecting the epitope of the target antigen to which the antibody binds.
In one embodiment said second antigen-binding region comprises a second heavy chain variable (VH) sequence, and a second light chain variable (VL) sequence and wherein said variable sequences each comprises three CDR sequences, CDR1, CDR2 and CDR3, respectively.
In one embodiment, said second antigen-binding region comprises a second heavy chain variable (VH) sequence, and a second light chain variable (VL) sequence and wherein said variable sequences each comprises three CDR sequences, CDR1, CDR2 and CDR3, respectively, and four framework sequences, FR1, FR2, FR3 and FR4, respectively.
In one embodiment, said VH sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of:
a) a VH sequence as set forth in SEQ ID NO:123 (humanized CD137 clone 009)
b) a VH sequence as set forth in SEQ ID NO:13 (CD137 clone 001)
c) a VH sequence as set forth in SEQ ID NO:20 (CD137 clone 002)
d) a VH sequence as set forth in SEQ ID NO:27 (CD137 clone 003)
e) a VH sequence as set forth in SEQ ID NO:34 (CD137 clone 004)
f) a VH sequence as set forth in SEQ ID NO:41 (CD137 clone 005)
g) a VH sequence as set forth in SEQ ID NO:48 (CD137 clone 006)
h) a VH sequence as set forth in SEQ ID NO:55 (CD137 clone 007)
i) a VH sequence as set forth in SEQ ID NO:62 (CD137 clone 008)
j) a VH sequence as set forth in SEQ ID NO:69 (CD137 clone 009)
k) a VH sequence as set forth in SEQ ID NO:76 (CD137 clone 010)
l) a VH sequence as set forth in SEQ ID NO:83 (CD137 clone 011)
m) a VH sequence as set forth in SEQ ID NO:90 (CD137 clone 012)
In one embodiment, said VL sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to an amino acid sequence selected from the group consisting of:
a) a VL sequence as set forth in SEQ ID NO:127 (humanized CD137 clone 009)
b) a VL sequence as set forth in SEQ ID NO:14 (CD137 clone 001)
c) a VL sequence as set forth in SEQ ID NO:21 (CD137 clone 002)
d) a VL sequence as set forth in SEQ ID NO:28 (CD137 clone 003)
e) a VL sequence as set forth in SEQ ID NO:35 (CD137 clone 004)
f) a VL sequence as set forth in SEQ ID NO:42 (CD137 clone 005)
g) a VL sequence as set forth in SEQ ID NO:49 (CD137 clone 006)
h) a VL sequence as set forth in SEQ ID NO:56 (CD137 clone 007)
i) a VL sequence as set forth in SEQ ID NO:63 (CD137 clone 008)
j) a VL sequence as set forth in SEQ ID NO:70 (CD137 clone 009)
k) a VL sequence as set forth in SEQ ID NO:77 (CD137 clone 010)
l) a VL sequence as set forth in SEQ ID NO:84 (CD137 clone 011)
m) a VL sequence as set forth in SEQ ID NO:91 (CD137 clone 012)
In one embodiment, said VH and VL sequences of the second antigen-binding region each comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to an amino acid sequence selected from the group consisting of:
a) a VH sequence as set forth in SEQ ID NO:123 and a VL sequence as set forth in SEQ ID NO:127 (humanized CD137 clone 009)
b) a VH sequence as set forth in SEQ ID NO:13 and a VL sequence as set forth in SEQ ID NO:14 (CD137 clone 001)
c) a VH sequence as set forth in SEQ ID NO:20 and a VL sequence as set forth in SEQ ID NO:21 (CD137 clone 002)
d) a VH sequence as set forth in SEQ ID NO:27 and a VL sequence as set forth in SEQ ID NO:28 (CD137 clone 003)
e) a VH sequence as set forth in SEQ ID NO:34 and a VL sequence as set forth in SEQ ID NO:35 (CD137 clone 004)
f) a VH sequence as set forth in SEQ ID NO:41 and a VL sequence as set forth in SEQ ID NO:42 (CD137 clone 005)
g) a VH sequence as set forth in SEQ ID NO:48 and a VL sequence as set forth in SEQ ID NO:49 (CD137 clone 006)
h) a VH sequence as set forth in SEQ ID NO:55 and a VL sequence as set forth in SEQ ID NO:56 (CD137 clone 007)
i) a VH sequence as set forth in SEQ ID NO:62 and a VL sequence as set forth in SEQ ID NO:63 (CD137 clone 008)
j) a VH sequence as set forth in SEQ ID NO:69 and a VL sequence as set forth in SEQ ID NO:70 (CD137 clone 009)
k) a VH sequence as set forth in SEQ ID NO:76 and a VL sequence as set forth in SEQ ID NO:77 (CD137 clone 010)
l) a VH sequence as set forth in SEQ ID NO:83 and a VL sequence as set forth in SEQ ID NO:84 (CD137 clone 011)
m) a VH sequence as set forth in SEQ ID NO:90 and a VL sequence as set forth in SEQ ID NO:91 (CD137 clone 012).
In one embodiment, said VH sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to an amino acid sequence selected from the group consisting of:
a) a VH sequence as set forth in SEQ ID NO:123 (humanized CD137 clone 009)
b) a VH sequence as set forth in SEQ ID NO:41 (CD137 clone 005)
c) a VH sequence as set forth in SEQ ID NO:69 (CD137 clone 009)
In one embodiment, said VL sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to an amino acid sequence selected from the group consisting of:
a) a VL sequence as set forth in SEQ ID NO:127 (humanized CD137 clone 009)
b) a VL sequence as set forth in SEQ ID NO:42 (CD137 clone 005)
c) a VL sequence as set forth in SEQ ID NO:70 (CD137 clone 009)
In one embodiment, said VH and VL sequences of the second antigen-binding region each comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to an amino acid sequence selected from the group consisting of:
a) a VH sequence as set forth in SEQ ID NO:123 and a VL sequence as set forth in SEQ ID NO:127 (humanized CD137 clone 009)
b) a VH sequence as set forth in SEQ ID NO:41 and a VL sequence as set forth in SEQ ID NO:42 (CD137 clone 005)
c) a VH sequence as set forth in SEQ ID NO:69 and a VL sequence as set forth in SEQ ID NO:70 (CD137 clone 009)
In one embodiment, said VH sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity to SEQ ID NO:41 (CD137 clone 005).
In one embodiment, said VH sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity to SEQ ID NO:69 (CD137 clone 009).
In one embodiment, said VH sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to SEQ ID NO:123 (humanized CD137 clone 009).
In one embodiment, said VL sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity to SEQ ID NO:42 (CD137 clone 005).
In one embodiment, said VL sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity to SEQ ID NO:70 (CD137 clone 009).
In one embodiment said VL sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to SEQ ID NO:127 (humanized CD137 clone 009).
In one embodiment, said VH and said VL sequence of the second antigen-binding region each comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity to SEQ ID NO:41; and SEQ ID NO:42 (CD137 clone 005), respectively.
In one embodiment, said VH and VL sequence of the second antigen-binding region each comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity to SEQ ID NO:69 and SEQ ID NO:70 (CD137 clone 009), respectively.
In one embodiment, said VH and VL sequence of the second antigen-binding region each comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to SEQ ID NO:123 and SEQ ID NO:127, respectively (humanized CD137 clone 009).
In one embodiment, said VH and VL sequences only deviate in the framework sequences.
In one embodiment, the respective FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the first and/or second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of said VH and VL sequences.
In one embodiment, the VH and VL sequences only deviate in the non-CDR sequences as set forth in SEQ ID NO:41 and 42, respectively, (CD137 clone 005).
In one embodiment the VH and VL sequences only deviate in the non-CDR sequences as set forth in SEQ ID NO:69 and 70, respectively, (CD137 clone 009).
In one embodiment the VH and VL sequences only deviate in the non-CDR sequences as set forth in SEQ ID NO:123 and 127, respectively, (humanized CD137 clone 009).
In one embodiment, the VH and VL sequences only deviate in the framework sequences.
In one embodiment, the respective FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the first and/or second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of said VH and VL sequences.
In one embodiment, the respective FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:41, and VL sequence as set forth in SEQ ID NO:42.
In one embodiment, the respective FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:41, and VL sequence as set forth in SEQ ID NO:42, and the heavy and light chain variable region CDR1, CDR2 and CDR3 of the second antigen-binding region have a total of one to twelve mutations compared to the heavy and light chain variable region CDR1, CDR2 and CDR3 having the sequences as set forth in SEQ ID NOs:36, 37, 38, 39, SAS and 40, respectively. In a further embodiment said mutations may be as described above.
In an even further embodiment, the respective FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:41, and VL sequence as set forth in SEQ ID NO:42, and the second antigen-binding region comprises heavy and light chain variable region CDR1, CDR2 and CDR3 having the sequences as set forth in SEQ ID NOs:36, 37, 38, 39, SAS and 40, respectively.
In one embodiment the respective FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:69, and VL sequence as set forth in SEQ ID NO:70.
In one embodiment the respective FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:69, and VL sequence as set forth in SEQ ID NO:70, and the heavy and light chain variable region CDR1, CDR2 and CDR3 of the second antigen-binding region have a total of one to twelve mutations compared to the heavy and light chain variable region CDR1, CDR2 and CDR3 having the sequences as set forth in SEQ ID NOs:64, 65, 66, 67, GAS and 68, respectively. In a further embodiment said mutations may be as described above.
In an even further embodiment the respective FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:69, and VL sequence as set forth in SEQ ID NO:70, and the second antigen-binding region comprises heavy and light chain variable region CDR1, CDR2 and CDR3 having the sequences as set forth in SEQ ID NOs:64, 65, 66, 67, GAS and 68, respectively.
In one embodiment, the respective FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:123, and VL sequence as set forth in SEQ ID NO:127.
In one embodiment the respective FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:123, and VL sequence as set forth in SEQ ID NO:127, and the heavy and light chain variable region CDR1, CDR2 and CDR3 of the second antigen-binding region have a total of one to twelve mutations compared to the heavy and light chain variable region CDR1, CDR2 and CDR3 having the sequences as set forth in SEQ ID NOs:64, 65, 66, 67, GAS and 68, respectively. In a further embodiment said mutations may be as described above.
In an even further embodiment the respective FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% amino acid sequence identity to the respective FR1, FR2, FR3 and FR4 framework sequences of the VH sequence as set forth in SEQ ID NO:123, and VL sequence as set forth in SEQ ID NO:127, and the second antigen-binding region comprises heavy and light chain variable region CDR1, CDR2 and CDR3 having the sequences as set forth in SEQ ID NOs:64, 65, 66, 67, GAS and 68, respectively.
In one embodiment, said VH sequence of the second antigen-binding region comprises SEQ ID NO:123 (humanized CD137 clone 009).
In one embodiment, said VL sequence of the second antigen-binding region comprises SEQ ID NO:127 (humanized CD137 clone 009).
In one embodiment, said VH and VL sequences of the second antigen-binding region comprise SEQ ID NO:123 and SEQ ID NO:127, respectively.
In one embodiment, said VH sequence of the second antigen-binding region comprises a VH sequence selected from the group consisting of:
a) SEQ ID NO:41 (CD137 clone 005)
b) SEQ ID NO:69 (CD137 clone 009)
In one embodiment, said VL sequence of the second antigen-binding region comprises a VL sequence selected from the group consisting of:
a) SEQ ID NO:42 (CD137 clone 005)
b) SEQ ID NO:70 (CD137 clone 009)
In one embodiment, said VH and VL sequences of the second antigen-binding are selected from the group consisting of:
a) a VH sequence as set forth in SEQ ID NO:41 and a VL sequence as set forth in SEQ ID NO:42 (CD137 clone 005),
b) a VH sequence as set forth in SEQ ID NO:69 and a VL sequence as set forth in SEQ ID NO:70 (CD137 clone 009).
In one embodiment, the multispecific antibody according to the present invention comprises a second binding arm comprising said second antigen-binding region.
In one embodiment, the multispecific antibody according to the present invention comprises a second binding arm comprising said second-antigen-binding region and a second heavy chain constant sequence.
In one embodiment, the multispecific antibody according to the present invention comprises a second binding arm comprising said second antigen-binding region, wherein the second binding arm comprises a second heavy chain comprising a second heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence, and a second light chain comprising a second light chain variable (VL) sequence.
In one embodiment said second light chain further comprises a second light chain constant sequence.
In a further embodiment, said second heavy chain comprises at least a hinge region, a CH2 and a CH3 region.
In a specific embodiment, the multispecific antibody according to the present invention comprises a second Fab-arm comprising said second antigen-binding region.
In one embodiment, the second antigen-binding region is derived from a rabbit antibody, such as any of anti-CD137 clones 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, in particular any of clones 5 and 9, disclosed herein.
In one embodiment, the second antigen-binding region is derived from a chimeric antibody, such as an antibody comprising a variable region from any of the anti-CD137 clones 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, in particular any of clones 5 and 9, disclosed herein.
In one embodiment, the second antigen-binding region is derived from a humanized antibody.
In one embodiment, the second binding arm is derived from a full-length antibody.
In one embodiment the second binding arm is derived from a full-length IgG1,λ (lambda) or IgG1,κ (kappa) antibody.
In one embodiment, the second binding arm is derived from a monoclonal antibody.
In one embodiment, said second heavy chain is of an IgG isotype, optionally having a subclass selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.
In one embodiment, the first binding arm may be derived from an antibody comprising a HC comprising SEQ ID NO:124 and an LC comprising SEQ ID NO:128, optionally with one or more mutations in the constant region of the HC, such as 1 to 10, such as 1 to 5, such as 1, 2, 3, 4 or 5 mutations.
In one embodiment, the first binding arm comprises a HC comprising SEQ ID NO:124, 125 or 126 and an LC comprising SEQ ID NO:128.
Binding to CD40 and CD137
In some embodiments, the present invention relates to a multispecific antibody comprising:
(I) a first antigen-binding region binding to human CD40, wherein said first antigen-binding region comprises heavy and light chain variable regions selected from the group consisting of:
a) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1, 2 and 3, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:4, YTS and 5, respectively,
b) heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) having a total of one to twelve mutations; and
c) heavy and light chain variable region CDR1, CDR2 and CDR3 of an antibody which (i) competes for human CD40 binding with an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) or b) and/or (ii) has the specificity for CD40 of an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) or b), and
(II) a second antigen-binding region binding to human CD137, wherein said second antigen-binding region comprises heavy and light chain variable regions selected from the group consisting of:
x) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:64, 65 and 66, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:67, GAS and 68, respectively, (CD137 clone 009),
y) heavy and light chain variable region CDR1, CDR2 and CDR3 according to x) having a total of one to twelve mutations; and
z) heavy and light chain variable region CDR1, CDR2 and CDR3 of an antibody which (i) competes for human CD137 binding with an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to x) or y) and/or (ii) has the specificity for CD137 of an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to x) or y).
In another embodiment, the present invention relates to a multispecific antibody comprising:
(I) a first antigen-binding region binding to human CD40, wherein said first antigen-binding region comprises heavy and light chain variable regions selected from the group consisting of:
a) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1, 2 and 3, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:4, YTS and 5, respectively,
b) heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) having a total of one to twelve mutations; and
c) heavy and light chain variable region CDR1, CDR2 and CDR3 of an antibody which (i) competes for human CD40 binding with an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) or b) and/or (ii) has the specificity for CD40 of an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to a) or b), and
(II) a second antigen-binding region binding to human CD137, wherein said second antigen-binding region comprises heavy and light chain variable regions selected from the group consisting of:
x) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:36, 37 and 38, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS and 40, respectively, (CD137 clone 005),
y) heavy and light chain variable region CDR1, CDR2 and CDR3 according to x) having a total of one to twelve mutations; and
z) heavy and light chain variable region CDR1, CDR2 and CDR3 of an antibody which (i) competes for human CD137 binding with an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to x) or y) and/or (ii) has the specificity for CD137 of an antibody comprising heavy and light chain variable region CDR1, CDR2 and CDR3 according to x) or y).
Hence, in one embodiment, said first antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NOs:1, 2 and 3, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NOs:4, YTS and 5, respectively; and said second antigen-binding region comprises
a) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:64, 65 and 66, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:67, GAS and 68, respectively (CD136 clone 009), or
b) heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:36, 37 and 38, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS and 40 (CD137 clone 005), respectively.
In another embodiment, said first antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1, 2 and 3, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:4, YTS and 5, respectively; and said second antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:64, 65 and 66, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:67, GAS and 68, respectively, (CD137 clone 009).
In another embodiment said first antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1, 2 and 3, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:4, YTS and 5, respectively; and said second antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:36, 37 and 38, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS and 40, respectively, (CD137 clone 005).
In a further embodiment, said first antigen-binding region of the multispecific antibody according to the present invention comprises a first heavy chain variable (VH) sequence, and a first light chain variable (VL) sequence, and said second antigen-binding region of the multispecific antibody according to the present invention comprises a second heavy chain variable (VH) sequence, and a second light chain variable (VL) sequence and wherein said variable sequences each comprise three CDR sequences, CDR1, CDR2 and CDR3, respectively, and four framework sequences, FR1, FR2, FR3 and FR4, respectively.
In a further embodiment said VH and VL sequence of the first antigen-binding region each comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity to the amino acid sequence of the VH sequence as set forth in SEQ ID NO:6 and the VL sequence as set forth in SEQ ID NO:7, respectively, and said VH and said VL sequence of the second antigen-binding region each comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity to the amino acid sequence of the VH sequence as set forth in SEQ ID NO:41 and the VL sequence as set forth in SEQ ID NO:42, respectively, (CD137 clone 005).
In another further embodiment, said VH and VL sequence of the first antigen-binding region each comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity to the amino acid sequence of the VH sequence as set forth in SEQ ID NO:6 and the VL sequence as set forth in SEQ ID NO:7, respectively, and said VH and said VL sequence of the second antigen-binding region each comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity to the amino acid sequence of the VH sequence as set forth in SEQ ID NO:69 and the VL sequence as set forth in SEQ ID NO:70, respectively, (CD137 clone 009).
In another further embodiment, said VH and VL sequence of the first antigen-binding region each comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to the amino acid sequence of the VH sequence as set forth in SEQ ID NO:117 and the VL sequence as set forth in SEQ ID NO:121, respectively, and said VH and said VL sequence of the second antigen-binding region each comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to the amino acid sequence of the VH sequence as set forth in SEQ ID NO:123 and the VL sequence as set forth in SEQ ID NO:127, respectively, (humanized CD137 clone 009).
In a particular embodiment, the present invention relates to a bispecific antibody comprising
(I) a first binding arm comprising a first heavy chain comprising a first heavy chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence and a first light chain constant (CL) sequence, and wherein said heavy first chain variable sequence comprises CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1, 2 and 3, respectively, and said first light chain sequence comprises CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:4, YTS and 5, respectively, and
(II) a second binding arm comprising a second heavy chain comprising a second heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence, and a second light chain further comprises a second light chain constant (CL) sequence, and a second light chain variable (VL) sequence, wherein said second heavy chain variable sequence comprises CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:64, 65 and 66, respectively, and said second light chain sequence comprises CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:67, GAS and 68, respectively, (CD137 clone 009);
wherein the first and second heavy chain are of a human IgG1 isotype and wherein the first and second light chain is of IgG1,κ, and wherein the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering of both the first and second constant heavy chain are F, E, and A, respectively, and wherein (a) the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the first constant heavy chain is L, and the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the second constant heavy chain is R; or (b) the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the first constant heavy chain is R, and the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the second constant heavy chain is L.
In a further particular embodiment, the present invention relates to a bispecific antibody comprising
(I) a first binding arm comprising a first heavy chain comprising a first heavy chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence and a first light chain constant (CL) sequence, and wherein said heavy first chain variable sequence comprises CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1, 2 and 3, respectively, and said first light chain sequence comprises CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:4, YTS and 5, respectively, and
(II) a second binding arm comprising a second heavy chain comprising a second heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence, and a second light chain further comprises a second light chain constant (CL) sequence, and a second light chain variable (VL) sequence, wherein said second heavy chain variable sequence comprises CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:36, 37 and 38, respectively, and said second light chain sequence comprises CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS and 40, respectively, (CD137 clone 005); and
wherein the first and second heavy chain are of a human IgG1 isotype and wherein the first and second light chain is of IgG1,κ, and wherein the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering of both the first and second constant heavy chain are F, E, and A, respectively, and wherein (a) the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the first constant heavy chain is L, and the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the second constant heavy chain is R; or (b) the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the first constant heavy chain is R, and the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the second constant heavy chain is L.
In a specific embodiment, the present invention relates to a bispecific antibody comprising
(I) a first binding arm comprising a first heavy chain comprising a first heavy chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence and a first light chain constant (CL) sequence, and wherein said first VH and VL sequences each comprises a sequence having at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to the amino acid sequence of the VH sequence as set forth in SEQ ID NO:117 and the VL sequence as set forth in SEQ ID NO:121, respectively, and
(II) a second binding arm comprising a second heavy chain comprising a second heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence, and a second light chain further comprises a second light chain constant (CL) sequence, and a second light chain variable (VL) sequence, wherein said second VH and VL sequences comprise a sequence having at least 70%, at least 75%, at least 80%, at least at least at least 95%, at least 97%, at least 99% or 100% identity to 85%,90%, the amino acid sequence of the VH sequence as set forth in SEQ ID NO:123 and the VL sequence as set forth in SEQ ID NO:127, respectively, (humanized CD137 clone 009).
In a further specific embodiment, the present invention relates to a bispecific antibody comprising
(I) a first binding arm comprising a first heavy chain comprising a first heavy chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence and a first light chain constant (CL) sequence, and wherein said first VH sequence comprises SEQ ID NO:117 and said first VL sequence comprises SEQ ID NO:121, and
(II) a second binding arm comprising a second heavy chain comprising a second heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence, and a second light chain further comprises a second light chain constant (CL) sequence, and a second light chain variable (VL) sequence, wherein said second VH sequence comprises SEQ ID NO:123 and said second VL sequence comprises SEQ ID NO:127, (humanized CD137 clone 009).
In a specific embodiment, the present invention relates to a bispecific antibody comprising
(I) a first binding arm comprising a first heavy chain comprising a first heavy chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence and a first light chain constant (CL) sequence, and wherein said first VH and VL sequences comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to the amino acid sequence of the VH sequence as set forth in SEQ ID NO:117 and the VL sequence as set forth in SEQ ID NO:121, respectively, and
(II) a second binding arm comprising a second heavy chain comprising a second heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence, and a second light chain further comprises a second light chain constant (CL) sequence, and a second light chain variable (VL) sequence, wherein said second VH and VL sequences comprise a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identity to the amino acid sequence of the VH sequence as set forth in SEQ ID NO:123 and the VL sequence as set forth in SEQ ID NO:127, respectively, (humanized CD137 clone 009),
wherein the first and second heavy chain are of a human IgG1 isotype and wherein the first and second light chain is of IgG1,κ, and wherein the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering of both the first and second constant heavy chain are F, E, and A, respectively, and wherein (a) the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the first constant heavy chain is L, and the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the second constant heavy chain is R; or (b) the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the first constant heavy chain is R, and the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the second constant heavy chain is L.
In a further specific embodiment, the present invention relates to a bispecific antibody comprising
(I) a first binding arm comprising a first heavy chain comprising a first heavy chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence and a first light chain constant (CL) sequence, and wherein said first VH sequence comprises SEQ ID NO:117 and said first VL sequence comprises SEQ ID NO:121, and
(II) a second binding arm comprising a second heavy chain comprising a second heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence, and a second light chain further comprises a second light chain constant (CL) sequence, and a second light chain variable (VL) sequence, wherein said second VH sequence comprises SEQ ID NO:123 and said second VL sequence comprises SEQ ID NO:127, (humanized CD137 clone 009),
wherein the first and second heavy chain are of a human IgG1 isotype and wherein the first and second light chain is of IgG1,κ, and wherein the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering of both the first and second constant heavy chain are F, E, and A, respectively, and wherein (a) the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the first constant heavy chain is L, and the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the second constant heavy chain is R; or (b) the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the first constant heavy chain is R, and the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the second constant heavy chain is L.
In another aspect, the present invention relates to a bispecific antibody comprising a first binding arm binding to human CD40 and a second binding arm binding to human CD137, wherein
(i) said first binding arm comprises a heavy chain (HC) amino acid sequence comprising or consisting of SEQ ID NO:118 and a light chain (LC) amino acid sequence comprising or consisting of SEQ ID NO:122, and
(ii) said second binding arm comprises a HC amino acid sequence comprising or consisting of SEQ ID NO:124 and a LC amino acid sequence comprising or consisting of SEQ ID NO:128,
optionally wherein SEQ ID NOS:118, SEQ ID NO:124 or both comprise one or more mutations in the constant region of the HC, such as 1 to 10, such as 1 to 5, such as 1, 2, 3, 4 or 5 mutations.
In another aspect, the present invention relates to a bispecific antibody comprising a first binding arm binding to human CD40 and a second binding arm binding to human CD137, wherein
(i) said first binding arm comprises a HC amino acid sequence comprising or consisting of SEQ ID NO:119 and a LC amino acid sequence comprising or consisting of SEQ ID NO:122, and
(ii) said second binding arm comprises a HC amino acid sequence comprising or consisting of SEQ ID NO:125 and a LC amino acid sequence comprising or consisting of SEQ ID NO:128.
In another aspect, the present invention relates to a bispecific antibody comprising a first binding arm binding to human CD40 and a second binding arm binding to human CD137, wherein
(i) said first binding arm comprises a HC amino acid sequence comprising or consisting of SEQ ID NO:120 and a LC amino acid sequence comprising or consisting of SEQ ID NO:122, and
(ii) said second binding arm comprises a HC amino acid sequence comprising or consisting of SEQ ID NO:126 and a LC amino acid sequence comprising or consisting of SEQ ID NO:128.
Bispecific Formats
In a particular embodiment the multispecific antibody according to the present invention is a bispecific antibody.
The present invention provides bispecific CD40×CD137 antibodies which are able of cross-linking cells expressing CD40 and cells expressing CD137; such as antigen-presenting cells and T cells, respectively. Depending on the desired functional properties for a particular use, particular antigen-binding regions can be selected from the set of antibodies or antigen-binding regions provided by the present invention. Many different formats and uses of bispecific antibodies are known in the art, and were reviewed by Kontermann; Drug Discov Today, 2015 July; 20(7):838-47 and; MAbs, 2012 March-April; 4(2):182-97.
A bispecific antibody according to the present invention is not limited to any particular bispecific format or method of producing it.
Examples of bispecific antibody molecules which may be used in the present invention comprise (i) a single antibody that has two binding arms comprising different antigen-binding regions; (ii) a single chain antibody that has specificity to two different epitopes, e.g., via two scFvs linked in tandem by an extra peptide linker; (iii) a dual-variable-domain antibody (DVD-Ig™), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (iv) a chemically-linkedbispecific (Fab′)2 fragment; (v) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vi) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (vii) a so-called “dock and lock” molecule, based on the “dimerization and docking domain” in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (viii) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fab-arm; and (ix) a diabody.
In one embodiment, the bispecific antibody of the present invention is a diabody, a cross-body, or a bispecific antibody obtained via a controlled Fab-arm exchange (such as described in WO2011131746 (Genmab)).
Examples of different classes of bispecific antibodies include, but are not limited to, (i) IgG-like molecules with complementary CH3 domains to force heterodimerization; (ii) recombinant IgG-like dual targeting molecules, wherein the two sides of the molecule each contain the Fab fragment or part of the Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, wherein full length IgG antibodies are fused to extra Fab fragment or parts of Fab fragment; (iv) Fc fusion molecules, wherein single chain Fv molecules or stabilized diabodies are fused to heavy-chain constant-domains, Fc-regions or parts thereof; (v) Fab fusion molecules, wherein different Fab-fragments are fused together, fused to heavy-chain constant-domains, Fc-regions or parts thereof; and (vi) ScFv- and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) wherein different single chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g. domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule fused to heavy-chain constant-domains, Fc-regions or parts thereof.
Examples of IgG-like molecules with complementary CH3 domain molecules include, but are not limited to, the Triomab/Quadroma molecules (Trion Pharma/Fresenius Biotech; Roche, WO2011069104), the so-called Knobs-into-Holes molecules (Genentech, WO9850431), CrossMAbs (Roche, WO2011117329) and the electrostatically-matched molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), the LUZ-Y molecules (Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52): 43331-9, doi: 10.1074/jbc.M112.397869. Epub 2012 Nov. 1), DIG-body and PIG-body molecules (Pharmabcine, WO2010134666, WO2014081202), the Strand Exchange Engineered Domain body (SEEDbody) molecules (EMD Serono, WO2007110205), the Biclonics molecules (Merus, WO2013157953), FcΔAdp molecules (Regeneron, WO201015792), bispecific IgG1 and IgG2 molecules (Pfizer/Rinat, WO11143545), Azymetric scaffold molecules (Zymeworks/Merck, WO2012058768), mAb-Fv molecules (Xencor, WO2011028952), bivalent bispecific antibodies (WO2009080254) and the DuoBody® molecules (Genmab A/S, WO2011131746).
Examples of recombinant IgG-like dual targeting molecules include, but are not limited to, Dual Targeting (DT)-Ig molecules (WO2009058383), Two-in-one Antibody (Genentech; Bostrom, et al 2009. Science 323, 1610-1614.), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013 March-April; 5(2):208-18), approaches with common light chain (Crucell/Merus, U.S. Pat. No. 7,262,028), κλBodies (NovImmune, WO2012023053) and CovX-body (CovX/Pfizer; Doppalapudi, V. R., et al 2007. Bioorg. Med. Chem. Lett. 17,501-506.).
Examples of IgG fusion molecules include, but are not limited to, Dual Variable Domain (DVD-Ig™) molecules (Abbott, U.S. Pat. No. 7,612,181), Dual domain double head antibodies (Unilever; Sanofi Aventis, WO20100226923), IgG-like Bispecific molecules (ImClone/Eli Lilly, Lewis et al. Nat Biotechnol. 2014 February; 32(2):191-8), Ts2Ab5 (MedImmune/AZ; Dimasi et al. 3 Mol Biol. 2009 Oct. 30; 393(3): 672-92) and BsAb molecules (Zymogenetics, WO2010111625), HERCULES molecules (Biogen Idec, U.S. Ser. No. 00/795,1918), scFv fusion molecules (Novartis), scFv fusion molecules (Changzhou Adam Biotech Inc, CN 102250246) and TvAb molecules (Roche, WO2012025525, WO2012025530).
Examples of Fc fusion molecules include, but are not limited to, ScFv/Fc Fusions (Pearce et al., Biochem Mol Biol Int. 1997 September; 42(6):1179-88), SCORPION molecules (Emergent BioSolutions/Trubion, Blankenship J W, et al. AACR 100th Annual meeting 2009 (Abstract #5465); Zymogenetics/BMS, WO2010111625), Dual Affinity Retargeting Technology (Fc-DART) molecules (MacroGenics, WO2008157379, WO2010080538) and Dual(ScFv)2-Fab molecules (National Research Center for Antibody Medicine—China).
Examples of Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 molecules (Medarex/AMGEN; Deo et al J Immunol. 1998 Feb. 15; 160(4):1677-86.), Dual-Action or Bis-Fab molecules (Genentech, Bostrom, et al 2009. Science 323, 1610-1614.), Dock-and-Lock (DNL) molecules (ImmunoMedics, WO2003074569, WO2005004809), Bivalent Bispecific molecules (Biotecnol, Schoonjans, J Immunol. 2000 Dec. 15; 165(12):7050-7.) and Fab-Fv molecules (UCB-Celltech, WO 2009040562 A1).
Examples of ScFv-, diabody-based and domain antibodies include, but are not limited to, Bispecific T Cell Engager (BiTE) molecules (Micromet, WO2005061547), Tandem Diabody molecules (TandAb) (Affimed) Le Gall et al., Protein Eng Des Sel. 2004 April; 17(4):357-66.), Dual Affinity Retargeting Technology (DART) molecules (MacroGenics, WO2008157379, WO2010080538), Single-chain Diabody molecules (Lawrence, FEBS Lett. 1998 Apr. 3; 425(3):479-84), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack, WO2010059315) and COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 August; 88(6):667-75.), dual targeting nanobodies (Ablynx, Hmila et al., FASEB J. 2010) and dual targeting heavy chain only domain antibodies.
In one embodiment, each of said first and second heavy chains comprises at least a hinge region, a CH2 and a CH3 region.
In a further embodiment, the CH3 regions of the first and second heavy chains comprise asymmetrical mutations, such as asymmetrical mutations (also referred to as modifications herein) yielding a stable heterodimeric antibody.
In one embodiment, the bispecific antibody of the invention comprises a first heavy chain comprising a first CH3 region, and a second heavy chain comprising a second CH3 region, wherein the sequences of the first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. More details on these interactions and how they can be achieved are provided in, e.g., WO 2011/131746 and WO 2013/060867 (Genmab), which are hereby incorporated by reference.
As described further herein, a stable bispecific CD40×CD137 antibody can be obtained at high yield using a particular method on the basis of one homodimeric parental CD40 antibody and one homodimeric parental CD137 antibody containing only a few, fairly conservative, asymmetrical mutations in the CH3 regions. Asymmetrical mutations mean that the sequences of said first and second CH3 regions contain amino acid substitutions at non-identical positions.
Accordingly, in one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the sequences of said first and second heavy chain CH3 regions contain asymmetrical mutations, e.g., a mutation at the position corresponding to position 405 in a human IgG1 heavy chain according to EU numbering in one of the CH3 regions, and a mutation at the position corresponding to position 409 in a human IgG1 heavy chain according to EU numbering in the other CH3 region.
In one aspect, the bispecific antibody as defined in any of the embodiments disclosed herein comprises first and second heavy chains, wherein in said first heavy chain at least one of the amino acids in the positions corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering has been substituted, and in said second heavy chain at least one of the amino acids in the positions corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering has been substituted, and wherein said first and said second heavy chains are not substituted in the same positions.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, 407 and 409 in a human IgG1 heavy chain according to EU numbering, and the second heavy chain has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, 407 and 409 in a human IgG1 heavy chain according to EU numbering, and wherein the first and second heavy chains are not substituted in the same positions.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid substitution at position 366, and said second heavy chain has an amino acid substitution at a position selected from the group consisting of: 368, 370, 399, 405, 407 and 409. In one embodiment the amino acid at position 366 is selected from Ala, Asp, Glu, His, Asn, Val, or Gln.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid substitution at position 368, and said second heavy chain has an amino acid substitution at a position selected from the group consisting of: 366, 370, 399, 405, 407 and 409.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid substitution at position 370, and said second heavy chain has an amino acid substitution at a position selected from the group consisting of: 366, 368, 399, 405, 407 and 409.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid substitution at position 399, and said second heavy chain has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 405, 407 and 409.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid substitution at position 405, and said second heavy chain has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 407 and 409.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid substitution at position 407, and said second heavy chain has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, and 409.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid substitution at position 409, and said second heavy chain has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, and 407.
Accordingly, in one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the sequences of said first and second CH3 regions contain asymmetrical mutations, i.e. mutations at different positions in the two CH3 regions, e.g. a mutation at position 405 in one of the CH3 regions and a mutation at position 409 in the other CH3 region.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain has an amino-acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405 and 407. In one such embodiment, said first heavy chain has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain has an amino acid other than Phe, e.g. Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Cys, Lys, or Leu, at position 405. In a further embodiment hereof, said first heavy chain has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain has an amino acid other than Phe, Arg or Gly, e.g. Leu, Ala, Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 405.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first heavy chain comprises a Phe at position 405 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain comprises an amino acid other than Phe, e.g. Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Leu, Met, or Cys, at position 405 and a Lys at position 409. In a further embodiment hereof, said first heavy chain comprises a Phe at position 405 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain comprises an amino acid other than Phe, Arg or Gly, e.g. Leu, Ala, Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 405 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first heavy chain comprises a Phe at position 405 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain comprises a Leu at position 405 and a Lys at position 409. In a further embodiment hereof, said first heavy chain comprises a Phe at position 405 and an Arg at position 409 and said second heavy chain comprises an amino acid other than Phe, Arg or Gly, e.g. Leu, Ala, Val, Ile, Ser, Thr, Lys, Met, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 405 and a Lys at position 409. In another embodiment, said first heavy chain comprises Phe at position 405 and an Arg at position 409 and said second heavy chain comprises a Leu at position 405 and a Lys at position 409.
In a further embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first heavy chain comprises an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405. In a further embodiment, said first heavy chain comprises an Arg at position 409 and said second heavy chain comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405.
In an even further embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first heavy chain comprises a Lys at position 370, a Phe at position 405 and an Arg at position 409 and said second heavy chain comprises a Lys at position 409, a Thr at position 370 and a Leu at position 405.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first heavy chain comprises an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain comprises a Lys at position 409 and: a) an Ile at position 350 and a Leu at position 405, or b) a Thr at position 370 and a Leu at position 405.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first heavy chain comprises an Arg at position 409 and said second heavy chain comprises a Lys at position 409 and: a) an Ile at position 350 and a Leu at position 405, or b) a Thr at position 370 and a Leu at position 405.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first heavy chain comprises a Thr at position 350, a Lys at position 370, a Phe at position 405 and an Arg at position 409 and said second heavy chain comprises a Lys at position 409 and: a) an Ile at position 350 and a Leu at position 405, or b) a Thr at position 370 and a Leu at position 405.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first heavy chain comprises a Thr at position 350, a Lys at position 370, a Phe at position 405 and an Arg at position 409 and said second heavy chain comprises an Ile at position 350, a Thr at position 370, a Leu at position 405 and a Lys at position 409.
In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first heavy chain has an amino acid other than Lys, Leu or Met at position 409 and said second heavy chain has an amino acid other than Phe at position 405, such as other than Phe, Arg or Gly at position 405; or said first CH3 region has an amino acid other than Lys, Leu or Met at position 409 and said second CH3 region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first heavy chain having an amino acid other than Lys, Leu or Met at position 409 and a second heavy chain having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first heavy chain having a Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409 and a second heavy chain having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and a Lys at position 409.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first heavy chain having a Tyr at position 407 and an Arg at position 409 and a second heavy chain having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and a Lys at position 409.
In another embodiment, said first heavy chain has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr, e.g. Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, at position 407. In another embodiment, said first heavy chain has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first heavy chain has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain has a Gly, Leu, Met, Asn or Trp at position 407.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first heavy chain has a Tyr at position 407 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr, e.g. Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, at position 407 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first heavy chain has a Tyr at position 407 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first heavy chain has a Tyr at position 407 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409 and said second heavy chain has a Gly, Leu, Met, Asn or Trp at position 407 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first heavy chain has a Tyr at position 407 and an Arg at position 409 and said second heavy chain has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr, e.g. Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, at position 407 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first heavy chain has a Tyr at position 407 and an Arg at position 409 and said second heavy chain has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first heavy chain has a Tyr at position 407 and an Arg at position 409 and said second heavy chain has a Gly, Leu, Met, Asn or Trp at position 407 and a Lys at position 409.
In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 409, and the second heavy chain has
(i) an amino acid other than Phe, Leu and Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 368, or
(ii) a Trp at position 370, or
(iii) an amino acid other than Asp, Cys, Pro, Glu or Gln, e.g. Phe, Leu, Met, Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asn, Trp, Tyr, or Cys, at position 399 or
(iv) an amino acid other than Lys, Arg, Ser, Thr, or Trp, e.g. Phe, Leu, Met, Ala, Val, Gly, Ile, Asn, His, Asp, Glu, Gln, Pro, Tyr, or Cys, at position 366.
In one embodiment, the first heavy chain has an Arg, Ala, His or Gly at position 409, and the second heavy chain has
(i) a Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or
(ii) a Trp at position 370, or
(iii) an Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr at position 399, or
(iv) an Ala, Asp, Glu, His, Asn, Val, Gln, Phe, Gly, Ile, Leu, Met, or Tyr at position 366.
In one embodiment, the first heavy chain has an Arg at position 409, and the second heavy chain has
(i) an Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or
(ii) a Trp at position 370, or
(iii) a Phe, His, Lys, Arg or Tyr at position 399, or
(iv) an Ala, Asp, Glu, His, Asn, Val, Gln at position 366.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises first and second heavy chains, wherein (i) the amino acid in the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L in said first heavy chain, and the amino acid in the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R in said second heavy chain, or (ii) the amino acid in the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R in said first heavy chain, and the amino acid in the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L in said second heavy chain.
In a further embodiment said first and second heavy chain are of a human IgG1 isotype.
In another further embodiment said first and second heavy chain are of a human IgG2 isotype.
In another further embodiment said first and second heavy chain are of a human IgG3 isotype.
In another embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises first and second heavy chains of the human IgG4 isotype, wherein (i) the amino acid in the position corresponding to S228 in a human IgG4 heavy chain according to EU numbering is P in said first heavy chain, and the amino acid in the position corresponding to S228, F405 and R409 in a human IgG4 heavy chain according to EU numbering is P, L and K, respectively, in said second heavy chain, or (ii) the amino acid in the position corresponding to S228, F405 and R409 in a human IgG4 heavy chain according to EU numbering is P, L and K, respectively, in said first heavy chain, and the amino acid in the position corresponding to S228 in a human IgG4 heavy chain according to EU numbering is P in said second heavy chain.
If reference is made herein to amino acids at certain positions of the first heavy chain and/or amino acids at certain positions of the second heavy chain, such reference is to be understood to include embodiments wherein the amino acids at certain positions of the first heavy chain are present at the corresponding positions of the second heavy chain rather than the first heavy chain and/or the amino acids at certain positions of the second heavy chain are present at the corresponding positions of the first heavy chain rather than the second heavy chain.
In addition to the above-specified amino-acid substitutions, said first and second heavy chains may contain further amino-acid substitutions, deletion or insertions relative to wild-type heavy chain sequences.
In a further embodiment, said first and second Fab-arms (or heavy chain constant domains) comprising the first and second heavy chains comprise, except for the specified mutations, a CH3 sequence independently selected from the following:
(IgG1m(a)) (SEQ ID NO:106), (IgG1m(f)) (SEQ ID NO:107), and (IgG1m(ax) (SEQ ID NO:108).
In one embodiment, neither said first nor said second heavy chain comprises a Cys-Pro-Ser-Cys sequence in the (core) hinge region.
In a further embodiment, both said first and said second heavy chain comprise a Cys-Pro-Pro-Cys sequence in the (core) hinge region.
In separate and specific embodiments, one or both Fab-arms comprise a heavy-chain constant region sequence independently selected from SEQ ID NO:109, 110, 111, 112, 113 and 116 (see Table 1).
Methods of Preparing Bispecific Antibodies
Traditional methods such as the hybrid hybridoma and chemical conjugation methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649) can be used in the preparation of the bispecific antibodies of the invention. Co-expression in a host cell of two antibodies, consisting of different heavy and light chains, leads to a mixture of possible antibody products in addition to the desired bispecific antibody, which can then be isolated by, e.g., affinity chromatography or similar methods.
Strategies favoring the formation of a functional bispecific product upon co-expression of different antibody constructs can also be used, e.g., by the method described by Lindhofer et al. (1995 J Immunol 155:219). Fusion of rat and mouse hydridomas producing different antibodies leads to a limited number of heterodimeric proteins because of preferential species-restricted heavy/light chain pairing. Another strategy to promote formation of heterodimers over homodimers is a “knob-into-hole” strategy in which a protuberance is introduced on a first heavy-chain polypeptide and a corresponding cavity in a second heavy-chain polypeptide, such that the protuberance can be positioned in the cavity at the interface of these two heavy chains so as to promote heterodimer formation and hinder homodimer formation. “Protuberances” are constructed by replacing small amino-acid side-chains from the interface of the first polypeptide with larger side chains. Compensatory “cavities” of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino-acid side-chains with smaller ones (U.S. Pat. No. 5,731,168). EP1870459 (Chugai) and WO 2009/089004 (Amgen) describe other strategies for favoring heterodimer formation upon co-expression of different antibody domains in a host cell. In these methods, one or more residues that make up the CH3-CH3 interface in both CH3 domains are replaced with a charged amino acid such that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable. WO2007110205 (Merck) describe yet another strategy, wherein differences between IgA and IgG CH3 domains are exploited to promote heterodimerization.
A preferred method for preparing the bispecific CD40×CD137 antibodies of the present invention includes the methods described in WO 2011/131746 and WO 2013/060867 (Genmab) comprising the following steps:
a) providing a first antibody comprising an Fc region, said Fc region comprising a first CH3 region;
b) providing a second antibody comprising a second Fc region, said Fc region comprising a second CH3 region,
wherein the first antibody is a CD40 antibody comprising two first antigen-binding regions as described herein and the second antibody is a CD137 antibody comprising two second antigen-binding regions as described herein, or vice versa; and wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions;
c) incubating said first antibody together with said second antibody under reducing conditions; and
d) obtaining said bispecific CD40×CD137 antibody.
In one embodiment, said first antibody is incubated together with said second antibody under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide-bond isomerization, wherein the heterodimeric interaction between said first and second antibodies in the resulting heterodimeric antibody is such that no Fab-arm exchange occurs at 0.5 mM GSH after 24 hours at 37° C.
Without being limited to theory, in step c), the heavy-chain disulfide bonds in the hinge regions of the parent antibodies (first and second antibody in step a) and b)) are reduced and the resulting cysteines are then able to form inter heavy-chain disulfide bond with cysteine residues of another parent antibody molecule (originally with a different specificity). In one embodiment of this method, the reducing conditions in step c) comprise the addition of a reducing agent, e.g. a reducing agent selected from the group consisting of: 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercapto-ethanol, preferably a reducing agent selected from the group consisting of: 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine. In a further embodiment, step c) comprises restoring the conditions to become non-reducing or less reducing, for example by removal of a reducing agent, e.g. by desalting. In one particular embodiment, bispecific antibodies are generated as follows: the two parental complementary antibodies, both in the same amount, are incubated with 75 mM 2-mercaptoethylamine-HCl (2-MEA) in buffer (e.g., PBS or Tris-EDTA) at 31° C. for 5 hours; the reduction reaction is stopped by removing the reducing agent 2-MEA using spin columns (e.g., Microcon centrifugal filters, 30 k, Millipore) (Labrijn et al. Nature Protocols, Vol 9 No 10, p2450-2463; 2014). In another particular embodiment, the method is that of Example 3.
For this method, any of the CD40 and CD137 antibodies disclosed herein may be used. In a particular embodiment the first and second antibodies, binding to human CD40 and CD137, respectively, may be chosen so as to obtain a bispecific antibody as described herein.
In one embodiment of this method, said first and/or second antibodies are full-length antibodies.
The Fc regions of the first and second antibodies may be of any isotype, including, but not limited to, an IgG isotype having a subclass selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. In one embodiment of this method, the Fc regions of both said first and said second antibodies are of the IgG1 isotype. In another embodiment, one of the Fc regions of said antibodies is of the IgG1 isotype and the other of the IgG4 isotype. In the latter embodiment, the resulting bispecific antibody comprises an Fc region of an IgG1 and an Fc region of IgG4.
In a further embodiment, one of the parental antibodies has been engineered to not bind Protein A, thus allowing separation of the heterodimeric antibody from said parental homodimeric antibodies by passing the product over a protein A column.
As described above, the sequences of the first and second CH3 regions of the homodimeric parental antibodies are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. More details on these interactions and how they can be achieved are provided in WO 2011/131746 and WO 2013/060867 (Genmab), which are hereby incorporated by reference in their entirety.
In particular, a stable bispecific CD40×CD137 antibody can be obtained at high yield using the above method of the invention on the basis of two homodimeric antibodies which bind CD40 and CD137, respectively, and contain only a few, fairly conservative, asymmetrical mutations in the CH3 regions. Asymmetrical mutations mean that the sequences of said first and second CH3 regions contain amino acid substitutions at non-identical positions.
The bispecific antibodies of the invention may also be obtained by co-expression of constructs encoding the first and second polypeptides in a single cell. Thus, in a further aspect, the invention relates to a method for producing a bispecific antibody, said method comprising the following steps:
a) providing a first nucleic-acid construct encoding a first polypeptide comprising a first Fc region and a first antigen-binding region binding to human CD40 according to any aspect or embodiment herein, said first Fc region comprising a first CH3 region,
b) providing a second nucleic-acid construct encoding a second polypeptide comprising a second Fc region and a second antigen-binding region binding to human CD137 according to any aspect or embodiment herein, said second Fc region comprising a second CH3 region,
wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions, and wherein in said first CH3 region at least one of the amino acids in the positions corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering has been substituted, and in said second CH3 region at least one of the amino acids in the positions corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering has been substituted, and wherein said first and said second heavy chains are not substituted in the same positions,
optionally wherein said first and second nucleic acid constructs encode light chain sequences of said first and second antibodies,
c) co-expressing said first and second nucleic-acid constructs in a host cell, and
d) obtaining said heterodimeric protein from the cell culture.
Thus, the present invention also relates to a recombinant eukaryotic or prokaryotic host cell which produces a bispecific antibody of the present invention.
In one embodiment of the present invention, the bispecific antibody is obtained by any of the methods according to the present invention.
Suitable expression vectors, including promoters, enhancers, etc., and suitable host cells for the production of antibodies are well-known in the art. Examples of host cells include yeast, bacterial and mammalian cells, such as CHO or HEK cells.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises first and second CH3 regions, except for the specified mutations, comprising the sequence of SEQ ID NO:107 (IgG1m(f)).
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a second Fc-region, wherein neither said first nor said second Fc-region comprises a Cys-Pro-Ser-Cys sequence in the hinge region.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a second Fc-region, wherein both of said first and said second Fc-region comprise a Cys-Pro-Pro-Cys sequence in the hinge region.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a second Fc-region, wherein the first and second Fc-regions are human antibody Fc-regions.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a second Fc-region, wherein said first and second Fc region, except for the specified mutations, comprise a sequence independently selected from the group consisting of SEQ ID NOS:109, 110, 111, 112, 113 and 116.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a second Fc-region, wherein the first and second antigen-binding regions are from heavy-chain antibodies.
In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc-region and a second Fc-region, wherein the first and second antigen-binding regions comprise a first and second light chain.
In further embodiments, the co-expression method according to the invention comprises any of the further features described under the in vitro method above.
Inert Format
The effector functions mediated by the Fc region of an antibody allow for the destruction of foreign entities, such as the killing of pathogens and the clearance and degradation of antigens. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are initiated by binding of the Fc region to Fc receptor (FcR)-bearing cells, whereas complement-dependent cytotoxicity (CDC) and complement-dependent cell-mediated cytotoxicity (CDCC) are initiated by binding of the Fc region to C1q, which initiates the classical route of complement activation.
Fc-mediated effector function, such as ADCC and complement activation, have been suggested to contribute to the therapeutic efficacy of monoclonal antibodies used for the treatment of cancer (Weiner et al. Cell 2012, 148:1081-1084).
The multispecific antibody, such as a bispecific antibody, according to the present invention binds to CD137 which is expressed on T-cells, e.g. CD4+ and/or CD8+ T-cells. By concomitant binding of the antibody to CD40, which is expressed on e.g. antigen-presenting cells (APCs), provides stimulation to both APCs expressing CD40 and T-cells expressing CD137 and thereby e.g. T-cell proliferation can be increased.
In general, binding of an antibody to a target antigen expressed by a cell may lead to interactions with effector molecules such as Fc receptors or complement proteins which may induce Fc-mediated effector functions, such as ADCC or complement activation, which may result in killing of the cell expressing said target antigen.
The use of the multispecific antibody, such as a bispecific antibody, according to the present invention is based on its ability to provide co-stimulation to APCs and T cells.
It is, in a particular embodiment, preferred that the multispecific antibody does not bind to FcRs, e.g. FcγRs, and therefore does not induce FcR-mediated cross-linking.
It is, in a further embodiment, preferred that the multispecific antibody does not engage effector functions so as to avoid killing of the CD40 and/or CD137 expressing cells.
In one aspect of the present invention, the multispecific CD40×CD137 antibody according to the present invention comprises (i) a first binding arm comprising a first heavy chain and a first antigen-binding region and (ii) a second binding arm comprising a second heavy chain and a second antigen-binding region, according to any aspect or embodiment described herein.
In one embodiment the multispecific antibody according to present invention comprises a first and a second heavy chain, wherein said antibody induces and/or enhances Fc-mediated effector function to a lesser extent compared to a multispecific antibody comprising the same first and second antigen-binding regions as said antibody, and comprising two heavy chains comprising a human IgG1 hinge, CH2 and CH3 regions.
In one embodiment, the multispecific antibody according to present invention comprises a first and a second antigen-binding region and a first and a second heavy chain, each of the first and second heavy chains comprising a human IgG1 hinge, CH2 and CH3 regions, wherein at least one of the first and second heavy chain comprises a modification so as to induce and/or enhance Fc-mediated effector function to a lesser extent compared to a reference multispecific antibody comprising the same first and second antigen-binding regions as said antibody, and comprising two heavy chains comprising a human IgG1 hinge, CH2 and CH3 regions without said modification.
In one embodiment, said first and second heavy chains are modified so that the multispecific antibody induces and/or enhances Fc-mediated effector function to a lesser extent compared to a multispecific antibody which is identical except for comprising non-modified first and second heavy chains.
In one embodiment, said Fc-mediated effector function may be measured by binding to Fcγy-receptors, binding to C1q, or induction of Fc-mediated cross-linking of FcRs.
In one embodiment, said Fc-mediated effector function is measured by binding to C1q.
In one embodiment, said first and second heavy and light chain constant sequences have been modified so that binding of C1q to said multispecific antibody is reduced compared to a wild-type multispecific antibody by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, wherein C1q binding is determined by ELISA.
Human IgG1 is known for its ability to induce Fc-mediated effector functions, while other human isotypes, such as IgG4, are less able to induce Fc-mediated effector functions.
The first and second heavy chains may each be of any isotype, including, but not limited to, an IgG1 isotype selected from the groups consisting of IgG1, IgG2, IgG3 and IgG4, and may optionally comprise one or more mutations or modifications. In one embodiment, each of the first and second heavy chains is of the IgG4 isotype or derived therefrom, optionally with one or more mutations or modifications. In one embodiment, each of the first and second heavy chains is of the IgG1 isotype or derived therefrom, optionally with one or more mutations or modifications. In another embodiment, one of the heavy chains is of the IgG1 isotype and the other of the IgG4 isotype, or is derived from such respective isotypes, optionally with one or more mutations or modifications.
In one embodiment, one or both of the first and heavy chains are such that an antibody comprising two first or two second heavy chains would be effector-function-deficient. For example, the first and second heavy chains may be of an IgG4 isotype, or a non-IgG4 type, e.g. IgG1, IgG2 or IgG3, which has been mutated such that the ability to mediate effector functions, such as ADCC, has been reduced or even eliminated compared to non-mutated heavy chains. Such mutations have e.g. been described in Dall'Acqua W F et al., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J Virol.; 75(24):12161-12168 (2001). The multispecific antibody according to the present invention may comprise modifications in the first and second heavy chains compared to a wild type human IgG1 sequence. A multispecific antibody comprising such modifications in the Fc region of the antibody may become an inert, or non-activating, multispecific antibody. The term “inertness”, “inert” or “non-activating” as used herein, refers to an Fc region which is at least not able to bind any Fcγ(gamma) receptors, bind to C1q, or induce Fc-mediated cross-linking of FcRs. The inertness of an Fc region, or the first and/or second heavy chain of a multispecific antibody of the present invention may be tested with a bivalent, monospecific antibody comprising said Fc region, or two first heavy chains or two second heavy chains. It may also be tested with a multispecific antibody comprising a first and a second heavy chain.
Several variants can be constructed to make the Fc region of an antibody inactive for interactions with Fcγ receptors and C1q for therapeutic antibody development. The present invention is not limited to any specific mutation relevant for reducing Fc-mediated effector functions. Examples of such variants are described herein.
Thus, amino acids in the Fc region that play a dominant role in the interactions with C1q and the Fcγ receptors may be modified. Examples of amino acid positions that may be modified include positions L234, L235 and P331.
Hence, in one embodiment, in at least one of said first and second heavy chains the amino acid in at least one position corresponding to L234, L235 and P331 in a human IgG1 heavy chain according to EU numbering, may be A, A and S, respectively. (Xu et al., 2000, Cell Immunol. 200(1):16-26; Oganesyan et al., 2008, Acta Cryst. (D64):700-4). Also, L234F and L235E amino acid substitutions can result in Fc regions with abrogated interactions with Fcγ receptors and C1q (Canfield et al., 1991, J. Exp.Med. (173):1483-91; Duncan et al., 1988, Nature (332):738-40). Hence, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to L234 and L235 in a human IgG1 heavy chain according to EU numbering, may be F and E, respectively. A D265A amino acid substitution can decrease binding to all Fc gamma receptors and prevent ADCC (Shields et al., 2001, J. Biol. Chem. (276):6591-604). Hence, in one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to D265 in a human IgG1 heavy chain according to EU numbering, may be A. Binding to C1q can be abrogated by mutating positions D270, K322, P329, and P331. Mutating these positions to either D270A or K322A or P329A or P331A can make the antibody deficient in CDC activity Idusogie E E, et al., 2000, J Immunol. 164: 4178-84). Hence, in one embodiment, in at least one of said first and second heavy chain, the amino acids in at least one position corresponding to D270, K322, P329 and P331 in a human IgG1 heavy chain according to EU numbering, may be A, A, A, and A, respectively.
An alternative approach to minimize the interaction of the Fc region with Fcγ receptors and C1q is by removal of the glycosylation site of an antibody. Mutating position N297 to e.g. Q, A, or E removes a glycosylation site which is critical for IgG-Fcγ receptor interactions. Hence, in one embodiment, in at least one of said first and second heavy chains, the amino acid in a position corresponding to N297, may be G, Q, A or E in a human IgG1 heavy chain according to EU numbering (Leabman et al., 2013, MAbs; 5(6):896-903). Another alternative approach to minimize interaction of the Fc region with Fcγ receptors may be obtained by the following mutations; P238A, A327Q, P329A or E233P/L234V/L235A/G236del (Shields et al., 2001, J. Biol. Chem. (276):6591-604).
Alternatively, human IgG2 and IgG4 subclasses are considered naturally compromised in their interactions with C1q and Fc gamma Receptors although interactions with Fcγ receptors were reported (Parren et al., 1992, J. Clin Invest. 90: 1537-1546; Bruhns et al., 2009, Blood 113: 3716-3725). Mutations abrogating these residual interactions can be made in both isotypes, resulting in reduction of unwanted side-effects associated with FcR binding. For IgG2, these include V234A and G237A, and for IgG4, L235E. Hence, in one embodiment, in at least one of said first and second heavy chains, such as in both said first and second heavy chains, the amino acid in a position corresponding to V234 and G237 in a human IgG2 heavy chain according to EU numbering, may be A and A, respectively. In one embodiment, the amino acid in a position corresponding to L235 in a human IgG4 heavy chain according to EU numbering, may be E.
Other approaches to further minimize the interaction with Fcγ receptors and C1q in IgG2 antibodies include those described in WO2011066501 and Lightle, S., et al., 2010, Protein Science (19):753-62.
The hinge region of the antibody can also be of importance with respect to interactions with Fcγ receptors and complement (Brekke et al., 2006, J Immunol 177:1129-1138; Dall'Acqua W F, et al., 2006, J Immunol 177:1129-1138). Accordingly, mutations in or deletion of the hinge region can influence effector functions of an antibody.
In one embodiment, the multispecific antibody comprises a first and a second heavy chain, wherein in at least one of said first and second immunoglobulin heavy chains one or more amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering, are not L, L, D, N, and P, respectively.
In one embodiment, in both the first and second heavy chains one or more amino acids in the position corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering, are not L, L, D, N, and P, respectively.
In another embodiment, in at least one of the first and second heavy chains one or more amino acids in the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain according to EU numbering, are not L, L and D, respectively, and the amino acids in the positions corresponding to N297 and P331 in a human IgG1 heavy chain according to EU numbering, are N and P, respectively.
In one embodiment, one or both of the heavy chains comprise a mutation removing the acceptor site for Asn-linked glycosylation or is otherwise manipulated to change the glycosylation properties. For example, in an IgG1 Fc-region, an N297Q mutation can be used to remove an Asn-linked glycosylation site. Accordingly, in a specific embodiment, one or both heavy chains comprise an IgG1 wildtype sequence with an N297Q mutation.
The term “amino acid corresponding to position” as used herein refers to an amino acid position number in a human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins may be found by alignment with human IgG1. Unless otherwise stated or contradicted by context, the amino acids of the constant region sequences are herein numbered according to the EU-index of numbering (described in Kabat, E. A. et al., 1991, Sequences of proteins of immunological interest. 5th Edition—US Department of Health and Human Services, NIH publication No. 91-3242, pp 662, 680, 689). Thus, an amino acid or segment in one sequence that “corresponds to” an amino acid or segment in another sequence is one that aligns with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW or similar, typically at default settings and has at least 50%, at least 80%, at least 90%, or at least 95% identity to a human IgG1 heavy chain. It is considered well-known in the art how to align a sequence or segment in a sequence and thereby determine the corresponding position in a sequence to an amino acid position according to the present invention.
In the context of the present invention, the amino acid position may be defined as described above.
The term “the amino acid is not” or similar wording when referring to amino acids in a heavy chain is to be understood to mean that the amino acid is any other amino acid than the specific amino acid mentioned. For example, the amino acid in the position corresponding to L234 in a human IgG1 heavy chain is not L, means that the amino acid may be any of the other naturally or non-naturally occurring amino acids than L.
In one embodiment, in said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering, is not D.
In one embodiment, in said first and second heavy chains the amino acid in the position corresponding to D265 in a human IgG1 heavy chain according to EU numbering, is not D, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering, are N and P, respectively.
In one embodiment, in said first and second heavy chains the amino acids in the positions corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is hydrophobic or polar amino acids.
The term “hydrophobic” as used herein in relation to an amino acid residue, refers to an amino acid residue selected from the group consisting of: A, C, F, G, H, I, L, M, R, T, V, W, and Y.
Thus, in one embodiment, in said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group of amino acids consisting of: A, C, F, G, H, I, L, M, R, T, V, W and Y.
The term “polar” as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of: C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: C, E, H, K, N, Q, R, S, and T.
In another embodiment, in said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is an aliphatic uncharged, aromatic or acidic amino acid.
The term “aliphatic uncharged” as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of: A, G, I, L, and V.
Thus, in one embodiment, in said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: A, G, I, L, and V.
The term “aromatic” as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of: F, T, and W.
Thus, in one embodiment, in said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: F, T, and W.
The term “acidic” as used herein in relation to amino acid residues, refers to any amino acid residue chosen from the group consisting of: D and E.
Thus, in one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: D and E.
Thus, in one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: D and E.
In a particular embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: A, E, F, G, I, L, T, V, and W.
In a particular embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: A, E, F, G, I, L, T, V, and W.
In one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering, is not D.
In one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering, is not D.
In one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to D265 in a human IgG1 heavy chain according to EU numbering, is not D, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering, are N and P, respectively.
In one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to D265 in a human IgG1 heavy chain according to EU numbering, is not D, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering, are N and P, respectively.
In one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is hydrophobic or polar amino acid.
In one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is hydrophobic or polar amino acid.
Thus, in one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group of amino acids consisting of: A, C, F, G, H, I, L, M, R, T, V, W and Y.
Thus, in one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human heavy chain according to EU numbering is selected from the group consisting of: C, E, H, K, N, Q, R, S, and T. In one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group of amino acids consisting of: A, C, F, G, H, I, L, M, R, T, V, W and Y.
In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to position D265 in a human heavy chain according to EU numbering is selected from the group consisting of: C, E, H, K, N, Q, R, S, and T.
In another embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is aliphatic uncharged, aromatic or acidic amino acids.
Thus, in one embodiment, in least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: A, G, I, L, and V.
Thus, in one embodiment, in least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: F, T, and W.
Thus, in one embodiment, in least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of: D and E.
In a particular embodiment, in least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: A, E, F, G, I, L, T, V, and W.
In another embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is aliphatic uncharged, aromatic or acidic amino acids.
Thus, in one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: A, G, I, L, and V.
Thus, in one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: F, T, and W.
Thus, in one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of: D and E.
In a particular embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: A, E, F, G, I, L, T, V, and W.
In further embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position N297 in a human IgG1 heavy chain according to EU numbering, is not N.
In one embodiment, in at least one of the first and second heavy chains the amino acid in the position corresponding to N297 in a human IgG1 heavy chain according to EU numbering, is not N, and the amino acid in the position corresponding to position P331 in a human IgG1 heavy chain according to EU numbering, is P.
In one embodiment, in said first and second heavy chains the amino acid in the position corresponding to positions N297 in a human IgG1 heavy chain according to EU numbering, is not N.
In one embodiment, in both the first and second heavy chains the amino acid in the position corresponding to N297 in a human IgG1 heavy chain according to EU numbering, is not N, and the amino acid in the position corresponding to position P331 in a human IgG1 heavy chain according to EU numbering, is P.
In further embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering, are not L and L, respectively.
In one embodiment, in at least one of the first and second heavy chains the amino acids in the positions corresponding to L234 and L235 in a human IgG1 heavy chain according to EU numbering, are not L and L, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering, are N and P, respectively.
In one embodiment, in at least one of said first and second heavy chains the amino acids corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of: A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, Y, V.
In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy according to EU numbering chain are hydrophobic or polar amino acids.
Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group consisting of: A, C, F, G, H, I, M, R, T, V, W, and Y.
Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group of amino acids consisting of: C, D, E, H, K, N, Q, R, S, and T.
In a particular embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group consisting of: A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y.
In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering, are not L and L, respectively.
In one embodiment, in both the first and second heavy chains the amino acids in the positions corresponding to L234 and L235 in a human IgG1 heavy chain according to EU numbering, are not L and L, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering, are N and P, respectively.
In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to L234 and L235 in a human IgG1 heavy chain according to EU numbering are hydrophobic or polar amino acids.
In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group consisting of: A, C, F, G, H, I, M, R, T, V, W, and Y.
In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group of amino acids consisting of: C, D, E, H, K, N, Q, R, S, and T.
In a particular embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group consisting of: A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y.
In another embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy according to EU numbering chain are aliphatic uncharged, aromatic or acidic amino acids.
Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group consisting of: A, G, I, and V.
Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group consisting of: F, T, and W.
Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group consisting of; D and E.
In a particular embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to L234 and L235 are each selected from the group consisting of: A, D, E, F, G, I, T, V, and W.
In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering, are F and E; or A and A, respectively.
In one embodiment, in at least one of the first and second heavy chains the amino acids in the positions corresponding to L234 and L235 in a human IgG1 heavy chain according to EU numbering, are F and E; or A and A, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering, are N and P, respectively.
In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering, are F and E; or A and A, respectively.
In one embodiment, in both the first and second heavy chains the amino acids in the positions corresponding to L234 and L235 in a human IgG1 heavy chain according to EU numbering, are F and E; or A and A, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering, are N and P, respectively.
In a particular embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering, are F and E, respectively.
In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering, are F and E, respectively.
In one embodiment, in at least one of said first and second heavy chains at least the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering, are A and A, respectively.
In one embodiment, in both said first and second heavy chains at least the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering, are A and A, respectively.
In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering, are not L, L, and D, respectively.
In one embodiment, in at least one of the first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering, are not L, L and D, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering, are N and P, respectively.
In one embodiment, in at least one of said first and second heavy chains the amino acids corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of: A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, Y, V, and W, and the amino acid corresponding to position D265 is selected from the group consisting of: A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Y, V, and W.
In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain according to EU numbering are hydrophobic or polar amino acids.
Thus, in one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group of amino acids consisting of: A, C, F, G, H, I, L, M, R, T, V, W and Y, and the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group consisting of: A, C, F, G, H, I, M, R, T, V, W, and Y.
Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group of amino acids consisting of: C, D, E, H, K, N, Q, R, S, and T, the amino acid in the position corresponding to position D265 in a human heavy chain according to EU numbering is selected from the group consisting of: C, E, H, K, N, Q, R, S, and T.
In a particular embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group consisting of: A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, and the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.
In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are hydrophobic or polar amino acids.
In one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group of amino acids consisting of: A, C, F, G, H, I, L, M, R, T, V, W and Y, and the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group consisting of: A, C, F, G, H, I, M, R, T, V, W, and Y.
In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group of amino acids consisting of: C, D, E, H, K, N, Q, R, S, and T, the amino acid in the position corresponding to position D265 in a human heavy chain according to EU numbering is selected from the group consisting of: C, E, H, K, N, Q, R, S, and T.
In a particular embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group consisting of: A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, and the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.
In another embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain according to EU numbering are aliphatic uncharged, aromatic or acidic amino acids.
Thus, in one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: A, G, I, L, and V, and the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group consisting of: A, G, I, and V.
Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain according to EU numbering are each selected from the group consisting of: F, T, and W.
Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are each selected from the group consisting of: D and E.
In a particular embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: A, E, F, G, I, L, T, V, and W, and the amino acids in the positions corresponding to L234 and L235 are each selected from the group consisting of: A, D, E, F, G, I, T, V, and W.
In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain according to EU numbering, are not L, L, and D, respectively.
In one embodiment, in both the first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering, are not L, L, and D, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering, are N and P, respectively.
In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are aliphatic uncharged, aromatic or acidic amino acids.
In one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of; A, G, I, L, and V, and the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group consisting of; A, G, I, and V.
In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are each selected from the group consisting of; D and E.
In a particular embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of: A, E, F, G, I, L, T, V, and W, and the amino acids in the positions corresponding to L234 and L235 are each selected from the group consisting of: A, D, E, F, G, I, T, V, and W.
In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering, are F, E, and A; or A, A, and A, respectively.
In one embodiment, in at least one of the first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering, are F, E, and A; or A, A, and A, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering, are N and P, respectively.
In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering, are F, E, and A; or A, A, and A, respectively.
In one embodiment, in both the first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering, are F, E, and A; or A, A, and A, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering, are N and P, respectively.
In a particular embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering, are F, E, and A, respectively.
In a particularly preferred embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering, are F, E, and A, respectively.
In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering, are A, A, and A, respectively.
In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering, are A, A, and A, respectively.
In another embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering, are F, E, A, Q, and S, respectively.
In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering, are F, E, A, Q, and S, respectively.
In a particular embodiment said first antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1, 2 and 3, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:4, YTS and 5, respectively; and said second antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:64, 65 and 66, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:67, GAS and 68, respectively, (CD137 clone 009), and in at least one of the first and second heavy chains, such as both said first and second heavy chain, the amino acids in positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.
In another embodiment said first antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:1, 2 and 3, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:4, YTS and 5, respectively; and said second antigen-binding region comprises heavy chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:36, 37 and 38, respectively, and light chain variable region CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs:39, SAS and 40, respectively, (CD137 clone 005), and in at least one of the first and second heavy chains, such as both said first and second heavy chain, the amino acids in positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.
A non-activating Fc region prevents the antibody from interacting with Fc-receptors present on blood cells, such as monocytes, or with C1q to activate the classical complement pathway. Reduction of the Fc activity was tested in antibody variants that contain different combinations of amino acid substitutions in the Fc region. Three amino acid substitutions were introduced in the parental antibodies of the present invention, which include the mutations L234F, L235E, and D265A. Substitutions in these three amino acid positions were introduced in the K409R and/or F405L IgG1 backbone. The resulting non-activating antibody variant is termed with the suffix “FEAR” or “FEAL”, respectively. Said parental antibodies were used in to generate bispecific antibodies of the present invention as described in the examples.
In one aspect, the multispecific antibodies according to the invention may be modified in the light chain and/or heavy chain to increase the expression level and/or production yield. In one embodiment, the antibodies according to the invention may be modified in the light chain. Such modifications are known in the art and may be performed according to the methods described in e.g. Zheng, L., Goddard, J. P., Baumann, U., & Reymond, J. L. (2004). Expression improvement and mechanistic study of the retro-Diels-Alderase catalytic antibody 10F11 by site-directed mutagenesis. Journal of Molecular Biology, 341(3), 807-14.
In a further embodiment of the invention, one or both of the antibodies forming part of the multispecific antibody of the invention have been engineered to reduce or increase the binding to the neonatal Fc receptor (FcRn) in order to manipulate the serum half-life of the multispecific antibody. Techniques for increasing or reducing the serum half-life are well-known in the art. See for example Dall'Acqua et al. 2006, J. Biol. Chem., 281:23514-24; Hinton et al. 2006, J. Immunol., 176:346-56; and Zalevsky et al. 2010 Nat. Biotechnol., 28:157-9.
In one aspect, the multispecific antibody as defined in any of the embodiments disclosed herein comprises a first constant heavy chain (HC) and a first constant light chain (LC), wherein the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain of SEQ ID NO:109 of both the first heavy chain and the second heavy chain are F, E, and A, respectively.
In one embodiment, the multispecific antibody as defined in any of the embodiments disclosed herein comprises a first and second constant heavy chain (HC) and a first and second constant light chain (LC), wherein the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain of SEQ ID NO:109 of both the first heavy chain and the second heavy chain are F and E, respectively.
In one embodiment, the multispecific antibody comprises a first and a second heavy chain, wherein the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering of both the first heavy chain and the second heavy chain are F and E, respectively, and wherein (i) the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the first heavy chain is L, and the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the first heavy chain is R, and the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the second heavy chain is L.
In one embodiment, the multispecific antibody comprises a first and a second heavy chain, wherein the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering of both the first and the second heavy chain are F, E, and A, respectively, and wherein the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the first heavy chain is L, and the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the second heavy chain is R. Thus in a further embodiment, said first heavy chain comprises the constant heavy chain sequence as set forth in SEQ ID NO:113; and the second heavy chain comprises the constant heavy chain sequence as set forth in SEQ ID NO:112.
In one embodiment, the multispecific antibody comprises a first and a second heavy chain, wherein the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering of both the first and second heavy chain are F, E, and A, respectively, and wherein the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering of the first heavy chain is R, and the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering of the second heavy chain is L. Thus in a further embodiment, said first heavy chain comprises the constant heavy chain sequence as set forth in SEQ ID NO:112; and the second heavy chain comprises the constant heavy chain sequence as set forth in SEQ ID NO:113.
Nucleic Acids
The present invention also relates to a nucleic acid encoding one or more amino acid sequences according to any aspect or embodiment disclosed herein.
The present invention also relates to a nucleic acid encoding a multispecific antibody as defined in any aspect or embodiment disclosed herein.
The present invention also relates to an expression vector comprising a nucleic acid of the present invention.
The present invention also relates to a host cell comprising a nucleic acid or an expression vector according to the present invention.
In one embodiment said host cell is a recombinant eukaryotic, recombinant prokaryotic, or recombinant microbial host cell.
In a further embodiment, the expression vector further comprises a nucleotide sequence encoding the constant region of a light chain, a heavy chain or both light and heavy chains of an antibody, e.g. a human antibody.
An expression vector in the context of the present invention may be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (a nucleic acid sequence comprising a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors obtained from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, a nucleic acid is comprised in a naked DNA or RNA vector, including, for example, a linear expression element (as described in for instance Sykes and Johnston, Nat Biotech 17, 355 59 (1997)), a compacted nucleic acid vector (as described in for instance U.S. Pat. No. 6,077,835 and/or WO 00/70087), a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sized nucleic acid vector (as described in for instance Schakowski et al., Mol Ther 3, 793 800 (2001)), or as a precipitated nucleic acid vector construct, such as a CaP04-precipitated construct (as described in for instance WO200046147, Benvenisty and Reshef, PNAS USA 83, 9551 55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such nucleic acid vectors and the usage thereof are well known in the art (see for instance U.S. Pat. Nos. 5,589,466 and 5,973,972).
In one embodiment, the vector is suitable for expression of the CD40 antibody and/or the CD137 antibody in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503 5509 (1989), pET vectors (Novagen, Madison Wis.) and the like).
An expression vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be employed. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), and Grant et al., Methods in Enzymol 153, 516 544 (1987)).
An expression vector may also or alternatively be a vector suitable for expression in mammalian cells, e.g. a vector comprising glutamine synthetase as a selectable marker, such as the vectors described in Bebbington (1992) Biotechnology (NY) 10:169-175.
A nucleic acid and/or vector may also comprises a nucleic acid sequence encoding a secretion/localization sequence, which can target a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or into cell culture media. Such sequences are known in the art, and include secretion leader or signal peptides.
The expression vector may comprise or be associated with any suitable promoter, enhancer, and other expression-facilitating elements. Examples of such elements include strong expression promoters (e. g., human CMV IE promoter/enhancer as well as RSV, SV40, SL3 3, MMTV, and HIV LTR promoters), effective poly (A) termination sequences, an origin of replication for plasmid product in E. coli, an antibiotic resistance gene as selectable marker, and/or a convenient cloning site (e.g., a polylinker). Nucleic acids may also comprise an inducible promoter as opposed to a constitutive promoter such as CMV IE.
In one embodiment, the CD40 and/or CD137 antibody-encoding expression vector may be positioned in and/or delivered to the host cell or host animal via a viral vector.
In an even further aspect, the invention relates to a host cell comprising the first and second nucleic-acid constructs specified herein above.
Thus the present invention also relates to a recombinant eukaryotic or prokaryotic host cell which produces a multispecific antibody of the present invention, such as a transfectoma.
The first, CD40-specific, antibody may be expressed in a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, which produces an antibody as defined herein. The second, CD137-specific, antibody may likewise be expressed in a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, which produces an antibody. Such antibodies may be used to prepare a multispecific antibody according to the present invention. A multispecific antibody according to the present invention may also be expressed in a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma.
Examples of host cells include yeast, bacterial, plant and mammalian cells, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F™, PER.C6® or NSO cells or lymphocytic cells. For example, in one embodiment, the host cell may comprise a first and second nucleic acid construct stably integrated into the cellular genome. In another embodiment, the present invention provides a cell comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a first and second nucleic acid construct as specified above.
In an even further aspect, the invention relates to a transgenic non-human animal or plant comprising nucleic acids encoding one or two sets of a human heavy chain and a human light chain, wherein the animal or plant produces a multispecific antibody of the invention.
The first, CD40-specific, antibody and/or second, CD137-specific, antibody may also be produced by a hybridoma, a transgenic non-human animal or plant comprising nucleic acids encoding one or two sets of a human heavy chain and a human light chain, wherein the animal or plant produces an antibody for use in a multispecific antibody or a multispecific antibody of the invention.
In one aspect, the invention relates to a nucleic acid encoding one or more amino acid sequences set out in Table 1.
In one aspect, the invention relates to an expression vector comprising
(i) a nucleic acid sequence encoding a heavy chain sequence of a first binding arm according to any one of the embodiments disclosed herein;
(ii) a nucleic acid sequence encoding a light chain sequence of a first binding arm according to any one of the embodiments disclosed herein;
(iii) a nucleic acid sequence encoding a heavy chain sequence of a second binding arm according to any one of the embodiments disclosed herein;
(iv) a nucleic acid sequence encoding a light chain sequence of a second binding arm according to any one of the of the embodiments disclosed herein;
(v) the nucleic acid set forth in (i) and the nucleic acid set forth in (ii);
(vi) the nucleic acid set forth in (iii) and the nucleic acid set forth in (iv).
(vii) the nucleic acid set forth in (i), (ii), (iii) and (iv).
In a particular embodiment, the nucleic acid may encode a heavy chain variable region comprising the VH CDR1, CDR2 and CDR3 of the CD40 antibody listed in Table 1 and encoding a human IgG1 heavy chain having a sequence selected from the group consisting of SEQ ID NO:110, 111, 112, 113 and 116.
In another embodiment, the nucleic acid may encode a heavy chain variable region comprising the VH CDR1, CDR2 and CDR3 of one the CD137 antibodies listed in Table 1, i.e. any one of clones 001-012, and encoding a human IgG1 heavy chain having a sequence selected from the group consisting of SEQ ID NO:110, 111, 112, 113 and 116.
In separate and specific embodiments, a nucleic acid, nucleic acid construct, a combination of a first and a second nucleic acid construct, an expression vector, or a combination of a first and a second expression vector according to the present invention may encode
(a) a HC comprising (i) a VH comprising the VH CDR1, CDR2 and CDR3 of the CD40 antibody in Table 1, and primarily human framework regions, optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and (ii) a human IgG1 heavy chain having a sequence selected from the group consisting of SEQ ID NO:110, 111, 112, 113 and 116;
(b) a HC comprising (i) a VH comprising the VH CDR1, CDR2 and CDR3 of one the CD137 antibodies listed in Table 1, i.e. any one of clones 001-012, and primarily human framework regions, optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and (ii) a human IgG1 heavy chain having a sequence selected from the group consisting of SEQ ID NO:110, 111, 112, 113 and 116;
(c) an LC comprising (i) a VL comprising the VL CDR1, CDR2 and CDR3 of the CD40 antibody in Table 1, and primarily human framework regions, optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and (ii) a light chain constant region having the sequence of SEQ ID NO:114;
(d) an LC comprising (i) a VL comprising the VL CDR1, CDR2 and CDR3 of one the CD137 antibodies listed in Table 1, i.e. any of clones 001-012, and primarily human framework regions, optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and (ii) a light chain constant region having the sequence of SEQ ID NO:114;
(e) both (a) and (b);
(f) both (a) and (c);
(g) both (b) and (d);
(h) both (c) and (d); or
(i) both (a), (b), (c) and (d).
In other separate and specific embodiments, a nucleic acid, nucleic acid construct, a combination of a first and a second nucleic acid construct, an expression vector, or a combination of a first and a second expression vector according to the present invention may encode
(a) a HC comprising (i) a VH comprising the VH CDR1, CDR2 and CDR3 of SEQ ID NOS:1, 2 and 3, and primarily human framework regions, optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and (ii) a human IgG1 heavy chain having a sequence selected from the group consisting of SEQ ID NO:110, 111, 112, 113 and 116;
(b) a HC comprising (i) a VH comprising the VH CDR1, CDR2 and CDR3 of SEQ ID NO: 64, 65 and 66, and primarily human framework regions, optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and (ii) a human IgG1 heavy chain having a sequence selected from the group consisting of SEQ ID NO:110, 111, 112, 113 and 116;
(c) an LC comprising (i) a VL comprising the VL CDR1, CDR2 and CDR3 of SEQ ID NO:4, YTS and SEQ ID NO:5, and primarily human framework regions, optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and (ii) a light chain constant region having the sequence of SEQ ID NO:114;
(d) an LC comprising (i) a VL comprising the VL CDR1, CDR2 and CDR3 of SEQ ID NO: 67, GAS and SEQ ID NO:68, and primarily human framework regions, optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and (ii) a light chain constant region having the sequence of SEQ ID NO:114;
(e) both (a) and (b);
(f) both (a) and (c);
(g) both (b) and (d);
(h) both (c) and (d); or
(i) both (a), (b), (c) and (d).
In other separate and specific embodiments, a nucleic acid, nucleic acid construct, a combination of a first and a second nucleic acid construct, an expression vector, or a combination of a first and a second expression vector according to the present invention may encode
(a) a HC comprising a VH comprising SEQ ID NO:117 and a human IgG1 heavy chain having a sequence selected from the group consisting of SEQ ID NO:110, 111, 112, 113 and 116;
(b) a HC comprising a VH comprising SEQ ID NO:123 and a human IgG1 heavy chain having a sequence selected from the group consisting of SEQ ID NO:110, 111, 112, 113 and 116;
(c) an LC comprising a VL comprising SEQ ID NO:121 and a light chain constant region having the sequence of SEQ ID NO:114;
(d) an LC comprising a VL comprising SEQ ID NO:127 and a light chain constant region having the sequence of SEQ ID NO:114;
(e) both (a) and (b);
(f) both (a) and (c);
(g) both (b) and (d);
(h) both (c) and (d); or
(i) both (a), (b), (c) and (d).
In other separate and specific embodiments, a nucleic acid, a nucleic acid construct, a combination of a first and a second nucleic acid construct, an expression vector, or a combination of a first and a second expression vector according to the present invention may encode
(a) a HC comprising SEQ ID NO:118 (CD40-001-HC6, IgG1);
(b) a HC comprising SEQ ID NO:119 (CD40-001-HC6-FEAL);
(c) a HC comprising SEQ ID NO:120 (CD40-001-HC6-FEAR);
(d) a HC comprising SEQ ID NO:124 (CD137-009-HC7);
(e) a HC comprising SEQ ID NO:125 (CD137-009-HC7-FEAR);
(f) a HC comprising SEQ ID NO:126 (CD137-009-HC7-FEAL);
(g) an LC comprising SEQ ID NO:122 (CD40-001-LC1);
(h) an LC comprising SEQ ID NO:128 (CD137-009-LC2);
(i) both (a) and (g);
(j) both (b) and (g);
(k) both (c) and (g);
(l) both (d) and (h);
(m) both (e) and (h);
(n) both (f) and (h);
(o) both (b) and (e);
(p) both (c) and (f);
(q) both (g) and (h);
(r) both (b), (e), (g) and (h);
(s) both (c), (f), (g) and (h).
In one aspect, the invention relates to a method for producing a bispecific antibody according to any one of the embodiments as disclosed herein, comprising the steps of
a) culturing a host cell as disclosed herein comprising an expression vector as disclosed herein expressing the first antibody as disclosed herein and purifying said antibody from the culture media;
b) culturing a host cell as disclosed herein comprising an expression vector as disclosed herein expressing the second antibody as disclosed herein and purifying said antibody from the culture media;
c) incubating said first antibody together with said second antibody under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide-bond isomerization, and
d) obtaining said bispecific antibody.
In one aspect, the invention relates to a host cell comprising an expression vector as defined above. In one embodiment, the host cell is a recombinant eukaryotic, recombinant prokaryotic, or recombinant microbial host cell.
Compositions
The present invention also relates to a composition comprising a multispecific antibody according to the present invention, a nucleic acid according to the present invention, an expression vector to the present invention or a host cell according to the present invention.
In a further embodiment the composition according to the present invention is a pharmaceutical composition.
In an even further embodiment, the pharmaceutical composition according to the present invention further comprises a pharmaceutically acceptable carrier.
In a further aspect, the invention relates to a pharmaceutical composition comprising:
a multispecific CD40×CD137 antibody as defined in any of the embodiments disclosed herein, and
a pharmaceutically acceptable carrier.
The pharmaceutical composition of the present invention may contain one multispecific antibody of the present invention or a combination of different multispecific antibodies of the present invention.
The pharmaceutical compositions may be formulated in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995. A pharmaceutical composition of the present invention may e.g. include diluents, fillers, salts, buffers, detergents (e.g., a nonionic detergent, such as Tween®-20 or Tween®-80), stabilizers (e. g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and/or other materials suitable for inclusion in a pharmaceutical composition.
Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible with a multispecific antibody of the present invention. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, carboxymethyl cellulose colloidal solutions, tragacanth gum and injectable organic esters, such as ethyl oleate, and/or various buffers. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
Pharmaceutical compositions of the present invention may also comprise pharmaceutically acceptable antioxidants for instance (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Pharmaceutical compositions of the present invention may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the compositions.
The pharmaceutical compositions of the present invention may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the pharmaceutical composition. The pharmaceutical composition of the present invention may be prepared with carriers that will protect the multispecific antibody against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid alone or with a wax, or other materials well known in the art. Methods for the preparation of such formulations are generally known to those skilled in the art.
Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients e.g. as enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients e.g. from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
The pharmaceutical composition may be administered by any suitable route and mode. In one embodiment, a pharmaceutical composition of the present invention is administered parenterally. “Administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion.
In one embodiment the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.
Uses
The present invention also relates to the multispecific antibody according to the present invention, the nucleic acid according to the present invention, the expression vector according to the present invention, the host cell according to the present invention, the composition according to the present invention, or the pharmaceutical composition according to the present invention for use as a medicament.
The present invention also relates to the multispecific antibody according to the present invention, the nucleic acid according to the present invention, the expression vector according to the present invention, the host cell according to the present invention, the composition according to the present invention, or the pharmaceutical composition according to the present invention for use in the treatment of a disease, such as cancer or an infectious disease.
According to the invention, the term “disease” refers to any pathological state, in particular cancer, infectious diseases, inflammatory diseases, metabolic diseases, autoimmune disorders, degenerative diseases, apoptosis-associated diseases and transplant rejections.
As used herein, the term “cancer” includes a disease characterized by aberrantly regulated cellular growth, proliferation, differentiation, adhesion, and/or migration. By “cancer cell” is meant an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease.
The term “cancer” according to the invention comprises leukemias, seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestine cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophagus cancer, colorectal cancer, pancreas cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, cancer of the uterus, ovarian cancer and lung cancer and the metastases thereof. Examples thereof are lung carcinomas, mamma carcinomas, prostate carcinomas, colon carcinomas, renal cell carcinomas, cervical carcinomas, or metastases of the cancer types or tumors described above.
The term “cancer” according to the invention also comprises cancer metastases. By “metastasis” is meant the spread of cancer cells from its original site to another part of the body. The formation of metastasis is a very complex process and depends on detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then, after being transported by the blood, infiltration of target organs. Finally, the growth of a new tumor, i.e. a secondary tumor or metastatic tumor, at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor because tumor cells or components may remain and develop metastatic potential. In one embodiment, the term “metastasis” according to the invention relates to “distant metastasis” which relates to a metastasis which is remote from the primary tumor and the regional lymph node system.
The term “infectious disease” refers to any disease which can be transmitted from individual to individual or from organism to organism, and is caused by a microbial agent (e.g. common cold).
Examples of infectious diseases include viral infectious diseases, such as AIDS (HIV), hepatitis A, B or C, herpes, herpes zoster (chicken-pox), German measles (rubella virus), yellow fever, dengue etc. flaviviruses, influenza viruses, hemorrhagic infectious diseases (Marburg or Ebola viruses), and severe acute respiratory syndrome (SARS), bacterial infectious diseases, such as Legionnaire's disease (Legionella), sexually transmitted diseases (e.g. chlamydia or gonorrhea), gastric ulcer (Helicobacter), cholera (Vibrio), tuberculosis, diphtheria, infections by E. coli, Staphylococci, Salmonella or Streptococci (tetanus); infections by protozoan pathogens such as malaria, sleeping sickness, leishmaniasis; toxoplasmosis, i.e. infections by Plasmodium, Trypanosoma, Leishmania and Toxoplasma; or fungal infections, which are caused, e.g., by Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis or Candida albicans.
The term “inflammatory disease” refers to any disease, which is characterized by or associated with high levels of inflammation in tissues, in particular connective tissues, or degeneration of these tissues. A chronic inflammatory disease is a medical condition which is characterized by persistent inflammation. Examples of (chronic) inflammatory diseases include celiac disease, vasculitis, lupus, chronic obstructive pulmonary disease (COPD), irritable bowel disease, atherosclerosis, arthritis, ankylosing spondylitis, Crohn's disease, colitis, chronic active hepatitis, dermatitis and psoriasis.
The term “metabolic disease” refers to any disease or disorder that disrupts normal metabolism. Examples include cystinosis, diabetes, dyslipidemia, hyperthyroidism, hypothyroidism, hyperlipidemia, hypolipidemia, galactosemia, Gaucher's disease, obesity and phenylketonuria.
The term “autoimmune disorder” refers to any disease/disorder in which the body produces an immunogenic (i.e. immune system) response to some constituent of its own tissue. In other words, the immune system loses its ability to recognize some tissue or system within the body as self and targets and attacks it as if it were foreign. Autoimmune diseases can be classified into those in which predominantly one organ is affected (e.g. hemolytic anemia and anti-immune thyroiditis), and those in which the autoimmune disease process is diffused through many tissues (e.g. systemic lupus erythematosus). For example, multiple sclerosis is thought to be caused by T cells attacking the sheaths that surround the nerve fibers of the brain and spinal cord. This results in loss of coordination, weakness, and blurred vision. Autoimmune diseases are known in the art and include, for instance, Hashimoto's thyroiditis, Grave's disease, lupus, multiple sclerosis, rheumatic arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, and the like.
The term “degenerative disease” refers to any disease in which the function or structure of the affected tissues or organs will increasingly deteriorate over time. Examples include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, macular degeneration, multiple sclerosis, muscular dystrophy, Niemann Pick disease, osteoporosis and rheumatoid arthritis.
The term “apoptosis-associated diseases” refers to any disease in which alterations of apoptosis are involved. Examples include cancer, neurological disorders, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS) and stroke, heart diseases, such as ischemia reperfusion and chronic heart failure, infectious diseases and autoimmune diseases.
The term “transplant rejection” refers to the rejection of a transplanted tissue or organ by the recipient's immune system, which may, ultimately, destroy the transplanted tissue or organ.
In one embodiment, the use of the multispecific antibody, nucleic acid, expression vector, host cell or composition for use according to the present invention may be for treating cancer.
In one embodiment, the use of the multispecific antibody, nucleic acid, expression vector, host cell or composition for use according to the present invention may be for treating an infectious disease.
The present invention also relates to a method of treatment of a disease, such as cancer or an infectious disease, comprising administering the multispecific antibody according to the present invention, the nucleic acid according to the present invention, the expression vector according to the present invention, the host cell according to claim the present invention, the composition according to the present invention, or the pharmaceutical composition according to the present invention to a subject in need thereof.
The present invention also relates to use of a multispecific antibody according to the present invention, the nucleic acid according to the present invention, the expression vector according to the present invention, the host cell according to the present invention, the composition according to the present invention, or the pharmaceutical composition according to the present invention for the manufacture of a medicament.
In one embodiment the method or use according to the present invention is for use in combination with one or more further therapeutic agent, such as a chemotherapeutic agent.
In one aspect, the invention relates to the multispecific antibody, such as a bispecific antibody, according to any one of the embodiments disclosed herein, the composition as disclosed herein, or the pharmaceutical composition as disclosed herein for use as a medicament.
In another aspect, the present invention relates to the use of a multispecific antibody according to the present invention in the manufacture of a medicament for the treatment of a disease, such as cancer or an infectious disease.
In one aspect, the invention relates to the multispecific antibody according to any one of the embodiments disclosed herein, the composition as disclosed herein, or the pharmaceutical composition as disclosed herein for use in the treatment of a disease, such as cancer or an infectious disease.
In one aspect, the invention relates to a method of treatment of a disease comprising administering the multispecific antibody according to any one of the embodiments disclosed herein, the composition as disclosed herein, or the pharmaceutical composition as disclosed herein to a subject in need thereof.
The multispecific antibodies of the invention may be used for a number of purposes. In particular, the multispecific antibodies of the invention may be used for the treatment of various forms of cancer, including metastatic cancer and refractory cancer.
In one embodiment the use according to the present invention is in combination with one or more further therapeutic agent, such as a chemotherapeutic agent.
In particular, the multispecific antibodies according to the invention may be useful in therapeutic settings in which increasing proliferation of T cells is relevant. An example of such a therapeutic setting includes but is not limited to cancer or tumors, such as hematological and solid tumors, e.g., melanoma, lung cancer, breast cancer, non-small cell lung cancer (NSCLC), colon cancer, renal cancer, cervical cancer and prostate cancer, such as melanoma or NSCLC. Examples thereof are lung carcinomas, mamma carcinomas, prostate carcinomas, colon carcinomas, renal cell carcinomas, cervical carcinomas, or metastases of such cancer types or tumors.
The present invention also relates to a method for treating cancer, comprising
a) selecting a subject suffering from a cancer, and
b) administering to the subject the multispecific antibody of the present invention or a pharmaceutical composition of the present invention.
Also, the invention relates to the use of a multispecific antibody that binds to human CD40 and human CD137 for the preparation of a medicament for the treatment of cancer, such as one of the specific cancer indications mentioned herein.
The invention further relates to a multispecific antibody for use in the treatment of cancer, such as one of the cancer indications mentioned above.
In one embodiment the method or use according to the present invention is for use in combination with one or more further therapeutic agent, such as a chemotherapeutic agent.
For the above mentioned uses any multispecific antibody, such as a bispecific antibody, of the present invention may be used.
In one aspect, the invention relates to a diagnostic composition comprising a multispecific antibody according to any one of the embodiments as disclosed herein.
In one embodiment, the diagnostic composition is a companion diagnostic which is used to screen and select those patients who will benefit from treatment with the multispecific antibody.
In a further embodiment of the methods of treatment of the present invention, the efficacy of the treatment is being monitored during the therapy, e.g. at predefined points in time, by determining tumor burden.
Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.
The efficient dosages and the dosage regimens for the multispecific antibodies depend on the disease or condition to be treated and may be determined by the persons skilled in the art. An exemplary, non-limiting range for a therapeutically effective amount of a multispecific antibody of the present invention is about 0.001-30 mg/kg.
A physician or veterinarian having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the multispecific antibody employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a multispecific antibody of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Administration may e.g. be parenteral, such as intravenous, intramuscular or subcutaneous. In one embodiment, the multispecific antibodies may be administered by infusion in a weekly dosage of calculated by mg/m2. Such dosages can, for example, be based on the mg/kg dosages provided above according to the following: dose (mg/kg)×70: 1.8. Such administration may be repeated, e.g., 1 to 8 times, such as 3 to 5 times. The administration may be performed by continuous infusion over a period of from 2 to 24 hours, such as from 2 to 12 hours. In one embodiment, the multispecific antibodies may be administered by slow continuous infusion over a long period, such as more than 24 hours, in order to reduce toxic side effects.
In one embodiment the multispecific antibodies may be administered in a weekly dosage of calculated as a fixed dose for up to 8 times, such as from 4 to 6 times when given once a week. Such regimen may be repeated one or more times as necessary, for example, after 6 months or 12 months. Such fixed dosages can, for example, be based on the mg/kg dosages provided above, with a body weight estimate of 70 kg. The dosage may be determined or adjusted by measuring the amount of multispecific antibody of the present invention in the blood upon administration by for instance taking out a biological sample and using anti-idiotypic antibodies which target the CD137 antigen antigen-binding region of the multispecific antibodies of the present invention.
In one embodiment, the multispecific antibodies may be administered as maintenance therapy, such as, e.g., once a week for a period of 6 months or more.
A multispecific antibody may also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and/or reduce the risk of recurrence when a cancer is in remission.
The multispecific antibodies of the invention may also be administered in combination therapy, i.e., combined with other therapeutic agents relevant for the disease or condition to be treated. Accordingly, in one embodiment, the multispecific antibody-containing medicament is for combination with one or more further therapeutic agents, such as a cytotoxic, chemotherapeutic or anti-angiogenic agent.
Such combined administration may be simultaneous, separate or sequential. For simultaneous administration the agents may be administered as one composition or as separate compositions, as appropriate. The present invention thus also provides methods for treating a disorder, which methods comprise administration of a multispecific antibody of the present invention combined with one or more additional therapeutic agents as described below.
In one embodiment, the present invention provides a method for treating a disorder, which method comprises administration of a therapeutically effective amount of a multispecific antibody of the present invention, and optionally at least one additional therapeutic agent, to a subject in need thereof.
In one embodiment, the present invention provides a method for treating or preventing cancer, which method comprises administration of a therapeutically effective amount of a multispecific antibody of the present invention and at least one additional therapeutic agent to a subject in need thereof.
Pharmaceutical compositions of the invention can also be administered in combination therapy, i.e., combined with other agents, or combined with other treatment regimen. For example the multispecific antibodies may be combined with radiotherapy and/or surgery and/or autologous or allogeneic peripheral stem cell or bone marrow transplantation.
Biomarkers
Thus, in one aspect, the present invention also relates to use of the multispecific antibody as a biomarker.
In another aspect, the invention relates to a kit for detecting cross-linking between CD40- and CD137-expressing cells, in a sample obtained from a patient, such as a blood sample, lymph node sample or bone marrow sample, comprising
i) a multispecific antibody according to any one of the embodiments as disclosed herein; and
ii) instructions for use of said kit.
In a further aspect, the invention relates to a method for detecting whether cross-linking between CD40- and CD137-expressing cells occurs in a sample obtained from a patient, such as a blood sample, lymph node sample or bone marrow sample, upon administration of a bispecific antibody according to any one of the embodiments as disclosed herein, comprising the steps of:
(i) contacting the sample with a multispecific antibody according to any one of the embodiments as disclosed herein under conditions that allow for formation of a complex between said multispecific antibody and the CD40- and CD137-expressing cells; and
(ii) analyzing whether a complex has been formed.
Detection of the complex can be done by methods known in the art, such as performed in Example 4, 5, 6, 10, 11 or 12.
Anti-Idiotypic Antibodies
In another aspect, the invention relates to an anti-idiotypic antibody which binds to the first and/or second antigen-binding region as defined in any one of the embodiments disclosed herein.
In a particular embodiment, the anti-idiotypic antibody binds to the first and/or second antigen-binding region of a multispecific antibody, wherein
the first antigen-binding region comprises a VH sequence as set forth in SEQ ID NO:117 and a VL sequence as set forth in SEQ ID NO:121, and
the second antigen-binding region comprises a VH sequence comprising SEQ ID NO:123 and a VL sequence comprising SEQ ID NO:127.
In one embodiment, the anti-idiotypic antibody binds to the first antigen-binding region defined in any one of the embodiments disclosed herein. In a specific embodiment, the first antigen-binding region comprises a VH sequence comprising SEQ ID NO:117 and a VL sequence comprising SEQ ID NO:121.
In another embodiment, the anti-idiotypic antibody binds to the second antigen-binding region defined in any one of the embodiments disclosed herein. In a specific embodiment, the second antigen-binding region comprises a VH sequence comprising SEQ ID NO:123 and a VL sequence comprising SEQ ID NO:127.
The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.
EXAMPLES
Example 1
Generation of Antibodies
The CD40 and each of the CD137 antibodies (i.e. clones 1-12) mentioned in Table 1 above were produced with the VH and VL sequences described in Table 1 and with a human κ light chain, and with a human IgG1 heavy chain. The CD40 antibody was produced with two different human IgG1 heavy chains; 1) a human IgG1 heavy chain containing the following amino acid mutations: L234F, L235E, D265A and F405L (FEAL) wherein the amino acid position number is according to EU numbering (corresponding to SEQ ID NO:113); and 2) a human IgG1 heavy chain containing the following amino acid mutations: L234F, L235E, D265A and K409R (FEAR) wherein the amino acid position number is according to EU numbering (corresponding to SEQ ID NO:112).
The CD137 antibodies were all produced with a human IgG1 heavy chain containing the following amino acid mutations: L234F, L235E, D265A and K409R (FEAR) wherein the amino acid position number is according to EU numbering (correspond to SEQ ID NO:112).
Similarly, a b12 antibody was produced comprising the VH and VL sequences mentioned in Table 1, and with a human IgG1 light chain and a human IgG heavy chain containing the following amino acid mutations: L234F, L235E, D265A and F405L (FEAL) wherein the amino acid position number is according to EU numbering (correspond to SEQ ID NO:113).
Antibodies were produced under serum-free conditions by co-transfecting relevant heavy and light chain expression vectors in Expi293F™ cells (ThermoFisher catalogue number A14527), using ExpiFectamine™ 293 (ThermoFisher catalogue number A14525), according to the manufacturer's instructions. Antibodies were purified by protein A affinity chromatography and buffer exchanged into 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 buffer (B.Braun or Thermo Fisher). After buffer exchange, samples were sterile filtered over 0.2 μm dead-end filters. Purified proteins were analyzed by CE-SDS and HP-SEC. Concentration was measured by absorbance at 280 nm. Purified antibodies were stored at 2-8° C.
Example 2
DNA Shuffling Between Wild Boar or Elephant and Human CD137 to Determine Domains Important for Binding of Anti CD137 Antibodies
To determine domains important for binding of anti CD137 antibodies to human CD137, DNA shuffling was performed between human and wild boar CD137 (sus scrofa; XP_005665023) or between human and African elephant CD137 (loxodonta africana; XP_003413533). Shuffle constructs were prepared from DNA encoding human CD137, by replacing human domains with wild boar (shuffle construct 1-4, 6) or elephant (shuffle construct 5) domains. If a domain in human CD137 is important for binding of an anti CD137 antibody, binding will be lost upon replacement of that domain with the wild boar or African elephant domain. Requirement is that the antibody does not bind to the whole CD137 elephant or wild boar sequence Homology between human and wild boar and between human and African elephant CD137 is 70.2 and 74.5%, respectively. FIG. 1 shows sequence alignments of human, wild boar and African elephant CD137. FIG. 2 shows the constructs for human CD137 containing wild boar CD137 or African elephant domains, as indicated.
3×106 HEK293T-17 cells were seeded in T75 culture flasks (Greiner Bio-One, cat. no. 658175) in 20 mL RPMI 1640 GlutaMAX medium containing 10% FCS (Biochrom, cat. no. S 0115). After O/N incubation, cells were transiently transduced with expression vectors encoding the shuffle constructs or the wild boar, African elephant or human CD137 downstream of a constitutively active human elongation factor-1 alpha (EF-1 alpha) promotor using TransIT®-LT1 Transfection Reagent, Mirus Bio (VWR International, cat. no. 731-0029), according to the manufacturer's instructions. The next day, cells were harvested using 1.5 mL Accutase (Sigma Aldrich, cat. no. A6964) (incubation at 37° C. for 5 min.) and flow cytometry was performed, essentially as described in Example 4, to measure surface expression of the shuffle constructs and the human, African elephant and wild boar CD137 and to measure binding of the antibody clones to the different shuffle constructs. To measure cell surface expression of the constructs, transduced cells were incubated with 1 μg/mL goat polyclonal anti-human CD137 (R&D Systems, cat. no. AF838) in FACS buffer (D-PBS supplemented with 5 mM EDTA [Sigma Aldrich, cat. no. 03690] and 5% (v/v) fetal bovine serum [FBS, Biochrom, cat. no. S 0115]) (4° C., 20 min.), followed by incubation with APC-labeled anti-goat IgG (H+L) (R&D Systems, cat. no. F0108) (4° C., 20 min.). Binding of the different CD137 antibody clones to cells expressing the shuffle constructs was measured by incubation of the transduced cells with 1 μg/mL of the antibody clones, followed by APC-labeled AffiniPure F(ab′)2 Fragment (1:50 final dilution; Jackson, cat. no. 109-136-127).
All CD137 shuffle constructs, as well as human, African elephant and wild boar CD137, were expressed on the cell surface with similar expression levels (FIG. 3).
Table 2 and FIG. 4 show that all clones, except for clone 1, showed loss of binding to at least African elephant or wild boar CD137. Clones 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 showed loss of binding to at least one of the shuffle constructs. Clone 1 showed reduced binding to African elephant and shuffle construct 5, as compared to binding to human CD137. Clone 12 did not show loss of binding to any of the shuffle constructs, but showed reduced binding to shuffle construct 5. None of the clones showed loss of binding or reduced binding to shuffle constructs 1 and 2.
TABLE 2
Summary of binding of the CD137 antibodies
to the shuffle constructs
Binding
decreased
Binding similar
compared
to human
to human
CD137 binding
CD137 binding
No binding
Wild boar CD137
Clone 1, 2, 6,
None
Clone 3, 4, 5,
10
7, 8, 9, 11, 12
African elephant
None
Clone 1, 3
Clone 2, 4, 5,
CD137
6, 7-12
Shuffle 1 (aa 162-196)
Clone 1-12
None
None
Shuffle 2 (139-161)
Clone 1-12
None
None
Shuffle 3 (115-138)
Clone 1, 2, 5,
Clone 3, 7, 8
Clone 4, 11
6, 9, 10, 12
Shuffle 4 (89-114)
Clone 1, 2, 5,
None
Clone 3, 4, 7,
6, 9, 10, 12
8, 11
Shuffle 5 (48-88)
Clone 3, 4, 5,
Clone 1, 12
Clone 2, 6, 9,
7, 8, 11
10
Shuffle 6 (aa 24-47)
Clone 1, 2, 3,
None
Clone 5
4, 6, 7-12
Example 3
Generation of Bispecific Antibodies by 2-MEA-Induced Fab-Arm Exchange
A method to produce stable IgG1-based bispecific antibodies is described in WO2011131746 (Genmab). The bispecific antibody product generated by this method described below will no longer participate in Fab-arm exchange. The basis for this method was the use of complimentary CH3 domains, which promote the formation of heterodimers under specific assay conditions. To enable the production of bispecific antibodies by this method, IgG1 molecules carrying certain mutations in the CH3 domain were generated: in one parental IgG1 antibody T350I, K370T and F405L mutations (or minimally F405L), in the other parental IgG1 antibody a K409R mutation.
The concentrations of parental IgG1 antibodies that minimally contained either an F405L or a K409R point mutation were measured using their absorption at 280 nm. Specific extinction coefficients based upon the amino acid sequence were used to infer the protein concentration.
The Cube system is Genmab's flexible robotic work cell. The system was designed and built in collaboration with Peak Analysis and Automation (PAA), Farnborough UK.
Bispecific antibodies were generated by combining the following antibodies from Example 1:
CD40-FEAL antibody combined with each of the CD137-FEAR antibodies,
CD40-FEAR antibody combined with the b12-FEAL antibody, and
Each of the CD137-FEAR antibodies combined with the b12-FEAL antibody
The bispecific antibody discovery process is performed in an automated fashion on the Cube system, as shown in FIG. 5 and described below.
To generate bispecific antibodies, the following (automated) steps are performed:
Depending on the volume required, deep well source plates (96 well clear V-bottom 2 mL polypropylene deep well plate, Corning, cat. no. 3960; 48 well Riplate® SW 5 mL, Ritter, cat. no. 43001-1062; 24 well Riplate SW 10 mL, Ritter, cat. no. 43001-1066) are filled with parental antibodies (F405L- and K409R-containing antibodies in different plates) at a concentration of 1.0 mg/mL (in 1× PBS, B.Braun) (FIG. 5, left plates).
From these source plates, the pre-grid plates (96 well V-bottom, Corning) are prepared by the Cube, according to FIG. 5, middle plates. For each combination with a parental antibody in the exchange grid, 67.5 μL parental antibody is added to the appropriate pre-grid plate.
After the pre-grid, the exchange is performed. Here, two parental antibodies (67.5 μL, 1.0 mg/mL each), each from a different pre-grid plate are added to an exchange plate (96 well round-bottom polypropylene plate, Greiner, cat. no. 650293), each antibody at a final concentration of 0.5 mg/mL (equimolar concentration) (FIG. 5, right plates).
The exchange reaction is started by adding 15 μL 75 mM 2-mercaptoethylamine-HCl (2-MEA) (in 1× PBS, B.Braun) to the exchange plate. The total volume in the exchange plate is now 150 μL (final concentration 2-MEA 7.5 mM)
The exchange plates are incubated at 31° C. for 5 hours in the Cytomat 6000 automated incubator (Thermo Scientific).
The reducing agent 2-MEA is removed by using desalting columns (PhyTip desalting columns, 600 μL resin, PhyNexus, cat. no. PDR 91-60-06), for which flow is based on gravity.
The columns are conditioned by placing an adapter with 96 columns on a waste position, adding two times 450 μL 1× PBS (B.Braun) and allowing the solutions to flow through the columns into the waste.
After conditioning, 100 μL sample from the exchange plate is added, thereby pushing the remaining PBS out of the columns.
After allowing the solutions to flow through the columns into the waste, the adapter with columns is placed on a desalting (or destination) plate (96 well round-bottom, Greiner).
The remaining sample from the exchange plate is added to the columns.
After allowing the samples to flow through the columns into the desalting plate, 225 μL 1× PBS (B.Braun) is added to the columns and the sample is eluted into the desalting plate.
The 2-MEA remains inside the columns. Where appropriate, the columns can be regenerated by washing with 1× PBS (B.Braun).
The desalting plates are stored in the Cytomat 6001 automated incubator (Thermo Scientific) at 8° C. These plates now contain the bispecific antibodies.
The final bispecific antibody samples were filtered over 0.2 μm dead-end filters and the absorbance at 280 nm (A280) of bispecific products was measured to determine the final concentration. Samples were stored at 2-8° C. for at least 24 hours before further use.
The bispecific antibody exchange efficiency was quantified using High Pressure Liquid Chromatography (HPLC)-hydrophobic interaction chromatography (HIC) using a Butyl-NPR, 2.5 μm, 4.6×35 mm HIC-HPLC column (Tosoh Bioscience) with a flow rate of 1 mL/min. Parental antibodies and analysis samples were normalized in concentration and diluted two-fold with HIC eluent A (15.4 mM K2HPO4, 9.6 mM KH2PO4, 1.5 M (NH4)2SO4; pH 7.0). 50 μL of sample was injected and elution was performed with a 12-min gradient of HIC eluent A to HIC eluent B (15.4 mM K2HPO4, 9.6 mM KH2PO4; pH 7.0) with detection at 280 nm. Alternatively, HPLC—analytical cation exchange chromatography (CIEX) was used to quantify the bispecific antibody exchange efficiency. Parental antibodies and analysis samples at 1 mg/mL in mobile Phase A (10 mM NaPO4, pH 7.0) were injected onto the HPLC. The differently charged IgG molecules were separated by using a ProPac WCX-10, 4 mm×250 mm, analytical column with a flow rate of 1 mL/min. 50 μL of sample was injected and elution was performed with a gradient of Mobile Phase A (10 mM NaPO4, pH 7.0; prepared from a 0.1 M stock of sodium phosphate buffer, that was obtained by adding 10.3 g Na2HPO4.2H2O and 5.07 g NaH2PO4 per liter Milli-Q) to Mobile Phase B (10 mM NaPO4, pH 7.0, 0.25 M NaCl) with detection at 280 nm. Empower 3 software (Waters) was used to assign peaks as parental antibodies or bispecific reaction products, and to integrate peak areas to quantify extent of the bispecific antibody exchange reaction. Bispecific antibody reaction products were further analyzed using analytical size exclusion chromatography, using a TSK HP-SEC column (G3000SWxl; Tosoh Biosciences, via Omnilabo, Breda, The Netherlands) and Capillary Electrophoresis-Sodium Dodecyl Sulfate (CE-SDS) using a LabChip GXII (Caliper Life Sciences, Mass.) on a HT Protein Express LabChip (Caliper Life Sciences, Mass.) under reducing and non-reducing conditions according to manufacturer's instructions.
Example 4
Reporter Assay Measuring Trans-Activation by Bispecific Antibodies Binding to CD40 and CD137
CD40 is predominantly expressed on antigen-presenting cells (APCs), such as dendritic cells, whereas CD137 is predominantly expressed on activated T cells. Thus, bispecific antibodies binding to CD40 and CD137 can bind simultaneously to APCs and T cells expressing these receptors. Thereby, these bispecific antibodies can mediate cell-cell contact between APCs and T cells by receptor binding and activate both receptors. This receptor activation can be induced by cross-linking and receptor clustering upon cell-cell interaction and is not necessarily dependent on agonistic activity of the parental monospecific bivalent antibodies. Thus, these trans-activating bispecific antibodies can exert co-stimulatory activity in the context of interactions between APCs and T cells.
A reporter assay system was established to measure activation of each receptor by the bispecific antibodies. NF-KB/293/GFP-Luc™ Transcriptional Reporter Cell Line (System Biosciences; cat. no. TR860A-1) is a reporter cell line designed for monitoring the NF-KB signal transduction pathway in vitro. Activation of the NF-KB pathway can be monitored by the detection of green fluorescent protein (GFP) fluorescence as well as luciferase activity for quantitative transcription activation reporter assays. NF-KB/293/GFP-Luc™ cells were stably transduced with expression vectors encoding full length human CD40 or CD137 downstream of a constitutively active human elongation factor-1 alpha (EF-1 alpha) promotor, using Trans1T®-LT1 Transfection Reagent, Minis Bio (VWR International, cat. no. 731-0029), according to the manufacturer's instructions. Stable clones were selected using 10 mg/mL blasticidin (Invivogen, cat. no. ant-bl-1). In addition, K562 cells were stably transduced, as described supra, with human CD40 and CD137 to generate cell lines that can provide the corresponding target antigen for the other arm of the bispecific antibody. Cell surface expression of the receptors was measured by flow cytometry. 0.3×106 cells were spun down (460 x g, 5 min.) and washed in FACS buffer (D-PBS supplemented with 5 mM EDTA [Sigma Aldrich, cat. no. 03690] and 5% (v/v) fetal bovine serum [FBS, Biochrom, cat. no. S 0115]) (460 x g, 5 min.). 50 pL of 1:50 diluted allophycocyanin (APC)-labeled anti-human CD40 (BD Biosciences, clone 5C3, cat. no. 555591) or phycoerythrin (PE)-labeled anti-human CD137 (BD Biosciences, clone 4B4-1, cat. no. 555956) was added to the cell pellet and incubated at 4° C. in the dark for 20 minutes. After washing three times with FACS buffer, cells were resuspended in 100 pL FACS buffer and binding of the antibodies was detected by flow cytometry on a FACSCanto™ II (BD Biosciences). Cell surface expression 10 of CD40 and CD137 on transduced NF-KB/293/GFP-Luc™ cells (FIG. 6A) and K562 cells (FIG. 6B) was nicely shown.
The reporter assay measuring trans-activation was set up as follows: NF-KB/293/GFP-Luc™ cells expressing one of the two indicated TNF receptors were seeded at 10,000 cells/well in 30 pL RPMI 1640 medium with GlutaMAX supplement (Life Technologies, cat. no. 61870) in white opaque 384-well cell culture plates (PerkinElmer, cat. no. 6007680). Bispecific antibodies binding with one arm to CD40 and with the other arm to CD137 and the corresponding monospecific, monovalent (containing one irrelevant control arm [b12]) control antibodies were added in 10 pL/well to the reporter cells in serial dilutions (in medium), ranging from 0.078 pg/mL to 10 pg/mL final concentration, including a buffer control. 17,000 K562 cells expressing the second TNF receptor or wildtype K562 (K562 wt) cells were added in 10 pL medium to each well and incubated at 37° C. and 5% CO2 for 18 hours. Thus, the bispecific antibodies are able to bind to the first TNF receptor on the NF-KB/293/GFP-Luc™ cell line and, at the same time, to the second TNF receptor on the K562 cell line. Only receptor activation of the first TNF receptor on NF-KB/293/GFP-Luc™ cells is measured by luciferase activity induced upon NF-KB signaling. Thus, bispecific antibodies targeting CD40 and CD137 were analyzed by two reporter assays: the first assay measuring CD137 activation induced by simultaneous binding of CD137 on the reporter cell line and CD40 on the K562 cells (HEK293 NFK CD137 gfp luc+K562 CD40) and the second assay measuring CD40 activation induced by simultaneous binding of CD40 on the reporter cell line and CD137 on the K562 cells (HEK293 NFK CD40 gfp luc+K562 CD137). Luciferase activity was measured as relative luminescence units on an Envision plate reader (PerkinElmer) after addition of 50 pL/well Steady-Glog reagent (Promega; cat. no. E2520) reconstituted in Glo Lysis Buffer (Promega; cat. no. E266A) and incubation at room temperature for 30 min.
Only the bispecific CD40xCD137 antibodies (FIGS. 7 A-L, lower panels, first and third graph) induced luciferase activity (at concentrations of about 100 ng/mL and higher) in NF-KB/293/GFP-Luc™ cells transduced either with CD137 or with CD40, under trans-activation conditions (incubation with K562-CD40 or K562-CD137, respectively). None of the monospecific, monovalent (containing one irrelevant control arm [b12]) control antibodies induced luciferase activity in the transduced NF-KB/293/GFP-Luc™ cells (upper panels). Furthermore, in the absence of trans-activation conditions (using wildtype K562 cells) no luciferase activity was induced by the bispecific CD40xCD137 antibodies (lower panels, second and fourth panel).
Example 5
Non-Antigen-Specific T-Cell Proliferation Assay to Measure Trans-Activation by Bispecific Antibodies Binding to CD40 and CD137
To measure non-antigen-specific proliferation, T cells in a peripheral blood mononuclear cell (PBMC) population were incubated with a sub-optimal concentration of anti-CD3 antibody (clone UCHT1), combined with CD40×CD137 bispecific or control antibodies. Within this PBMC population, antigen-presenting cells expressing CD40 can be bound by the CD40-specific arm of the bispecific antibody, whereas the T cells in the population can be bound by the CD137-specific arm. Trans-activation of the T cells induced by cross-linking to the antigen-presenting cells via the bispecific antibody is measured as T-cell proliferation.
PBMCs were obtained from buffy coats of healthy donors (Transfusionszentrale, University Hospital, Mainz, Germany) using a Ficoll gradient (VWR, cat. no. 17-5446-02). PBMCs were labeled using 1.6 μM carboxyfluorescein succinimidyl ester (CFSE) (Thermo Fisher, cat. no. C34564) in PBS, according to the manufacturer's instructions. 75,000 CFSE-labeled PBMCs were seeded per well in a 96-well round-bottom plate (Sigma Aldrich, CLS3799-50EA) and incubated with a sub-optimal concentration of anti-CD3 antibody (R&D Systems, clone UCHT1, cat. no. MAB100; 0.01-0.1 μg/mL final concentration) that was pre-determined for each donor, and bispecific or control antibodies, in 150 μL IMDM GlutaMAX supplemented with 5% human AB serum, at 37° C., 5% CO2, for four days. Proliferation of CD4+ and CD8+ T cells was analyzed by flow cytometry, essentially as described supra. 30 μL containing PE-labeled CD4 antibody (BD Biosciences, cat. no. 555347; 1:80 final dilution), PE-Cy7-labeled CD8α antibody (clone RPA-T8, eBioscience, cat. no. 25-0088-41; 1:80 final dilution) APC-labeled CD56 antibody (eBiosciences, cat. no. 17-0567; 1:80 final dilution) and 7-AAD (Beckman Coulter, cat. no. A07704; 1:80 final dilution) in FACS buffer was used to stain the cells and exclude natural killer (NK) cells (CD56) and dead cells (7-AAD) from the analysis.
Samples were measured on a FACSCanto™ II (BD Biosciences). Detailed analyses of T-cell proliferation based on CFSE-peaks indicating cell divisions were made by FlowJo 7.6.5 software. Mean percentages of T cells that went into division (% divided cells) and the average number of divisions of cells that went into division (proliferation index) were calculated.
FIG. 8A shows that only the CD40×CD137 bispecific antibodies efficiently enhanced proliferation of CD8+ T cells. The control monospecific, monovalent antibodies (b12×CD40; b12×CD137) and the combination of monospecific, monovalent CD40 with monospecific, monovalent CD137 antibodies (b12×CD40+b12×CD137) did not induce more proliferation than observed in the control (only the weakly activated PBMCs, ctrl, w/o). The flow cytometry histograms, for the different antibodies at different concentrations, were quantified to indicate percentage of divided cells (FIG. 8B) and proliferation index (FIG. 8C), as described supra. These figures show that only the bispecific antibodies were capable of inducing proliferation of CD8+ cells, with an optimum between 0.04 and 0.2 μg/mL.
Example 6
Antigen-Specific CD8+ T Cell Proliferation Assay to Measure Trans-Activation by Bispecific Antibodies Binding to CD40 and CD137
To measure induction of proliferation by the bispecific antibodies in an antigen-specific assay, dendritic cells (DCs) were transfected with claudin 6 in vitro-transcribed RNA (IVT-RNA,) to express the claudin 6 antigen. T cells were transfected with the claudin-6-specific, HLA-A2-restricted T cell receptor (TCR). This TCR can recognize the claudin-6-derived epitope presented in HLA-A2 on the DC. The CD40×CD137 bispecific antibody can cross-link CD40 on the dendritic cell and CD137 on the T cell, leading to activation of the DC and a co-stimulatory signal to the T cell, resulting in T-cell proliferation.
HLA-A2+ PBMCs were obtained from healthy donors (Transfusionszentrale, University Hospital, Mainz, Germany). Monocytes were isolated from PBMCs by magnetic-activated cell sorting (MACS) technology using anti-CD14 MicroBeads (Miltenyi; cat. no. 130-050-201), according to the manufacturer's instructions. The peripheral blood lymphocytes (PBLs, CD14-negative fraction) were frozen for future T-cell isolation. For differentiation into immature DCs (iDCs), 1×106 monocytes/mL were cultured in RPMI GlutaMAX (Life technologies GmbH, cat. no. 61870-044) containing 5% human AB serum (Sigma-Aldrich Chemie GmbH, cat. no. H4522-100ML), sodium pyruvate (Life technologies GmbH, cat. no. 11360-039), non-essential amino acids (Life technologies GmbH, cat. no. 11140-035), 100 IU/mL penicillin-streptomycin (Life technologies GmbH, cat. no. 15140-122), 1000 IU/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; Miltenyi, cat. no. 130-093-868) and 1000 IU/mL interleukin 4 (IL-4; Miltenyi, cat. no. 130-093-924), for five days. Once during these five days, half of the medium was replaced with fresh medium. iDCs were harvested by collecting non-adherent cells; adherent cells were detached by incubation with PBS containing 2 mM EDTA for 10 min at 37°. After washing, iDCs were frozen in RPMI GlutaMAX containing 10% v/v DMSO (AppliChem GmbH, cat. no A3672,0050) and 50% v/v human AB serum for future antigen-specific T cell assays.
One day before T-cell assays were started, frozen PBLs and iDCs, from the same donor, were thawed. PBLs were used for isolation of CD8+ T cells by MACS technology using anti-CD8 MicroBeads (Miltenyi, cat. no. 130-045-201), according to the manufacturer's instructions. About 10-15×106 CD8+ T cells were electroporated with 10 μg IVT-RNA encoding the alpha-chain plus 10 μg of IVT-RNA encoding the beta-chain of a claudin-6-specific murine TCR (HLA-A2-restricted; described in WO 2015150327 A1) in 250 μL X-Vivo15 (Biozym Scientific GmbH, cat. no. 881026) in a 4-mm electroporation cuvette (VWR International GmbH, cat. no. 732-0023) using the BTX ECM® 830 Electroporation System device (BTX; 500 V, 1×3 ms pulse). Immediately after electroporation, cells were transferred into fresh IMDM medium (Life Technologies GmbH, cat. no. 12440-061) supplemented with 5% human AB serum and rested at 37° C., 5% CO2 for at least 1 hour. T cells were labeled using 1.6 μM carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, cat. no. C34564) in PBS, according to the manufacturer's instructions, and incubated in IMDM medium supplemented with 5% human AB serum, O/N.
Up to 5×106 thawed iDCs were electroporated with 0.4-5 μg IVT-RNA encoding full length claudin-6 (Uniprot P56747), in 250 μL X-Vivo15 medium, using the electroporation system as described above (300 V, 1×12 ms pulse) and incubated in IMDM medium supplemented with 5% human AB serum, O/N.
The next day, cells were harvested. Cell surface expression of claudin-6 on DCs and TCR on T cells were checked by flow cytometry. Therefore, DCs were stained with an Alexa647-conjugated CLDN6-specific antibody (not commercially available; in-house production) and T cells were stained with an anti-mouse TCR β Chain antibody (Becton Dickinson GmbH, cat. no. 553174). 5,000 electroporated DCs were incubated with 50,000 electroporated, CFSE-labeled T cells in the presence of bispecific or control antibodies in IMDM GlutaMAX (Life Technologies, cat. no. 12440-061) supplemented with 5% human AB serum in a 96-well round-bottom plate. T-cell proliferation was measured after 5 days by flow cytometry. Detailed analyses of T-cell proliferation based on CFSE-peaks indicating cell divisions were made by FlowJo 7.6.5 software. Mean percentages of T cells that went into division (% divided cells) and the average number of divisions of cells that went into division (proliferation index) were calculated.
FIG. 9A shows that only the CD40×CD137 bispecific antibodies efficiently enhanced proliferation of CD8+ T cells. The control monospecific, monovalent antibodies (b12×CD40; b12×CD137) and the combination of monospecific, monovalent CD40 with monospecific, monovalent CD137 antibodies (b12×CD40+b12×CD137) did not induce more proliferation than observed in the control (only the weakly activated PBMCs, ctrl, w/o). The same is also reflected in the percentage of divided cells (FIG. 9B) and is very clear from the proliferation index (FIG. 9C). FIG. 9D shows that the induction of antigen-specific proliferation by the CD40×CD137 bispecific antibodies was concentration dependent, with an optimum around 0.1 μg/mL in this assay.
Example 7
Humanization of Murine and Rabbit Antibodies
Humanized antibody sequences from the antibodies mouse anti-CD40-001 and rabbit anti-CD137-009 were generated at Antitope (Cambridge, UK). Humanized antibody sequences were generated using germline humanization (CDR-grafting) technology. Humanized V region genes were designed based upon human germline sequences with closest homology to the VH and VK amino acid sequences of the murine and rabbit antibodies. A series of four to six VH and four or five Vκ (VL) germline humanized V-region genes were designed for each of the non-human parental antibodies. Structural models of the non-human parental antibody V regions were produced using Swiss PDB and analyzed in order to identify amino acids in the V region frameworks that may be important for the binding properties of the antibody. These amino acids were noted for incorporation into one or more variant CDR-grafted antibodies. The closest matching germline sequences used as the basis for the humanized designs are shown in Table 3.
TABLE 3
Closest matching human germline V
segment and J segment sequences.
Heavy chain
Light chain (κ)
Human V
Human J
Human V
Human J
region
region
region
region
germline
germline
germline
germline
Antibody
segment
segment
segment
segment
Mouse anti-
hIGHV1-
hIGHJ4
hIGKV1-33*01
IGKJ4
CD40-001
46*01
Rabbit anti-
hIGHV3-
hIGHJ4
hIGKV1-33*01
IGKJ4
CD137-009
49*04
Variant sequences with the lowest incidence of potential T cell epitopes were then selected using Antitope's proprietary in silico technologies, iTope™ and TCED™ (T Cell Epitope Database) (Perry, L. C. A, Jones, T. D. and Baker, M. P. New Approaches to Prediction of Immune Responses to Therapeutic Proteins during Preclinical Development (2008). Drugs in R&D 9 (6): 385-396; 20; Bryson, C. J., Jones, T. D. and Baker, M. P. Prediction of Immunogenicity of Therapeutic Proteins (2010). Biodrugs 24 (1):1-8). Finally, the nucleotide sequences of the designed variants were codon-optimized for expression in human cells.
The variable region sequences of the humanized CD40 and CD137 antibodies are shown in the Sequence Listing and in Table 1 above.
Example 8
Expression Constructs for Antibodies, Transient Expression and Purification
For antibody expression the VH and VL sequences were cloned in expression vectors (pcDNA3.3) containing, in case of the VH, the relevant constant heavy chain 10 (HC), in certain cases containing a F405L or K409R mutation, and/or L234F, L235E and D265A, and, in case of the VL, light chain (LC) regions. Antibodies were expressed as IgG1,K. Plasmid DNA mixtures encoding both heavy and light chains of antibodies were transiently transfected in Expi293F™ cells (Life technologies, USA) using 293fectin (Life technologies) essentially as described by Vink et al. (Vink et al., Methods, 65 (1), 5-10 15 2014). Next, antibodies were purified by immobilized protein G chromatography.
Example 9
Non-Specific T-Cell Proliferation Assay to Test the Functionality of a Humanized Bispecific Antibody Binding to CD40 and CD137
To measure the functionality of a humanized bispecific antibody binding to CD40 and CD137, a non-antigen-specific T-cell proliferation assay was performed as described supra. In short, PBMCs of one donor were CFSE-labeled and incubated with a sub-optimal concentration of anti-CD3 antibody (clone UCHT1; 0.01 μg/mL as determined for this donor) and 0.008, 0.04, 0.2 or 1 μg/mL humanized CD40×CD137 bispecific antibody, the parental bispecific antibody or IgG1 control antibody.
Proliferation of CD8+ T cells was analyzed by flow cytometry, essentially as described supra. Detailed analyses of T-cell proliferation based on CFSE-peaks indicating cell divisions were made by FlowJo 7.6.5 software. Mean percentages of T cells that went into division (% divided cells) and the average number of divisions of cells that went into division (proliferation index) were calculated.
FIG. 10 shows that the humanized CD40×CD137 bispecific antibody (BisG1-CD40-001-HC6LC1-FEAL×CD137-009-HC7LC2-FEAR) efficiently enhanced proliferation of CD8+ T cells. The humanized bispecific antibody enhanced both the percentage of divided cells and the average number of divisions of CD8+ cells. Efficacy of the humanized bispecific antibody was comparable to that of the parental bispecific antibody (CD40-001×CD137-009).
Example 10
Antigen-Specific CD8+ T-Cell Proliferation Assay to Test the Functionality of the Humanized Bispecific Antibody Binding to CD40 and CD137
To measure the functionality of the humanized bispecific antibody binding to CD40 and CD137, an antigen-specific CD8+ T-cell proliferation assay was performed as described supra. In short, CFSE-labeled, CLDN6-TCR transfected CD8+ T-cells were incubated with CLDN6 RNA-electroporated DCs in the presence of humanized CD40×CD137 bispecific antibody, the parental antibody or IgG1 control antibody. T-cell proliferation was measured by flow cytometry after 4 days. Detailed analyses of T-cell proliferation based on CFSE-peaks indicating cell divisions were made by FlowJo 7.6.5 software. Mean percentages of T cells that went into division (% divided cells) and the average number of divisions of cells that went into division (proliferation index) were calculated.
FIG. 11 shows that the humanized CD40×CD137 bispecific antibody (BisG1-CD40-001-H6LC1-FEAL×CD137-009-HC7LC2-FEAR) efficiently enhanced proliferation of CD8+ T cells. Efficacy of the humanized bispecific antibody was comparable to that of the parental bispecific antibody (CD40-001×CD137-009). Both the humanized and the parental bispecific antibody enhanced the percentage of divided cells as well as the proliferation index of the CD8+ cells in this assay.
Example 11
Ex Vivo TIL Expansion Assay to Evaluate the Effects of CD40×CD137 Bispecific Antibodies on Tumor Infiltrating Lymphocytes
To evaluate the effects of CD40×CD137 bispecific antibody (BisG1-CD40-001-FEAL/CD137-009-FEAR) on tumor infiltrating lymphocytes (TIL), ex vivo culture of human tumor tissue was performed as follows. Freshly human tumor tissue resections were washed three times by transferring the isolated tumor chunks from one wash medium-containing well of a six-well plate (Fisher Scientific cat. no. 10110151) to the next using a spatula or serological pipette. Wash medium was composed of X-VIVO 15 (Biozym, cat. no. 881024) supplemented with 1% Pen/Strep (Thermo Fisher, cat. no. 15140-122) and 1% Fungizone (Thermo Fisher, cat. no. 15290-026). Next, the tumor was dissected with a surgical knife (Braun/Roth, cat. no. 5518091 BA223) and cut into tumor pieces with a diameter of about 1-2 mm. Two pieces each were put into one well of a 24-well plate (VWR international, cat. no. 701605) containing 1 mL TIL medium (X-VIVO 15, 10% Human Serum Albumin (HSA, CSL Behring, cat. no. PZN-6446518), 1% Pen/Strep, 1% Fungizone and IL-2 (Proleukin®S, Novartis Pharma, cat. no. 02238131) at the indicated concentration. Bispecific antibody binding to CD40 and CD137 was added at the indicated final concentrations. Culture plates were incubated at 37° C. and 5% CO2 for 72 hours and 1 mL fresh TIL medium containing the indicated IL-2 concentration and the indicated concentration of bispecific antibody was added to each well. Wells were monitored for the occurrence of TIL clusters using a Leica DMi1 microscope equipped with a 5.0 megapixel camera, every other day. Wells were split on an individual basis, when more than 25 TIL microclusters were detected. To split TIL cultures, cells were re-suspended and transferred to a well of a 6-well plate and supplemented with another 2 mL of TIL medium.
After a total culture period of 10-14 days, TILs were harvested and analyzed by flow cytometry. To allow for quantitative comparison of the different treatment groups, cell pellets were re-suspended after the last washing step with FACS-buffer supplemented with BD™ CompBeads (BD biosciences, cat. no. 51-90-9001291). Flow cytometric analysis was performed on a BD FACSCanto™ II flow cytometer (Becton Dickinson) and acquired data was analyzed using FlowJo 7.6.5 software. The relative viable TIL count (7-AAD-negative cells) per 1,000 beads was calculated for each well.
FIG. 12 shows the analysis of a TIL expansion from a human melanoma tissue. Here, 100 U/mL IL-2 was used as supplement for the TIL medium. Moreover, the following concentrations of the bispecific antibody binding to CD40 and CD137 (BisG1-CD40-001-FEAL/CD137-009-FEAR) were added: 0.016, 0.08, 0.4, 2.0 and 10.0 μg/mL; wells without antibody addition served as negative control. After 14 days of culture, TILs were harvested and analyzed by flow cytometry. Five samples for each antibody concentration, derived from different wells of the 24-well plate, were measured. In all samples cultured with the bispecific antibody binding to CD40 and CD137 the viable count of TIL was substantially increased in comparison to the control samples without antibody. Overall, about a 100-fold increase of the mean relative viable TIL count was observed (FIG. 12).
FIG. 13 shows the analysis of a TIL expansion from a non-small cell lung cancer (NSCLC) tissue. Here, 10 U/mL IL-2 was used as supplement for the TIL medium. In addition, the following final concentrations of the bispecific antibody binding to CD40 and CD137 (BisG1-CD40-001-FEAL/CD137-009-FEAR) were administered: 0.01, 0.1, and 1.0 μg/mL; wells without antibody addition served as negative control. After 10 days of culture, TILs were harvested and analyzed by flow cytometry. Five samples for each antibody concentration, derived from different wells of the 24-well plate, were measured. In all samples cultured with the bispecific antibody binding to CD40 and CD137, the viable count of TIL was substantially increased in comparison to the control samples without antibody. Overall, an up to 10-fold increase of the mean relative viable TIL count was observed at 0.1 or 1 μg/mL (FIG. 13).
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16855703
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genmab a/s
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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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Apr 1st, 2022 06:04PM
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Apr 1st, 2022 06:04PM
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Genmab A/S
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Health Care
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Pharmaceuticals & Biotechnology
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cph:gen
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Genmab A/S
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Oct 4th, 2016 12:00AM
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Oct 16th, 2013 12:00AM
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https://www.uspto.gov?id=US09458236-20161004
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Human monoclonal antibodies to epidermal growth factor receptor (EGFR)
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Isolated human monoclonal antibodies which specifically bind to human EGFR, and related antibody-based compositions and molecules, are disclosed. The human antibodies can be produced by a transfectoma or in a non-human transgenic animal, e.g., a transgenic mouse, capable of producing multiple isotypes of human monoclonal antibodies by undergoing V-D-J recombination and isotype switching. Also disclosed are pharmaceutical compositions comprising the human antibodies, non-human transgenic animals and hybridomas which produce the human antibodies, and therapeutic and diagnostic methods for using the human antibodies.
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9458236
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1. An isolated human monoclonal antibody which binds to human EGFR comprising heavy and light chain variable regions, wherein the heavy chain variable region is encoded by the nucleic acid set forth in SEQ ID NO: 1, or the light chain variable region is encoded by the nucleic acid sequence set forth in SEQ ID NO: 3.
2. An isolated human monoclonal antibody which binds to human EGFR comprising heavy and light chain variable regions, wherein the heavy chain variable region comprises the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 5, 6, and 7, respectively, and the light chain variable region comprises the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 8, 9, and 10, respectively, wherein the antibody binds to human EGFR with an equilibrium association constant (KA) of at least 108M−1.
3. The antibody of claim 1, wherein the antibody is an IgG1 antibody.
4. The antibody of claim 1, wherein the antibody binds to human EGFR with an equilibrium association constant (KA) of at least 108M−1.
5. The antibody of claim 1, wherein the antibody has a dissociation constant (KD) from EGFR of about 10−3s−1 or less.
6. The antibody of claim 1, wherein the antibody: (a) inhibits EGFR ligand binding to EGFR; (b) has a binding equilibrium association constant (KA) to human EGFR of at least 107M−1; and (c) inhibits growth of a cell expressing EGFR.
7. The antibody of claim 6, wherein the cell is a tumor cell selected from the group of tumor cells consisting of a bladder cell, a breast cell, a colon cell, a kidney cell, an ovarian cell, a prostate cell, a squamous cell, and a non-small lung cell.
8. The antibody of claim 6, wherein the cell is selected from the group consisting of a synovial fibroblast cell and a keratinocyte.
9. The antibody of claim 7, which binds to a cell expressing EGFR and induces lysis (ADCC) of the cell in the presence of human effector cells and/or does not induce complement-mediated lysis of the cell in vivo.
10. The antibody of claim 1, which binds to EGFR and inhibits EGF- or TGF-α-induced autophosphorylation of EGFR.
11. An antigen-binding portion of the antibody of claim 1 comprising a Fab fragment or a single chain antibody.
12. The antibody of claim 1 produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal having a genome comprising a human heavy chain transgene and a human light chain transgene fused to an immortalized cell.
13. A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds the same epitope on EGFR as the antibody of claim 1, and wherein the second antigen-binding region binds a second target epitope.
14. A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds the same epitope on EGFR as the antibody of claim 1, and wherein the second antigen-binding region binds a human Fc receptor.
15. The bispecific antibody of claim 14, wherein the human Fc receptor is a human FcγRI or a human Fcα receptor.
16. The bispecific antibody of claim 14, wherein the bispecific antibody binds to the human Fc receptor at a site which is distinct from the immunoglobulin Fc binding site of the receptor.
17. A composition comprising the antibody of claim 1 and a carrier.
18. A composition comprising the antibody of claim 1 and a chemotherapeutic agent.
19. An immunotoxin comprising the antibody of claim 1 linked to a cytotoxic agent.
20. A method of detecting the presence of EGFR antigen, or a cell expressing EGFR, in a sample, comprising:
contacting the sample with an antibody comprising heavy and light chain variable region sequences which comprise the amino acid sequences as set forth in SEQ ID NO: 2 and SEQ ID NO: 4, respectively, under conditions that allow for formation of a complex between the antibody and EGFR; and
detecting the formation of the complex.
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20
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RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 12/464,057, filed on May 11, 2009 (now U.S. Pat. No. 8,586,041 B2), which is a divisional of U.S. patent application Ser. No. 10/320,094, filed on Dec. 16, 2002, issued on Sep. 29, 2009 as U.S. Pat. No. 7,595,378, which is a continuation-in-part of U.S. patent application Ser. No. 10/172,317, filed on Jun. 13, 2002, issued on Jul. 24, 2007 as U.S. Pat. No. 7,247,301, which claims priority to U.S. Provisional Patent Application No. 60/298,172, filed on Jun. 13, 2001, the contents of which are incorporated herein in their entirety by this reference.
BACKGROUND OF THE INVENTION
The EGF receptor (EGFR) is a 170 kDa type 1 transmembrane molecule. Its expression is found to be upregulated in many human tumors including carcinoma of the head and neck, breast, colon, prostate, lung, and ovaries. The degree of over-expression is correlated to poor clinical prognosis (Baselga, et al. (1994) Pharmac. Therapeut. 64:127-154; Modjtahedi, et al. (1994) Int. J. Oncology 4:277-296). Furthermore, its expression is frequently accompanied by the production of EGFR-ligands, TGF-α and EGF among others, by EGFR-expressing tumor cells which suggests that an autocrine loop participates in the progression of these cells (Baselga, et al. (1994) Pharmac. Therapeut. 64:127-154; Modjtahedi, et al. (1994) Int. J. Oncology. 4:277-296). Blocking the interaction between such EGFR ligands and EGFR therefore can inhibit tumor growth and survival (Baselga, et al. (1994) Pharmac. Therapeut. 64:127-154).
Monoclonal antibodies (MAbs) directed to the ligand-binding domain of EGFR can block the interaction with EGF and TGF-α and, concomitantly, the resultant intracellular signaling pathway. Several murine monoclonal antibodies have been generated which achieve such a block in vitro and which have been evaluated for their ability to affect tumor growth in mouse xenograft models (Masui, et al. (1986) Cancer Res. 46: 5592-5598; Masui, et al. (1984) Cancer Res. 44: 1002-1007; Goldstein, et al. (1995) Clin. Cancer Res. 1: 1311-1318). When administered one day after the human tumor cells, most of the anti-EGFR MAbs were efficacious in preventing tumor formation in athymic mice (Baselga, et al. (1994) Pharmac. Therapeut. 64:127-154). However, when injected into mice bearing established human tumor xenografts, these murine MAbs (e.g., MAbs 225 s and 528) caused only partial tumor regression. Co-administration of chemotherapeutic agents was needed to fully eradicate the tumors (Baselga, et al. (1994) Pharmac. Therapeut. 64:127-154; Fan, et al. (1993) Cancer Res. 53: 4322-4328; Baselga, et al. (1993) J. Natl. Cancer Inst. 85: 1327-1333).
Therefore, while the results obtained to date clearly establish EGFR as a target for immunotherapy, they also show that murine antibodies do not constitute ideal therapeutic agents. Moreover, treatment with murine antibodies generally triggers severe immune reactions in patients. To circumvent the immunogenicity of mouse antibodies, therapeutics should ideally be fully human. As a step towards this goal, a chimeric version of the 225 MAb (C225), in which the mouse antibody variable regions are linked to human constant regions, has been developed. While C225 exhibited an improved anti-tumor activity in the treatment of established xenograft tumors in vivo, this was only achieved at high doses (Goldstein, et al. (1995) Clin. Cancer Res. 1:1311-1318). Currently C225 is being evaluated in clinical trials for treatment of various types of solid tumors (Baselga, J. (2000) J. Clin. Oncol. 18: 54S-59S; Baselga, J. (2000) Ann. Oncol. 11 Suppl 3: 187-190, 2000).
Accordingly, the need exists for improved therapeutic antibodies against EGFR which are effective at treating and/or preventing diseases related to overexpression of EGFR when administered at low dosages, and which do not elicit immune reactions in patients. As described above, monoclonal antibodies (MAb) play a prominent role in many diagnostic and therapeutic approaches to diseases and have become even more attractive agents with the recent advent of technologies that allow development of fully human antibodies. Antibodies and antibody derivatives constitute twenty five percent of biological drugs currently under development and many of these are being developed as cancer therapeutics. Antibodies combine target specificity with the capacity to effectively engage the immune system. The combination of these properties and their long biological half-life alerted researchers to the therapeutic potential of antibodies. This has recently culminated in the U.S. Food and Drug Administration (FDA) approval of several antibodies for cancer treatment.
SUMMARY OF THE INVENTION
The present invention provides improved antibody therapeutics for treating and preventing diseases related to expression of EGFR, particularly EGFR-expressing tumors and autoimmune diseases. The antibodies are improved in that they are fully human (referred to herein as “HuMAbs™”) and, thus, are less immunogenic in patients. The antibodies are also therapeutically effective (e.g., at preventing growth and/or function of EGFR-expressing cells) at lower dosages than previously reported for other anti-EGFR antibodies. In addition, in certain embodiments, the antibodies have the added benefit of not activating complement (e.g., not inducing complement mediated lysis of target cells) which reduces adverse side-effects during treatment.
Accordingly, in one embodiment, the present invention provides isolated human monoclonal antibodies which specifically bind to human epidermal growth factor receptor (EGFR), as well as compositions containing one or a combination of such antibodies. The human antibodies inhibit (e.g., block) binding of EGFR ligands, such as EGF and TGF-α, to EGFR. For example, binding of EGFR ligand to EGFR can be inhibited by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% and preferably results in the prevention of EGFR-mediated cell signaling.
Preferred human antibodies of the invention inhibit the growth and/or mediate the killing (e.g., lysis or phagocytosis) of cells expressing EGFR (in vitro or in vivo) in the presence of human effector cells (e.g., polymorphonuclear cells, monocytes, macrophages and dendritic cells), yet they do not activate complement mediated lysis of cells which express EGFR. Accordingly, human monoclonal antibodies of the invention can be used as diagnostic or therapeutic agents in vivo and in vitro.
In one embodiment exemplified herein, human antibodies of the invention are IgG1 (e.g., IgG1k) antibodies having an IgG1 heavy chain and a kappa light chain. However, other antibody isotypes are also encompassed by the invention, including IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE. The antibodies can be whole antibodies or antigen-binding fragments of the antibodies, including Fab, F(ab′)2, Fv and chain Fv fragments.
In another aspect, the invention relates to an isolated human monoclonal antibody which binds to human EGFR, wherein the antibody is an IgG1,κ or IgG1,λ isotype.
In another aspect, the invention relates to an isolated human monoclonal antibody which binds to human EGFR, wherein the antibody is an IgG4 antibody.
In another aspect, the invention relates to an isolated human monoclonal antibody which binds to human EGFR, wherein the antibody is an IgG4,κ or IgG4,λ isotype.
In another aspect, the invention relates to an isolated human monoclonal antibody which binds to human EGFR, wherein the antibody is selected from the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgD antibodies, and wherein the antibody comprises a variable heavy chain amino acid sequence as set forth in SEQ ID NO:2.
In another aspect, the invention relates to an isolated human monoclonal antibody which binds to human EGFR, wherein the antibody is selected from the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgD antibodies, and wherein the antibody comprises a variable heavy chain amino acid sequence which is at least 90% homologous, preferably at least 95% homologous, and more preferably at least 98%, or at least 99% homologous to the amino acid sequence as set forth in SEQ ID NO:2.
In another aspect, the invention relates to an isolated human monoclonal antibody which binds to human EGFR, wherein the antibody is selected from the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgD antibodies, and wherein the antibody comprises a variable light chain amino acid sequence as set forth in SEQ ID NO:4.
In another aspect, the invention relates to an isolated human monoclonal antibody which binds to human EGFR, wherein the antibody is selected from the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgD antibodies, and wherein the antibody comprises a variable light chain amino acid sequence which is at least 90% homologous, preferably at least 95% homologous, and more preferably at least 98%, or at least 99% homologous to the amino acid sequence as set forth in SEQ ID NO:4.
In another aspect, the invention relates to an isolated human monoclonal antibody which binds to human EGFR, wherein the antibody is selected from the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgD antibodies, and wherein the antibody comprises human heavy chain and human kappa light chain variable regions which are at least 90% homologous, preferably at least 95% homologous, and more preferably at least 98%, or at least 99% homologous to the amino acid sequences as set forth in SEQ ID NO:2 and SEQ ID NO:4, respectively.
In another aspect, the invention relates to an isolated human monoclonal antibody which binds to human EGFR, wherein the antibody is selected from the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgD antibodies, and wherein the antibody comprises at least one CDR sequence selected from the group consisting of:
(i) the CDR1, CDR2, and CDR3 regions shown in FIG. 15 (SEQ ID NOs:5, 6, 7, 8, 9, and 10);
(ii) sequences which are at least 90% homologous, preferably at least 95% homologous, and more preferably at least 98%, or at least 99% homologous to the sequences defined in (i); and
(iii) fragments of any one of the sequences defined in (i) or (ii), which retain the ability to bind to human EGFR.
In another aspect, the invention relates to an isolated human monoclonal antibody which binds to human EGFR, wherein the antibody is selected from the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgD antibodies, and wherein the antibody comprises the heavy chain CDR3 region shown in FIG. 15 (SEQ ID NO:7), a sequence which is at least 90% homologous, preferably at least 95% homologous, and more preferably at least 98%, or at least 99% homologous to SEQ ID NO:7, or a fragment thereof, which retains the ability to bind to human EGFR.
In another aspect the invention relates to an isolated human monoclonal antibody which binds to human EGFR, wherein the antibody is selected from the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgD antibodies, and wherein the antibody comprises at least four CDRs selected from (i) the CDR regions shown in FIG. 15 (SEQ ID NOs:5, 6, 7, 8, 9, or 10); (ii) sequences which are at least 90% homologous, preferably at least 95% homologous, and more preferably at least 98%, or at least 99% homologous to the sequences defined in (i); and (iii) fragments of the sequences defined in (i) or (ii), which retain the ability to bind to human EGFR.
In another aspect the invention relates to an isolated human monoclonal antibody which binds to human EGFR, wherein the antibody is selected from the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgD antibodies, and wherein the antibody comprises (i) the 6 CDR regions shown in FIG. 15 (SEQ ID NOs:5, 6, 7, 8, 9, and 10); (ii) sequences which are at least 90% homologous, preferably at least 95% homologous, and more preferably at least 98%, or at least 99% homologous to the sequences defined in (i); or (iii) fragments of the sequences defined in (i) or (ii), which retain the ability to bind to human EGFR.
In another aspect, the invention relates to an isolated human monoclonal antibody which binds to human EGFR, wherein the antibody is an intact antibody selected from the group consisting of an intact IgG1 antibody, an intact IgG4 antibody, an intact IgM antibody, an intact IgA1 antibody, an intact IgA2 antibody, an intact secretory IgA antibody, an intact IgD antibody, and an intact IgE antibody, wherein the antibody is glycosylated in a eukaryotic cell.
In another aspect, the invention relates to an isolated human monoclonal antibody which binds to human EGFR, wherein the antibody is an intact antibody selected from the group consisting of an intact IgG1,κ antibody, an intact IgG1,λ antibody, an intact IgG4,κ antibody, and an intact IgG4,κ antibody, wherein the antibody is glycosylated in a eukaryotic cell.
In another aspect, the invention relates to an isolated human monoclonal antibody which binds to human EGFR, wherein the antibody is selected from the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgD antibodies, and wherein the antibody comprises a heavy chain variable region amino acid sequence derived from a human VH3-33 germline sequence (SEQ ID NO:12) and a light chain variable region amino acid sequence derived from a human VκL18 germline sequence (SEQ ID NO:11).
In a particular embodiment, the human antibody is encoded by human IgG heavy chain and human kappa light chain nucleic acids comprising nucleotide sequences in their variable regions as set forth in SEQ ID NO:1 and SEQ ID NO:3, respectively, and conservative sequence modifications thereof. In another embodiment, the human antibody include IgG heavy chain and kappa light chain variable regions which comprise the amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4, respectively, and conservative sequence modifications thereof.
In another particular embodiment, the human antibody corresponds to antibody 2F8 or an antibody that binds to the same epitope as (e.g., competes with) or has the same functional binding characteristics as antibody 2F8.
Human antibodies of the invention can be produced recombinantly in a host cell, such as a transfectoma (e.g., a transfectoma consisting of immortalized CHO cells or lymphocytic cells) containing nucleic acids encoding the heavy and light chains of the antibody, or be obtained directly from a hybridoma which expresses the antibody (e.g., which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a human light chain transgene that encode the antibody, fused to an immortalized cell). In a particular embodiment, the antibodies are produced by a hybridoma referred to herein as 2F8 or by a host cell (e.g., a CHO cell) transfectoma containing human heavy chain and human light chain nucleic acids which comprise nucleotide sequences in their variable regions as set forth in SEQ ID NOs:1 and 3, respectively, and conservative modifications thereof.
In another embodiment, human anti-EGFR antibodies of the present invention can be characterized by one or more of the following properties:
a) specificity for the EGFR;
b) a binding affinity to EGFR with an equilibrium association constant (KA) of at least about 107 M−1, preferably about, 108 M−1, 109 M−1, and more preferably, about 1010 M−1 to 1011 M−1 or higher;
c) a dissociation constant (KD) from EGFR of about 10−3 s−1, preferably about 10−4 s−1, more preferably, 10−5 s−1, and most preferably, 10−6 s−1;
d) the ability to opsonize a cell expressing EGFR; or
e) the ability to inhibit growth and/or mediate phagocytosis and killing of cells expressing EGFR (e.g., a tumor cell) in the presence of human effector cells at a concentration of about 10 μg/ml or less (e.g., in vitro).
Examples of EGFR-expressing tumor cells which can be targeted (e.g., opsonized) by human antibodies of the present invention include, but are not limited to, bladder, breast, colon, kidney, ovarian, prostate, renal cell, squamous cell, lung (non-small cell), or head and neck tumor cells. Other EGFR-expressing cells include synovial fibroblast cells and keratinocytes which can be used, for example, as target cells in the treatment of arthritis and psoriasis, respectively.
In another embodiment, human antibodies of the invention bind to EGFR antigen with an affinity constant of at least about 108 M−1, more preferably at least about 109 M−1 or 1010 M−1, and are capable of inhibiting growth and/or mediating phagocytosis and killing of cells expressing EGFR by human effector cells, e.g., polymorphonuclear cells (PMNs), monocytes and macrophages, with an IC50 of about 1×10−7 M or less, or at a concentration of about 10 μg/ml or less in vitro.
In yet another embodiment, human antibodies of the invention inhibit EGFR-mediated cell signaling. For example, the antibodies can inhibit EGFR ligand (e.g., EGF or TGF-α) induced autophosphorylation of EGFR. The antibodies also can inhibit autocrine EGF or TGF-α induced cell activation or by inducing lysis (ADCC) of EGFR expressing cells in the presence of human polymorphonuclear cells. Cells which express EGFR include, among others, a bladder cell, a breast cell, a colon cell, a kidney cell, an ovarian cell, a prostate cell, a renal cell, a squamous cell, a non-small lung cell, a synovial fibroblast cell, and a keratinocyte.
In another aspect, the present invention provides nucleic acid molecules encoding the antibodies, or antigen-binding portions, of the invention. Recombinant expression vectors which include nucleic acids encoding antibodies of the invention, and host cells transfected with such vectors, are also encompassed by the invention, as are methods of making the antibodies of the invention by culturing such host cells, e.g., an expression vector comprising a nucleotide sequence encoding the variable and constant regions of the heavy and light chains of antibody 2F8 produced by the hybridoma.
In yet another aspect, the invention provides isolated B-cells from a transgenic non-human animal, e.g., a transgenic mouse, which express human anti-EGFR antibodies of the invention. Preferably, the isolated B cells are obtained from a transgenic non-human animal, e.g., a transgenic mouse, which has been immunized with a purified or enriched preparation of EGFR antigen and/or cells expressing the EGFR. Preferably, the transgenic non-human animal, e.g., a transgenic mouse, has a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or a portion of an antibody of the invention. The isolated B-cells are then immortalized to provide a source (e.g., a hybridoma) of human anti-EGFR antibodies.
Accordingly, the present invention also provides a hybridoma capable of producing human monoclonal antibodies of the invention that specifically bind to EGFR. In one embodiment, the hybridoma includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse having a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or a portion of an antibody of the invention, fused to an immortalized cell. Particular hybridomas of the invention include 2F8.
In another aspect the invention relates to a hybridoma comprising a B cell obtained from a transgenic nonhuman animal in which V-(D)-J gene segment rearrangements have resulted in the formation of a functional human heavy chain transgene and a functional light chain transgene fused to an immortalized cell, wherein the hybridoma produces a detectable amount of the monoclonal antibody of the invention as defined in any of the claims or embodiments herein.
In another aspect the invention relates to a transfectoma comprising nucleic acids encoding a human heavy chain and a human light chain, wherein the transfectoma produces a detectable amount of the monoclonal antibody of the invention as defined in any of the claims or embodiments herein.
In yet another aspect, the invention provides a transgenic non-human animal, such as a transgenic mouse (also referred to herein as a “HuMAb”), which express human monoclonal antibodies that specifically bind to EGFR. In a particular embodiment, the transgenic non-human animal is a transgenic mouse having a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or a portion of an antibody of the invention. The transgenic non-human animal can be immunized with a purified or enriched preparation of EGFR antigen and/or cells expressing EGFR. Preferably, the transgenic non-human animal, e.g., the transgenic mouse, is capable of producing multiple isotypes of human monoclonal antibodies to EGFR (e.g., IgG, IgA and/or IgM) by undergoing V-D-J recombination and isotype switching. Isotype switching may occur by, e.g., classical or non-classical isotype switching.
In another aspect, the present invention provides methods for producing human monoclonal antibodies which specifically react with EGFR. In one embodiment, the method includes immunizing a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or a portion of an antibody of the invention, with a purified or enriched preparation of EGFR antigen and/or cells expressing EGFR. B cells (e.g., splenic B cells) of the animal are then obtained and fused with myeloma cells to form immortal, hybridoma cells that secrete human monoclonal antibodies against EGFR.
In yet another aspect, human anti-EGFR antibodies of the invention are derivatized, linked to or co-expressed with another functional molecule, e.g., another peptide or protein (e.g., an Fab′ fragment). For example, an antibody or antigen-binding portion of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., to produce a bispecific or a multispecific antibody), a cytoxin, a cellular ligand or an antigen. Accordingly, present invention encompasses a large variety of antibody conjugates, bi- and multispecific molecules, and fusion proteins, all of which bind to EGFR expressing cells and which target other molecules to the cells, or which bind to EGFR and to other molecules or cells.
In a particular embodiment, the invention includes a bispecific or multispecific molecule comprising at least one first binding specificity for EGFR (e.g., a human anti-EGFR antibody or fragment or mimetic thereof), and a second binding specificity for an Fc receptor, e.g., human FcγRI or a human Fcα receptor, or another antigen on an antigen presenting cell (APC). Typically, bispecific and multispecific molecules of the invention comprise at least one antibody, or fragment thereof (e.g., an Fab, Fab′, F(ab′)2, Fv, or a single chain Fv), preferably a human antibody or a portion thereof, or a “chimeric” or a “humanized” antibody or a portion thereof (e.g., has a variable region, or at least a complementarity determining region (CDR), derived from a non-human antibody (e.g., murine) with the remaining portion(s) being human in origin).
Accordingly, the present invention includes bispecific and multispecific molecules that bind to both human EGFR and to an Fc receptor, e.g., a human IgG receptor, e.g., an Fc-gamma receptor (FcγR), such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). Other Fc receptors, such as human IgA receptors (e.g., FcαRI), also can be targeted. The Fc receptor is preferably located on the surface of an effector cell, e.g., a monocyte, macrophage or an activated polymorphonuclear cell. In a preferred embodiment, the bispecific and multispecific molecules bind to an Fc receptor at a site which is distinct from the immunoglobulin Fc (e.g., IgG or IgA) binding site of the receptor. Therefore, the binding of the bispecific and multispecific molecules is not blocked by physiological levels of immunoglobulins.
In another aspect, the present invention provides a conjugate comprising a human anti-EGFR antibody of the invention linked to a therapeutic moiety, e.g., a cytotoxic drug, an enzymatically active toxin, or a fragment thereof, a radioisotope, or a small molecule anti-cancer drug.
Alternatively, human antibodies of the invention can be co-administered with such therapeutic and cytotoxic agents, but not linked to them. They can be coadministered simultaneously with such agents (e.g., in a single composition or separately) or can be administered before or after administration of such agents. Such agents can include chemotherapeutic agents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, and cyclophosphamide hydroxyurea. Human antibodies of the invention also can be administered in conjunction with radiation therapy.
In another aspect, the present invention provides compositions, e.g., pharmaceutical and diagnostic compositions/kits, comprising a pharmaceutically acceptable carrier and at least one human monoclonal antibody of the invention, or an antigen-binding portion thereof, which specifically binds to EGFR. In one embodiment, the composition comprises a combination of the human antibodies or antigen-binding portions thereof, preferably each of which binds to a distinct epitope. For example, a pharmaceutical composition comprising a human monoclonal antibody that mediates highly effective killing of target cells in the presence of effector cells can be combined with another human monoclonal antibody that inhibits the growth of cells expressing EGFR. Thus, the combination provides multiple therapies tailored to provide the maximum therapeutic benefit. Compositions, e.g., pharmaceutical compositions, comprising a combination of at least one human monoclonal antibody of the invention, or antigen-binding portions thereof, and at least one bispecific or multispecific molecule of the invention, are also within the scope of the invention.
In another aspect the invention relates to a pharmaceutical composition comprising the human antibody of the invention as defined in any of the claims or embodiments herein and a pharmaceutically acceptable carrier.
In another aspect the pharmaceutical composition is in a form suitable for injection or infusion.
In another aspect the pharmaceutical composition is a liposome formulation.
In yet another aspect, the invention provides a method for inhibiting the proliferation and/or growth of a cell expressing EGFR, and/or inducing killing of a cell expressing EGFR, using human antibodies of the invention and related compositions as described above. In one embodiment, the method comprises contacting a cell expressing EGFR either in vitro or in vivo with one or a combination of human monoclonal antibodies of the invention in the presence of a human effector cell. The method can be employed in culture, e.g., in vitro or ex vivo (e.g., cultures comprising cells expressing EGFR and effector cells). For example, a sample containing cells expressing EGFR and effector cells can be cultured in vitro, and combined with an antibody of the invention, or an antigen-binding portion thereof (or a bispecific or multispecific molecule of the invention). Alternatively, the method can be performed in a subject, e.g., as part of an in vivo (e.g., therapeutic or prophylactic) protocol.
For use in in vivo treatment and prevention of diseases related to EGFR expression (e.g., over-expression), human antibodies of the invention are administered to patients (e.g., human subjects) at therapeutically effective dosages (e.g., dosages which result in growth inhibition, phagocytosis and/or killing of tumor cells expressing EGFR) using any suitable route of administration, such as injection and other routes of administration known in the art for antibody-based clinical products.
Typical EGFR-related diseases which can be treated and/or prevented using the human antibodies of the invention include, but are not limited to, autoimmune diseases and cancers. For example, cancers which can be treated and/or prevented include cancer of the bladder, breast, uterine/cervical, colon, kidney, ovarian, prostate, renal cell, pancreatic, colorectal, stomach, squamous cell, lung (non-small cell), esophageal, head and neck. Autoimmune diseases which can be treated include, for example, psoriasis and inflammatory arthritis, e.g., rheumatoid arthritis, systemic lupus erythematosus-associated arthritis, psoriatic arthritis, Menetrier's disease, systemic sclerosis, Sjögren's syndrome, pulmonary fibrosis, bronchial asthma, myelofibrosis, diabetic nephropathy, chronic allograft rejection, chronic glomerulonephritis, Crohn's disease, ulcerative colitis, hepatic cirrhosis, sclerosing cholangitis, chronic uveitis, or cicatricial pemphigoid.
In another aspect the invention relates to a method of treating or preventing Alzheimer's disease or other forms of dementia.
In one embodiment, the patient is additionally treated with one or more further therapeutic agents and/or physical therapies (e.g., radiation therapy, hyperthermia, transplantation (e.g., bone marrow transplantation), surgery, sunlight, or phototherapy), such as one or more further therapeutic agents and/or physical therapies as disclosed in the following. The patient can also be additionally treated with a chemotherapeutic agent, radiation, or an agent that modulates, e.g., enhances or inhibits, the expression or activity of an Fc receptor, e.g., an Fcα receptor or an Fcγ receptor, such as a cytokine. Typical cytokines for administration during treatment include granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumor necrosis factor (TNF). Typical therapeutic agents include, among others, anti-neoplastic agents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, and cyclophosphamide hydroxyurea.
The additional therapeutic agents and/or physical therapies may be administered either prior to, simultaneously with, or following administration of the human antibody.
In another aspect the pharmaceutical composition comprises one or more further therapeutic agents, such as one or more agents selected from chemotherapeutic agents, immunosuppressive agent, anti-inflammatory agents, and anti-psoriasis agents, and/or physical therapies (e.g., radiation therapy, hyperthermia, transplantation (e.g., bone marrow transplantation), surgery, sunlight, or phototherapy).
In another aspect the pharmaceutical composition comprises one or more further chemotherapeutic agents selected from the group consisting of nitrogen mustards (e.g., cyclophosphamide and ifosfamide), aziridines (e.g., thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine and streptozocin), platinum complexes (e.g., carboplatin and cisplatin), non-classical alkylating agents (e.g., dacarbazine and temozolamide), folate analogs (e.g., methotrexate), purine analogs (e.g., fludarabine and mercaptopurine), adenosine analogs (e.g., cladribine and pentostatin), pyrimidine analogs (e.g., fluorouracil (alone or in combination with leucovorin) and gemcitabine), substituted ureas (e.g., hydroxyurea), antitumor antibiotics (e.g., bleomycin and doxorubicin), epipodophyllotoxins (e.g., etoposide and teniposide), microtubule agents (e.g., docetaxel and paclitaxel), camptothecin analogs (e.g., irinotecan and topotecan), enzymes (e.g., asparaginase), cytokines (e.g., interleukin-2 and interferon-α), monoclonal antibodies (e.g., trastuzumab and bevacizumab), recombinant toxins and immunotoxins (e.g., recombinant cholera toxin-B and TP-38), cancer gene therapies, and cancer vaccines (e.g., vaccine against telomerase). Other treatments may include hyperthermia, radiation therapy, transplantation and surgery.
In another aspect the pharmaceutical composition comprises one or more further therapeutic agents selected from the group consisting of immunosuppressive antibodies (e.g., antibodies against MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40, CD45, IFN-γ TNF-α, IL-4, IL-5, IL-6R, IL-7, IL-8, IL-10, CD11a, CD20, or CD58, or antibodies against their ligands) and other immunomodulatory compounds (e.g., soluble IL-15R or IL-10).
In another aspect the pharmaceutical composition comprises one or more further immunosuppressive agents selected from the group consisting of cyclosporine, azathioprine, mycophenolic acid, mycophenolate mofetil, corticosteroids (e.g., prednisone), methotrexate, gold salts, sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine, 15-deoxyspergualine, 6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, and anti-thymocyte globulin.
In another aspect the pharmaceutical composition comprises one or more further anti-inflammatory agents selected from the group consisting of aspirin and other salicylates, steroidal drugs, NSAIDs (nonsteroidal anti-inflammatory drugs) (e.g., ibuprofen, fenoprofen, naproxen, sulindac, diclofenac, piroxicam, ketoprofen, diflunisal, nabumetone, etodolac, oxaprozin, and indomethacin), Cox-2 inhibitors (e.g., rofecoxib and celecoxib), and DMARDs (disease modifying antirheumatic drugs) (e.g., methotrexate, hydroxychloroquine, sulfasalazine, azathioprine, pyrimidine synthesis inhibitors (e.g., leflunomide), IL-1 receptor blocking agents (e.g., anakinra), TNF-α blocking agents (e.g., etanercept, infliximab and adalimumab), anti-IL-6R antibodies, CTLA4Ig, and anti-IL-15 antibodies).
In another aspect the pharmaceutical composition comprises one or more further anti-psoriasis agents selected from the group consisting of coal tar, A vitamin, anthralin, calcipotrien, tarazotene, corticosteroids, methotrexate, retinoids (e.g., acitretin), cyclosporine, etanercept, alefacept, efaluzimab, 6-thioguanine, mycophenolate mofetil, tacrolimus (FK-506), and hydroxyurea.
Other treatments may include exposure to sunlight or phototherapy, including UVB (broad-band and narrow-band ultraviolet B), UVA (ultraviolet A) and PUVA (psoralen methoxalen plus ultraviolet A).
In one embodiment the antibodies of the invention are administered in combination with two or more of the above therapies, such as in combination with methotrexate+phototherapy (PUVA or UVA); methotrexate+acitretin; acitretin+phototherapy (PUVA or UVA); methotrexate+acitretin+phototherapy (PUVA or UVB); hydroxyurea+phototherapy (PUVA or UVB); hydroxyurea+acitretin; cyclosporine+methotrexate; or calcipotrien+phototherapy (UVB).
In another aspect, the invention relates to an immunoconjugate comprising an antibody according to the invention linked to a radioisotope, cytotoxic agent (e.g., calicheamicin and duocarmycin), a cytostatic agent, or a chemotherapeutic drug.
In another aspect the invention relates to an immunoconjugate, wherein the antibody is linked to a radioisotope (e.g., iodine-131, yttrium-90 or indium-111).
In another aspect the invention relates to an immunoconjugate, wherein the antibody is linked to any one of the chemotherapeutic agents as defined above.
To increase the therapeutic efficacy of human anti-EGFR antibodies of the invention against cancer cells which do not highly express EGFR, the antibodies can be co-administered with an agent which upregulates or otherwise effects expression of EGFR, such as a lymphokine preparation which cause upregulated and more homogeneous expression of EGFR on tumor cells. Lymphokine preparations suitable for administration include interferon-gamma, tumor necrosis factor, and combinations thereof. These can be administered intravenously. Suitable dosages of lymphokine typically range from 10,000 to 1,000,000 units/patient.
In another aspect, the invention relates to a pharmaceutical composition comprising an expression vector comprising a nucleotide sequence encoding the variable region of a light chain, heavy chain or both light and heavy chains of a human antibody which binds EGFR.
In another aspect, the invention relates to a pharmaceutical composition comprising an expression vector comprising a nucleotide sequence encoding the variable region of a light chain, heavy chain or both light and heavy chains of a human antibody which binds EGFR, and further comprising a nucleotide sequence encoding the constant region of a light chain, heavy chain or both light and heavy chains of a human antibody which binds EGFR.
In another aspect, the invention relates to a pharmaceutical composition comprising an expression vector comprising a nucleotide sequence encoding heavy chain and light chain variable regions which comprise the amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4, respectively, and conservative sequence modifications thereof.
In yet another aspect, the present invention provides a method for detecting in vitro or in vivo the presence of EGFR antigen in a sample, e.g., for diagnosing an EGFR-related disease. In one embodiment, this is achieved by contacting a sample to be tested, optionally along with a control sample, with a human monoclonal antibody of the invention (or an antigen-binding portion thereof) under conditions that allow for formation of a complex between the antibody and EGFR. Complex formation is then detected (e.g., using an ELISA). When using a control sample along with the test sample, complex is detected in both samples and any statistically significant difference in the formation of complexes between the samples is indicative the presence of EGFR antigen in the test sample.
Other features and advantages of the instant invention be apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing competitive ELISA with hybridoma supernatants from mouse 20241 versus murine monoclonal anti-EGFR MAbs 225, 528, and AB5.
FIG. 2 is a graph showing competitive ELISA with human antibody supernatants from mouse 20242 and 20243 versus murine monoclonal anti-EGFR MAbs 225, 528, and AB5.
FIG. 3 is a graph showing competitive ELISA with purified human antibodies versus murine monoclonal anti-EGFR MAbs 225 and 528.
FIGS. 4A, 4B, 4C, and 4D are graphs showing competitive ELISA with HuMabs (A) 6B3, (B) 5F12, (C) 2F8, and (D) 2A2.
FIGS. 5A and 5B are graphs showing inhibition of EGF-biotin binding to EGFR by anti-EGFR HuMabs and murine MAbs (ELISA format).
FIG. 6 is a graph showing inhibition of EGF-biotin binding to EGFR on A431 cells by anti-EGFR HuMabs and murine MAbs.
FIG. 7 is a graph showing titration of anti-EGFR HuMabs on A431 cells.
FIG. 8 is a graph showing the ability of 2F8 to inhibit the binding of EGF binding to purified and native EGFR. The effect of 2F8 (diamonds), murine 225 (squares), EGF (triangles) or human IgG1 kappa isotype control (bullets) is measured on the binding of EGF-biotin to immobilized EGFR. As depicted in FIG. 8, 2F8 is able to inhibit EGF-biotin binding with an IC50 of 17 nM, significantly lower than 225 (IC50 of 30 nM).
FIG. 9 is a bar graph showing the ability of 2F8 to inhibit the binding of EGF and TGF-α to A431 cells. A431 cells are derived from an ovarian epidermoid carcinoma and express in excess of 1×106 EGFR molecules on their cell surface. Inhibition of 2F8-binding to A431 cells was determined using flow cytometer analysis. Cells were pre-incubated with either 5 (open bars) or 50 μg/ml (closed bars) ligand before adding 2F8. Binding of antibody without ligand (PBS group) was designated as 100%. As shown, EGF and TGF-α binding to A431 cells is efficiently blocked by 2F8. These results indicate that 2F8 binds close to, or at the same site, on EGFR as the ligands.
FIGS. 10A and 10B show the effect of monoclonal anti EGFR on autophosphorylation of A431 cells. Serum deprived subconfluent A431 cells were treated with different antibodies (10 μg/ml) as indicated in the methods, stimulated with either EGF (A) or TGF-α (B), and extracted. The EGFR phosphorylation was analyzed by SDS-PAGE and immunoblotting with antiphosphotyrosine antibodies.
FIGS. 11A, 11B, and 11C are graphs showing growth inhibition of EGFR-expressing tumor cell lines by anti-EGFR human antibodies. The EGFR-expressing tumor cell lines A431 (A), HN5 (B) and MDA-MB-468 (C) were incubated with various concentrations of HuMab 2F8 (squares), 5C5 (triangles), 6E9 (crosses), 2A2 (diamonds) antibody negative control anti-CTLA4 (open circles), antibody positive control 225 (closed circles) or with medium only (control) for seven (7) days. Thereafter, cell growth was evaluated using crystal violet staining of fixed cells. The percentage growth inhibition was calculated as the amount of protein left after seven (7) days incubation compared to the amount of protein present in the medium only control. The data represent triplicate measurements, and are representative of three experiments performed on different days.
FIG. 12 is a graph showing human PMN mediated antibody dependent cellular cytotoxicity. PMN were isolated as described. 51Chromium labeled A431 cells were plated in 96 wells flat bottom plates. PMN were added in effector:target ratio 100:1 and antibodies were added in different concentrations. After overnight incubation, the 51Cr release was measured.
FIG. 13 is a graph showing the prevention of tumor formation by HuMab 2F8 in an athymic murine model. Groups of six (6) mice were injected subcutaneously in the flank with 3×106 tumor cells in 200 μl PBS at day zero (0). Subsequently, mice were injected i.p. on days 1 (75 μg/200 μl), 3 (25 μg/200 μl), and 5 (25 μg/200 μl) (arrows) with either HuMab 2F8 (closed squares) i.p. of human IgG1-κ MAb as a control (open circles). The data are presented as mean tumor volume±SEM, and are representative of 3 individual experiments, yielding similar results.
FIG. 14 is a graph showing the eradication of established A431 tumor xenografts by HuMab 2F8 in comparison to murine anti-EGFR MAb (m225). Mice were injected subcutaneously in the flank with 3×106 tumor cells in 200 μl PBS on day zero (0). At day 10, mice were randomly allocated to treatment groups and treated on days 12 (75 μg/200 μl), 14 (25 μg/200 μl), and 16 (25 μg/200 μl) (arrows) with HuMab 2F8 (closed squares, 2F8 short-term) or with murine anti-EGFR MAb 225 (closed triangles, m225 short-term). Furthermore, groups were included receiving 75 μg/200 μl HuMab 2F8 or m225 on day 12, continued by 25 μg/200 μl HuMab 2F8 or m225 on days 14, 16, 19, 22, 26, 29, 33, 36, and 40 (open squares, 2F8 long-term; open triangles, m225 long-term). The data are presented as mean tumor volume+SEM, and are representative of 3 individual experiments, yielding similar results. Black arrows indicate treatment days for the short-term treatment, open arrows indicate treatment days for the long-term treatment.
FIGS. 15A and 15B show the nucleotide (SEQ ID NOs:1 and 3) and amino acid (SEQ ID NOs:2, and 4) sequences of the VH- and VL-regions of 2F8, respectively, with CDR regions designated (SEQ ID NOs:5, 6, 7, 8, 9, and 10).
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel antibody-based therapies for treating and diagnosing diseases characterized by expression, particularly over-expression, of epidermal growth factor receptor antigen (referred to herein as “EGFR”). Therapies of the invention employ isolated human IgG monoclonal antibodies, or antigen-binding portions thereof, which bind to an epitope present on EGFR. Other isolated human monoclonal antibodies encompassed by the present invention include IgA, IgG1-4, IgE, IgM, and IgD antibodies, e.g., IgG1,κ or IgG1,λ isotypes, or IgG4,κ or IgG4,λ isotypes. In one embodiment, the human antibodies are produced in a non-human transgenic animal, e.g., a transgenic mouse, capable of producing multiple isotypes of human monoclonal antibodies to EGFR (e.g., IgG, IgA and/or IgE) by undergoing V-D-J recombination and isotype switching. Accordingly, aspects of the invention include not only antibodies, antibody fragments, and pharmaceutical compositions thereof, but also non-human transgenic animals, B-cells, host cell transfectomas, and hybridomas which produce monoclonal antibodies. Methods of using the antibodies of the invention to detect a cell expressing EGFR or a related, cross-reactive growth factor receptor, or to inhibit growth, differentiation and/or motility of a cell expressing EGFR, either in vitro or in vivo, are also encompassed by the invention.
In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.
The terms “epidermal growth factor receptor,” “EGFR,” and “EGFR antigen” are used interchangeably herein, and include variants, isoforms and species homologs of human EGFR. In a preferred embodiment, binding of an antibody of the invention to the EGFR-antigen inhibits the growth of cells expressing EGFR (e.g., a tumor cell) by inhibiting or blocking binding of EGFR ligand to EGFR. The term “EGFR ligand” encompasses all (e.g., physiological) ligands for EGFR, including EGF, TGF-α, heparin binding EGF (HB-EGF), amphiregulin (AR), and epiregulin (EPI). In another preferred embodiment, binding of an antibody of the invention to the EGFR-antigen mediates effector cell phagocytosis and/or killing of cells expressing EGFR.
As used herein, the term “inhibits growth” (e.g., referring to cells) is intended to include any measurable decrease in the growth of a cell when contacted with an anti-EGFR antibody as compared to the growth of the same cell not in contact with an anti-EGFR antibody, e.g., the inhibition of growth of a cell by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
As used herein, the terms “inhibits binding” and “blocks binding” (e.g., referring to inhibition/blocking of binding of EGFR ligand to EGFR) are used interchangeably and encompass both partial and complete inhibition/blocking. The inhibition/blocking of EGFR ligand to EGFR preferably reduces or alters the normal level or type of cell signaling that occurs when EGFR ligand binds to EGFR without inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in the binding affinity of EGFR ligand to EGFR when in contact with an anti-EGFR antibody as compared to the ligand not in contact with an anti-EGFR antibody, e.g., the blocking of EGFR ligands to EGFR by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
The term “antibody” as referred to herein includes whole antibodies and any antigen binding fragment (i.e., “antigen-binding portion”) or single chain thereof. An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
The term “antigen-binding portion” of an antibody (or simply “antibody portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., EGFR). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
The term “epitope” means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
The term “bispecific molecule” is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has two different binding specificities. For example, the molecule may bind to, or interact with, (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell. The term “multispecific molecule” or “heterospecific molecule” is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has more than two different binding specificities. For example, the molecule may bind to, or interact with, (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Accordingly, the invention includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules which are directed to cell surface antigens, such as EGFR, and to other targets, such as Fc receptors on effector cells.
The term “bispecific antibodies” also includes diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).
As used herein, the term “heteroantibodies” refers to two or more antibodies, antibody binding fragments (e.g., Fab), derivatives therefrom, or antigen binding regions linked together, at least two of which have different specificities. These different specificities include a binding specificity for an Fc receptor on an effector cell, and a binding specificity for an antigen or epitope on a target cell, e.g., a tumor cell. The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term “human monoclonal antibody” refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
The term “recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or a hybridoma prepared therefrom (described further in Section I, below), (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
As used herein, a “heterologous antibody” is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal.
As used herein, a “heterohybrid antibody” refers to an antibody having a light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody. Examples of heterohybrid antibodies include chimeric and humanized antibodies, discussed supra.
An “isolated antibody,” as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to EGFR is substantially free of antibodies that specifically bind antigens other than EGFR). An isolated antibody that specifically binds to an epitope, isoform or variant of human EGFR may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., EGFR species homologs). Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment of the invention, a combination of “isolated” monoclonal antibodies having different specificities are combined in a well defined composition.
As used herein, “specific binding” refers to antibody binding to a predetermined antigen. Typically, the antibody binds with an affinity of at least about 1×107 M−1, and binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen”.
As used herein, the term “high affinity” for an IgG antibody refers to a binding affinity of at least about 107M−1, preferably at least about 108M−1, more preferably at least about 109M−1, and still more preferably at least about 1010 M−1, 1011 M−1, 1012 M−1 or greater, e.g., up to 1013 M−1 or greater. However, “high affinity” binding can vary for other antibody isotypes. For example, “high affinity” binding for an IgM isotype refers to a binding affinity of at least about 1×107M−1.
The term “KA”, as used herein, is intended to refer to the association constant of a particular antibody-antigen interaction.
The term “KD”, as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction.
As used herein, “isotype” refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes.
As used herein, “isotype switching” refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes.
As used herein, “nonswitched isotype” refers to the isotypic class of heavy chain that is produced when no isotype switching has taken place; the CH gene encoding the nonswitched isotype is typically the first CH gene immediately downstream from the functionally rearranged VDJ gene. Isotype switching has been classified as classical or non-classical isotype switching. Classical isotype switching occurs by recombination events which involve at least one switch sequence region in the transgene. Non-classical isotype switching may occur by, for example, homologous recombination between human σμ and human Σμ (δ-associated deletion). Alternative non-classical switching mechanisms, such as intertransgene and/or interchromosomal recombination, among others, may occur and effectuate isotype switching.
As used herein, the term “switch sequence” refers to those DNA sequences responsible for switch recombination. A “switch donor” sequence, typically a μ switch region, will be 5′ (i.e., upstream) of the construct region to be deleted during the switch recombination. The “switch acceptor” region will be between the construct region to be deleted and the replacement constant region (e.g., γ, ε, etc.). As there is no specific site where recombination always occurs, the final gene sequence will typically not be predictable from the construct.
As used herein, “glycosylation pattern” is defined as the pattern of carbohydrate units that are covalently attached to a protein, more specifically to an immunoglobulin protein. A glycosylation pattern of a heterologous antibody can be characterized as being substantially similar to glycosylation patterns which occur naturally on antibodies produced by the species of the nonhuman transgenic animal, when one of ordinary skill in the art would recognize the glycosylation pattern of the heterologous antibody as being more similar to said pattern of glycosylation in the species of the nonhuman transgenic animal than to the species from which the CH genes of the transgene were derived.
The term “naturally-occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
The term “rearranged” as used herein refers to a configuration of a heavy chain or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete VH or VL domain, respectively. A rearranged immunoglobulin gene locus can be identified by comparison to germline DNA; a rearranged locus will have at least one recombined heptamer/nonamer homology element.
The term “unrearranged” or “germline configuration” as used herein in reference to a V segment refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment.
The term “nucleic acid molecule”, as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
The term “isolated nucleic acid molecule,” as used herein in reference to nucleic acids encoding antibodies or antibody portions (e.g., VH, VL, CDR3) that bind to EGFR, is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than EGFR, which other sequences may naturally flank the nucleic acid in human genomic DNA. In one embodiment, the human anti-EGFR antibody, or portion thereof, includes the nucleotide or amino acid sequence of 2F8, as well as heavy chain (VH) and light chain (VL) variable regions having the sequences shown in SEQ ID NOs:1 and 3, and 2 and 4, respectively.
As disclosed and claimed herein, the sequences set forth in SEQ ID NOs: 1-12 include “conservative sequence modifications”, i.e., nucleotide and amino acid sequence modifications which do not significantly affect or alter the binding characteristics of the antibody encoded by the nucleotide sequence or containing the amino acid sequence. Such conservative sequence modifications include nucleotide and amino acid substitutions, additions and deletions. Modifications can be introduced into SEQ ID NOs:1-12 by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a human anti-EGFR antibody is preferably replaced with another amino acid residue from the same side chain family.
Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a anti-EGFR antibody coding sequence, such as by saturation mutagenesis, and the resulting modified anti-EGFR antibodies can be screened for binding activity.
Accordingly, antibodies encoded by the (heavy and light chain variable region) nucleotide sequences disclosed herein and/or containing the (heavy and light chain variable region) amino acid sequences disclosed herein (i.e., SEQ ID NOs:1-12) include substantially similar antibodies encoded by or containing similar sequences which have been conservatively modified. Further discussion as to how such substantially similar antibodies can be generated based on the partial (i.e., heavy and light chain variable regions) sequences disclosed herein as SEQ ID NOs:1-12 is provided below.
For nucleic and amino acids, the term “substantial homology” indicates that two nucleic/amino acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide/amino acid residue insertions or deletions, in at least about 80% of the nucleotides/amino acid residues, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides/amino acid residues. Alternatively, substantial homology exists for nucleic acids when the segments will hybridize under selective hybridization conditions, to the complement of the strand.
For nucleotide acid and amino acid sequences, the term “homology” indicates the degree of identity between two sequences, when optimally aligned and compared, with appropriate nucleotide insertions or deletions.
The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at the GCG website), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at the GCG website), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See NIH website.
The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).
The nucleic acid compositions of the present invention, while often in a native sequence (except for modified restriction sites and the like), from either cDNA, genomic or mixtures may be mutated, thereof in accordance with standard techniques to provide gene sequences. For coding sequences, these mutations, may affect amino acid sequence as desired. In particular, DNA sequences substantially homologous to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated (where “derived” indicates that a sequence is identical or modified from another sequence).
A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcription regulatory sequences, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. For switch sequences, operably linked indicates that the sequences are capable of effecting switch recombination.
The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Recombinant host cells include, for example, CHO cells and lymphocytic cells.
The term “transfectoma”, as used herein, includes recombinant eukaryotic host cell expressing the antibody, such as CHO cells or NS/0 cells.
As used herein, the term “subject” includes any human or nonhuman animal. The term “nonhuman animal” includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.
The terms “transgenic, nonhuman animal” refers to a nonhuman animal having a genome comprising one or more human heavy and/or light chain transgenes or transchromosomes (either integrated or non-integrated into the animal's natural genomic DNA) and which is capable of expressing fully human antibodies. For example, a transgenic mouse can have a human light chain transgene and either a human heavy chain transgene or human heavy chain transchromosome, such that the mouse produces human anti-EGFR antibodies when immunized with EGFR and/or cells expressing EGFR. The human heavy chain transgene can be integrated into the chromosomal DNA of the mouse, as is the case for transgenic, e.g., HuMAb mice, or the human heavy chain transgene can be maintained extrachromosomally, as is the case for transchromosomal (e.g., KM) mice as described in WO 02/43478. Such transgenic and transchromosomal mice are capable of producing multiple isotypes of human monoclonal antibodies to EGFR (e.g., IgG, IgA and/or IgE) by undergoing V-D-J recombination and isotype switching.
Various aspects of the invention are described in further detail in the following subsections.
I. Production of Human Antibodies to EGFR
The monoclonal antibodies (MAbs) of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology e.g., the standard somatic cell hybridization technique of Kohler and Milstein (1975) Nature 256: 495. Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed e.g., viral or oncogenic transformation of B lymphocytes.
The preferred animal system for preparing hybridomas is the murine system. Hybridoma production in the mouse is a very well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.
In a preferred embodiment, human monoclonal antibodies directed against EGFR can be generated using transgenic mice carrying parts of the human immune system rather than the mouse system. These transgenic mice, referred to herein as “HuMAb” mice, contain a human immunoglobulin gene miniloci that encodes unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg, et al. (1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGκ monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). The preparation of HuMAb mice is described in detail Section II below and in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Lonberg et al., (1994) Nature 368(6474): 856-859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Taylor, L. et al. (1994) International Immunology 6: 579-591; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93; Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851, the contents of all of which are hereby incorporated by reference in their entirety. See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay, and GenPharm International; U.S. Pat. No. 5,545,807 to Surani et al.; International Publication Nos. WO 98/24884, published on Jun. 11, 1998; WO 94/25585, published Nov. 10, 1994; WO 93/1227, published Jun. 24, 1993; WO 92/22645, published Dec. 23, 1992; WO 92/03918, published Mar. 19, 1992, the disclosures of all of which are hereby incorporated by reference in their entity. Alternatively, the HCO12 transgenic mice described in Example 2, can be used to generate human anti-EGFR antibodies.
Human Antibody Immunizations
To generate fully human monoclonal antibodies to EGFR, HuMAb mice can be immunized with a purified or enriched preparation of EGFR antigen and/or cells expressing EGFR, as described by Lonberg, N. et al. (1994) Nature 368(6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851 and WO 98/24884. Preferably, the mice will be 6-16 weeks of age upon the first infusion. For example, a purified or enriched preparation (5-20 μg) of EGFR antigen (e.g., purified from EGFR-expressing LNCaP cells) can be used to immunize the HuMAb mice intraperitoneally. In the event that immunizations using a purified or enriched preparation of EGFR antigen do not result in antibodies, mice can also be immunized with cells expressing EGFR, e.g., a tumor cell line, to promote immune responses.
Cumulative experience with various antigens has shown that the HuMAb transgenic mice respond best when initially immunized intraperitoneally (IP) with antigen in complete Freund's adjuvant, followed by every other week i.p. immunizations (up to a total of 6) with antigen in incomplete Freund's adjuvant. The immune response can be monitored over the course of the immunization protocol with plasma samples being obtained by retroorbital bleeds. The plasma can be screened by ELISA (as described below), and mice with sufficient titers of anti-EGFR human immunoglobulin can be used for fusions. Mice can be boosted intravenously with antigen 3 days before sacrifice and removal of the spleen. It is expected that 2-3 fusions for each antigen may need to be performed. Several mice will be immunized for each antigen. For example, a total of twelve HuMAb mice of the HC07 and HC012 strains can be immunized.
Generation of Hybridomas Producing Human Monoclonal Antibodies to EGFR
The mouse splenocytes can be isolated and fused with PEG to a mouse myeloma cell line based upon standard protocols. The resulting hybridomas are then screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from immunized mice are fused to one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cells are plated at approximately 2×105 in flat bottom microtiter plate, followed by a two week incubation in selective medium containing 20% fetal Clone Serum, 18% “653” conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM L˜glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and 1×HAT (Sigma; the HAT is added 24 hours after the fusion). After two weeks, cells are cultured in medium in which the HAT is replaced with HT. Individual wells are then screened by ELISA for human anti-EGFR monoclonal IgM and IgG antibodies. Once extensive hybridoma growth occurs, medium is observed usually after 10-14 days. The antibody secreting hybridomas are replated, screened again, and if still positive for human IgG, anti-EGFR monoclonal antibodies, can be subcloned at least twice by limiting dilution. The stable subclones are then cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.
Generation of Transfectomas Producing Human Monoclonal Antibodies to EGFR
Human antibodies of the invention can also be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (Morrison, S. (1985) Science 229:1202).
For example, to express the antibodies, or antibody fragments thereof, DNAs encoding partial or full-length light and heavy chains, can be obtained by standard molecular biology techniques (e.g., PCR amplification, site directed mutagenesis) and can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term “operatively linked” is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). The light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term “regulatory sequence” is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter or β-globin promoter.
In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, and most preferably mammalian host cells, is the most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.
Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NS/0 myeloma cells, COS cells and SP2.0 cells. In particular for use with NS/0 myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338 841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
Use of Partial Antibody Sequences to Express Intact Antibodies
Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al., 1998, Nature 332:323-327; Jones, P. et al., 1986, Nature 321:522-525; and Queen, C. et al., 1989, Proc. Natl. Acad. See. U.S.A. 86:10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V(D)J joining during B cell maturation. Germline gene sequences will also differ from the sequences of a high affinity secondary repertoire antibody at individual evenly across the variable region. For example, somatic mutations are relatively infrequent in the amino-terminal portion of framework region. For example, somatic mutations are relatively infrequent in the amino terminal portion of framework region 1 and in the carboxy-terminal portion of framework region 4. Furthermore, many somatic mutations do not significantly alter the binding properties of the antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody in order to recreate an intact recombinant antibody having binding properties similar to those of the original antibody (see PCT/US99/05535 filed on Mar. 12, 1999, which is herein incorporated by referenced for all purposes). Partial heavy and light chain sequence spanning the CDR regions is typically sufficient for this purpose. The partial sequence is used to determine which germline variable and joining gene segments contributed to the recombined antibody variable genes. The germline sequence is then used to fill in missing portions of the variable regions. Heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. For this reason, it is necessary to use the corresponding germline leader sequence for expression constructs. To add missing sequences, cloned cDNA sequences cab be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized as a set of short, overlapping, oligonucleotides and combined by PCR amplification to create an entirely synthetic variable region clone. This process has certain advantages such as elimination or inclusion or particular restriction sites, or optimization of particular codons.
The nucleotide sequences of heavy and light chain transcripts from a hybridomas are used to design an overlapping set of synthetic oligonucleotides to create synthetic V sequences with identical amino acid coding capacities as the natural sequences. The synthetic heavy and kappa chain sequences can differ from the natural sequences in three ways: strings of repeated nucleotide bases are interrupted to facilitate oligonucleotide synthesis and PCR amplification; optimal translation initiation sites are incorporated according to Kozak's rules (Kozak, 1991, J. Biol. Chem. 266:19867-19870); and, HindIII sites are engineered upstream of the translation initiation sites.
For both the heavy and light chain variable regions, the optimized coding, and corresponding non-coding, strand sequences are broken down into 30-50 nucleotide approximately the midpoint of the corresponding non-coding oligonucleotide. Thus, for each chain, the oligonucleotides can be assemble into overlapping double stranded sets that span segments of 150-400 nucleotides. The pools are then used as templates to produce PCR amplification products of 150-400 nucleotides. Typically, a single variable region oligonucleotide set will be broken down into two pools which are separately amplified to generate two overlapping PCV products. These overlapping products are then combined by PCT amplification to form the complete variable region. It may also be desirable to include an overlapping fragment of the heavy or light chain constant region (including the BbsI site of the kappa light chain, or the AgeI site if the gamma heavy chain) in the PCR amplification to generate fragments that can easily be cloned into the expression vector constructs.
The reconstructed heavy and light chain variable regions are then combined with cloned promoter, translation initiation, constant region, 3′ untranslated, polyadenylation, and transcription termination, sequences to form expression vector constructs. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected, or separately transfected into host cells which are then fused to form a host cell expressing both chains.
Plasmids for use in construction of expression vectors for human IgGκ are described below. The plasmids were constructed so that PCR amplified V heavy and V kappa light chain cDNA sequences could be used to reconstruct complete heavy and light chain minigenes. These plasmids can be used to express completely human, or chimeric IgG1κ or IgG4κ antibodies. Similar plasmids can be constructed for expression of other heavy chain isotypes, or for expression of antibodies comprising lambda light chains.
Thus, in another aspect of the invention, the structural features of an human anti-EGFR antibodies of the invention, e.g., 2F8, are used to create structurally related human anti-EGFR antibodies that retain at least one functional property of the antibodies of the invention, such as binding to EGFR. More specifically, one or more CDR regions of 2F8 can be combined recombinantly with known human framework regions and CDRs to create additional, recombinantly-engineered, human anti-EGFR antibodies of the invention.
Accordingly, in another embodiment, the invention provides a method for preparing an anti-EGFR antibody comprising:
preparing an antibody comprising (1) human heavy chain framework regions and human heavy chain CDRs, wherein at least one of the human heavy chain CDRs comprises an amino acid sequence selected from the amino acid sequences of CDRs shown in FIG. 15 (SEQ ID NOs:5, 6, and 7); and (2) human light chain framework regions and human light chain CDRs, wherein at least one of the human light chain CDRs comprises an amino acid sequence selected from the amino acid sequences of CDRs shown in FIG. 15 (SEQ ID NOs:8, 9, and 10); wherein the antibody retains the ability to bind to EGFR.
The ability of the antibody to bind EGFR can be determined using standard binding assays, such as those set forth in the Examples (e.g., an ELISA). Since it is well known in the art that antibody heavy and light chain CDR3 domains play a particularly important role in the binding specificity/affinity of an antibody for an antigen, the recombinant antibodies of the invention prepared as set forth above preferably comprise the heavy and light chain CDR3s of 2F8. The antibodies further can comprise the CDR2s of 2F8. The antibodies further can comprise the CDR1s of 2F8. Accordingly, the invention further provides anti-EGFR antibodies comprising: (1) human heavy chain framework regions, a human heavy chain CDR1 region, a human heavy chain CDR2 region, and a human heavy chain CDR3 region, wherein the human heavy chain CDR3 region is the CDR3 of 2F8 as shown in FIG. 15 (SEQ ID NO:7); and (2) human light chain framework regions, a human light chain CDR1 region, a human light chain CDR2 region, and a human light chain CDR3 region, wherein the human light chain CDR3 region is the CDR3 of 2F8 as shown in FIG. 15 (SEQ ID NO:10), wherein the antibody binds EGFR. The antibody may further comprise the heavy chain CDR2 and/or the light chain CDR2 of 2F8. The antibody may further comprise the heavy chain CDR1 and/or the light chain CDR1 of 2F8.
Preferably, the CDR1, 2, and/or 3 of the engineered antibodies described above comprise the exact amino acid sequence(s) as those of 2F8 disclosed herein. However, the ordinarily skilled artisan will appreciate that some deviation from the exact CDR sequences of 2F8 may be possible while still retaining the ability of the antibody to bind EGFR effectively (e.g., conservative substitutions). Accordingly, in another embodiment, the engineered antibody may be composed of one or more CDRs that are, for example, 90%, 95%, 98% or 99.5% identical to one or more CDRs of 2F8.
In addition or alternative, to simply binding EGFR, engineered antibodies such as those described above may be selected for their retention of other functional properties of antibodies of the invention, such as:
(1) binding to live cells expressing EGFR;
(2) high affinity binding to EGFR;
(3) binding to a unique epitope on EGFR (to eliminate the possibility that monoclonal antibodies with complimentary activities when used in combination would compete for binding to the same epitope);
(4) opsonization of cells expressing EGFR; and/or
(5) mediation of growth inhibition, phagocytosis and/or killing of cells expressing EGFR in the presence of human effector cells.
Characterization of Binding of Human Monoclonal Antibodies to EGFR
To characterize binding of human monoclonal EGFR antibodies of the invention, sera from immunized mice can be tested, for example, by ELISA. Briefly, microtiter plates are coated with purified EGFR at 0.25 μg/ml in PBS, and then blocked with 5% bovine serum albumin in PBS. Dilutions of plasma from EGFR-immunized mice are added to each well and incubated for 1-2 hours at 37° C. The plates are washed with PBS/Tween and then incubated with a goat-anti-human IgG Fc-specific polyclonal reagent conjugated to alkaline phosphatase for 1 hour at 37° C. After washing, the plates are developed with pNPP substrate (1 mg/ml), and analyzed at OD of 405-650. Preferably, mice which develop the highest titers will be used for fusions.
An ELISA assay as described above can also be used to screen for hybridomas that show positive reactivity with EGFR immunogen. Hybridomas that bind with high avidity to EGFR will be subcloned and further characterized. One clone from each hybridoma, which retains the reactivity of the parent cells (by ELISA), can be chosen for making a 5-10 vial cell bank stored at −140° C., and for antibody purification.
To purify human anti-EGFR antibodies, selected hybridomas can be grown in two-liter spinner-flasks for monoclonal antibody purification. Supernatants can be filtered and concentrated before affinity chromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.). Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using 1.43 extinction coefficient. The monoclonal antibodies can be aliquoted and stored at −80° C.
To determine if the selected human anti-EGFR monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, Ill.). Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using EGFR coated-ELISA plates as described above. Biotinylated MAb binding can be detected with a strep-avidin-alkaline phosphatase probe.
To determine the isotype of purified antibodies, isotype ELISAs can be performed. Wells of microtiter plates can be coated with 10 g/ml of anti-human Ig overnight at 4° C. After blocking with 5% BSA, the plates are reacted with 10 g/ml of monoclonal antibodies or purified isotype controls, at ambient temperature for two hours. The wells can then be reacted with either human IgGl or human IgM-specific alkaline phosphatase-conjugated probes. Plates are developed and analyzed as described above.
In order to demonstrate binding of monoclonal antibodies to live cells expressing the EGFR, flow cytometry can be used. Briefly, cell lines expressing EGFR (grown under standard growth conditions) are mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% Tween 80 and 20% mouse serum, and incubated at 37° C. for 1 hour. After washing, the cells are reacted with Fluorescein-labeled anti-human IgG antibody under the same conditions as the primary antibody staining. The samples can be analyzed by FACScan instrument using light and side scatter properties to gate on single cells. An alternative assay using fluorescence microscopy may be used (in addition to or instead of) the flow cytometry assay. Cells can be stained exactly as described above and examined by fluorescence microscopy. This method allows visualization of individual cells, but may have diminished sensitivity depending on the density of the antigen.
Anti-EGFR human IgGs can be further tested for reactivity with EGFR antigen by Western blotting. Briefly, cell extracts from cells expressing EGFR can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes, blocked with 20% mouse serum, and probed with the monoclonal antibodies to be tested. Human IgG binding can be detected using anti-human IgG alkaline phosphatase and developed with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).
Phagocytic and Cell Killing Activities of Human Monoclonal Antibodies to EGFR
In addition to binding specifically to EGFR, human monoclonal anti-EGFR antibodies can be tested for their ability to mediate phagocytosis and killing of cells expressing EGFR. The testing of monoclonal antibody activity in vitro will provide an initial screening prior to testing in vivo models. Briefly, polymorphonuclear cells (PMN), or other effector cells, from healthy donors can be purified by Ficoll Hypaque density centrifugation, followed by lysis of contaminating erythrocytes. Washed PMNs, can be suspended in RPMI supplemented with 10% heat-inactivated fetal calf serum and mixed with 51Cr labeled cells expressing EGFR, at various ratios of effector cells to tumor cells (-effector cells:tumor cells). Purified human anti-EGFR IgGs can then be added at various concentrations. Irrelevant human IgG can be used as negative control. Assays can be carried out for 0-120 minutes at 37° C. Samples can be assayed for cytolysis by measuring 51Cr release into the culture supernatant. Anti-EGFR monoclonal can also be tested in combinations with each other to determine whether cytolysis is enhanced with multiple monoclonal antibodies.
Human monoclonal antibodies which bind to EGFR also can be tested in an in vivo model (e.g., in mice) to determine their efficacy in mediating phagocytosis and killing of cells expressing EGFR, e.g., tumor cells. These antibodies can be selected, for example, based on the following criteria, which are not intended to be exclusive:
1.) binding to live cells expressing EGFR;
2.) high affinity of binding to EGFR;
3.) binding to a unique epitope on EGFR (to eliminate the possibility that monoclonal antibodies with complimentary activities when used in combination would compete for binding to the same epitope);
4.) opsonization of cells expressing EGFR;
5.) mediation of growth inhibition, phagocytosis and/or killing of cells expressing EGFR in the presence of human effector cells.
Preferred human monoclonal antibodies of the invention meet one or more, and preferably all, of these criteria. In a particular embodiment, the human monoclonal antibodies are used in combination, e.g., as a pharmaceutical composition comprising two or more anti-EGFR monoclonal antibodies or fragments thereof. For example, human anti-EGFR monoclonal antibodies having different, but complementary activities can be combined in a single therapy to achieve a desired therapeutic or diagnostic effect. An illustration of this would be a composition containing an anti-EGFR human monoclonal antibody that mediates highly effective killing of target cells in the presence of effector cells, combined with another human anti-EGFR monoclonal antibody that inhibits the growth of cells expressing EGFR.
II. Production of Transgenic Nonhuman Animals which Generate Human Monoclonal Anti-EGFR Antibodies
In yet another aspect, the invention provides transgenic non-human animals, e.g., a transgenic mice, which are capable of expressing human monoclonal antibodies that specifically bind to EGFR, preferably with high affinity. In a preferred embodiment, the transgenic non-human animals, e.g., the transgenic mice (HuMAb mice), have a genome comprising a human heavy chain transgene and a light chain transgene. In one embodiment, the transgenic non-human animals, e.g., the transgenic mice, have been immunized with a purified or enriched preparation of EGFR antigen and/or cells expressing EGFR. Preferably, the transgenic non-human animals, e.g., the transgenic mice, are capable of producing multiple isotypes of human monoclonal antibodies to EGFR (e.g., IgG, IgA and/or IgE) by undergoing V-D-J recombination and isotype switching. Isotype switching may occur by, e.g., classical or non-classical isotype switching.
The design of a transgenic non-human animal that responds to foreign antigen stimulation with a heterologous antibody repertoire, requires that the heterologous immunoglobulin transgenes contain within the transgenic animal function correctly throughout the pathway of B-cell development. In a preferred embodiment, correct function of a heterologous heavy chain transgene includes isotype switching. Accordingly, the transgenes of the invention are constructed so as to produce isotype switching and one or more of the following: (1) high level and cell-type specific expression, (2) functional gene rearrangement, (3) activation of and response to allelic exclusion, (4) expression of a sufficient primary repertoire, (5) signal transduction, (6) somatic hypermutation, and (7) domination of the transgene antibody locus during the immune response.
Not all of the foregoing criteria need be met. For example, in those embodiments wherein the endogenous immunoglobulin loci of the transgenic animal are functionally disrupted, the transgene need not activate allelic exclusion. Further, in those embodiments wherein the transgene comprises a functionally rearranged heavy and/or light chain immunoglobulin gene, the second criteria of functional gene rearrangement is unnecessary, at least for that transgene which is already rearranged. For background on molecular immunology, see, Fundamental Immunology, 2nd edition (1989), Paul William E., ed. Raven Press, N.Y., which is incorporated herein by reference.
In certain embodiments, the transgenic non-human animals used to generate the human monoclonal antibodies of the invention contain rearranged, unrearranged or a combination of rearranged and unrearranged heterologous immunoglobulin heavy and light chain transgenes in the germline of the transgenic animal. Each of the heavy chain transgenes comprises at least one CH gene. In addition, the heavy chain transgene may contain functional isotype switch sequences, which are capable of supporting isotype switching of a heterologous transgene encoding multiple CH genes in the B-cells of the transgenic animal. Such switch sequences may be those which occur naturally in the germline immunoglobulin locus from the species that serves as the source of the transgene CH genes, or such switch sequences may be derived from those which occur in the species that is to receive the transgene construct (the transgenic animal). For example, a human transgene construct that is used to produce a transgenic mouse may produce a higher frequency of isotype switching events if it incorporates switch sequences similar to those that occur naturally in the mouse heavy chain locus, as presumably the mouse switch sequences are optimized to function with the mouse switch recombinase enzyme system, whereas the human switch sequences are not. Switch sequences may be isolated and cloned by conventional cloning methods, or may be synthesized de novo from overlapping synthetic oligonucleotides designed on the basis of published sequence information relating to immunoglobulin switch region sequences (Mills et al., Nucl. Acids Res. 15:7305-7316 (1991); Sideras et al., Intl. Immunol. 1:631-642 (1989), which are incorporated herein by reference). For each of the foregoing transgenic animals, functionally rearranged heterologous heavy and light chain immunoglobulin transgenes are found in a significant fraction of the B-cells of the transgenic animal (at least 10 percent).
The transgenes used to generate the transgenic animals of the invention include a heavy chain transgene comprising DNA encoding at least one variable gene segment, one diversity gene segment, one joining gene segment and at least one constant region gene segment. The immunoglobulin light chain transgene comprises DNA encoding at least one variable gene segment, one joining gene segment and at least one constant region gene segment. The gene segments encoding the light and heavy chain gene segments are heterologous to the transgenic non-human animal in that they are derived from, or correspond to, DNA encoding immunoglobulin heavy and light chain gene segments from a species not consisting of the transgenic non-human animal. In one aspect of the invention, the transgene is constructed such that the individual gene segments are unrearranged, i.e., not rearranged so as to encode a functional immunoglobulin light or heavy chain. Such unrearranged transgenes support recombination of the V, D, and J gene segments (functional rearrangement) and preferably support incorporation of all or a portion of a D region gene segment in the resultant rearranged immunoglobulin heavy chain within the transgenic non-human animal when exposed to EGFR antigen.
In an alternate embodiment, the transgenes comprise an unrearranged “mini-locus.” Such transgenes typically comprise a substantial portion of the C, D, and J segments as well as a subset of the V gene segments. In such transgene constructs, the various regulatory sequences, e.g., promoters, enhancers, class switch regions, splice-donor and splice-acceptor sequences for RNA processing, recombination signals and the like, comprise corresponding sequences derived from the heterologous DNA. Such regulatory sequences may be incorporated into the transgene from the same or a related species of the non-human animal used in the invention. For example, human immunoglobulin gene segments may be combined in a transgene with a rodent immunoglobulin enhancer sequence for use in a transgenic mouse. Alternatively, synthetic regulatory sequences may be incorporated into the transgene, wherein such synthetic regulatory sequences are not homologous to a functional DNA sequence that is known to occur naturally in the genomes of mammals. Synthetic regulatory sequences are designed according to consensus rules, such as, for example, those specifying the permissible sequences of a splice-acceptor site or a promoter/enhancer motif. For example, a minilocus comprises a portion of the genomic immunoglobulin locus having at least one internal (i.e., not at a terminus of the portion) deletion of a non-essential DNA portion (e.g., intervening sequence; intron or portion thereof) as compared to the naturally-occurring germline Ig locus.
In a preferred embodiment of the invention, the transgenic animal used to generate human antibodies to EGFR contains at least one, typically 2-10, and sometimes 25-50 or more copies of the transgene described in Example 12 of WO 98/24884 (e.g., pHC1 or pHC2) bred with an animal containing a single copy of a light chain transgene described in Examples 5, 6, 8, or 14 of WO 98/24884, and the offspring bred with the JH deleted animal described in Example 10 of WO 98/24884, the contents of which are hereby expressly incorporated by reference. Animals are bred to homozygosity for each of these three traits. Such animals have the following genotype: a single copy (per haploid set of chromosomes) of a human heavy chain unrearranged mini-locus (described in Example 12 of WO 98/24884), a single copy (per haploid set of chromosomes) of a rearranged human K light chain construct (described in Example 14 of WO 98/24884), and a deletion at each endogenous mouse heavy chain locus that removes all of the functional JH segments (described in Example 10 of WO 98/24884). Such animals are bred with mice that are homozygous for the deletion of the JH segments (Examples 10 of WO 98/24884) to produce offspring that are homozygous for the JH deletion and hemizygous for the human heavy and light chain constructs. The resultant animals are injected with antigens and used for production of human monoclonal antibodies against these antigens.
B cells isolated from such an animal are monospecific with regard to the human heavy and light chains because they contain only a single copy of each gene. Furthermore, they will be monospecific with regards to human or mouse heavy chains because both endogenous mouse heavy chain gene copies are nonfunctional by virtue of the deletion spanning the JH region introduced as described in Example 9 and 12 of WO 98/24884. Furthermore, a substantial fraction of the B cells will be monospecific with regards to the human or mouse light chains because expression of the single copy of the rearranged human κ light chain gene will allelically and isotypically exclude the rearrangement of the endogenous mouse κ and lambda chain genes in a significant fraction of B-cells.
The transgenic mouse of the preferred embodiment will exhibit immunoglobulin production with a significant repertoire, ideally substantially similar to that of a native mouse. Thus, for example, in embodiments where the endogenous Ig genes have been inactivated, the total immunoglobulin levels will range from about 0.1 to 10 mg/ml of serum, preferably 0.5 to 5 mg/ml, ideally at least about 1.0 mg/ml. When a transgene capable of effecting a switch to IgG from IgM has been introduced into the transgenic mouse, the adult mouse ratio of serum IgG to IgM is preferably about 10:1. The IgG to IgM ratio will be much lower in the immature mouse. In general, greater than about 10%, preferably 40 to 80% of the spleen and lymph node B cells express exclusively human IgG protein.
The repertoire will ideally approximate that shown in a non-transgenic mouse, usually at least about 10% as high, preferably 25 to 50% or more. Generally, at least about a thousand different immunoglobulins (ideally IgG), preferably 104 to 106 or more, will be produced, depending primarily on the number of different V, J and D regions introduced into the mouse genome. These immunoglobulins will typically recognize about one-half or more of highly antigenic proteins, e.g., staphylococcus protein A. Typically, the immunoglobulins will exhibit an affinity for preselected antigens of at least about 107M−1, preferably at least about 109M−1, more preferably at least about 1010 M−1, 1011 M−1, 1012 M−1, or greater, e.g., up to 1013M−1 or greater.
In some embodiments, it may be preferable to generate mice with predetermined repertoires to limit the selection of V genes represented in the antibody response to a predetermined antigen type. A heavy chain transgene having a predetermined repertoire may comprise, for example, human VH genes which are preferentially used in antibody responses to the predetermined antigen type in humans. Alternatively, some VH genes may be excluded from a defined repertoire for various reasons (e.g., have a low likelihood of encoding high affinity V regions for the predetermined antigen; have a low propensity to undergo somatic mutation and affinity sharpening; or are immunogenic to certain humans). Thus, prior to rearrangement of a transgene containing various heavy or light chain gene segments, such gene segments may be readily identified, e.g., by hybridization or DNA sequencing, as being from a species of organism other than the transgenic animal.
The transgenic mice of the present invention can be immunized with a purified or enriched preparation of EGFR antigen and/or cells expressing EGFR as described previously. The mice will produce B cells which undergo class-switching via intratransgene switch recombination (cis-switching) and express immunoglobulins reactive with EGFR. The immunoglobulins can be human sequence antibodies, wherein the heavy and light chain polypeptides are encoded by human transgene sequences, which may include sequences derived by somatic mutation and V region recombinatorial joints, as well as germline-encoded sequences; these human sequence immunoglobulins can be referred to as being substantially identical to a polypeptide sequence encoded by a human VL or VH gene segment and a human JL or JL segment, even though other non-germline sequences may be present as a result of somatic mutation and differential V-J and V-D-J recombination joints. With respect to such human sequence antibodies, the variable regions of each chain are typically at least 80 percent encoded by human germline V, J, and, in the case of heavy chains, D, gene segments; frequently at least 85 percent of the variable regions are encoded by human germline sequences present on the transgene; often 90 or 95 percent or more of the variable region sequences are encoded by human germline sequences present on the transgene. However, since non-germline sequences are introduced by somatic mutation and VJ and VDJ joining, the human sequence antibodies will frequently have some variable region sequences (and less frequently constant region sequences) which are not encoded by human V, D, or J gene segments as found in the human transgene(s) in the germline of the mice. Typically, such non-germline sequences (or individual nucleotide positions) will cluster in or near CDRs, or in regions where somatic mutations are known to cluster.
The human sequence antibodies which bind to the predetermined antigen can result from isotype switching, such that human antibodies comprising a human sequence γ chain (such as γ1, γ2a, γ2B, or γ3) and a human sequence light chain (such as K) are produced. Such isotype-switched human sequence antibodies often contain one or more somatic mutation(s), typically in the variable region and often in or within about 10 residues of a CDR) as a result of affinity maturation and selection of B cells by antigen, particularly subsequent to secondary (or subsequent) antigen challenge. These high affinity human sequence antibodies may have binding affinities of at least 1×109 M−1, typically at least 5×109 M−1, frequently more than 1×1010 M−1, and sometimes 5×1010 M−1 to 1×1011 M−1 or greater.
Another aspect of the invention pertains to the B cells from such mice which can be used to generate hybridomas expressing human monoclonal antibodies which bind with high affinity (e.g., greater than 2×109 M−1) to EGFR. Thus, in another embodiment of the invention, these hybridomas are used to generate a composition comprising an immunoglobulin having an affinity constant (KA) of at least 2×109 M−1 for binding EGFR, wherein said immunoglobulin comprises:
a human sequence light chain composed of (1) a light chain variable region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human VL gene segment and a human JL segment, and (2) a light chain constant region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human CL gene segment; and
a human sequence heavy chain composed of a (1) a heavy chain variable region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human VH gene segment, optionally a D region, and a human JH segment, and (2) a constant region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human CH gene segment.
The development of high affinity human monoclonal antibodies against EGFR is facilitated by a method for expanding the repertoire of human variable region gene segments in a transgenic mouse having a genome comprising an integrated human immunoglobulin transgene, said method comprising introducing into the genome a V gene transgene comprising V region gene segments which are not present in said integrated human immunoglobulin transgene. Often, the V region transgene is a yeast artificial chromosome comprising a portion of a human VH or VL (VK) gene segment array, as may naturally occur in a human genome or as may be spliced together separately by recombinant methods, which may include out-of-order or omitted V gene segments. Often at least five or more functional V gene segments are contained on the YAC. In this variation, it is possible to make a transgenic mouse produced by the V repertoire expansion method, wherein the mouse expresses an immunoglobulin chain comprising a variable region sequence encoded by a V region gene segment present on the V region transgene and a C region encoded on the human Ig transgene. By means of the V repertoire expansion method, transgenic mice having at least 5 distinct V genes can be generated; as can mice containing at least about 24 V genes or more. Some V gene segments may be non-functional (e.g., pseudogenes and the like); these segments may be retained or may be selectively deleted by recombinant methods available to the skilled artisan, if desired.
Once the mouse germline has been engineered to contain a functional YAC having an expanded V segment repertoire, substantially not present in the human Ig transgene containing the J and C gene segments, the trait can be propagated and bred into other genetic backgrounds, including backgrounds where the functional YAC having an expanded V segment repertoire is bred into a mouse germline having a different human Ig transgene. Multiple functional YACs having an expanded V segment repertoire may be bred into a germline to work with a human Ig transgene (or multiple human Ig transgenes). Although referred to herein as YAC transgenes, such transgenes when integrated into the genome may substantially lack yeast sequences, such as sequences required for autonomous replication in yeast; such sequences may optionally be removed by genetic engineering (e.g., restriction digestion and pulsed-field gel electrophoresis or other suitable method) after replication in yeast in no longer necessary (i.e., prior to introduction into a mouse ES cell or mouse prozygote). Methods of propagating the trait of human sequence immunoglobulin expression, include breeding a transgenic mouse having the human Ig transgene(s), and optionally also having a functional YAC having an expanded V segment repertoire. Both VH and VL gene segments may be present on the YAC. The transgenic mouse may be bred into any background desired by the practitioner, including backgrounds harboring other human transgenes, including human Ig transgenes and/or transgenes encoding other human lymphocyte proteins. The invention also provides a high affinity human sequence immunoglobulin produced by a transgenic mouse having an expanded V region repertoire YAC transgene. Although the foregoing describes a preferred embodiment of the transgenic animal of the invention, other embodiments are contemplated which have been classified in four categories:
I. Transgenic animals containing an unrearranged heavy and rearranged light immunoglobulin transgene;
II. Transgenic animals containing an unrearranged heavy and unrearranged light immunoglobulin transgene;
III. Transgenic animal containing rearranged heavy and an unrearranged light immunoglobulin transgene; and IV. Transgenic animals containing rearranged heavy and rearranged light immunoglobulin transgenes.
Of these categories of transgenic animal, the preferred order of preference is as follows II>I>III>IV where the endogenous light chain genes (or at least the K gene) have been knocked out by homologous recombination (or other method) and I>II>III>IV where the endogenous light chain genes have not been knocked out and must be dominated by allelic exclusion.
III. Bispecific/Multispecific Molecules which Bind to EGFR
In yet another embodiment of the invention, human monoclonal antibodies to EGFR, or antigen-binding portions thereof, can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., an Fab′ fragment) to generate a bispecific or multispecific molecule which binds to multiple binding sites or target epitopes. For example, an antibody or antigen-binding portion of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic.
Accordingly, the present invention includes bispecific and multispecific molecules comprising at least one first binding specificity for EGFR and a second binding specificity for a second target epitope. In a particular embodiment of the invention, the second target epitope is an Fc receptor, e.g., human FcγRI (CD64) or a human Fcα receptor (CD89). Therefore, the invention includes bispecific and multispecific molecules capable of binding both to FcγR, FcαR or FcεR expressing effector cells (e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)), and to target cells expressing EGFR. These bispecific and multispecific molecules target EGFR expressing cells to effector cell and, like the human monoclonal antibodies of the invention, trigger Fc receptor-mediated effector cell activities, such as phagocytosis of a EGFR expressing cells, antibody dependent cell-mediated cytotoxicity (ADCC), cytokine release, or generation of superoxide anion.
Bispecific and multispecific molecules of the invention can further include a third binding specificity, in addition to an anti-Fc binding specificity and an anti-EGFR binding specificity. In one embodiment, the third binding specificity is an anti-enhancement factor (EF) portion, e.g., a molecule which binds to a surface protein involved in cytotoxic activity and thereby increases the immune response against the target cell. The “anti-enhancement factor portion” can be an antibody, functional antibody fragment or a ligand that binds to a given molecule, e.g., an antigen or a receptor, and thereby results in an enhancement of the effect of the binding determinants for the Fc receptor or target cell antigen. The “anti-enhancement factor portion” can bind an Fc receptor or a target cell antigen. Alternatively, the anti-enhancement factor portion can bind to an entity that is different from the entity to which the first and second binding specificities bind. For example, the anti-enhancement factor portion can bind a cytotoxic T-cell (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell that results in an increased immune response against the target cell).
In one embodiment, the bispecific and multispecific molecules of the invention comprise as a binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., an Fab, Fab′, F(ab′)2, Fv, or a single chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in Ladner et al. U.S. Pat. No. 4,946,778, issued Aug. 7, 1990, the contents of which is expressly incorporated by reference.
In one embodiment bispecific and multispecific molecules of the invention comprise a binding specificity for an FcαR or an FcγR present on the surface of an effector cell, and a second binding specificity for a target cell antigen, e.g., EGFR.
In one embodiment, the binding specificity for an Fc receptor is provided by a human monoclonal antibody, the binding of which is not blocked by human immunoglobulin G (IgG). As used herein, the term “IgG receptor” refers to any of the eight γ-chain genes located on chromosome 1. These genes encode a total of twelve transmembrane or soluble receptor isoforms which are grouped into three Fcγ receptor classes: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). In one preferred embodiment, the Fcγ receptor a human high affinity FcγRI. The human FcγRI is a 72 kDa molecule, which shows high affinity for monomeric IgG (108-109 MA).
The production and characterization of these preferred monoclonal antibodies are described by Fanger et al. in PCT application WO 88/00052 and in U.S. Pat. No. 4,954,617, the teachings of which are fully incorporated by reference herein. These antibodies bind to an epitope of FcγRI, FcγRII or FcγRIII at a site which is distinct from the Fcγ binding site of the receptor and, thus, their binding is not blocked substantially by physiological levels of IgG. Specific anti-FcγRI antibodies useful in this invention are MAb 22, MAb 32, MAb 44, MAb 62 and MAb 197. The hybridoma producing MAb 32 is available from the American Type Culture Collection, ATCC Accession No. HB9469. Anti-FcγRI MAb 22, F(ab′)2 fragments of MAb 22, and can be obtained from Medarex, Inc. (Annandale, N.J.). In other embodiments, the anti-Fcγ receptor antibody is a humanized form of monoclonal antibody 22 (H22). The production and characterization of the H22 antibody is described in Graziano, R. F. et al. (1995) J. Immunol. 155 (10): 4996-5002 and PCT/US93/10384. The H22 antibody producing cell line was deposited at the American Type Culture Collection on Nov. 4, 1992 under the designation HA022CL1 and has the accession no. CRL 11177.
In still other preferred embodiments, the binding specificity for an Fc receptor is provided by an antibody that binds to a human IgA receptor, e.g., an Fc-alpha receptor (FcαRI (CD89)), the binding of which is preferably not blocked by human immunoglobulin A (IgA). The term “IgA receptor” is intended to include the gene product of one α-gene (FcαRI) located on chromosome 19. This gene is known to encode several alternatively spliced transmembrane isoforms of 55 to 110 kDa. FcαRI (CD89) is constitutively expressed on monocytes/macrophages, eosinophilic and neutrophilic granulocytes, but not on non-effector cell populations. FcαRI has medium affinity (≈5×107 M−1) for both IgA1 and IgA2, which is increased upon exposure to cytokines such as G-CSF or GM-CSF (Morton, H. C. et al. (1996) Critical Reviews in Immunology 16:423-440). Four FcαRI-specific monoclonal antibodies, identified as A3, A59, A62 and A77, which bind FcαRI outside the IgA ligand binding domain, have been described (Monteiro, R. C. et al., 1992, J. Immunol. 148:1764).
FcαRI and FcγRI are preferred trigger receptors for use in the invention because they are (1) expressed primarily on immune effector cells, e.g., monocytes, PMNs, macrophages and dendritic cells; (2) expressed at high levels (e.g., 5,000-100,000 per cell); (3) mediators of cytotoxic activities (e.g., ADCC, phagocytosis); (4) mediate enhanced antigen presentation of antigens, including self-antigens, targeted to them.
In other embodiments, bispecific and multispecific molecules of the invention further comprise a binding specificity which recognizes, e.g., binds to, a target cell antigen, e.g., EGFR. In a preferred embodiment, the binding specificity is provided by a human monoclonal antibody of the present invention.
An “effector cell specific antibody” as used herein refers to an antibody or functional antibody fragment that binds the Fc receptor of effector cells. Preferred antibodies for use in the subject invention bind the Fc receptor of effector cells at a site which is not bound by endogenous immunoglobulin.
As used herein, the term “effector cell” refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, e.g., lymphocytes (e.g., B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Some effector cells express specific Fc receptors and carry out specific immune functions. In preferred embodiments, an effector cell is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC. For example, monocytes, macrophages, which express FcR are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. In other embodiments, an effector cell can phagocytose a target antigen, target cell, or microorganism. The expression of a particular FcR on an effector cell can be regulated by humoral factors such as cytokines. For example, expression of FcγRI has been found to be up-regulated by interferon gamma (IFN-γ). This enhanced expression increases the cytotoxic activity of FcγRI-bearing cells against targets. An effector cell can phagocytose or lyse a target antigen or a target cell.
“Target cell” shall mean any undesirable cell in a subject (e.g., a human or animal) that can be targeted by a composition (e.g., a human monoclonal antibody, a bispecific or a multispecific molecule) of the invention. In preferred embodiments, the target cell is a cell expressing or overexpressing EGFR. Cells expressing EGFR typically include tumor cells, such as bladder, breast, colon, kidney, ovarian, prostate, renal cell, squamous cell, lung (non-small cell), and head and neck tumor cells. Other EGFR-expressing cells include synovial fibroblast cells and keratinocytes which can be used as targets in the treatment of arthritis and psoriasis, respectively.
While human monoclonal antibodies are preferred, other antibodies which can be employed in the bispecific or multispecific molecules of the invention are murine, chimeric and humanized monoclonal antibodies.
Chimeric mouse-human monoclonal antibodies (i.e., chimeric antibodies) can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted. (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).
The chimeric antibody can be further humanized by replacing sequences of the Fv variable region which are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General reviews of humanized chimeric antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207 and by Oi et al., 1986, BioTechniques 4:214. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from 7E3, an anti-GPIIbIIIa antibody producing hybridoma. The recombinant DNA encoding the chimeric antibody, or fragment thereof, can then be cloned into an appropriate expression vector. Suitable humanized antibodies can alternatively be produced by CDR substitution U.S. Pat. No. 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; and Beidler et al. 1988 J. Immunol. 141:4053-4060.
All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to the Fc receptor.
An antibody can be humanized by any method, which is capable of replacing at least a portion of a CDR of a human antibody with a CDR derived from a non-human antibody. Winter describes a method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987), the contents of which is expressly incorporated by reference. The human CDRs may be replaced with non-human CDRs using oligonucleotide site-directed mutagenesis as described in International Application WO 94/10332 entitled,
Humanized Antibodies to Fc Receptors for Immunoglobulin G on Human Mononuclear Phagocytes.
Also within the scope of the invention are chimeric and humanized antibodies in which specific amino acids have been substituted, deleted or added. In particular, preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, in a humanized antibody having mouse CDRs, amino acids located in the human framework region can be replaced with the amino acids located at the corresponding positions in the mouse antibody. Such substitutions are known to improve binding of humanized antibodies to the antigen in some instances. Antibodies in which amino acids have been added, deleted, or substituted are referred to herein as modified antibodies or altered antibodies.
The term modified antibody is also intended to include antibodies, such as monoclonal antibodies, chimeric antibodies, and humanized antibodies which have been modified by, e.g., deleting, adding, or substituting portions of the antibody. For example, an antibody can be modified by deleting the constant region and replacing it with a constant region meant to increase half-life, e.g., serum half-life, stability or affinity of the antibody. Any modification is within the scope of the invention so long as the bispecific and multispecific molecule has at least one antigen binding region specific for an FcγR and triggers at least one effector function.
Bispecific and multispecific molecules of the present invention can be made using chemical techniques (see e.g., D. M. Kranz et al. (1981) Proc. Natl. Acad. Sci. USA 78:5807), “polydoma” techniques (See U.S. Pat. No. 4,474,893, to Reading), or recombinant DNA techniques.
In particular, bispecific and multispecific molecules of the present invention can be prepared by conjugating the constituent binding specificities, e.g., the anti-FcR and anti-EGFR binding specificities, using methods known in the art and described in the examples provided herein. For example, each binding specificity of the bispecific and multispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-5-acetyl-thioacetate (SATA), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus (Behring Ins. Mitt. (1985) No. 78, 118-132); Brennan et al. (Science (1985) 229:81-83), and Glennie et al. (J. Immunol. (1987) 139: 2367-2375). Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).
When the binding specificities are antibodies (e.g., two humanized antibodies), they can be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.
Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific and multispecific molecule is a MAb×MAb, MAb×Fab, Fab×F(ab′)2 or ligand×Fab fusion protein. A bispecific and multispecific molecule of the invention, e.g., a bispecific molecule can be a single chain molecule, such as a single chain bispecific antibody, a single chain bispecific molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific and multispecific molecules can also be single chain molecules or may comprise at least two single chain molecules. Methods for preparing bi- and multispecific molecules are described for example in U.S. Pat. No. 5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat. No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S. Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No. 5,482,858.
Binding of the bispecific and multispecific molecules to their specific targets can be confirmed by enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), FACS analysis, a bioassay (e.g., growth inhibition), or a Western Blot Assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, the FcR-antibody complexes can be detected using e.g., an enzyme-linked antibody or antibody fragment which recognizes and specifically binds to the antibody-FcR complexes. Alternatively, the complexes can be detected using any of a variety of other immunoassays. For example, the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a γ counter or a scintillation counter or by autoradiography.
IV. Antibody Conjugates/Immunotoxins
In another aspect, the present invention features a human anti-EGFR monoclonal antibody, or a fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug or a radioisotope. When conjugated to a cytotoxin, these antibody conjugates are referred to as “immunotoxins.” A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). Other examples of therapeutic cytotoxins that can be conjugated to an antibody of the invention include calicheamicin and duocarmycin. An antibody of the present invention can be conjugated to a radioisotope, e.g., radioactive iodine, to generate cytotoxic radiopharmaceuticals for treating a EGFR-related disorder, such as a cancer
The antibody conjugates of the invention can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-γ; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58 (1982).
V. Pharmaceutical Compositions
In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, containing one or a combination of human monoclonal antibodies, or antigen-binding portion(s) thereof, of the present invention, formulated together with a pharmaceutically acceptable carrier. In a preferred embodiment, the compositions include a combination of multiple (e.g., two or more) isolated human antibodies or antigen-binding portions thereof of the invention. Preferably, each of the antibodies or antigen-binding portions thereof of the composition binds to a distinct, pre-selected epitope of EGFR.
In one embodiment, human anti-EGFR monoclonal antibodies having complementary activities are used in combination, e.g., as a pharmaceutical composition, comprising two or more human anti-EGFR monoclonal antibodies. For example, a human monoclonal antibody that mediates highly effective killing of target cells in the presence of effector cells can be combined with another human monoclonal antibody that inhibits the growth of cells expressing EGFR.
In another embodiment, the composition comprises one or a combination of bispecific or multispecific molecules of the invention (e.g., which contains at least one binding specificity for an Fc receptor and at least one binding specificity for EGFR).
Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e., combined with other agents. For example, the combination therapy can include a composition of the present invention with at least one anti-tumor agent or other conventional therapy.
As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, bispecific and multispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
A “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
A composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
For the therapeutic compositions, formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 percent to about ninety-nine percent of active ingredient, preferably from about 0.1 percent to about 70 percent, most preferably from about 1 percent to about 30 percent.
Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forms for the topical or transdermal administration of compositions of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.01 to 99.5% (more preferably, 0.1 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a compositions of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the site of the target. If desired, the effective daily dose of a therapeutic compositions may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).
Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.
In certain embodiments, the human monoclonal antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134), different species of which may comprise the formulations of the inventions, as well as components of the invented molecules; p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273. In one embodiment of the invention, the therapeutic compounds of the invention are formulated in liposomes; in a more preferred embodiment, the liposomes include a targeting moiety. In a most preferred embodiment, the therapeutic compounds in the liposomes are delivered by bolus injection to a site proximal to the tumor or infection. The composition must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
A “therapeutically effective dosage” preferably inhibits tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. The ability of a compound to inhibit cancer can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
The composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
When the active compound is suitably protected, as described above, the compound may be orally administered, for example, with an inert diluent or an assimilable edible carrier.
VI. Uses and Methods of the Invention
The compositions (e.g., human monoclonal antibodies to EGFR and derivatives/conjugates thereof) of the present invention have in vitro and in vivo diagnostic and therapeutic utilities. For example, these molecules can be administered to cells in culture, e.g., in vitro or ex vivo, or in a subject, e.g., in vivo, to treat, prevent or diagnose a variety of disorders. As used herein, the term “subject” is intended to include human and non-human animals. Preferred human animals include a human patient having disorder characterized by expression, typically aberrant expression (e.g., overexpression) of EGFR. For example, the methods and compositions of the present invention can be used to treat a subject with a tumorigenic disorder, e.g., a disorder characterized by the presence of tumor cells expressing EGFR including, for example, bladder, breast, colon, kidney, ovarian, prostate, renal cell, squamous cell, lung (non-small cell), and head and neck tumor cells. The methods and compositions of the present invention can be also be used to treat other disorders, e.g., autoimmune diseases, cancer, psoriasis, or inflammatory arthritis, e.g., rheumatoid arthritis, systemic lupus erythematosus-associated arthritis, or psoriatic arthritis. The term “non-human animals” of the invention includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.
The compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can be initially tested for binding activity associated with therapeutic or diagnostic use in vitro. For example, compositions of the invention can be tested using the ELISA and flow cytometric assays described in the Examples below. Moreover, the activity of these molecules in triggering at least one effector-mediated effector cell activity, including cytolysis of cells expressing EGFR can be assayed. Protocols for assaying for effector cell-mediated phagocytosis are described in the Examples below.
The compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention have additional utility in therapy and diagnosis of EGFR-related diseases. For example, the human monoclonal antibodies, the multispecific or bispecific molecules can be used, for example, to elicit in vivo or in vitro one or more of the following biological activities: to opsonize a cell expressing EGFR; to mediate phagocytosis or cytolysis of a cell expressing EGFR in the presence of human effector cells; to inhibit EGF or TGF-α induced autophosphorylation in a cell expressing EGFR; to inhibit autocrine EGF or TGF-α-induced activation of a cell expressing EGFR; or to inhibit the growth of a cell expressing EGFR, e.g., at low dosages.
In another embodiment, the human monoclonal antibodies of the present invention are unable to induce complement-mediated lysis of cells and, therefore, has fewer side effects in triggering complement-activated afflictions, e.g., acne. The primary cause of acne is an alteration in the pattern of keratinization within the follicle that produce sebum. Since keratinocytes express EGFR, interference with EGFR signaling processes in the skin can alter the growth and differentiation of the keratinocytes in the follicles which results in the formation of acne. Direct immunofluorescent studies have shown that in early non-inflamed and inflamed acne lesions there is activation of the classical and alternative complement pathways.
In a particular embodiment, the human antibodies and derivatives thereof are used in vivo to treat, prevent or diagnose a variety of EGFR-related diseases. Examples of EGFR-related diseases include a variety of cancers, such as bladder, breast, uterine/cervical, colon, pancreatic, colorectal, kidney, stomach, ovarian, prostate, renal cell, squamous cell, lung (non-small cell), esophageal, and head and neck cancer.
In another aspect the invention relates to a method of treating or preventing psoriasis, rheumatoid arthritis, systemic lupus erythematosus, psoriatic arthritis, Menetrier's disease, systemic sclerosis, Sjögren's syndrome, pulmonary fibrosis, bronchial asthma, myelofibrosis, diabetic nephropathy, chronic allograft rejection, chronic glomerulonephritis, Crohn's disease, ulcerative colitis, hepatic cirrhosis, sclerosing cholangitis, chronic uveitis, or cicatricial pemphigoid, as well as methods of treating or preventing Alzheimer's disease or other forms of dementia.
Methods of administering the compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention are known in the art. Suitable dosages of the molecules used will depend on the age and weight of the subject and the particular drug used. The molecules can be coupled to radionuclides, such as 131I, 90Y, 105Rh, indium-111, etc., as described in Goldenberg, D. M. et al. (1981) Cancer Res. 41: 4354-4360, and in EP 0365 997. In another aspect the invention relates to an immunoconjugate comprising an antibody according to the invention linked to a radioisotope, cytotoxic agent (e.g., calicheamicin and duocarmycin), a cytostatic agent, or a chemotherapeutic drug. The compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can also be coupled to anti-infectious agents.
In another embodiment, the human anti-EGFR antibodies, or antigen binding fragments thereof, can be co-administered with a therapeutic agent, e.g., a chemotherapeutic agent, an immunosuppressive agent, an ant-inflammatory agent, or an ant-psoriasis agent, or can be co-administered with other known therapies, such as physical therapies, e.g., radiation therapy, hyperthermia, transplantation (e.g., bone marrow transplantation), surgery, sunlight, or phototherapy. Such therapeutic agents include, among others, anti-neoplastic agents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, and cyclophosphamide hydroxyurea which, by themselves, are only effective at levels which are toxic or subtoxic to a patient. Cisplatin is intravenously administered as a 100 mg/m2 dose once every four weeks and adriamycin is intravenously administered as a 60-75 mg/m2 dose once every 21 days.
Pharmaceutical compositions of the present invention can include one or more further chemotherapeutic agents selected from the group consisting of nitrogen mustards (e.g., cyclophosphamide and ifosfamide), aziridines (e.g., thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine and streptozocin), platinum complexes (e.g., carboplatin and cisplatin), non-classical alkylating agents (e.g., dacarbazine and temozolamide), folate analogs (e.g., methotrexate), purine analogs (e.g., fludarabine and mercaptopurine), adenosine analogs (e.g., cladribine and pentostatin), pyrimidine analogs (e.g., fluorouracil (alone or in combination with leucovorin) and gemcitabine), substituted ureas (e.g., hydroxyurea), antitumor antibiotics (e.g., bleomycin and doxorubicin), epipodophyllotoxins (e.g., etoposide and teniposide), microtubule agents (e.g., docetaxel and paclitaxel), camptothecin analogs (e.g., irinotecan and topotecan), enzymes (e.g., asparaginase), cytokines (e.g., interleukin-2 and interferon-α), monoclonal antibodies (e.g., trastuzumab and bevacizumab), recombinant toxins and immunotoxins (e.g., recombinant cholera toxin-B and TP-38), cancer gene therapies, physical therapies (e.g., hyperthermia, radiation therapy, and surgery) and cancer vaccines (e.g., vaccine against telomerase).
In another aspect the pharmaceutical composition comprises one or more further therapeutic agents selected from the group consisting of immunosuppressive antibodies (e.g., antibodies against MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40, CD45, IFN-γ TNF-α, IL-4, IL-5, IL-6R, IL-7, IL-8, IL-10, CD11a, CD20, or CD58, or antibodies against their ligands) and other immunomodulatory compounds (e.g., soluble IL-15R or IL-10).
In another aspect the pharmaceutical composition comprises one or more further immunosuppressive agents selected from the group consisting of cyclosporine, azathioprine, mycophenolic acid, mycophenolate mofetil, corticosteroids (e.g., prednisone), methotrexate, gold salts, sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine, 15-deoxyspergualine, 6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, anti-thymocyte globulin.
In another aspect the pharmaceutical composition comprises one or more further anti-inflammatory agents selected from the group consisting of aspirin and other salicylates, steroidal drugs, NSAIDs (nonsteroidal anti-inflammatory drugs) (e.g., ibuprofen, fenoprofen, naproxen, sulindac, diclofenac, piroxicam, ketoprofen, diflunisal, nabumetone, etodolac, oxaprozin, and indomethacin), Cox-2 inhibitors (e.g., rofecoxib and celecoxib), and DMARDs (disease modifying antirheumatic drugs) (e.g., methotrexate, hydroxychloroquine, sulfasalazine, azathioprine, pyrimidine synthesis inhibitors (e.g., leflunomide), IL-1 receptor blocking agents (e.g., anakinra), TNF-α, blocking agents (e.g., etanercept, infliximab and adalimumab), anti-IL-6R antibodies, CTLA4Ig, and anti-IL-15 antibodies).
In another aspect the pharmaceutical composition comprises one or more further anti-psoriasis agents selected from the group consisting of coal tar, A vitamin, anthralin, calcipotrien, tarazotene, corticosteroids, methotrexate, retinoids (e.g., acitretin), cyclosporine, etanercept, alefacept, efaluzimab, 6-thioguanine, mycophenolate mofetil, tacrolimus (FK-506), and hydroxyurea.
Co-administration of the human anti-EGFR antibodies, or antigen binding fragments thereof, of the present invention with chemotherapeutic agents provides two anti-cancer agents which operate via different mechanisms which yield a cytotoxic effect to human tumor cells. Such co-administration can solve problems due to development of resistance to drugs or a change in the antigenicity of the tumor cells which would render them unreactive with the antibody.
Target-specific effector cells, e.g., effector cells linked to compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can also be used as therapeutic agents. Effector cells for targeting can be human leukocytes such as macrophages, neutrophils or monocytes. Other cells include eosinophils, natural killer cells and other IgG- or IgA-receptor bearing cells. If desired, effector cells can be obtained from the subject to be treated. The target-specific effector cells, can be administered as a suspension of cells in a physiologically acceptable solution. The number of cells administered can be in the order of 108-109 but will vary depending on the therapeutic purpose. In general, the amount will be sufficient to obtain localization at the target cell, e.g., a tumor cell expressing EGFR, and to effect cell killing by, e.g., phagocytosis. Routes of administration can also vary.
Therapy with target-specific effector cells can be performed in conjunction with other techniques for removal of targeted cells. For example, anti-tumor therapy using the compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention and/or effector cells armed with these compositions can be used in conjunction with chemotherapy. Additionally, combination immunotherapy may be used to direct two distinct cytotoxic effector populations toward tumor cell rejection. For example, anti-EGFR antibodies linked to anti-Fc-gammaRI or anti-CD3 may be used in conjunction with IgG- or IgA-receptor specific binding agents.
Bispecific and multispecific molecules of the invention can also be used to modulate FcαR or FcγR levels on effector cells, such as by capping and elimination of receptors on the cell surface. Mixtures of anti-Fc receptors can also be used for this purpose.
The compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention which have complement binding sites, such as portions from IgG1, -2, or -3 or IgM which bind complement, can also be used in the presence of complement. In one embodiment, ex vivo treatment of a population of cells comprising target cells with a binding agent of the invention and appropriate effector cells can be supplemented by the addition of complement or serum containing complement. Phagocytosis of target cells coated with a binding agent of the invention can be improved by binding of complement proteins. In another embodiment target cells coated with the compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can also be lysed by complement. In yet another embodiment, the compositions of the invention do not activate complement.
The compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can also be administered together with complement. Accordingly, within the scope of the invention are compositions comprising human antibodies, multispecific or bispecific molecules and serum or complement. These compositions are advantageous in that the complement is located in close proximity to the human antibodies, multispecific or bispecific molecules. Alternatively, the human antibodies, multispecific or bispecific molecules of the invention and the complement or serum can be administered separately.
Compositions of the present invention can also include an expression vector comprising a nucleotide sequence encoding the variable region of a light chain, heavy chain or both light and heavy chains of a human antibody which binds EGFR, and further comprising a nucleotide sequence encoding the constant region of a light chain, heavy chain or both light and heavy chains of a human antibody which binds EGFR. In a particular embodiment, the invention relates to a pharmaceutical composition comprising an expression vector comprising a nucleotide sequence encoding heavy chain and light chain variable regions which comprise the amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4, respectively, and conservative sequence modifications thereof.
Also within the scope of the invention are kits comprising the compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention and instructions for use. The kit can further contain a least one additional reagent, such as complement, or one or more additional human antibodies of the invention (e.g., a human antibody having a complementary activity which binds to an epitope in EGFR antigen distinct from the first human antibody).
In other embodiments, the subject can be additionally treated with an agent that modulates, e.g., enhances or inhibits, the expression or activity of Fcα or Fcγ receptors by, for example, treating the subject with a cytokine. Preferred cytokines for administration during treatment with the multispecific molecule include of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumor necrosis factor (TNF).
In another embodiment, the subject can be additionally treated with a lymphokine preparation. Cancer cells which do not highly express EGFR can be induced to do so using lymphokine preparations. Lymphokine preparations can cause a more homogeneous expression of EGFRs among cells of a tumor which can lead to a more effective therapy. Lymphokine preparations suitable for administration include interferon-gamma, tumor necrosis factor, and combinations thereof. These can be administered intravenously. Suitable dosages of lymphokine are 10,000 to 1,000,000 units/patient.
The compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can also be used to target cells expressing FcγR or EGFR, for example for labeling such cells. For such use, the binding agent can be linked to a molecule that can be detected. Thus, the invention provides methods for localizing ex vivo or in vitro cells expressing Fc receptors, such as FcγR, or EGFR. The detectable label can be, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
In one embodiment, the invention provides methods for detecting the presence of EGFR antigen in a sample, or measuring the amount of EGFR antigen, comprising contacting the sample, and a control sample, with a human monoclonal antibody, or an antigen binding portion thereof, which specifically binds to EGFR, under conditions that allow for formation of a complex between the antibody or portion thereof and EGFR. The formation of a complex is then detected, wherein a difference complex formation between the sample compared to the control sample is indicative the presence of EGFR antigen in the sample.
In still another embodiment, the invention provides a method for detecting the presence or quantifying the amount of Fc-expressing cells in vivo or in vitro. The method comprises (i) administering to a subject a composition (e.g., a multi- or bispecific molecule) of the invention or a fragment thereof, conjugated to a detectable marker; (ii) exposing the subject to a means for detecting said detectable marker to identify areas containing Fc-expressing cells.
The present invention is further illustrated by the following examples which should not be construed as further limiting. The contents of all figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.
EXAMPLES
Materials and Methods
Antigen:
Transgenic mice were immunized with the A431 Human epidermoid carcinoma cell line (CRL-1555, Lot 203945, ATCC Manassas, Va.) and with soluble epidermal growth factor receptor (EGFR) obtained from Sigma Chemical Co (product E 3641 lot 109H4108 and 20K4079). Soluble EGFR was stored at −20° to −80° C. until use.
Media Formulations:
(A) High Glucose DMEM (Mediatech Cellgro #10013) containing 10% FBS, Pennicillin-Streptomycin (Sigma P-7539), and 2-mercaptoethanol (GibcoBRL 21985-023) was used to culture A431 cells and myeloma cells. Additional media supplements were added to the Hybridoma growth media, which included: Origin-Hybridoma Cloning Factor (Igen 21001), OPI supplement (Sigma O-5003), HAT or HT (Sigma H 0262, H 0137). (B) Serum Free Medium contains DMEM, antibiotics and 2-mercaptoethanol only.
Cells for Immunization:
Cells for immunization were grown in DMEM (see above) to confluence on T-75 cell culture flasks, and were harvested with Trypsin EDTA (Cellgrow, Cat #25-053-Cl) solution 5-10 ml per flask. Cells recovered from flasks were resuspended in 50 ml of complete medium and then washed by three cycles of centrifugation (1000 G) and resuspended in 50 ml of sterile PBS. Mice were injected with 1×107 cells suspended in 0.5 ml of sterile PBS.
EGFR:
Soluble EGFR was mixed with Ribi adjuvant (Sigma, M 6536) in sterile PBS at a concentration of 25 μg EGFR/100 μl. Final tail vein immunizations were performed with soluble EGFR in sterile PBS.
Transgenic Mice:
Mice were housed in filter cages and were evaluated to be in good physical condition on the date of the fusion. Mice that produced the selected hybridomas were males 6-8 weeks old of the (CMD)++; (HCo7)11952+; (JKD)++; (KCoS)9272+ genotype (see Table 1).
TABLE 1
Genotype Data*
Mouse
Sex
Born
Genotype
20241
Male
Sep. 21, 1999
CMD++
(HCo7) 11952 +
(JKD) ++
(KCo5) 9272
20242
Male
Sep. 21, 1999
CMD++
(HCo7) 11952 +
(JKD) ++
(KCo5) 9272
20243
Male
Sep. 21, 1999
CMD++
(HCo7) 11952 +
(JKD) ++
(KCo5) 9272
*Individual transgene designations are in parentheses, followed by line numbers for randomly integrated transgenes. The symbol ++ and + indicate homozygous or hemizygous; however, because the mice are routinely screened using a PCR-based assay that does not allow us to distinguish between heterozygosity and homozygosity for the randomly integrated human Ig transgenes, a + designation may be given to mice that are actually homozygous for these elements.
Antibodies:
The following anti-EGFR MAbs were used in vitro and in vivo: 2F8 (also referred to as “Humax-EGFR”), a human IgG1 anti-EGFR antibody (Genmab, Utrecht, The Netherlands); the hybridoma producing m225, a mouse IgG2a anti-EGFR antibody, was obtained from American Type Culture Collection (ATCC, Rockville, Md., HB-8508); irrelevant human IgG isotype control (Genmab) which was used as an irrelevant IgG1 antibody; and fluorescein isothiocynate (FITC)-conjugated F(ab′)2 fragment of goat anti-mouse IgG (H+L) which was used as the secondary antibody for indirect immunofluorescence (Protos, San Francisco, Calif.), FITC-conjugated F(ab′)2 rabbit-α-human IgG (DAKO, Glostrup, Denmark).
The 2F8 hybridoma was cultured in DMEM (Gibco BRL, Life Technologies, Paisley, Scotland) supplemented with 10% Fetal bovine serum (FBS) (Hyclone, Logan, Utah) and 100 U/ml penicillin and 100 U/ml streptomycin (both Gibco BRL) (pen/strep). The m225 hybridoma was cultured in RPMI1640 (Gibco BRL) supplemented with 15% FBS (Hyclone) and pen/strep (both Gibco BRL). All cell lines were kept at 37° C. in humidified atmosphere containing 5% carbon dioxide. Humax antibody was purified using protein-A affinity chromatography followed by size exclusion on a HR200 column (Pharmacia, New Jersey). Mouse antibodies were purified using protein-G chromatography followed by size exclusion on a HR200 column. The purity of all antibodies was >95% as determined by dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). F(ab′) fragments were made via pepsin or β-mercaptoethanol treatment followed by protein-A/G purification. Isolated F(ab′) fragments were >95% pure as determined by SDS-PAGE.
Cell Lines:
A431, an epidermoid carcinoma which highly over-expresses EGFR, was obtained from the ATCC (Rockville, Md., CRL-155). The cells were cultured in RPMI 1640 medium (Gibco BRL), supplemented with 10% heat-inactivated FBS (Hyclone), 50 μg/ml streptomycin, 50 IU/ml penicillin, and 4 mM L-glutamine (all Gibco BRL). As the cells grow adherent they were detached by using trypsin-EDTA in PBS (Life Technologies, Paisley, Scotland). In tumor models, the cells are always used in log-phase. The cells are tested for stable EGFR expression and mycoplasma contamination before each experiment.
Fusion Procedure:
Spleens were aseptically harvested from freshly euthanized mice, and placed in 20-30 ml cold serum free media (SFM) in a petri plate. Adherent tissue was removed and spleens were rinsed twice in SFM. Spleen cells were gently harvested by homogenization in a tissue grinder in SFM.
Cells were centrifuged at 1000 g for 10 minutes and the red blood cells in the cell pellet were lysed by suspending the spleen cell pellet in 5 ml of ice cold 0.17 M NH4Cl for 2-5 minutes. The cell mix was then diluted with 20 ml of SFM and centrifuged at 1000 g for 10 minutes. Myeloma cells were harvested into 50 ml centrifuge tubes. Spleen cells and myeloma cells were then washed by three cycles of centrifugation at 1000 g and resuspended in 30-40 ml of SFM.
After a cell count, spleen and myeloma cells were mixed at a 1:1 to 4:1 ratio spleen/myeloma. The spleen cell/myeloma cell mix was pelleted by centrifugation and the supernatant was removed by aspiration. The fusion was done by adding 1-2 ml of PEG solution (Sigma # P-7181) drop wise to the cell pellet over 45 seconds and gently mixing the solution for 75 seconds. The PEG solution was slowly diluted by adding 2 ml SFM drop wise over a minute. This was repeated with another 2 ml of SFM and then the solution was allowed to stand for 1 minute.
The solution was then slowly diluted with an additional 30 ml of SFM over 90 seconds. Cells were centrifuged at 1000 g and for 10 minutes and resuspended in 30 Ml of HAT medium. The fusion mix was diluted to 200-300 ml in HAT supplemented medium containing 3% Origin Hybridoma Cloning Factor, and dispersed into 96 well plates at >200 μl/well (˜10-15 plates/spleen). Hybridoma plates were examined after 3-7 days for hybridomas. Plates were fed at 7 days by replacing half the medium in each well with fresh HT medium supplemented with 3% Origin. Plates were fed every 3-4 days thereafter with HT Medium.
ELISA Reagents:
1. Phosphate buffered saline (PBS), D-PBS without Ca and Mg, Hyclone D-PBS # SH30013.03, or Sigma P 3813.
2. PBS-T (wash buffer), PBS containing 0.05% Tween 20, Sigma P-1379.
3. PBS-T plus 1% BSA (Sigma A 9647). This serves as the blocking buffer and sample buffer.
4. ELISA plates, Nunc Immuno-plate F96 Maxisorp 442404.
5. Anti-human IgG γ-chain specific antibody, Jackson ImmunoResearch #109-006-098.
6. Alkaline phosphatase labeled Goat anti-human γ-chain specific IgG, Jackson Immuno Research #109-056-098. An alternate is to use alkaline phosphatase labeled anti-human κ, Sigma A 3813.
7. Alkaline phosphatase labeled anti-human IgG1, or IgG3, Southern Biotechnology #9050-04 & 9210-04, for use in isotype specific ELISA.
8. p nitrophenyl (pNPP), Sigma N2765, or Sigma Fast tablet kit N-2770.
9. pNPP Substrate and buffer—Two options:
A. Diethanolamine buffer: Mix 97 ml of diethanolamine, Sigma D-2286, plus 0.1 g MgCl26H2O and 800 ml Di Water. Adjust the pH to 9.8 and adjust final volume to 1.0 L with Di water. Add one 20 mg tablet of pNPP, Sigma N-2765, per 20 ml diethanolamine buffer.
B. Sigma Fast pNPP Tablet Set, Sigma N-2770: Dissolve 1 buffer tablet and 1 pNPP tablet in 20 ml H2O.
10. ELISA plate reader with 405 nm filter.
11. Epidermal growth factor receptor (EGFR), Sigma E 3641, Biotin labeled EGF (EGF-B), Molecular Probes E-3477.
12. Biotin labeled anti EGFR MAbs or human antibodies.
13. Nonspecific human antibodies for negative controls, or purified human IgG1 κ, Sigma 1-13889.
14. Automated ELISA plate washer: Titertek MAP C.
Anti Human IgG, κ ELISA:
To screen hybridoma plates for human IgG, κ producing MAbs, ELISA plates were coated with 1 μg/ml of anti-human IgG γ-chain specific antibody, Jackson ImmunoResesarch #109-006-098, overnight or longer at 4° C. Plates were washed in a plate washer and 100 μl/well PBS-T plus 1% BSA was added. Plates were incubated at least 15 minutes and 10-50 μl of cell culture supernatant was added to ELISA plate wells with a few wells in each plate included with IgG as a positive control and cell culture medium as a negative control. Plates were incubated 1-2 hr at room temperature, washed, and alkaline phosphatase labeled anti-Human κ antibody (Sigma A-3813) 1:5000 in PBS-T plus 1% BSA was added. Plates were incubated for 1 hr at room temperature, washed 4 times in a plate washer, and pNPP substrate was added. Plates were incubated 10-60 minutes and absorbance was read at 405 nm in an ELISA plate reader.
ELISA Procedure for Testing Specificity of Anti-EGFR Human Antibodies—Direct Binding of Antibody to EGFR Coated ELISA Plates:
To verify that anti-EGFR antibodies specifically bind to EGFR, Nunc Maxisorp plates were coated with 100 μl/well of EGFR at 0.4 μg/ml in PBS overnight at 4° C. or for 2 hr at room temperature. Plates were washed in PBS-T three times, 100 μl/well of PBS-T plus 1% BSA was added to block nonspecific sites on the plastic surface, and incubated at least 15 minutes before loading samples. Dilutions of samples to be tested were loaded in PBS-T plus 1% BSA. Supernatants were diluted a minimum of 1:3 in PBS-T 1% BSA for loading into ELISA plates. Samples and standards were loaded at 100 μl well, incubated for 1 hr at room temperature, and plates were washed three times in PBS-T. 100 μl/well of PBS-T 1% BSA containing alkaline phosphatase labeled goat antihuman γ specific antibody at a 1:3000 to 1:5000 dilution was added. Alternatively, alkaline phosphatase labeled anti-human κ can be used. Plates were incubated 1 hr at room temp, washed 4 times, and pNPP substrate was added. A absorbance was read at 405 nm.
ELISA Procedure for Testing Specificity of anti-EGFR Human Antibodies—ELISA EGF/EGFR Blocking Assay:
To verify that anti-EGFR antibody binds to EGFR and additionally blocks the binding of biotin labeled epidermal growth factor to the Epidermal growth factor receptor (EGFR), Nunc Maxisorp plates were coated with 100 μl/well of EGFR at 0.4 μg/ml in PBS overnight at 4° C. or for 2 hr at room temperature. Plates were washed in PBS-T three times, 100 μl/well of PBS-T plus 1% BSA was added to block nonspecific sites on the plastic surface, and incubated at least 15 minutes before loading samples. Dilutions of samples to be tested were loaded in PBS-T plus 1% BSA. Supernatants were diluted a minimum of 1:3 in PBS-T 1% BSA for loading into ELISA plates. Samples and standards were loaded at 100 μl well, incubated for 30 minutes at room temperature, and 20 μl/well of Biotin labeled EGF at 0.5 μg/ml was added and plates were incubated for 1 hr (this is added to the sample solution already on the plates). Alternatively, samples can be incubated for 1 hr, washed, and 100 μl/well EGF-biotin at 0.1 μg/ml added and incubated for 1 hr. Plates were washed 3 times, 100 μl/well of PBS-T 1% BSA containing streptavidin alkaline phosphatase at 1:2000 dilution was added, and incubated 1 hr. Plates were washed 4 times, pNPP substrate was added, and absorbance was read at 405 nm.
Competitive ELISA for Determining Epitope Specificity of Anti-EGFR Human Antibodies—Competition with Commercial Murine MAbs 225, 528, AB5, and 29.1:
This assay was performed to determine which MAbs are most like antibodies 225, 528, AB5 and 29.1. MAbs 225, 528, and AB5 block EGF binding to its receptor and inhibit in-vivo endogenous tyrosine kinase activity of EGFR. MAb 29.1 is a non blocking MAb that binds to a carbohydrate residue of EGFR. Plates were coated for at least 2 hr at room temperature, or overnight at 4° C., with 0.4 μg/ml of EGFR in PBS and washed and blocked with 100 μl/well of PBS-T 1% BSA. The blocking solution was flicked out and 100 μl/well of PBS-T-1% BSA was added to columns 1-6 on the left side of the plate while an unlabeled mouse MAb at 1 μg/ml (100 μl/well) was added to the right side of the plate in columns 7-12. Plates were incubated at room temperature for 1 hour and 25 μl of cell culture supernatant was added to the equivalent position of each half of the plate so that each supernatant is loaded onto one well with PBS-T-1% BSA and one well with mouse MAb. Plates were incubated 1 hr, washed, and alkaline phosphatase labeled anti-Human IgG Fc antibody was added. Plates were incubated 1 hr. Plates were washed and substrate was added. Absorbence was read at 405 nm. The % competition from MAb was determined by the following formula: (OD supernatant without competition−OD supernatant with MAb competition/OD supernatant without competition)×100.
Competitive ELISA for Determining Epitope Specificity of Anti-EGFR Human Antibodies—Competitive ELISA with Biotin Labeled Human Antibodies:
Competitive ELISA assays were also performed to determine the specificity of the anti-EGFR human antibodies. Plates were coated for at least 2 hr at room temperature, or overnight at 4° C., with 0.4 μg/ml of EGFR in PBS. Plates were washed and blocked with 100 μl/well of PBS-T 1% BSA. 50 μl of (10-30 μg/ml) of unlabeled human antibodies or mouse MAbs was added to the top well(s) of the plate column and 50 μl was sequentially transferred and mixed serially down each column to create a three fold dilution series of each antibody. 50 μl was discarded from the bottom well after mixing. Plates were incubated for 1 hr and 20 μl/well of biotinylated anti-EGFR human antibody or unlabeled mouse anti-Human EGFR antibodies was added to the entire plate so that the final concentration of competing antibody was approximately 0.1-0.2 μg/ml. Plates were incubated for 1 hour at room temperature, washed, and 100 μl/well streptavidin alkaline phosphatase (1:2000 in PBS-T-BSA) or Alkaline phosphatase labeled goat anti-Mouse IgG was added. Plates were incubated 1 hour, washed, and substrate was added. Absorbence was read at 405 nm.
FACS Procedure for Testing Specificity of Anti-EGFR Human Antibodies—EGF/EGFR Blocking:
This assay was used to verify that anti-EGFR antibody binds to EGFR on the cell surface, and by doing so, blocks the binding of biotin labeled epidermal growth factor (EGF-B) to the Epidermal growth factor receptor (EGFR). This FACS based method uses the human epidermal carcinoma cell line A431 which expresses about 106 EGFR molecules/cell.
Materials for EGFR FACS Assays:
1. A431 cells (ATCC CRL 1555) confluent in one or more T-175 flasks. A 431 cells are cultured in DMEM plus 10% FCS.
2. Trypsin-EDTA solution, Sigma T-3924.
3. Biotin labeled EGF. Prepare a stock solution of about 5 μg/ml, use 10 μl/well.
4. Round bottomed 96 well plates.
5. PBS, sterile.
6. PBS plus 1% BSA plus 0.02% sodium azide (FACS buffer).
7. PE-labeled Streptavidin, Sigma S 3402. Dilute 1:20 in FACS buffer.
8. PE-labeled or FITC labeled anti human IgG, FC γ specific, Pharminigen 34164X, 34165X.
9. Low speed centrifuge with swinging buckets and adapter for 96 well plates (Beckman).
10. FACS
11. BD FACS tubes.
Procedure: A431 cells were harvested by trypsin EDTA treatment. Medium from tissue culture flask was removed and flask was rinsed briefly with 10-20 ml sterile PBS or HBSS. 5-10 ml of trypsin EDTA was added and flask was returned to incubator for a few minutes. As cells began to detach from the plastic surface, a 10 ml pipette was used to gently syringe the cells from the plastic surface and to generate a single cell suspension without too many cell clumps. Cells were transferred to a 50 ml tube with 20-30 ml of cell culture medium (with FBS), centrifuged for 10 min at 1000 g, and washed twice by centrifugation and resuspension of cells in cold FACS buffer. Cell solution was filtered through a nylon mesh to remove cell clumps (the top of BD FACS tubes are equipped for this). Cells were counted and the volume was adjusted so that there are between 1 to 5×106 cells ml. Cells were dispensed into a round bottom 96 well plate at about 200,000 cells/well and centrifuged for about 1 min at 1000 g and then the liquid was flicked out (cells should remain in well bottom). Plates were kept on ice or at 4° C. In a separate 96-well plate, antibody sample dilutions in FACS buffer was prepared by preparing a three fold dilution series of antibody starting at 10 μg/ml and decreasing to 4.5 ng/ml. 100 μl of each antibody dilution, isotype controls, and buffer controls was added to the round bottom plate. The antibody samples and controls were mixed with the cells and incubated for 30 minutes on ice. 10 μl of biotin labeled EGF was added to the antibody cell solution and incubated an additional 30 minutes. The cells were washed three times by centrifugation and resuspension in FACS buffer. 50 μl well of Streptavadin PE was added, mixed, and incubated for 30 minutes on ice. The cells were washed three times and resuspended in 50 μl FACS buffer. The contents of each well were transferred to a tube containing 300-400 μl FACS buffer. 5000-10000 cells were analyzed in each sample by FACS in the FL-2 channel. MCF versus Antibody concentration was plotted.
Human or Animal Derived Materials:
A431 Human epidermoid carcinoma cell line (CRL-1555, Lot 203945, ATCC Manassas, Va.). Trypsin EDTA (Cellgrow Cat #25-053-Cl). P3 X63 ag8.653 myeloma cell line: ATCC CRL 1580, lot F-15183 Origin-Hybridoma Cloning Factor (Igen 21001). OPI supplement (Sigma O-5003) Fetal bovine serum (SH30071 lot #s ALE10321, and AGH6843) from Hyclone, Logan, Utah. Origen Freeze Medium (Igen, #210002)
ELISA:
For determining the binding of human antibodies to EGFR, an ELISA with EGFR (Sigma, St Louis, M) coated overnight in a concentration of 1 μg/ml in PBS on a 96-wells microtiter plate (Greiner, Frickenhausen, Germany) was used. After blocking the plate with ELISA buffer (PBS/0.05% Tween 20 and 1% chicken serum (Gibco BRL)) at a concentration of 100 μl/well, monoclonal antibody diluted in ELISA buffer was added and incubated for 1 hour at 37° C. The plates were subsequently washed 3 times and incubated with peroxidase labeled goat anti-human IgG Fc specific (Jackson, West Grace, P) for 1 hour at 37° C. The assay was developed with ABTS (Roche Diagnostics, Mannheim, Germany) for 30 minutes. Absorbance was measured with a microplate reader (Biotek, Winooski, Canada) at 415 nm. With regard to blocking studies, the plates were pre-incubated for 10 minutes with 50 μl blocking agent in ELISA buffer before adding 50 μl fully human antibody. For determining human IgG in mouse serum ELISA plates were coated with rabbit anti-human kappa, light chains (DAKO) overnight in PBS in a 96-wells microtiter plate (Greiner). After blocking the plate with ELISA buffer (PBS/0.05% Tween20 and 1% chicken serum) 100 μl/well, mouse serum diluted in ELISA buffer was added and incubated for 1 hour at 37° C. The plates were subsequently washed 3 times and incubated with peroxidase labeled rabbit F(ab′)2 fragments anti human IgG (DAKO) for 1 hour at 37° C. The assay was developed with ABTS (Roche) for 30 minutes. Absorbance was measured with a microplate reader (Biotek) at 415 nm.
Flow Cytometry:
EGFR over expressing tumor cells were incubated with MAb for 30 minutes at 4° C. Cells were washed three times in phosphate buffered saline supplemented with 1% bovine serum albumin (Roche) and 0.01% azide. Counter-staining was performed with FITC-conjugated F(ab′)2 fragments of a goat anti-mouse antibody or with FITC-conjugated F(ab′)2 fragments of a rabbit anti-human IgG antibody. With regard to inhibition experiments, the cells were pre incubated with EGF or TGF-α, for 10 minutes at 4° C. All samples were analyzed on a FACScan flowcytometer (Becton-Dickinson, San Jose, Calif.).
Phosphorylation Studies:
Sub-confluent cultures of A431 cells in 24-wells plates (NUNC, Kamstrup, Denmark) were treated overnight with low serum conditions (0.5%). Different antibody dilutions were added to the wells and incubated for 30 minutes at 37° C. and 5% carbon dioxide. Cells were stimulated with or without 5 ng/ml EGF (Prepotech, Rocky Hill, N.J.) for 5 min at 37° C. and 5% carbon dioxide. Cell extracts were prepared as described by Tomic et al. (Tomic et al, 1995) using 100 μl of lysis buffer per well. Fifty μl of A431 cell extract was analyzed by sodium SDS-PAGE and immunoblotting with anti-phospho-tyrosine antibodies (PY20, Transduction Laboratories, Kentucky), goat anti mouse IgG-HRP antibodies (Transduction Laboratories), and ECL detection. For stimulation with TGF-α (Prepotech, Rocky Hill, N.J.) sub-confluent cultures of A431 cells in 24 well plates (Nunc) were treated overnight with low-serum medium (0.5%). Antibodies were added in a fixed dose of 10 or 0 μg/ml and incubated as described as above. The cells were stimulated with an increasing amount TGF-α. Cells were treated as above.
In Vitro Cell Growth Inhibition:
Cell growth inhibition features of fully human antibodies were evaluated with a non-radioactive inhibition assay. Briefly, 100 μl of 2×104/ml A431 cells was added to flat-bottomed tissue culture plates and placed in a cell culture incubator. After 2 hours 100 μl antibody dilution was added and placed back in the cell culture incubator. The cells were incubated for 6-7 days, supernatants were decanted, and 100 μl 0.25% glutaraldehyde in PBS was added to each well. After incubation for 45 minutes at room temperature the wells were washed two times with demi-water. 50 μl of 1% crystal violet in demi-water was added and incubated for 15 minutes at room temperature. After washing the plate twice with demi-water, the plates were developed with 100% methanol during 30 minutes on a plate shaker. Absorbance was measured with a microplate reader using a 550 nm filter with a 650 nm reference filter. Inhibition is measured in triplicates. Percentage of relative cell proliferation was determined by dividing the average absorbance from the triplicate of a particular antibody concentration by the average absorbance from wells which had no antibody added, then times 100.
Effector Cell Isolation:
Peripheral white blood cells were isolated by a method slightly modified from that described in Repp, et al. (1991) Blood 78: 885-889. Briefly, heparin-anticoagulated blood was layered over a ficoll gradient. After centrifugation, effector cells were harvested from the interphase and the remaining erythrocytes were removed by hypotonic lysis. Cytospin preparations were used to assess the purity of isolated cells which was higher than 95%. The viability of cells, determined by trypan blue exclusion, exceeded 95%.
ADCC Assays:
The capacity of fully human antibodies to lyse tumor cells was evaluated in 51Chromium release assays (Valerius, et al. (1993) Blood, 82: 931-939). Isolated human white blood cells were used as effector source. In brief, tumor targets were incubated with 100 μCi 51Cr for two hours. After a three times wash with culture medium, 5×103 target cells were added to round-bottomed tissue culture plates containing 50 μl of isolated effector cells and sensitizing MAb in different concentrations and diluted in culture medium. The final volume was 200 μl and the effector to target cell ratio (E:T) 80:1. The assays were incubated overnight at 37° C. and stopped by centrifugation. The chromium release was measured in supernatants in triplicates. Percentage of cellular cytotoxity was calculated using the formula:
% Specific Lysis=Experimental cpm−Spontaneous cpm/Maximum cpm−Spontaneous cpm×100
with maximal 51Cr release determined by adding ZAP-oglobin® (10% final concentration) to target cells and basal release measured in the absence of sensitizing antibodies and effector cells. Only very low levels of antibody mediated, non-cellular cytotoxicity (without effector cells) was observed under these assay conditions (<5% specific lysis).
Affinity Measurements Using SPR Technology:
Binding affinity of anti EGFR antibodies was determined using BIAcore 300 (Biacore, Upsula, Sweden). EGFR purified from A431 cells purchased from Sigma was immobilized on a CMS chip according to the manufacturer's instructions. Measurements were done with antibody F(ab′) fragments at different concentrations. Association and dissociation constants were determined using BIAevaluation software (version 3.1).
Mice and Tumor Models:
Nude Balb/c mice (NuNu) were purchased from Harlan (Horst, The Netherlands). All experiments described were performed with female mice of eight to twelve weeks old. Mice were housed in the Transgenic Mouse Facility of the Central Laboratory Animal Facility (Utrecht, The Netherlands) and experiments were approved by the Utrecht University animal ethics committee. When participating in an experiment, mice were checked thrice a week for signs of toxicity and discomfort including level of activity, skin abnormalities, diarrhea, and general appearance. A well-established subcutaneous (s.c.) tumor model was used. Briefly the high EGFR expressing A431 cells were inoculated, on the right side of the mouse, at a dose of 3×106 cells. The tumors grow uniform and can be easily measured by vernier calipers. The tumor volume is reported as length×width×height (in mm3). The monoclonal antibodies were injected intraperitoneally (i.p.) according to the study protocol. The tumor cells were tested for stable EGFR expression after in vivo passage by flow cytometry and immunohistochemistry. In order to determine pharmacokinetics, mice, with and without tumors, were injected i.p. with 2F8 antibody. Weekly blood samples were taken via the tail vein before and for six weeks after the injection. The samples were analyzed by human IgG ELISA.
Statistical Analysis:
Group data are reported as mean±standard error of the mean (SEM). Differences between groups are analyzed by unpaired (or, where appropriate, paired) Student's t-test. Levels of significance are indicated. Significance was accepted at the p<0.05 level.
Example 1
Generation of Cmu Targeted Mice for the Production of Anti-EGFR Human Antibodies, Also Referred to as “HuMabs”
Construction of a CMD Targeting Vector
The plasmid pICEmu contains an EcoRI/XhoI fragment of the murine Ig heavy chain locus, spanning the mu gene, that was obtained from a Balb/C genomic lambda phage library (Marcu et al. Cell 22: 187, 1980). This genomic fragment was subcloned into the XhoI/EcoRI sites of the plasmid pICEMI9H (Marsh et al; Gene 32, 481-485, 1984). The heavy chain sequences included in pICEmu extend downstream of the EcoRI site located just 3′ of the mu intronic enhancer, to the XhoI site located approximately 1 kb downstream of the last transmembrane exon of the mu gene; however, much of the mu switch repeat region has been deleted by passage in E. coli. The targeting vector was constructed as follows. A 1.3 kb HindIII/SmaI fragment was excised from pICEmu and subcloned into HindIII/SmaI digested pBluescript (Stratagene, La Jolla, Calif.). This pICEmu fragment extends from the HindIII site located approximately 1 kb 5′ of Cmu1 to the SmaI site located within Cmu1. The resulting plasmid was digested with SmaI/SpeI and the approximately 4 kb SmaI/XbaI fragment from pICEmu, extending from the SmaI site in Cmu1 3′ to the XbaI site located just downstream of the last Cmu exon, was inserted. The resulting plasmid, pTAR1, was linearized at the SmaI site, and a neo expression cassette inserted. This cassette consists of the neo gene under the transcriptional control of the mouse phosphoglycerate kinase (pgk) promoter (XbaI/TaqI fragment; Adra et al. (1987) Gene 60: 65-74) and containing the pgk polyadenylation site (PvuII/HindIII fragment; Boer et al. (1990) Biochemical Genetics 28: 299-308). This cassette was obtained from the plasmid pKJ1 (described by Tybulewicz et al. (1991) Cell 65: 1153-1163) from which the neo cassette was excised as an EcoRI/HindIII fragment and subcloned into EcoRI/HindIII digested pGEM-7Zf (+) to generate pGEM-7 (KJ1). The neo cassette was excised from pGEM-7 (KJ1) by EcoRI/SalI digestion, blunt ended and subcloned into the SmaI site of the plasmid pTAR1, in the opposite orientation of the genomic Cmu sequences. The resulting plasmid was linearized with Not I, and a herpes simplex virus thymidine kinase (tk) cassette was inserted to allow for enrichment of ES clones bearing homologous recombinants, as described by Mansour et al. (1988) Nature 336: 348-352. This cassette consists of the coding sequences of the tk gene bracketed by the mouse pgk promoter and polyadenylation site, as described by Tybulewicz et al. (1991) Cell 65: 1153-1163. The resulting CMD targeting vector contains a total of approximately 5.3 kb of homology to the heavy chain locus and is designed to generate a mutant mu gene into which has been inserted a neo expression cassette in the unique SmaI site of the first Cmu exon. The targeting vector was linearized with PvuI, which cuts within plasmid sequences, prior to electroporation into ES cells.
Generation and Analysis of Targeted ES Cells
AB-1 ES cells (McMahon, A. P. and Bradley, A., (1990) Cell 62: 1073-1085) were grown on mitotically inactive SNL76/7 cell feeder layers (ibid.) essentially as described (Robertson, E. J. (1987) in Teratocarcinomas and Embryonic Stem Cells: a Practical Approach (E. J. Robertson, ed.) Oxford: IRL Press, p. 71-112). The linearized CMD targeting vector was electroporated into AB-1 cells by the methods described Hasty et al. (Hasty, P. R. et al. (1991) Nature 350: 243-246). Electroporated cells were plated into 100 mm dishes at a density of 1−2×106 cells/dish. After 24 hours, G418 (200 micrograms/ml of active component) and FIAU (5×10−7 M) were added to the medium, and drug-resistant clones were allowed to develop over 8-9 days. Clones were picked, trypsinized, divided into two portions, and further expanded. Half of the cells derived from each clone were then frozen and the other half analyzed for homologous recombination between vector and target sequences.
DNA analysis was carried out by Southern blot hybridization. DNA was isolated from the clones as described Laird et al. (Laird, P. W. et al., (1991) Nucleic Acids Res. 19: 4293). Isolated genomic DNA was digested with SpeI and probed with a 915 by SacI fragment, probe A, which hybridizes to a sequence between the mu intronic enhancer and the mu switch region. Probe A detects a 9.9 kb SpeI fragment from the wild type locus, and a diagnostic 7.6 kb band from a mu locus which has homologously recombined with the CMD targeting vector (the neo expression cassette contains a SpeI site). Of 1132 G418 and FIAU resistant clones screened by Southern blot analysis, 3 displayed the 7.6 kb SpeI band indicative of homologous recombination at the mu locus. These 3 clones were further digested with the enzymes BglI, BstXI, and EcoRI to verify that the vector integrated homologously into the mu gene. When hybridized with probe A, Southern blots of wild type DNA digested with BglI, BstXI, or EcoRI produce fragments of 15.7, 7.3, and 12.5 kb, respectively, whereas the presence of a targeted mu allele is indicated by fragments of 7.7, 6.6, and 14.3 kb, respectively. All 3 positive clones detected by the SpeI digest showed the expected BglI, BstXI, and EcoRI restriction fragments diagnostic of insertion of the neo cassette into the Cmu1 exon.
Generation of Mice Bearing the Mutated Mu Gene
The three targeted ES clones, designated number 264, 272, and 408, were thawed and injected into C57BL/6J blastocysts as described by Bradley (Bradley, A. (1987) in Teratocarcinomas and Embryonic Stem Cells: a Practical Approach. (E. J. Robertson, ed.) Oxford: IRL Press, p. 113-151). Injected blastocysts were transferred into the uteri of pseudopregnant females to generate chimeric mice representing a mixture of cells derived from the input ES cells and the host blastocyst. The extent of ES cell contribution to the chimera can be visually estimated by the amount of agouti coat coloration, derived from the ES cell line, on the black C57BL/6J background. Clones 272 and 408 produced only low percentage chimeras (i.e. low percentage of agouti pigmentation) but clone 264 produced high percentage male chimeras. These chimeras were bred with C57BL/6J females and agouti offspring were generated, indicative of germline transmission of the ES cell genome. Screening for the targeted mu gene was carried out by Southern blot analysis of BglI digested DNA from tail biopsies (as described above for analysis of ES cell DNA). Approximately 50% of the agouti offspring showed a hybridizing BglI band of 7.7 kb in addition to the wild type band of 15.7 kb, demonstrating a germline transmission of the targeted mu gene.
Analysis of Transgenic Mice for Functional Inactivation of Mu Gene
To determine whether the insertion of the neo cassette into Cmu1 has inactivated the Ig heavy chain gene, a clone 264 chimera was bred with a mouse homozygous for the JHD mutation, which inactivates heavy chain expression as a result of deletion of the JH gene segments (Chen et al, (1993) Immunol. 5: 647-656). Four agouti offspring were generated. Serum was obtained from these animals at the age of 1 month and assayed by ELISA for the presence of murine IgM. Two of the four offspring were completely lacking IgM (see Table 2). Genotyping of the four animals by Southern blot analysis of DNA from tail biopsies by BglI digestion and hybridization with probe A (see FIG. 1), and by StuI digestion and hybridization with a 475 bp EcoRI/StuI fragment (ibid.) demonstrated that the animals which fail to express serum IgM are those in which one allele of the heavy chain locus carries the JHD mutation, the other allele the Cmu1 mutation. Mice heterozygous for the JHD mutation display wild type levels of serum Ig. These data demonstrate that the Cmu1 mutation inactivates expression of the mu gene.
TABLE 2
Serum IgM
Mouse
(micrograms/ml)
Ig H chain genotype
42
<0.002
CMD/JHD
43
196
+/JHD
44
<0.002
CMD/JHD
45
174
+/JHD
129 × BL6 F1
153
+/+
JHD
<0.002
JHD/JHD
Table 2 shows the levels of serum IgM, detected by ELISA, for mice carrying both the CMD and JHD mutations (CMD/JHD), for mice heterozygous for the JHD mutation (+/JHD), for wild type (129Sv×C57BL/6J)F1 mice (+/+), and for B cell deficient mice homozygous for the JHD mutation (JHD/JHD).
Example 2
Generation of HCO12 Transgenic Mice for the Production of Anti-EGFR Human Antibodies
The HCO12 Human Heavy Chain Transgene
The HCO12 transgene was generated by coinjection of the 80 kb insert of pHC2 (Taylor et al., 1994, Int. Immunol., 6: 579-591) and the 25 kb insert of pVx6. The plasmid pVx6 was constructed as described below.
An 8.5 kb HindIII/SalI DNA fragment, comprising the germline human VH1-18 (DP-14) gene together with approximately 2.5 kb of 5′ flanking, and 5 kb of 3′ flanking genomic sequence was subcloned into the plasmid vector pSP72 (Promega, Madison, Wis.) to generate the plasmid p343.7.16. A 7 kb BamHI/HindIII DNA fragment, comprising the germline human VH5-51 (DP-73) gene together with approximately 5 kb of 5′ flanking and 1 kb of 3′ flanking genomic sequence, was cloned into the pBR322 based plasmid cloning vector pGP1f (Taylor et al. 1992, Nucleic Acids Res. 20: 6287-6295), to generate the plasmid p251f. A new cloning vector derived from pGP1f, pGP1k, was digested with EcoRV/BamHI, and ligated to a 10 kb EcoRV/BamHI DNA fragment, comprising the germline human VH3-23 (DP47) gene together with approximately 4 kb of 5′ flanking and 5 kb of 3′ flanking genomic sequence. The resulting plasmid, p112.2RR.7, was digested with BamHI/SalI and ligated with the 7 kb purified BamHI/SalI insert of p251f. The resulting plasmid, pVx4, was digested with XhoI and ligated with the 8.5 kb XhoI/SalI insert of p343.7.16.
A clone was obtained with the VH1-18 gene in the same orientation as the other two V genes. This clone, designated pVx6, was then digested with NotI and the purified 26 kb insert coinjected—together with the purified 80 kb NotI insert of pHC2 at a 1:1 molar ratio—into the pronuclei of one-half day (C57BL/6J×DBA/2J)F2 embryos as described by Hogan et al. (B. Hogan et al., Manipulating the Mouse Embryo, A Laboratory Manual, 2nd edition, 1994, Cold Spring Harbor Laboratory Press, Plainview N.Y.). Three independent lines of transgenic mice comprising sequences from both Vx6 and HC2 were established from mice that developed from the injected embryos. These lines are designated (HCO12)14881, (HCO12)15083, and (HCO12)15087. Each of the three lines were then bred with mice comprising the CMD mutation described in Example 1, the JKD mutation (Chen et al. 1993, EMBO J. 12: 811-820), and the (KCoS)9272 transgene (Fishwild et al. 1996, Nature Biotechnology 14: 845-851). The resulting mice express human immunoglobulin heavy and kappa light chain transgenes in a background homozygous for disruption of the endogenous mouse heavy and kappa light chain loci.
Example 3
Production of Human Monoclonal Antibodies Against EGFR
Two different strains of mice were used to generate EGFR reactive human monoclonal antibodies. Strain ((CMD)++; (JKD)++; (HCo7)11952+/++; (KCoS)9272+/++) (referred to herein as “HCO7 mice”, and strain ((CMD)++; (JKD)++; (HCo12)15087+/++; (KCoS)9272+/++) (referred to herein as “HCO12 mice”). Each of these strains are homozygous for disruptions of the endogenous heavy chain (CMD) and kappa light chain (JKD) loci. Both strains also comprise a human kappa light chain transgene (HCo7), with individual animals either hemizygous or homozygous for insertion #11952. The two strains differ in the human heavy chain transgene used. Mice were hemizygous or homozygous for either the HCo7 or the HCo12 transgene. The CMD mutation is described above in Example 1. The generation of (HCo12)15087 mice is described in Example 2. The JKD mutation (Chen et al. 1993, EMBO J. 12: 811-820) and the (KCoS)9272 (Fishwild et al. 1996, Nature Biotechnology 14: 845-851) and (HCo7)11952 mice, are described in U.S. Pat. Nos. 5,770,429 and 5,545,806 (Lonberg & Kay, Jun. 23, 1998).
The immunization schedule used is listed in Table 3 below. Mice were immunized twice with A 431 cells followed by soluble antigen in Ribi Adjuvant. The EGFR specific serum titer was determined by ELISA after the third immunization. Three different immunizations were done for the final boosts before the fusion. These included two or three sequential intravenous (iv) boosts via the tail vein with 10 μg of antigen in 50 μl PBS or two sequential intraperitoneal (i.p.) boosts with 25 μg soluble EGFR in Ribi adjuvant (see Table 3). The three mice that were used in the fusion were part of a larger cohort of mice that included both HCo7 and HCo12 genotypes.
TABLE 3
Immunization Schedule
EGFR
ELISA
in Ribi
ELISA
EGFR
A431 cells
A431 cells
Titer
ip
Titer
Fusion
in RIBI ip
Fusion
Mouse
Day 1
Day 20
Day 30
Day 33
Day 43
Day 46
Day 50
Day 53
20241
2 × 106
1 × 107
0
25 μg
4050
25 μg
Ribi
2 × 25 μg***
20242
2 × 106
1 × 107
0
25 μg
4050
25 μg
2 iv × 10 μg*
20243
2 × 106
1 × 107
450
25 μg
12150
3 iv × 10 μg*
*EGFR in PBS (10 μg) iv on days −4, −3, and −2
**EGFR in PBS (10 μg) iv on day −4, and −3
***EGFR in Ribi (25 μg) i.p. on day −4 and −3
Immunization strategy used for the first two injections, 2−10×106 live A431 cells i.p., resulted in poor anti-EGFR titers (see Table 2). However, when these mice were given a third immunization with 25 μg/mouse of soluble EGFR in Ribi adjuvant, serum titers increased more than 30 fold. These results clearly demonstrate that cells expressing a large amount of EGFR on the cell surface are very effective at initiating a primary immune response that then was greatly enhanced with only one dose of purified antigen in adjuvant.
The final boost before fusion for mouse 20243 was done as i.v. tail vein boosts with 10 μg soluble EGFR in PBS on days −4, −3, and −2. The Triton X-100 in the soluble EGFR caused an irritation to the tail of the mouse. Therefore, to reduce the possibility of irritation, mouse 20242 received only two i.v. vein boosts with soluble EGFR on days −4 and −3, and mouse 20241 received two i.p. immunizations on days −4 and −3 with 25 μg EGFR in Ribi adjuvant. The three fusions resulted in 46 human γ, κ-antigen positive hybridomas (see Table 4). Mouse 20241 alone, which received the i.p. boosts with adjuvant, produced 35 antigen specific Human Gamma Kappa antibodies.
TABLE 4
γ/κ+
γ1κ+
γ3κ+
Mouse
γκ+
EGFR+
EGFR+
EGFR+
20243
120
14
13
1
20242
35
2
2
0
20241
*
30
28
2
Example 4
Hybridoma Preparation
The P3 X63 ag8.653 myeloma cell line (ATCC CRL 1580, lot F-15183) was used for the fusions. The original ATCC vial was thawed and expanded in culture. A seed stock of frozen vials was prepared from this expansion. A fresh vial of cells was thawed one to two weeks before the fusions.
High Glucose DMEM (Mediatech, Cellgro #10013) containing 10% FBS, Pennicillin-Streptomycin (Sigma, P-7539), and 5.5×10−5M 2-mercaptoethanol (GibcoBRL, 21985-023) was used to culture A431 cells and myeloma cells. Additional media supplements were added to the Hybridoma growth media, which included: 3% Origin-Hybridoma Cloning Factor (Igen, 21001), OPI supplement (Sigma, 0-5003), 1.1×10−3 M Oxalo acetic acid, 4.5×104 M sodium Pyruvate, and 24 international units/L bovine Insulin, HAT (Sigma, H 0262) 1.0×104 M Hypoxanthine, 4.0×10−7 M Aminopterin, 1.6×10−5 M Thymidine, or HT (Sigma, H0137) 1.0×104 M Hypoxanthine, 1.6×10−5 M Thymidine.
Characterized Fetal bovine serum (SH30071 lot #s AJE10321 and AGH6843) was obtained from Hyclone, Logan, Utah. Serum Free medium contained DMEM, antibiotics and 2-mercaptoethanol only.
Spleens from all three mice were normal in size and yielded from 2×107 to 1×108 splenocytes. The splenocytes were fused.
The initial ELISA screen for human IgG κ antibodies was performed 7-10 days post fusion. Human IgG, κ positive wells were screened on soluble EGFR coated ELISA plates. Antigen positive hybridomas were transferred to 24 well plates and eventually to tissue culture flasks. EGFR specific hybridomas were subcloned by limiting dilution to assure monoclonality. Antigen positive hybridomas were preserved at several stages in the development process by freezing cells in DMEM 10% FBS plus 10% DMSO (Sigma, D2650) or in Origen Freeze Medium (Igen, #210002). Cells were stored at −80° C. or in LN2.
Initial EGFR specific hybridomas were subsequently evaluated for epitope specificity and their ability to block the binding of EGF to the EGFR receptor. Mouse monoclonal anti-EGFR antibodies 225 and 528 have previously been shown to bind to EGFR, block binding of EGF to EGFR and to be anti-cancer immunotherapeutic agents in animal and human studies. Therefore these antibodies were used, in addition to a non-blocking antibody, in a competitive ELISA format to identify human antibodies that have immunotherapeutic characteristics.
Example 5
Binding Affinity
Binding affinity for hybridoma 2F8 was determined using BIAcore 3000 (Biacore, Upsula, Sweden). EGFR purified from A431 cells purchased from Sigma was immobilized on a CMS chip according to the manufacturer's instructions. Antibody 2F8 had an equilibrium association constant (KA) of 5.47 (±0.52)×108 M−1.
Example 6
Competitive ELISA Assays
Competitive ELISA assays were used as the initial qualifying assay as soon as antigen positive hybridomas were established in 24 well plates. In general, strong competition (80-100%) indicates that an antibody binds to the same epitope or to a region of the antigen in close proximity to the competing antibody. Weaker competition of less than 50% indicates that the antibody and its competitor bind to regions of the antigen not in close proximity. Initial assays were done with supernatants from uncloned hybridomas many of which contained more than one hybridoma per well. Later assays were done with subclones of the original wells. FIGS. 1 and 2 show (the data in FIGS. 1 and 2 are arranged based on degree of competition with MAb 225) that even with crude cell culture supernatants, antibodies can be identified that bind to similar or identical epitopes as the 225 and 528 antibodies. Also evident in this experiment is the different distribution in competitive binding patterns of antibodies derived from mouse 20241 or from mouse 20242 and 20243. For example, the first seven antibodies from the #20241 mouse (FIG. 1) compete strongly with both MAb 225 and 528. The remainder of the antibodies from 20241 competed moderately or weakly with the 225 and 528 antibodies. Five antibodies (1H6, 2F8, 1A8, 5C5, and 8E1) from the 20242 and 20243 mice showed strong competition with antibody 225 and no competition or weak competition from MAb 528 (FIG. 2). Antibodies 2F6, 8A12, 5F12, 6B3, and 6D9 from mouse 20242 and 20243 competed with both MAb 225 and 528, although the competition was stronger against the 225 antibody. Other antibodies from these mice did not compete or were weakly competitive with the commercial MAbs.
These initial competitive ELISA results were verified with purified antibodies produced by sub-cloned cells. FIGS. 3 and 4 show that antibodies 5F12 and 6B3 compete strongly with both MAb 225 and 528 and also demonstrate reciprocal competition with each other. This data indicate that these antibodies bind to the same epitope or to a region of the EGFR molecule in close proximity to the 225 or 528 binding site. Antibody 2F8 competes moderately with MAb 225 and does not significantly compete with antibody 528 (FIGS. 3 and 4). However, antibody 2F8, 6B3 and 5F12 show strong cross competition. This data suggests that antibody 2F8 is binding to a separate epitope from the 225 and 528 antibodies and binds to a region of the EGFR receptor that is adjacent to or overlaps with the epitope to which HuMabs 6B3 and 5F12 bind. Antibodies 2A2 and 6E9 do not compete with either MAb and bind to EGFR epitopes unrelated to the binding sites of the 225 and 528 MAbs (FIGS. 3 and 4).
Example 7
EGF/EGFR Blocking Assays
Antigen positive subclones were further evaluated in EGF/EGFR blocking assays. These assays included subclones of antibodies that compete strongly with MAb 225 and/or 528, as well as, antibodies that are weak or non competitive with 225 or 528. Several antibodies were expanded in culture medium and purified by protein A chromatography. FIGS. 5 and 6 show that antibodies 2F8, 5F12, and 6B3, which are moderate to strong competitors of the 225 antibody in ELISA, are strong blockers of EGF binding to EGFR. This is evident in assays done in ELISA format or by FACS on human A431 epidermoid cancer cells. In both assays, the human antibodies were as good as or better than MAb 225. Antibodies 2F8, 5F12, 6B3, and 6E9 also have similar binding characteristics on the surface of A431 cells (FIG. 7).
The in vitro EGF/EGFR blocking and ELISA competition studies demonstrated that the 2F8, 5F12, and 6B3 antibodies have similar properties to other anti-EGFR murine and human antibodies that have been shown to be immunotherapeutic agents (Sato, et al. (1983) Mol. Biol. Med. 511-529; Gill, et al. (1984) J. of Biol. Chem. 259(12):7755-7760). The 2F8 antibody was equivalent to or better than the 6B3 and 5F12 antibodies overall in the various evaluations.
Example 8
Inhibition of EGF/TGF-α Binding to the EGF Receptor Using Human Monoclonal Antibodies to the EGF Receptor
Inhibition studies were performed on A431 cells using flow cytometry, ELISA, and inhibition of ligand-induced autophosphorylation. Murine MAbs 225 or 525 were used as positive controls. An irrelevant human IgG isotype control, was used as an isotype control. A single human antibody, 2F8, was chosen for all the further studies. This antibody is also referred to herein as “Humax-EGFR™”. FIG. 8 shows the EGF blocking capacity of 2F8 in a concentration dependent manner. 2F8 and m225 block to the same extent while the blocking capacity of EGF is less.
FIG. 9 further shows the blocking capacity of 2F8 in that it efficiently inhibits the binding of EGF and TGF-α to A431 cells (cells derived from an ovarian epidermoid carcinoma and express in excess of 1×106 EGFR molecules on their cell surface). Inhibition of 2F8-binding to A431 cells was determined using flow cytometer analysis. Cells were pre-incubated with either 5 (open bars) or 50 μg/ml (closed bars) ligand before adding 2F8. Binding of antibody without ligand (PBS group) was designated as 100%. These results indicate that 2F8 binds close to, or at the same site, on EGFR as the ligands.
Example 9
Inhibition of Tumor Cell Activation Using Human Monoclonal Antibodies to the EGF Receptor
To evaluate the ability of 2F8 to inhibit tumor cell activation, the effect of 2F8 on EGF-triggered cellular responses, such as activation of the intrinsic tyrosine kinase activity and concomitant cell proliferation, was examined. One of the first events after EGF or TGF-α binding to the EGFR is the induction of autophosphorylation of the receptor. Incubation of EGF with A431 cells results in tyrosine phosphorylation of the EGFR (Mr 170,000) (FIG. 10A). While 2F8 did not activate the receptor kinase activity by itself, the antibody blocked EGF-triggered EGFR tyrosine phosphorylation in a dose-dependent manner with a complete inhibition at a concentration of 16.6 nM (antibody:EGF molar ratio, 20:1, FIG. 10A). Cells were treated with antibody and TGF-α showed that tyrosine phosphorylation was fully blocked by 2F8 at a concentration of 66 nM (antibody: TGF-α molar ratio, 7, 3:1, FIG. 10B).
Engagement of EGF/TGF-α with the receptor results in cell activation which is reflected in cell proliferation. Therefore the inhibitory effect of 2F8 on growth of tumor cells (A431, MDA-MD-468 and HN5 cells) was evaluated. The experiments were carried out in the absence of exogenous EGF. Mouse antibodies were used as a comparison. Humax-EGFR inhibited the growth of A431 cells in a concentration dependent manner with a maximal inhibition of 50%, a level similar to that obtained with mouse antibody 225 (FIG. 14). The control antibody had no effect on the cell proliferation (FIG. 14). Growth inhibition was also obtained with two other cell lines at similar levels (HN5 and MDA-MB468, panels B and C). As no exogenous EGF was added to the culture, these results indicate the ability of 2F8 to block autocrine stimulation and thus to inhibit autocrine EGF/TGF-α induced tumor cell activation.
Example 10
Human Monoclonal Antibodies to the EGF Receptor Induce ADCC
ADCC is a potent immune effector mechanism triggered by the recognition of tumor cells by antibodies. To evaluate the ability of human PMN cells to kill A431 cells in the presence of 2F8, A431 cells were loaded with 51Cr and subsequently incubated with antibody and effector cells (PMN) overnight. After incubation, chromium release was measured. As shown in FIG. 14, 2F8 is capable of inducing ADCC against A431 cells using human PMN. 2F8 is capable of mediating PMN-induced lysis of 45% of the A431 target cells, which is higher then observed with the MAb 425 (FIG. 14).
Importantly, while capable of recruiting immune effector cells and inducing ADCC, 2F8 is unable to induce complement-mediated lysis of tumor cells.
Example 11
Human Monoclonal Antibodies to the EGF Receptor Prevent Tumor Formation
To show the ability of HuMab 2F8 to prevent tumor formation in an athymic murine model, groups of six (6) mice were injected subcutaneously in the flank with 3×106 tumor cells in 200 μl PBS at day zero (0). Subsequently, mice were injected i.p. on days 1 (75 μg/200 μl), 3 (25 μg/200 μl), and 5 (25 μg/200 μl) (arrows) with either HuMab 2F8 (closed squares) i.p. of human IgG1-κ MAb as a control (open circles) (FIG. 14). The data are presented as mean tumor volume+SEM, and are representative of 3 individual experiments, yielding similar results.
Eradication of established A431 tumor xenografts by HuMab 2F8 in comparison to m225 is shown in FIG. 14. Mice were injected subcutaneously in the flank with 3×106 tumor cells in 200 μl PBS on day zero (0). At day 10, mice were randomly allocated to treatment groups and treated on days 12 (75 μg/200 μl), 14 (25 μg/200 μl), and 16 (25 μg/200 μl) (arrows) with HuMab 2F8 (closed squares, 2F8 short-term) or with murine anti-EGFR MAb m225 (closed triangles, m225 short-term). Furthermore, groups were included receiving 75 μg/200 μl HuMab 2F8 or m225 on day 12, continued by 25 μg/200 μl HuMab 2F8 or m225 on days 14, 16, 19, 22, 26, 29, 33, 36, and 40 (open squares, 2F8 long-term; open triangles, m225 long-term). The data are presented as mean tumor volume+SEM, and are representative of 3 individual experiments, yielding similar results. Black arrows indicate treatment days for the short-term treatment, open arrows indicate treatment days for the long-term treatment.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
INCORPORATION BY REFERENCE
All patents, pending patent applications and other publications cited herein are hereby incorporated by reference in their entirety. Any combination of the embodiments disclosed in the dependent claims are contemplated to be within the scope of the invention.
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14055222
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genmab a/s
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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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Apr 1st, 2022 06:04PM
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Apr 1st, 2022 06:04PM
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Genmab A/S
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Health Care
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Pharmaceuticals & Biotechnology
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